Ayman Abou El-Fetouh Gouda

ANALYTICAL STUDIES USING SOME 

PHARMACEUTICAL AND BIOCHEMICAL 

COMPOUNDS 

A Thesis Presented by 

Ayman Abou El-Fetouh Gouda 

B.Sc. (Chemistry) 

Under Supervision of 

Prof. Dr. Ragaa El-Sheikh Shaheeb Prof. Dr. Mounir Zaky Saad 

Prof. of Analytical Chemistry 

Chemistry Department 

Faculty of Science, 

Zagazig University 

Prof. Dr. Faten Zahran Mohammed 

Prof. of Biochemistry 


Faculty of Science 

Zagazig University 

2005 

Prof. of Analytical Chemistry 


Faculty of Science, 

Zagazig University

ACKNOWLEDGMENT 

I am deeply thankful to Allah by the grace whom the progress and 

success of this work possible. 

I am also indebted with sincere gratitude to Prof. Dr. Ragaa El- 

Sheikh Shaheeb, Professor of Analytical Chemistry, Faculty of Science, 

Zagazig University, for suggesting the problem, critical continuous 

supervision and help during the work, discussion and help during the 

scope of this work and in preparing the thesis. 

I wish to express my great indebtedness and sincere gratitude to 

Prof. Dr. Mounir Zaky Saad, Professor of Analytical Chemistry, Faculty 

of Science, Zagazig University, for the critical continuous supervision 

and help during the work. 

My great indebtedness and sincere gratitude are also to Prof. Dr. 

Faten Zahran Mohammed, Professor of Biochemistry, Faculty of 

Science, Zagazig University, for the critical continuous supervision, help 

during the work and preparing the thesis. 

I wish to express my great indebtedness and sincere gratitude to 

Prof. Dr. Alaa El-Sayed Amin, Professor of Analytical Chemistry, 

Faculty of Science, Benha, Zagazig University, for the critical help 

during the work. 

Lastly my thanks to all stuff members in Department of Chemistry, 

Faculty of Science, Zagazig University for their help. 

Ayman Abou El-Fetouh Gouda

ABSTRACT 

Four simple, sensitive and accurate spectrophotometric methods 

have been developed for the determination of dextromethorphan 

hydrobromide (DEX), Ketamine hydrochloride (KET) and Silymarin 

(SIL) in pharmaceutical formulations. These methods are based on the 

formation of ion-pair complexes with bromocresol green (BCG), 

bromocresol purple (BCP), bromothymol blue (BTB) and bromophenol 

blue (BPB). The coloured ion-pair products are extracted into chloroform 

or methylene chloride and measured at 419, 409, 413 and 417 nm in case 

of DEX, 417, 408, 412 and 416 nm in case of KET and the coloured ion- 

pair products are measured at 423, 418, 420 and 422 nm in case of SIL 

for BCG, BCP, BTB and BPB, respectively. The composition of the ion- 

pair were established by the continuous variation and molar ratio 

methods. The proposed methods were applied successfully for the 

determination of the studied drugs in pharmaceutical formulations and 

biological samples applying the standard addition method and compared 

statistically with the official methods. The molar absorpitivity, Sandell 

sensitivity, detection and quantification limits were also calculated.

iii 

CONTENTS 

Subject Page 

INTRODUCTION 

1. Introduction...........................................………………….…………………..........….. 1 

1.1. Knowledge of the studied drugs…………………………..………………….. 5 

1.1.1. Ketamine hydrochloride ……………………………………..…………………. 5 

1.1.2. Dextromethorphan hydrobromide ......…………….……................................ 6 

1.1.3. Silymarin .............……………………………..................…......................................... 7 

2. REVIEW OF LITERATURE 

2.1 Literature survey for determination of ketamine hydrochloride… 8 

2.1.1. Spectrophotometric methods ……………………………………….……......... 8 

2.1.2. High-performance liquid-chromatography (HPLC) methods ………….. 9 

2.1.3. Gas chromatographic (GC) methods ……………………….……………… 12 

2.1.4. Liquid chromatographic (LC) methods ……………………...…………… 14 

2.2. Literature survey for determination of dextromethorphan 

2.2.1. 

2.2.2. 

hydrobromide …………………………………………………….………………… 

Spectrophotometric methods ………………………………………………… 

Capillary electrophoresis methods …………………………………………. 

2.2.3. High performance liquid chromatography (HPLC) method …….. 21 

2.2.4. Gas chromatographic (GC) methods ………………………………………. 26 

2.2.5. Liquid chromatographic (LC) methods ………………………..…………. 28 

2.2.6. Thin-layer chromatography (TLC) methods .........…………………........ 30 

2.3. Literature survey for determination of silymarin …………………… 32 

2.3.1. Spectrophotometric methods ………………………………….……………… 32 

2.3.2. Electrokinetic capillary methods ……………………………….…………… 33 

2.3.3. High-performance liquid chromatography (HPLC) methods .….. 34 

15 

15 

19

iv 

2.3.4. Thin-layer chromatography (TLC) methods ……………………….….. 36 

2.4. Spectrophotometric determination of some drugs using acid 

dyes technique ……………………………………………………………………… 37 

2.4.1. Structure of the studied acid dyes ………………………………...………… 37 

2.4.2. Literature survey for spectrophotometric determination of some 

drugs using acid dyes technique …………………………………..………… 

Aim of the Work ……………………………………...……………………………. 47 

3. MATERIALS AND METHODS 

3. Material and methods ……………………………….…………………………… 48 

3.1. Apparatus ……………………………………….…………………………………….. 48 

3.2. Materials ………………………………………………………………………………. 48 

3.2.1. Drugs ……………………………………..…………………………………………….. 48 

3.2.1.1. Ketamine hydrochloride ………………………….…………………………….. 48 

3.2.1.1.A. Official method for ketamine hydrochloride (USP 23, 2000) ….. 48 

3.2.1.2. Dextromethorphan hydrobromide ………………………..………………… 49 

3.2.1.2.A. Official method (BP, 1998) for dextromethorphan 

hydrobromide ……………………………………………………………………….. 

3.2.1.3. Silymarin …………………………………………..………………………………….. 49 

3.3. Reagents ……………………………………...………………………………………... 50 

3.4. Market samples ………………………………..…………………………………… 50 

3.5. Determination of the molecular structure ……………………………….. 51 

3.5.A. The molar ratio method ………………………….……………………………… 51 

3.5.B. The continuous variation method …………………………………………… 51 

3.6. Spectrophotometric determination of the drugs under 

investigation using ion-pair complex formation with acid dyes .. 

3.6.1. Determination of the studied drugs in authentic powder using 

BCG ……………………………………………………………………………………… 

38 

49 

52 

52


v 

BCP ………………………………………………………………...……………….... 


BTB …………………………………………………………………………………….. 


BPB …........................…………………………………………………………............... 

3.7. Dosage Forms ……………………………………………………………………… 

54 

54 

3.7.1. Determination of ketamine hydrochloride in ketamar ampoules . 54 

3.7.1.A. Official method (USP 25, 2002) for the determination of 

ketamine hydrochloride injection …………………………………………… 

3.7.2. Determination of dextromethorphan hydrobromide in tussilar 

tablets …………………………………………………………………..….…………… 


drops ……………………………………………………………………..……………… 

3.7.4. Determination of dextromethorphan hydrobromide in codiphan 

syrup ……………………………………………………………………………………. 

3.7.4.A. Official method (USP 25, 2002) for the determination of 

dextromethorphan hydrobromide Syrup …………………………………. 57 

3.7.5. Determination of silymarin in hepamarin capsules …………………. 57 

3.7.6. Determination of silymarin in legalex and legalon tablets ………. 58 

3.7.7. Determination of the studied drugs in spiked urine samples …….. 58 

3.7.8. Determination of the studied drugs in serum samples………………. 59 

3.7.8.A. Determination of ketamine hydrochloride in spiked serum 

samples (in vitro) ……………………………………..…………………………. 

3.7.8.B. Biochemical studies for determination of ketamine 

hydrochloride in serum samples (in vivo) ..........………………........... 

3.7.8.B.1. Determination of KET by the proposed methods in serum 

samples (in vivo) ……………………………………….……………………….... 

52 

53 

55 

56 

56 

56 

59 

60 

67

vi 

Statistical Analysis ………………..………………………………………… 67 

4. RESULTS AND DISCUSSION 

4. Determination of the studied drugs by ion-pair complex 

formation with acid dyes ………………………………………………….. 

4.1. Absorption spectra of the studied drugs with BCG ……………….…. 

69 

69 

4.1.1. Effect of pH ……………………………………….…………………………………. 69 

4.1.2. Effect of time ………………………………………….…………………………….. 69 

4.1.3. Effect of the extracting solvent ………………………………...…………….. 70 

4.1.4. Effect of reagent concentration ………………………………...…………….. 70 

4.1.5. Molecular ratio of the complexes ……………………...……………………. 71 

4.1.6. Sequence of addition ……………………………………………...……………… 71 

4.1.7. Suggested mechanism ……………………………………..…………………….. 71 

4.1.8. Interference ……………………………………….…………………………………. 71 

4.1.9. Evaluation of the stability constants of the ion-pair complexes ... 72 

4.1.10. Validity of Beer ' s law ……………………………….…………………………… 73 

4.1.11. Accuracy and precision …………………………………….…………………… 74 

4.1.12. Determination of the studied drugs in spiked urine samples 

using BCG ……………………………………………………………………………. 

4.1.13.A. 

Determination of ketamine hydrochloride in spiked serum 

samples in vitro using BCG ……………………………….………..………… 

4.1.13.B. Determination of ketamine hydrochloride in serum samples in 

vivo using BCG ………………………………………………..…………………… 75 

4.1.14. Analytical applications …………………………………………………………. 76 

4.2. Absorption spectra of the studied drugs with BCP ……………….…. 91 

4.2.1. Effect of pH …………………………………………………………………………. 91 

4.2.2. Effect of time ……………………………………………….……………………….. 91 

4.2.3. Effect of the extracting solvent ……………………………….……………... 92 

74 

74

vii 

4.2.4. Effect of reagent concentration………………………………..…………….. 92 

4.2.5. Molecular ratio of the complexes……………………………….…………… 92 

4.2.6. Sequence of addition……………………………………………………………… 93 

4.2.7. Suggested mechanism…………………………………………..……………….. 93 

4.2.8. Interference…………………………………………………………………………… 93 

4.2.9. Validity of Beer ' s law………………………………………….………………… 94 

4.2.10. Accuracy and precision……………………………………..…………………… 94 


4.2.12.A. 

using BCP………………………………….………………………………………….. 


samples in vitro using BCP……………………………………………..……… 


vivo using BCP……………………………………………..……………..………… 96 

4.2.13. Analytical applications……………………………………..……………………. 96 

4.3.1. Absorption spectra of the studied drugs with BTB…………………… 112 

4.3.1. Effect of pH…………………………………………………………………..………. 112 

4.3.2. Effect of time………………………………………………………………………… 112 

4.3.3. Effect of the extracting solvent…………………………….…………………. 113 

4.3.4. Effect of reagent concentration……………………………………………….. 113 


4.3.6. Sequence of addition……………………………………..………………………. 114 

4.3.7. Suggested mechanism…………………………………….……………………... 114 

4.3.8. Interference…………………………………………………………………………… 114 

4.3.9. Validity of Beer ' s law…………………………………...………………………… 115 

4.3.10. Accuracy and precision……………………………………..…………………… 116 


using BTB………………………………….……………………………………….. 

95 

95 

116

4.3.12.A. 

4.3.12.B. 

viii 


samples in vitro using BTB………………………………….………….……… 

Determination of ketamine hydrochloride in serum samples in 

vivo using BTB……………………………………………….…………..………… 

4.3.14. Analytical applications……………………………………….…………………. 118 

4.4.1. Absorption spectra of the studied drugs with BPB……………….….. 133 

4.4.1. Effect of pH………………………………………………………………...……........ 133 

4.4.2. Effect of time…………………………………………………………..…………… 133 

4.4.3. Effect of the extracting solvent…………………………………….………… 134 

4.4.4. Effect of reagent concentration……………………………………………… 134 


4.4.6. Sequence of addition……………………………………………………...………. 135 

4.4.7. Suggested mechanism…………………………………………………………….. 135 

4.4.8. Interference…………………………………………………………………………… 135 

4.4.9. Validity of Beer ' s law………………………………………………..…………… 136 

4.4.10. Accuracy and precision…………………………………………….…………… 136 


4.4.12.A. 

using BPB…………………………………………………..……………………….. 


samples in vitro using BPB…………………………………..………………… 


vivo using BPB……………………………………………………………………… 138 

4.4.13. Analytical applications…………………………..………………………………. 138 

Results and discussion of biochemical studies 

SUMMARY AND CONCLUSION…..…………………...……… 

REFERENCES…………………………..……………………………….…… 

ARABIC SUMMARY 

116 

117 

137 

137 

154 

158 

164

ix 

LIST OF TABLES 

Table No. Table title 

Table. 1 Analytical data and characteristics of coloured product, 

precision and accuracy of the studied drugs using BCG…….. 

Table. 2 Evaluation of the accuracy and precision of the proposed 

method using BCG…………………………………………… 


method for investigated KET and DEX using BCG in urine 

samples……………………………………………………… 


method for investigated SIL using BCG in spiked urine 

samples………………………………………………………. 

Table. 5-A Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BCG in spiked serum 

samples (in vitro)……………………………………………… 

Table. 5-B Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BCG in serum samples (in 

vivo)………………………………………………………….. 

Table. 6 Evaluation of the accuracy and precision of the proposed and 

the official methods for determination of KET, DEX and SIL 

in it ' s pharmaceutical forms using BCG……………………… 

Table. 7 Determination of the studied drugs KET and DEX in it ' s 

pharmaceutical dosage forms applying the standard addition 

method using BCG………………………………………… 

Table. 8 Determination of SIL in it ' s pharmaceutical dosage forms 

applying the standard addition method using BCG………… 


precision and accuracy of the studied drugs using BCP……… 


method using BCP……………………………………………. 

Page 

83 

84 

85 

86 

87 

87 

88 

89 

90 

104 

105


method for investigated KET and DEX using BCP in spiked 

urine samples…………………………………………………. 


method for investigated SIL using BCP in spiked urine 

samples……………………………………………………… 

Table.13-A Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BCP in spiked serum 

samples, (in vitro)…………………………………………………… 

Table.13-B Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BPB in serum samples, (in 

vivo)………………………………………………………… 



in it ' s pharmaceutical forms using BCP……………………….. 



method using BCP………………………………………… 

Table. 16 Determination of Sil in it ' s pharmaceutical dosage forms 

applying the standard addition method using BCP…………. 


precision and accuracy of the studied drugs using BTB………. 


method using BTB……………………………………………. 


method for investigated KET and DEX using BTB in spiked 

urine samples………………………………………….……. 


method for investigated SIL using BTB in spiked urine 

samples……………………………………………………… 


method for investigated KET using BTB in spiked serum 

samples, (in vitro)……………………………………………. 

x 

106 

107 

108 

108 

109 

110 

111 

125 

126 

127 

128 

129

Table.21-B Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BTB in serum samples, (in 

vivo)……………………………………………………….. 



in it ' s pharmaceutical forms using BTB……………………….. 



method using BTB………………………………………….. 

Table. 24 Determination of SIL in it ' s pharmaceutical dosage forms 

applying the standard addition method using BTB………….... 


precision and accuracy of the studied drugs using BPB………. 


method using BPB……………………………………………. 


method for investigated KET and DEX using BPB in spiked 

urine samples………………………………………………. 


method for investigated SIL using BPB in spiked urine 

samples…………………………………………………….. 


method for investigated KET using BPB in spiked serum 

samples, (in vitro)……………………………..……………. 

Table. 29-B Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BPB in serum samples, (in 

vivo)…………………………………………………………. 



in it ' s pharmaceutical forms using BPB…………………….. 



method using BPB…………………………………………. 

xi 

129 

130 

131 

132 

146 

147 

148 

149 

150 

150 

151 

152

Table. 32 

Table. A. 

Table. B. 

xii 

Determination of SIL in it ' s pharmaceutical dosage forms 

applying the standard addition method using BPB………... 

Serum Alanine Transferase Level (ALT) (IU/l)………………. 154 

Serum Aspartate Transferase Level (AST) (IU/l)………………. 154 

Table. C. Serum Urea (mg /dl)………………………………………….. 155 

Table. D. Serum Creatinine (mg /dl)................................................................... 155 

153

xiii 

LIST OF FIGURES 

Figure No. Figure title 

Fig. 1 Absorption spectra studied drugs using BCG (5.0 x 10 -4 

M) at the optimum conditions…………… 

Fig. 2 Effect of pH on the absorbance of the studied drugs using 

(5.0 x 10 -4 M) BCG…………………. 

Fig. 3 Effect of ml added of buffer on the absorbance of the 

studied drugs using (5.0 x 10 -4 M) BCG……… 

Fig. 4 Effect of shaking time on the absorbance of the studied 

drugs solution using (5.0 x 10 -4 M) BCG………………… 

Fig. 5 Effect of reagent concentration on the absorbance of the 

studied drugs solution using (5.0 x 10 -4 M) BCG ………… 

Fig. 6 Molar ratio for BCG-Drugs (5.0 x 10 -4 M) under 

consideration……………………………………………………. 

Fig. 7 continuous variation using (5.0 x 10 -4 M) BCG reagent 

with (5.0 x 10 -4 M) of the drugs under consideration. 

Fig. 8 Proposed mechanism of the reaction between ketamine 

hydrochloride and bromocresol green sodium salt……… 

Fig. 9 Application of Beer ' s law for the studied drugs using the 

optimum volume of (5.0 x 10 -4 M) BCG………………….. 

Fig. 10 Ringbom plotting for the studied drugs solution using (5.0 

x 10 -4 M) BCG……………………………………………. 

Fig. 11 Absorption spectra of the studied drugs using BCP (5.0 x 

10 -4 M) at the optimum conditions……………. 


(5.0 x 10 -4 M) BCP………………….. 


studied drugs using (5.0 x 10 -4 M) BCP………. 

Page 

77 

78 

78 

79 

79 

80 

80 

81 

82 

82 

98 

99 

99

xiv 


drugs solution using (5.0 x 10 -4 M) BCP…………….. 


studied drugs solution using (5.0 x 10 -4 M) BCP ………… 

Fig. 16 Molar ratio for BCP-Drugs (5.0 x 10 -4 M) under 

consideration……………………………………………………. 

Fig. 17 continuous variation using (5.0 x 10 -4 M) BCP reagent 

with (5.0 x 10 -4 M) of the drugs under consideration……... 

Fig. 18 Proposed mechanism of the reaction between dextro- 

methorphan hydrobromide and bromocresol purple sodium 

salt. ……………………………………………… 

Fig. 19 

Application of Beer ' s law for the studied drugs using the 

optimum volume of (5.0 x 10 -4 M) BCP………………….. 


x 10 -4 M) BCP…………………………………………… 

Fig. 21 Absorption spectra the studied drugs using BTB (5.0 x 10 -4 

M) at the optimum conditions…………… 


(5.0 x 10 -4 M) BTB………………….. 


studied drugs using (5.0 x 10 -4 M) BTB………. 


drugs solution using (5.0 x 10 -4 M) BTB …………….….. 


studied drugs solution using (5.0 x 10 -4 M) BTB ……….. 

Fig. 26 Molar ratio for BTB-Drugs (5.0 x 10 -4 M) under 

consideration…………………………………………………… 

Fig. 27 continuous variation using (5.0 x 10 -4 M) BTB reagent 


Fig. 28 Proposed mechanism of the reaction between ketamine 

hydrochloride and bromothymol blue sodium salt………... 

100 

100 

101 

101 

102 

103 

103 

119 

120 

120 

121 

121 

122 

122 

123


optimum volume of (5.0 x 10 -4 M) BTB…………………... 


x 10 -4 M) BTB…………………………………………… 

Fig. 31 Absorption spectra the studied drugs using BPB (5.0 x 10 -4 

M) at the optimum conditions……………. 


(5.0 x 10 -4 M) BPB………………….. 

Fig. 33 Effect of ml added of buffer on the absorbance the studied 

drugs using (5.0 x 10 -4 M) BPB………. 


drugs solution using (5.0 x 10 -4 M) BPB …………. 


studied drugs solution using (5.0 x 10 -4 M) BCG ……….. 

Fig. 36 Molar ratio for BPB-Drugs (5.0 x 10 -4 M) under 

consideration………………………………………………….. 

Fig. 37 continuous variation using (5.0 x 10 -4 M) BPB reagent 


Fig. 38 Proposed mechanism of the reaction between 

dextromethorphan hydrobromide and bromophenol blue 

sodium salt. ……………………………………………… 


optimum volume of (5.0 x 10 -4 M) BPB………………….. 


x 10 -4 M) BPB…………………………………………… 

xv 

124 

124 

140 

141 

141 

142 

142 

143 

143 

144 

145 

145

1. INTROUDUCTION 

The quality of pharmaceutical products, is commonly expressed as an 

amount of active ingredient (measured by some assay procedure) 

compared with the amount that is claimed to be present. For some 

products, where there is the possibility of toxic substances related to the 

ingredient being present, additional tests may be carried out to ensure that 

they are not present beyond acceptable limits, Berridge (1993). These tests 

do not, however, give any indication of the cause of poor quality. A batch 

of any formulated product may be assayed for active ingredient and falled 

because appreciably less amount of drug is present. The results do not tell 

the analyst why this poor quality is observed. The drug may have degraded 

due to adverse storage conditions or inappropriate processing conditions; 

the raw material used for the manufacture of the batch may have been 

impure; or the manufacture may have inadvertently of deliberately 

produced the formulation containing the incorrect amount of active 

ingredient, which may point to poor quality assurance during the 

manufacture, or to counter feinting. 

Drug analysis in various phases of pharmaceutical development, such 

as formulations and stability studies, quality control, toxicological testing 

in animals and humans was described, Baselt and Cravey (1995). In 

hospitals, drug analysis is performed, Uges et al. (1996), Lai et al. (1997) 

and Mehta (1997) on patients samples on support of clinical 

(pharmacokinetic and bioavailability studies) and in monitoring 

therapeutic drugs and drugs of abuse. All these investigations require 

reliable and validated analytical methods in order to measure drugs

2 

___________________________________________________ Introduction 

concentration in complex media such as pharmaceutical products and 

biofluids. 

Because of their selectivity, sensitivity and overall versatility 

techniques such as gas chromatography, high performance liquid 

chromatography, supercritical fluid chromatography and capillary 

electrophoresis coupled with selective detectors [diode-array detectors] are 

frequently used to analyze multicomponent drug mixtures. Immunoassay, 

enzyme multiplied immunoassay technique are mainly used in drug 

monitoring in patients because of their speed and simplicity. 

Spectrophotometry (e.g. ultraviolet and visible, infrared as well as atomic 

absorption spectrometry), ion selective electrodes, polarographic, stripping 

voltammetric, conductometric and titrimetric techniques and more recently 

near-infrared spectrometry have been used for the rapid identification of 

impurities and degradation products in pharmaceutical analysis. 

A good planning is essential in the selection and development of the 

method for drug analysis. It is necessary to be able to quantify the active 

components in the samples rapidly with acceptable precision, accuracy and 

reliability within the cost and other constraints. It often happens that during 

product development or any other long-term work, analytical methods 

evolve. As the nature of the sample changes, (different dosage form or 

biological fluid), methods can be revised. A logical and systematic 

approach is required in method selection and development. This can be 

outlined in four basic steps: 1- generate background data, 2- review the 

literature, 3- develop the method (optimize the experimental conditions) 

and 4- validate the performance of the method before using it routinely. 

Before starting any work, all the initial information on the drugs 

(physico-chemical properties such as solubility, dissociation constant and

3 

___________________________________________________ Introduction 

UV absorption) and pharmaceutical formulations or the intended dosage 

forms (strength, presence of preservatives, type of container, stability, ect.) 

should be selected. All those information are very useful in setting up the 

analytical method, including sample clean-up. Some of the information 

mentioned above, Lund (1994), Wade and Weller (1994), Trissel (1996) 

and Wallingford (1997). 

Methods can be searched for (abstracts, journals, books, pharma- 

ceutical copoeias), recommended by colleagues or developed in 

laboratories. The choice of the method depends on factors such as the 

nature of the drug, the complexity of the sample and the intended use. The 

choice of the method will also be governed by practical conditions such as 

the availability of the equipment and specialist skills. If these are not 

available or prove costly then the work can be contracted out. 

The analytical method used in drug monitoring in patients must be 

simple, reliable and rapid because results are needed urgently. It must be 

specific for the drug in question and free of interference from other drugs 

and metabolites. As the therapeutic or toxicological drug monitoring is 

usually performed at comparatively high drug concentration, extreme 

sensitivity in the method is not often needed. Because most of the routinely 

analysed used drugs have narrow therapeutic ranges (i.e. the difference 

between therapeutic and toxic concentrations is very small), it is essential 

that the method is accurate and precise, since the imprecision of the 

method could lead to errors in interpretation and dose adjustment. 

Once the analytical method has been developed, it is validated before 

or during its use. Validation of the method means that its performance 

characteristics are adequate for the intended use. It builds quality and 

reliability into the method. In pharmaceutical industry, validation of

4 

___________________________________________________ Introduction 

analytical method is required in support of product registration 

applications, Clarke (1994). 

Many of principles, procedures and requirements of validation are 

common to the majority of analytical method. Validation is performed by 

conducting a series of experiments using the specific conditions of the 

method and the same type of matrix as intended sample. It entails 

evaluation of various parameters of the method such as accuracy, precision 

(reproducibility), linearity (concentration-detector response relationship), 

sensitivity, limits of detection and determination, recovery from the matrix 

and specificity (selectivity). The definitions and procedures used to 

calculate these parameters are adequately described in many publications 

related to pharmaceutical analysis. 

Validation does not imply that the method is free from errors. It only 

confirms that it is suitable for the purpose. Any modification to a method 

during its use requires its validation. For example, if a new instrument is 

brought into use, or the method is applied to a different type of sample, it 

will require revalidation. The greater the modification, the greater the need 

of revalidation. Some revalidation may also be required when transferring 

the method between laboratories or when changes are made in the 

manufacturing process for the drug. Other factors, which can be considered 

when validating a method, are cost per analysis, ease and speed of 

operation and potential for automation.

5 

___________________________________________________ Introduction 

1.1. Knowledge of the studied drugs: 

1.1.1. Ketamine hydrochloride: 

O 

H3C NH 

Cl 

Ketamine hydrochloride (Ket) USP, 25 (2002) is 2-(2-Chloro- 

phenyl)-2-methyl amino cyclohexanone hydrochloride [C13H16ClNO.HCl, 

with M.W.= 274.19]. Ketamine hydrochloride is an analgesic and 

anaesthetic, a white, crystalline powder, freely soluble in water and in 

methanol, soluble in alcohol; practically insoluble in ether. Ket has been 

advocated for maintaining or increased cardiovascular performance in 

selected patients during induction of anaesthesia as it may increased blood 

pressure and heart rate. However, there have been reported of reduced 

cardiac and pulmonary in severally patients and of arrhythmias. Ket is 

administered by intravenous injection, intravenous infusion, or 

intramuscular injection. It produces dissociative anaesthesia characterised 

by a trance-like state, amnesia, and marked analgesia which may persist 

into the recovery period. There is often an increase in muscle tone and the 

patient's eye may remain open for all or part of the period of anaesthesia. 

For induction the dose given by intravenous injection may range from 1.0 

to 4.5 mg of Ket per kg body-weight; a dose of 2.0 mg of Ket per kg body- 

weight given intravenously over 60 seconds usually produces surgical 

anaesthesia within 30 seconds of the end of the injection and lasting for 5.0 

to 10 minutes. The initial intramuscular dose may range from 6.5 mg to 13 

HCl

6 

___________________________________________________ Introduction 

mg of Ket per kg. For diagnostic or other procedures not involving intense 

pain an initial intramuscular dose of 4.0 mg of Ket per kg has been used. 

1.1.2. Dextromethorphan hydrobromide: 

MeO 

H 

HBr 

N Me 

Dextromethorphan hydrobromide, (Dex), (+)-3-Methoxy-17-methyl- 

9α, 13α, 14α-morphinan hydrobromide monohydrate. USP, 25 (2002), 

[C18H25NO, HBr, H2O, with M.W.= 370.30]. A white, crystalline powder 

with a faint odour, sparingly soluble in water , freely soluble in alcohol 

practically insoluble in ether. Dex is a cough suppressant, used for the 

relief of non-productive cough; it has a central action on the cough centre 

in the medulla. Dex is rapidly adsorbed from the gastro-intestinal tract. It is 

metabolized in the liver and excreted in the urine as unchanged Dex and 

demethylated metabolites including Dex, which has some cough 

suppressant activity. Dex is reported to act within half an hour of 

administration by mouth and to exert an effect for up to 6.0 hours. It is 

given by mouth in doses of 10 to 20 mg every 4.0 hours, or 30 mg every 

6.0 to 8.0 hours to an usual maximum of 120 mg in 24 hours.

7 

___________________________________________________ Introduction 

1.1.3. Silymarin: 

HO 

OH 

O 

O 

H 

H 

OH 

Silymarin (SIL) is a mixture of the isomers silibinin (SBN), silicristin 

(SCN), silidianin (SDN) and isosilibine (ISBN) which are the main active 

flavanoids, BP (1998). Chemical name is 3, 5, 7- trihydroxy-2-(3-(4- 

hydroxy-3-methoxyphenyl)- 2-(hydroxymethyl)-1, 4-benzodioxan-6-yl)-4- 

chromanone [C25H22O10, M. W.= 482.4]. Silymarin is an antihepatotoxic 

substance isolated from fruits of silybum marianum and has been used for 

the treatment of hepatic disorders. Silymarin is poorly water soluble and 

has been given by mouth, Silymarin in doses of up to 140 mg two or three 

times daily by mouth has been suggested for hepatic disorders. 

O 

O 

H 

H 

OH 

O 

OH 

CH 3

2. REVIEW OF LITERATURE 

2.1. Literature Survey for Determination of Ketamine Hydro- 

chloride 

2.1.1. Spectrophotometric methods 

Matsouka et al. (1982) studied the spectrophotometric determination 

of ketamine HCl and quinine HCl with ion-association reagent, the sample 

of quinine HCl or ketamine HCl was treated with 1.0 ml of 2.72 mM- 

tropaeolin 00 (C.I. acid orange 5.0 ), or 2.28 mM-Erio green B (C.I. acid 

green 16 ), 2.0 ml of Britton - Robinson buffer and 5.0 ml of CH2 Cl2. The 

mixture was shaken for 3.0 min and set a side for 30 min, then the 

absorbance of the CH2 Cl2 layer was measured at 637 nm for determination 

of quinine HCl . For determination of ketamine HCl, 0.5 ml of methanolic 

10 % HCl was added to 2.0 ml of the CH2Cl2 layer and the absorbance was 

measured at 543 nm. Many cations, NO3 - , Cl - , glucose and lactose do not 

interfere . 

Parkhomenko et al. (1992) studied the determination of ketamine 

HCl and sibazon (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4- 

benzodiaz-epin-2-one) simultaneously in aq. 95% ethanol solution by UV 

spectrophotometry. The calibration graphs were described by D = epsilon 

C + a (where D is the absorbance, C is the conc. and a is an additive term ), 

and were constructed at 268 nm for ketamine HCl and 365 nm for sibazon. 

D is determined as the difference between the absorbance of a solution in 

HCl and the absorbance in ammonical solution The relative error does not 

exceed 3% .

9 

______________________________________________ Review of Literature 

Okide and Odoh (1998) studied spectrophotometric determination of 

ketamine HCl by charge transfer complexation with p-chloranilic acid in a 

non-aqueous medium, conformity to Beer ' s law enabled the assay of 

dosage forms of continuous variation, chloranilic acid was found to form a 

charge-transfer complexation in a stoichiometry with maximum absorption 

at 528 nm. The method has been successfully applied to the analysis of 

commercially available ketamine HCl dosage forms without interference 

from their excipient. 

2.1.2. High-performance liquid-chromatography method 

The Enantiomers of ketamine HCl in human serum and pharmaceutical 

formulation were separated by Aboul-Enien and Islam (1992) using 

HPLC in a chiral column (25 cm 4.6 mm) of Daicel CA-1 (10 μm diam.; 

cellulose triacetate) with ethanol as mobile phase (1.0 ml min -1 ) and 

detection at 269 nm. Samples of ketalar in aqueous solution were analysed 

directly. Serum was treated with aqueous NaHCO3 and ketamine was 

extracted into CH2Cl2; the extract was dried and evaporated . The residue 

was acidified with ethanolic HCl, dried under N and dissolved in methanol 

for analysis. The (+)-(S)-isomer eluted first. Amounts of 20 nM of 

ketamine or 10 nM of each enantiomer were injected. 

A simple, rapid and sensitive HPLC procedure has been developed for 

the determination of ketamine and dehydronorketamine in equine serum by 

Seay et al. (1993). Sample preparation consisted of mixing equal volumes 

of serum and acetonitrile-phosphoric acid (85%)-water (20:2:78, v/v/v), 

followed by ultrafiltration through a 10.000 molecular mass cut-off filter. 

Separation of these two analytes in the ultra filtrate was accomplished on a 

reversed-phase phenyl column eluted with methanol-acetonitrile-phosphate

10 


buffer solution. Ketamine and dehydronorketamine were detected by a 

variable photometric UV-Vis detector set at 215 nm, and confirmed by a 

photodiode array detector operated in the 200-320 nm range. The limit of 

detection for ketamine was 5.0-15 ng ml -1 in equine serum. 

An HPLC method for determination of sub-anaesthetic concentrations 

of the enantiomers of ketamine and its metabolite norketamine in plasma 

was described by Svensson and Gustafsson (1996). The samples are 

purified by reversed-phase solid-phase extraction. The enantiomers are 

separated on a Chiral AGP column with a mobile phase containing 16% 

methanol and a 10 mM phosphate buffer at pH 7.0, and measured by UV- 

detection at a wavelength of 220 nm. Linear calibration curves with 

correlation coefficients better than 0.995 have been obtained in the range 

10-320 ng ml -1 . Minimum detectable concentrations were about 2 ng ml -1 . 

An isocratic, selective, and very sensitive HPLC method for the 

determination of ketamine and its two main metabolites in plasma was 

described by Bolze and Boulieu (1998). The compounds were extracted 

from plasma by a LLE with a dichloromethane : ethyl acetate mixture 

followed by an acidic back-extraction. Separation was achieved on a new 

stationary phase, Purospher RP-18 end capped, with a mobile phase 

containing acetonitrile : 0.03 M phosphate buffer (23 : 77; V/V) adjusted 

to pH 7.2. Because of the high column efficiency and the significant 

improvement of peak symmetry, the quantification limit could be down to 

0.005 μg ml -1 for ketamine and norketamine. The intraday and interday 

CVs ranged from 1.7% to 5.8% and 3.1% to 10.2% for all compounds 

respectively. 

An RPHPLC technique for the simultaneous measurement of both 

bupivacaine and ketamine in plasma was described by Gross et al. (1999).

11 


Plasma samples (0.5 ml) were prepared using a rapid and simple back- 

extraction technique. Resolution of both analytes and the internal standard, 

desipramine, from medicines co administered to surgical paediatric 

patients was obtained using a 5.0 μm cyano (CN) (250 4.6 mm) column 

and a mobile phase comprising methanol-acetonitrile-ortho phosphoric 

acid-0.01 M sodium dihydrogen phosphate (200:80:2:718). Good 

sensitivity for both analytes was observed using UV detection at a 

wavelength of 215 nm. 

A stereo selective HPLC method for the determination of the 

enantiomers of ketamine and its active metabolite, norketamine, in human 

plasma was described by Yanagihara et al. (2000) . The compounds were 

extracted from plasma by LLE three times in a combination of 

cyclohexane with 2.5 M NaOH, 1.0 mM HCl and 1.0 M carbonate buffer. 

Stereoselective separation was achieved on a Chiralcel OD column with a 

mobile phase of n-hexane-2-propanol (98:2, v/v). The detection 

wavelength was 215 nm. The lower limits of the determination of the 

method were 5.0 ng ml -1 for ketamine and 10 ng ml -1 for norketamine. The 

intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and 

from 3.4 to 10.7% for all compounds, respectively. 

A method of detecting and resolving cocaine, its metabolites and 

ketamine. A new precise, accurate and sensitive RPHPLC method has been 

developed and validated by Rofael and Abeld-Rahman (2002). This 

assay employed a phosphate-buffered aqueous mobile phase (pH 6.9) with 

an organic component consisting of acetonitrile and methanol and a C-18 

column as stationary phase at 225 nm wavelength. Minimum detection 

limits were 5.0 ng ml -1 for cocaine and 10 ng ml -1 for benzoylecgonine, 

norcocaine and ketamine. Linearity was demonstrated over a broad range

12 


of concentration in plasma, with good sensitivity for ketamine, cocaine and 

cocaine metabolites. 

An isocratic RPHPLC method for the simultaneous determination of 

ketamine and xylazine in canine plasma was described by Niedorf et al. 

(2003). Plasma samples (500 μL) are cleaned up via LLE. The analytes 

and the internal standard clonidine were separated on a cyano (CN) 

column using a mobile phase containing acetonitrile-0.005 M phosphate 

buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. 

2.1.3. Gas chromatographic (GC) methods 

An accurate and simplified method has been developed for 

determination of ketamine in human plasma using GC and electron impact 

mode MS with selected ion recording from 0.5 ml of plasma by Kudo et 

al. (1990). Standards and samples of plasma underwent the same 

procedure of two step extraction by methanol. Ketamine concentrations in 

the plasma were determined from the peak in the selected ion profile of 

ketamine (m/e: 237). Standard curve was linear with the increasing amount 

of ketamine (0.63-5.0 μg ml -1 ) in plasma with mean CV = 5.2 % mean RR 

= 63.8% and r = 0.998. The concentration of ketamine in plasma ranged 

from 1.6 μg ml -1 to 2.5 μg ml -1 during ketamine anesthesia (2.0 mg.kg -1 .h -1 ) 

in a surgical patient. 

A sensitive and precise GC-MS method with selected ion monitoring 

has been developed for identification and quantification of the phen 

cyclidine derivative ketamine in human plasma by Feng et al. (1995). The 

assay was based on an alkaline extraction from aqueous to organic solvent 

from plasma and an efficient GC separation on a DB-5 capillary column. 

The analytical procedure has a coefficient of variation of 0.7-6.2% and

13 


from 1.3 to 8.7% within-day and from day-to-day analysis, respectively. 

The low level of sensitivity was 10 ng ml -1 . It was used to measure low 

plasma concentrations in volunteers during ketamine-induced experimental 

psychosis. 

A method for analysis of veterinary tranquillizers in urine using GC- 

MS was described by Olmos-Carmona and Hernandez-Carrasquilla 

(1999). Detection limits are 5.0 μg L -1 for ketamine, azaperone and the 

phenothiazines (chlor-, aceto- and propionylpromazine), 10 μg L -1 for 

haloperidol, 20 μg L -1 for xylazine and 50 μg L -1 for azaperol, recoveries for 

all analytes were higher than 70%. Method performance in terms of 

within-batch, between-days and between-analysts reproducibility was 

studied and found to be acceptable. 

An analytical scheme using GC-isotope dilution MS assisted by 

precedent LLE and chemical derivatization (ChD) was described by 

Chou et al. (2004) for the simultaneous determination of ketamine and its 

major metabolite, norketamine, in urine. The simultaneous ChD of the two 

analytes, one primary amine and one secondary amine, with 

pentafluorobenzoyl chloride (PFBC) has not only enhanced their 

instrumental responses and mass-spectrum uniqueness but also afforded 

more proper yet easier selection of qualifier and quantifier ions and hence 

achieved more evidential identification and quantitation. Thus, the 

regression calibration curves for KT and NK in urine are linear within 100- 

5000 ng ml -1 , with correlation coefficients typically exceeding 0.99 and 

NK curves exclusively showing larger slopes than KT curves. The method 

limits of detection (LODs) determined by two definitions for KT and NK 

range from 3.0 to 75 ng ml -1 and limits of quantitation (LOQs) from 9.0 to 

100 ng ml -1 . The mean recoveries (N = 3.0) calculated for the LLE/ChD of

14 


KT and NK from 50 and 100 l, respectively, of a 100μg ml -1 urinary spike 

vary from 71.0 to 97.8%, with NK consistently giving better recoveries 

than KT. The precisions (N = 3.0) calculated for the total analyses of four 

real-case samples are typically below 12.3%. 

2.1.4. Liquid chromatographic (LC) methods 

A sensitive enantioselective LC-MS detection has been developed and 

validated for the simultaneous determination of plasma concentrations of 

(R)- and (S)-ketamine, and (R)- and (S)-norketamine by Rodriguez Rosas 

et al. (2003). The compounds were extracted from human plasma using 

solid-phase extraction and then directly injected into the LC-MS system 

for detection and quantification. Enantioselective separations were 

achieved on a LC chiral stationary phase based upon immobilized α1-acid 

glycoprotein (the Chiral AGP column). The separations were achieved 

using a mobile phase composed of 2-propanol-ammonium acetate buffer 

(10 mM, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml min -1 and a temperature 

of 25 ºC. Under these conditions, the analysis time was 20 min. Detection 

of the ketamine, norketamine and bromoketamine (internal standard) 

enantiomers was achieved using selected ion monitoring at m/z 238.1, 

224.1 and 284.0, respectively. Extracted calibration curves were linear 

from 1.0 to 125 ng ml -1 per enantiomer for each analyte with correlation 

coefficients better than 0.9993 and intra- and inter-day RSDs of less than 

8.0%.

15 


2.2. Literature Survey for Determination of Dextromethorphan 

Hydrobromide 

2.2.1. Spectrophotometric methods 

The proposed method was a rapid and validated analytical method that 

does not required prior separation for the simultaneous determination of 

the three drugs, pseudoephedrine HCl, chlorpheniramine maleate, and 

dextromethorphan HBr, in a tablet formulation. A diode array spectro- 

photometer, capable of multicomponent analysis, was used for the 

quantitation by Murtha et al. (1988). The utility of this method was 

demonstrated in two ways: the analysis of a chewable pediatric tablet 

(formulation CP) containing 7.5 mg of pseudoephedrine HCl, 0.5 mg of 

chlorpheniramine maleate, and 2.5 mg of dextromethorphan HBr. The 

dissolution analysis of a hydroxypropyl methylcellulose-based sustained- 

release tablet (formulation SR) containing 120 mg of pseudoephedrine 

HCl, 8.0 mg of chlorpheniramine maleate, and 60 mg of dextromethorphan 

HBr. The sensitivity of the assay was 7.5 μg ml -1 for pseudoephedrine HCl, 

1.0 μg ml -1 for chlorpheniramine maleate, and 5.0 μg ml -1 for 

dextromethorphan HBr, using the second-derivative spectra of the 

absorbance with respect to wavelength. Determinations were made in 0.1 

M sodium acetate buffer at pH 5.0 using a 1.0-cm quartz cell. Absorbance 

spectra, and their first and second derivatives, from 240 to 300 nm were 

used for the determination. The results obtained by the examined method 

compared favorably with the results obtained by a validated HPLC 

method. 

Tan et al. (1989) described a second-derivative spectrophotometric 

method for determination of mixtures of phenylpropanolamine HCl and

16 


dextromethorphan HBr in some pharmaceutical preparations. A portion 

of the contents of capsules or a portion of powdered lozenges was mixed 

with methanol, and the second-derivative absorption of the filtered solution 

was recorded from 220 to 320 nm. Peak amplitudes were measured 

between the maximum at 260 nm and its minimum at 257 nm for 

phenylpropanolamine HCl and corresponding measurments were made at 

291.5 and 287.5 nm for dextromethorphan HBr. Calibration graphs were 

rectilinear from 20 to 350 μg ml -1 for the examined drugs, and recoveries 

from simulated capsules and lozenges were 98.0 ± 2.0%. 

Tantishaiyakul et al. (1998) determined simultanously dextro- 

methorphen HBr and bromhexine HCl in tablets by first-derivative 

spectrophotometry. Powdered tablets, containing about 15 mg 

bromhexine HCl, were transferred into a 50 ml volumetric flask and 

dispersed in 30 ml methanol for 5.0 min by ultrasonic vibration. The 

suspension was diluted to volume with methanol and filtered. A 25 ml was 

transferred to a 50 ml volumetric flask and adjusted to volume with 

phosphate buffer solution (pH 6.0). A 3.0 ml aliquot was diluted to 25 ml 

with 50 % methanol in phosphate buffer. UV determination by first- 

derivative spectroscopy of bromhexine HCl and dextromethorphan HBr 

was carried out using zero-crossing and peak-to-base measurment at 234 

and 324 nm, respectivly. The method was linear in the range 6.0-30 and 

12-60 μg ml -1 for bromhexine HCl and dextromethorphan HBr with 

correlation coefficients of 0.9999 for both drugs. The limit of detection 

was 0.103 μg ml -1 for bromhexine HCl and 0.033 μg ml -1 for 

dextromethorphan HBr. Details of a HPLC method developed as the 

reference method were given. Results obtained by first-derivative 

spectrophotometry agree well with those obtained by HPLC method.

17 


Ajay and Piyush (1999) studied simultaneous spectrophotometric 

determination of pseudoephedrine HCl, dextromethorphan HBr and chlor- 

pheniramine maleate from multicomponent liquid orals using multi- 

wavelength and derivative spectroscopy. Volume of syrup equivalent to 10 

mg dextromethorphan HBr was extracted with 2 x 20 ml ether. Aqueous 

layer was retained, shaken with 5.0 ml of 1.0 M- NaOH, extracted with 4 x 

20 ml CHCl3. Organic layer was combined, evaporated to dryness, and the 

residue reconstituted in 0.1 N-HCl to give final concentration of 50 μg ml -1 

dextromethorphan HBr. For the multi wavelength approach, sample 

solutions were scanned from 220-350 nm and the data compared to that for 

mixed standard; absorption maxima was 278 nm for dextromethorphan 

HBr. For derivative spectroscopy, first derivative order was most suitable. 

Absorbance was measured at the optimum wavelength of 279.4 nm, and 

analyte concentration determined by simultaneous equation. Beer ' s law 

was obeyed from 0.0-100 μg ml -1 dextromethorphan HBr. RSD (n = 5) 

ranged from 0.2-1.9 % for the two methods, and recoveries from 98- 

102.2%. 

Grangwal and Trivedi (1999) determined dextromethorphan HBr and 

pseudoephedrine HCl from liquid oral dosage forms by two spectro- 

photometric methods, viz. seconed–order derivative spectrophotometry 

(2DS) and multi-wavelength spectrophotometry (MWS), for the deter- 

mination of dextromethorphan HBr and pseudoephedrine HCl in liquid 

dosage forms. Portion (5.0 ml) of a commercial syrup sample containing 

dextromethorphan HBr (5.0 or 7.5 mg) was extracted with 2.0 x 10 ml 

diethyl ether. The ethereal layer was discarded, the aqueous layer was 

made alkaline with 5.0 ml of 1.0 M- NaOH and extracted with 3.0 x 20 ml 

and 10 ml portions CHCl3. The combined organic extract was dried over

18 


anhydrous Na2SO4 and evaporated to dryness. The residue was dissolved 

in 0.1 M-HCl and the volume was made up to 100 ml. For 2DS, the 

absorbance was measured at 285.9 and 293 nm for dextromethorphan HBr 

(wavelengths of zero absorbance for pseudoephedrine HCl). For MWS, the 

absorbances were measured at 278, 263, 257 and 251 nm, along those of 

mixed standard solutions. Beer ' s law was obeyed from 5.0-20 μg ml -1 for 

dextromethorphan HBr. Recovery of dextromethorphan HBr were 98.40- 

101.90% and 99.70-101.60%, for 2DS and MWS methods, respectively. 

Bratio et al. (1999) determined dextromethorphan HBr, chlorphenir- 

amine maleate and bromhexine HCl by simultaneous analysis from liquid 

formulations. A suitable portion of the formulation was spiked with 5.0 

mg dextromethorphan HBr and diluted to 25 ml with 0.2 M –NaOH. The 

mixture was repeatedly extracted with chloroform and the combined 

extract was evaporated to dryness and reconstituted in 0.1 M-HCl (100 

ml). Determination of dextromethorphan HBr was measured at 245 nm. 

Two simple and economical methods requiring no prior separation for 

simultaneous analysis of phenylpropanolamine hydrochloride, 

chloropheniramine maleate and dextromethorphan hydrobromide in 

combination in pharmaceutical formulations was studied by Sahu and 

Sharma (2000). The methods employ multicomponent analysis procedure 

and simultaneous equations after spectral manipulation for quantification. 

In 0.1N hydrochloric acid, phenylpropanolamine hydrochloride has an 

absorbance maxima at 257nm, chloropheniramine maleate at 265nm and 

dextromethorphan hydrobromide at 278nm. All three drugs obey Beer's 

law in the concentration range employed for these methos. The linearity 

was validated by Least Squares method. The results of analysis have been

19 


validated statistically and by recovery studies. Both methods are simple, 

accurate, reproducible and rapid. 

2.2.2. Capillary electrophoresis methods 

Yururi et al. (1994) studied simultaneous determination of ingredients 

in anti-cold preparations by capillary electrophoresis, compounds that are 

etenzamide, guaiphesin paracetamol and potassium guaiacol sulfonate, 

were determined in 50 mM sodium dodecyl sulfate (SDS) in 15 mM- 

phosphate buffer of pH=11 as carrier, whereas those that were negatively 

charged in alkaline media, e.g., chloropheniramine maleate, dextromethor- 

phan HBr, dihydrocodeine phasphate, (±)-methylephedrine HCl and 

noscapine, were determined in 50 mM-phosphate buffer of pH 3.0 as 

carrier. Hydrostatic injection for 10 s and on-column detection at 185 nm 

were used. Recoveries were 92.7-109% and RSD (n = 3) were 0.5-6.3%. 

Lui and Hou (1997) studied the analysis of compound common cold 

preparations by high pressure capillary electrophoresis (HPCE). In this 

method, the syrup was treated with 2.0 ml aqueous theophylline (1.2 mg 

ml -1 ; internal standard) and electrophoresis buffer was added to 50 ml. 

Dextromethorphan HBr in the filtered solution was determined on a quartz 

column (72 cm x 50 μm i.d.; effective length 52 cm) operated at 30 °C, 

with NaH2PO4/Na2B4O7 buffer of pH 8.5 ± 0.2 as electrophoresis buffer, at 

voltage 30 kV and detection at 200 nm. The calibration graph was linear 

from 20.6-185 μg ml -1 . The within and between day RSD was 0.3-2.4 and 

0.8-3.3%, respectively. The recovery was 99-101.2 %. The method was 

also applied to tablets. 

Ji et al. (1998) described the separation and determination of 

dextromethorphan HBr in cold medicine by micellar electrokinetic

20 


capillary chromatography. Sample equivalent to one tablet, obtained from 

ten ground tablets, was sonicated with 5.0 ml anhydrous caffeine (internal 

standard) and some H2O for 20 min and then H2O was added to 5.0 ml. 

The separation and determination by micellar electrokinetic capillary 

(MEKC) on a fused-silica capillary column (60 cm x 75 μm i.d.;) operated 

at 30 °C and a separation voltage of 18 kV, with 20 mM-H3BO4/20 mM- 

NaOH of pH 10 containing 50 mM-sodium deoxycholate as running 

buffer, and detection at 214 nm. The calibration graphs for 

dextromethorphan HBr 120-360 μg ml -1 . The recovery was 97.9-101.5%. 

Within and between day RSD (n = 4) based on migration time was 0.7- 

1.32 and 0.92-1.87%, respectively. 

The separation of cold medicine ingredients (e.g., phenyl- 

propanolamine, dextromethorphan, chlorpheniramine maleate, and 

paracetamol) by capillary zone electrophoresis and micellar electrokinetic 

chromatography was described by Suntornsuk (2001). Factors affecting 

their separations were the buffer pH and the concentration of buffer, 

surfactant and organic modifiers. Optimum results were obtained with a 10 

mM sodium dihydrogen-phosphate-sodium tetraborate buffer containing 

50 mM sodium dodecyl sulfate (SDS) and 5.0% methanol (MeOH), pH 

9.0. The carrier electrolyte gave a baseline separation of phenylpropanol- 

amine, dextromethorphan, chlorpheniramine maleate, and paracetamol 

with a resolution of 1.2, and the total migration time was 11.38 min. 

The separation of basic nitrogenous compounds commonly used as 

active ingredients in cold medicine formulations by micellar electrokinetic 

capillary chromatography and capillary zone electrophoresis with direct 

absorptiometric detection was investigated by Gomez et al. (2002). The 

type and composition of the background electrolyte (BGE) were

21 


investigated with respect to separation selectivity and BGE stability. BGE 

of 10 mM sodium dihydrogen phosphate-sodium tetraborate buffer 

containing 10 mM SDS and 10% acetonitrile, pH 9.0 was found to be 

optimal. Dextromethorphan HBr, diphenhydramine HCl and phenylephrine 

HCl were baseline-separated in less than 11 min, giving separation 

efficiencies of up to 494,000. Theoretical plates, reproducibility of 

corrected peaks areas below 3.0% relative standard deviation and 

concentration detection limits from 2.5 to 5.5 μg ml -1 were obtained. 

Detection was performed at 196 and 214 nm. 

2.2.3. High performance liquid chromatography (HPLC) methods 

An HPLC assay for the simultaneous quantitation of dextromethor- 

phan and its o-demethylated metabolite dextrorphan from urine was 

described by Marshall et al. (1992). A cyano analytical column was used 

with a mobile phase consisting of CH3OH 16%, acetonitrile 3.0%, and 

triethylamine 0.06% at pH 2.8 and a flow rate of 1.0 ml min -1 . Betaxolol 

was used as the internal standard. Standard curves from 50 ng ml -1 to 

10,000 ng ml -1 (dextrorphan), and from 50 ng ml -1 to 8,000 ng ml -1 

(dextromethorphan) were developed. The peaks eluted at 7.8 min 

(dextrorphan), 12.2 min (betaxolol), and 17.8 min (dextromethorphan). 

The coefficients of variance ranged from 1.3 to 4.5% at 250 ng ml -1 and 

0.9 to 2.5% at 5,000 ng ml -1 . This assay was used to determine 

dextromethorphan /dextrorphan molar ratios in healthy male volunteers for 

the purpose of determining phenotype status for the P450IID6 isozyme. 

To establish the usefulness of fluorescence detection to quantify 

urinary concentrations of dextromethorphan and dextrorphan for oxidation 

phenotyping, the determination of the molar concentration ratio of dextro- 

methorphan to dextrorphan in 38 subjects by UV and fluorescence

22 


detection was described by Lam and Rodriguez (1993). Dextro- 

methorphan and dextrorphan concentrations were quantified after 

overnight hydrolysis of urine samples and organic solvent extraction with 

heptane and butanol. The compounds were separated by HPLC using a 

phenyl column and a mobile phase consisting of acetonitrile and an 

aqueous mixture of 0.01 M heptane sulfonic acid and 0.01 M phosphate 

buffer. The eluents were detected in series by a UV detector (280 nm) and 

fluorescence detector (excitation 280 nm and emission 310 nm). The 

dextromethorphan to dextrorphan molar concentration ratio by UV and 

fluorescence detection was highly correlated (r = 0.997) and not 

statistically different (p = 0.1036). However, increased sensitivity with 

fluorescence detection enabled detection of lower dextromethorphan and 

dextrorphan concentrations when compared with UV detection. 

Fluorescence detection was able to detect dextromethorphan as low as 0.02 

μg ml -1 , which may be helpful in phenotyping individuals with extremely 

rapid metabolism of dextromethorphan. Fluorescence detection also 

produced chromatograms with significantly less interference and allows a 

more accurate quantitation of dextromethorphan and dextrorphan 

concentrations. 

An HPLC determination of dextromethorphan HBr in cough-cold 

syrup with indirect conductometric detection was studied by Lau and 

Mok (1995). Dextromethorphan HBr as active ingredient in cough cold 

syrup was determined by HPLC following 25-50 fold dilution with the 

mobile phase. A 20 μL volume of the diluted samples was analysed using 

an ultrasphere 5.0 μm Spherical 80 Ǻ. Pore CN column (2.5 cm x 4.6 mm 

i.d.) with water/acetonitrile/ethanol (19: 30: 1) containing 1.0 mM-HClO4 

as the mobile phase (1.0 ml min -1 ) and conductivity detection. The

23 


retention time for dextromethorphan HBr was < 22 min. The calibration 

graph was linear up to 500 μg ml -1 . The RSD (n = 10) was < 3.0%. 

Abdel-Moety et al. (1995) determined dextromethorphan HBr in bulk 

form and dosage formulations by HPLC. The drug was analysed on a 10 

μm μ-Bondapak C18 column (30 cm x 3.9 mm i.d.) with acetonitrile/40 

mM-acetate buffer of pH 4.3 (3:1) as a mobile phase (1.5 ml min -1 ) and 

detection at 278 nm. The method was applied to the analysis of powdered 

tablets, syrup or drops, which were extracted and dilution with mobile 

phase. Labetalol (2.0 mg ml -1 in mobile phasse) was added as internal 

standard before HPLC. The recovery from tablets was 100.9 ± 0.29% (n = 

4) and the RSD was 0.41% (n = 6). 

Rauha et al. (1996) studied the determination of dextromethorphan 

HBr in cough-cold syrup by HPLC. Syrup (2.0 ml) was diluted to 50 ml 

with 0.1 M-HCl. Apportion (20 μL) was analysed on a 4.0 μm phenyl 

column (15 cm x 3.9 mm i.d.) with a mixture (47:53) of 3% ammonium 

format buffer adjusted to pH 3.9 with formic acid and methanol as mobile 

phase (1.0 ml min -1 ) and detection at 278 nm. The calibration graphs were 

linear for 0.025-0.20 mg ml -1 , the RSD was 0.2-1.0% and the recoveries 

were 85.1-96.6%. 

An RPHPLC determination of dextromethorphan HBr in oral solution 

was carried out by Zhang et al. (1997). Sample (5.0 ml) was diluted with 

H2O to 10 ml and then analysed for dextromethorphan HBr by HPLC. A 

portion (10 μL) was injected onto a μ-Bondapak C18 column (30 cm x 3.9 

mm i.d.) operated with a mixture of 0.5% triethylamine and H3PO4/aceto- 

nitrile/methanol as a mobile phase (1.0 ml min -1 ) and detection at 264 nm. 

The calibration graph was linear from 200-1000 μg ml -1 . Recovery was 

99.3-100.1% and RSD was 1.16-1.58%.

24 


A quantitative method for determination of dextromethorphan HBr in 

cough syrup by HPLC was described by Shervington (1997). Standard 

solution and cough syrup were analysed on a 5.0 μm Hypersil BDS C18 

column (25 cm x 4.6 i.d.) with methanol/H2O/-ammonium formate buffer 

(45:54:1) as mobile phase (1.0 ml min -1 after 19 min) and detection at 257 

nm. Calibration graph was linear from 300-540 μg ml -1 . RSD was 0.45 % 

and recovery was 99.13 %. 

Li et al. (2000) described a HPLC determination of dextromethorphan 

HBr in Redentai oral solutions on a 5.0 μm Spherisorb C8 column (25 cm x 

4.6 mm i.d.), with methanol/2.5 mM-hexane sulfonic acid/H2O (containing 

2.0 % triethylamine/3.0 M-H3PO4) (1400:1000:68) as mobile phase (1.0 ml 

min -1 ) and detection at 257 nm. The calibration graph was linear from 0.15 

- 0.65% with recovery of 99.7-100.8%. 

An HPLC procedure has been developed by Wilcox and Stewart 

(2000) for the simultaneous determination of guaifenesin pseudo- 

ephedrine, dextromethorphan and guaifenesin-pseudoephedrine in 

commercially available capsule dosage forms and guaifenesin-codeine in a 

commercial cough syrup dosage form . The separation and quantitation are 

achieved on a 25-cm underivatized silica column using a mobile phase of 

(60:40 %; v/v) 6.25 mM phosphate buffer, pH 3.0 - acetonitrile at a flow 

rate of 1.0 ml min -1 with detection of all analytes at 216 nm. The 

separation is achieved within 10 min for each drug mixture. The method 

showed linearity for the guaifenesin-pseudoephedrine-dextromethorphan 

mixture in the 50-200, 7.5-30 and 2.5-10 μg ml -1 ranges, respectively. The 

intra- and inter-day RSDs ranged from 0.23 to 4.20%, 0.18 to 2.85%, and 

0.13 to 5.04% for guaifenesin, pseudoephedrine, and dextromethorphan, 

respectively. The guaifenesin pseudoephedrine mixture yielded linear

25 


ranges of 25-100 and 3.75-15 μg ml -1 and intra- and inter-day RSDs 

ranged from 0.65 to 4.18% and 0.23 to 3.00% for guaifenesin and 

pseudoephedrine, respectively. The method showed linearity for the 

guaifenesin-codeine mixture in the 25-100 and 2.5-10 μg ml -1 ranges and 

RSDs ranged from 0.37 to 4.25% and 0.14 to 2.08% for guaifenesin and 

codeine, respectively. 

An HPLC method has been developed and validated by Bendriss et al. 

(2001) for the determination of dextromethorphan, dextrorphan, 3- 

methoxymorphinan and 3-hydroxymorphinan in urine samples. 

Deconjugated compounds were extracted on silica cartridges using 

dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic 

separation was accomplished on a phenyl analytical column serially 

connected with a nitrile analytical column. The mobile phase consisted of a 

mixture of an aqueous solution, containing 1.5% acetic acid and 0.1 % 

triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored 

using a fluorescence detector. Calibration curves were linear over the 

range investigated (0.2-8.0 μM) with correlation coefficients > 0.999. The 

method was reproducible and precise. Coefficients of variation and 

deviations from nominal values were both below 10 %. For all the 

analytes, recoveries exceeded 77% and the limits of detection were 0.01 

μM. 

A Simple and cost effective reversed-phase high performance liquid 

chromatography method which has been developed for the simultaneous 

determination of phenylpropanolamine hydrochlorides, dextromethorphan 

hydrobromide and chlorpheniramine maleate in expectorant formulations 

was described by Shenoy et al. (2002). Water's symmetry C18 column (5 

miu, 4.6 x 250 mm) was used with a mobile phase consisting of water,

26 


methanol and glacial acid in the ratio 70:30:1, with a flow rate of 1.0 

ml/min isocratically. Linearity coefficients, assay values, recovery studies 

showed that the method is accurate and precise. 

2.2.4. Gas chromatographic (GC) methods 

A GC-MS method for the analysis of dextromethorphan and its 

metabolites was described by Xu et al. (1993). The urine sample was 

hydrolyzed with HCl, extracted with diethyl ether and derivatized with 

MSTFA (N-methyl-N-trimethylsilyl-trifluoroacetamide) MBTFA (N- 

methyl-bistrifluoroacetamide). Dextromethorphan and its three metabolites 

were detected in urine samples within 2.0-60 h after administration of the 

drug. Their structures and the variation of their concentration in urine were 

determined. The detection limit of dextromethorphan is 10 ng. 

Argekar et al. (1998) described simultaneous determination of 

dextromethorphan HBr from expectorant by gas chromatography (GC). 

Cough syrup (10 g) was mixed with 1.0 ml aqueous bromohexine HCl (5.0 

mg ml -1 ; internal standard) and 20 ml 1.0 M-NaOH. After shaking for 2.0 

min, the mixture was allowed to stand for 5.0 min then extracted by 

shaking with CHCl3 (3 x 20 ml) for 5.0 min. The combined extracts were 

evaporated to dryness and the residue reconstituted in 2.0 ml CHCl3. A 2.0 

μL portion of the resulting solution was analysed by GC on a column of 

5.0 % SE 30 on Chromosorb W HP (80-100 mesh) temperature 

programmed from 135 °C (held for 1.0 min) to 250 °C (held for 5.0 min) at 

8.0 °C/min and operated with N2 as carrier gas at 30 ml min -1 and FID. 

Calibration graph was linear for 0.5-5.0 mg ml -1 dextromethorphan HBr. 

Detection limit and quantification limits were 0.3 mg ml -1 and 0.15 mg 

ml -1 . Recovery was 100.78% with RSD (n = 5) of 1.72-2.25%.

27 


Statheropoulos et al. (1998) studied short column GC-MS and 

principle component analysis for the identification of co eluted substances 

in doping control analysis. A mixture of 50 μg ml -1 dextromethorphan 

HBr, 50 μg ml -1 methadone HCl, 100 μg ml -1 dextropropoxyphene HCl 

and 500 μg ml -1 cocaine HCl in methanol was analysed by GC on a fused- 

silica column (1.8 m x 0.2 mm i.d.) coated with ULTRA 2 (0.33 μm) 

operated isothermally at 130 °C with He as carrier gas (1.2 ml/min) and 70 

eV EIMS detection operated in full scan mode from m/z 40 to 310. 

Principle component analysis was applied to the spectrometric data to 

resolve overlapping components in the single chromatographic peak 

obtained at the higher isothermal temperature. The analysis time could be 

reduced to less than one minute using this approach. 

A sensitive, simple and accurate method was developed by Wu et al. 

(2003) for determination of dextromethorphan (DM) and dextrorphan (DT) 

in human urine by capillary gas chromatography without derivatization. 

After an oral dose of 30 mg DM, urine samples were collected and 

extracted, then analyzed on 0.22 mm x 17 m HP-1 capillary column. DM 

and its metabolite DT were analyzed simultaneously with good separation. 

Docosane was used as the internal standard (I.S.). The detector used was 

flame ionization detector (FID). There was a linear relationship between 

peak area ratios of analytes to I.S. and concentration of analytes over the 

concentration range 0.37-7.38 μM L -1 for DM and 0.39-77.8 μM L -1 for 

DT. The recovery was 88.1 approximately 103.9% for DM and 86.7 

approximately 96.8% for DT. The within-day and between-day 

coefficients of variation were less than 7.4 and 7.3% (RSD) for the assay 

of DM and DT in urine, respectively. The limits of detection (LOD) were 

0.30 μM L -1 for DM and 0.16 μM L -1 for DT. The limits of quantitation

28 


(LOQ) were 0.37 μM L -1 (RSD < 6.0%) for DM and 0.39 μM L -1 (RSD < 

7.0 %) for DT. The method has been applied to determine the oxidative 

phenotypes of cytochrome P450 2D6 (CYP2D6) in a Chinese population 

with metabolic ratio of DM in human urine. 

2.2.5. Liquid chromatographic (LC) methods 

Thomase et al. (1994) described mixed ion-pair LC method for the 

simultaneous assay of dextromethorphan HBr in frenadol sachets. The 

contents of a sachet of the cited cold medication were dissolved in water 

and, after sonicated for 15 min, the solution was filtered. A 50 μL portion 

of the filtrate was analysed on a Shandon Hypersil phenyl-2 (5.0 μm ) end- 

capped column (25 cm x 4.6 mm i.d.) with a mobile phase (2.0 ml/min) of 

aquoeus 5.0 % acetonitrile containing 50 mM-monosodium phosphate, 125 

mM-tetrabutyl ammonium hydrogen sulfate and 1.0 mM-1-pentane- 

sulfonic acid and adjusted to pH 2.5 with 1.0 M-NaOH; detection was at 

210 and 290 nm. Recovery was closed to 100 %, calibration graph was 

linear and the minimum amounts of active drug substance that could be 

quantified were 0.1 % of the 100 % label claim with detection at 210 nm, 

and 1.0 % with detection at 290 nm. The minimum detection limit was ≈10 

ng, and the RSD (n = 20) obtained by two analysts on different days was 

0.4-2.0 %. 

Rapid, sensitive and selective methods were developed by Eichhold 

et al. (1997) for the determination of dextromethorphan and its major 

metabolite, dextrorphan, in human plasma using liquid chroma- 

tography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked 

with stable-isotope internal standards were prepared for analysis by (LLE) 

procedure. Dextromethorphan and dextrorphan were chromatographed on 

a short reversed-phase column, using separate isocratic mobile phase

29 


conditions optimized to elute each compound in approximately 1.1 min. 

For both analytes, calibration curves were obtained over four orders of 

magnitude and the limit of quantitation was 5.0 g ml -1 using a 1.0 ml 

plasma sample volume. The accuracy across the entire range of spiked DM 

and DOR concentrations was, in general, within 10 % of the spiked value. 

The precision was generally better than 6.0% for replicate sample 

preparations at levels of 50 g ml -1 or higher and typically better than 12 % 

at levels below 50 g ml -1 . The method was applied for the evaluation of 

the pharmacokinetic profiles of dextromethorphan and dextrorphan in a 

human volunteer following peroral administration of a commercially 

available cough formulation. 

The commonly used antitussive dextromethorphan can be used to 

simultaneously assess potential cytochrome P450 3A (CYP3A) and 

CYP2D6 inhibition during drug development. The metabolism of 

dextromethorphan to dextrorphan and subsequently to 3-hydroxy- 

morphinan were via the 2D6 pathway, while the metabolism of 

dextromethorphan to 3-methoxymorphinan is via the 3A pathway. A 

sensitive and specific LC-MS/MS assay has been developed by 

Vengurlekar et al. (2002) to determine the human urine concentrations of 

dextromethorphan and three metabolites (dextrorphan, 3-methoxy- 

morphinan and 3-hydroxymorphinan) in support of drug interaction 

studies. Urine samples (0.5 ml), after enzymatic hydrolysis of the 

conjugates and containing 3-ethylmorphine as an internal standard, were 

extracted with chloroform under basic conditions. Following concentration 

and reconstitution, the samples were analyzed by LC-MS/MS. The assay 

was linear over the range of 5.00-500 ng ml -1 for dextromethorphan and 3- 

methoxymorphinan; and 200-3000 ng ml -1 for dextrorphan and 3-

30 


hydroxymorphinan using a Perkin-Elmer Sciex triple quadrupole mass 

spectrometer (API 300). The intra- and inter-day relative standard 

deviation (RSD) across three validation runs over the The entire 

concentration range for all analytes was less than 15%. Accuracy 

determined at three or four concentrations (9.00, 200, and 400 ng ml -1 for 

dextromethorphan and 3-methoxymorphinan; 250, 400, 1300 and 2500 ng 

ml -1 for dextrorphan and 3-hydroxymorphinan) ranged between 96.3 and 

113.8 %. The stability of analytes in urine was demonstrated for 9 months 

at -20 ºC, 24 h under ambient conditions and for up to three freeze/thaw 

cycles. The method described herein was suitable for the rapid and 

efficient measurement of dextromethorphan and different metabolites to 

estimate potential CYP3A inhibition by drug candidates and for screening 

of extensive and poor metabolizers of CYP2D6 in the heterogeneous 

population. The method has subsequently been validated on a Sciex API 

3000 with lower limit of quantitation; 1.00 ng ml -1 for dextromethorphan 

and 3-methoxymorphinan; 60.0 ng ml -1 for dextrorphan and 100 ng ml -1 for 

3-hydroxymorphinan. 

2.2.6. Thin-layer chromatography method 

The o-demethylation of dextromethorphan (DM) to dextrorphan (DR) 

was studied in 241 unrelated, healthy Jordanian volunteers (171 males, 70 

females). Urine was collected for 8.0 h following a single oral dose of DM 

bromhydrate 30 mg. TLC technique was used to identify the metaboliser 

phenotype by Irshaid et al. (1993). The frequency of the poor metaboliser 

phenotype was found to be 2.9% (approximate 95 confidence interval 0.8 

- 5.0%). Applying the Hardy-Weinberg Law, the frequency of the 

recessive autosomal gene controlling poor metabolism was 0.17 (95% 

confidence interval 0.108 - 0.232).

31 


Indrayanto (1996) described simultaneous densitometric 

determination of dextromethorphan HBr in syrup and its validation. 

Dextromethorphan HBr syrup (3.0 ml) was mixed with 3.0 ml 25 % 

ammonia for 10 min, ultrasonicated for 10 min and extracted with 4.0 ml 

and 2.0 x2.5 ml hexane. The hexane extract was diluted to 10 ml, a 8.0 ml 

portion was evaporated to dryness and the residue to dissolved in 1.0 ml 

hexane. The resulting solution was spotted onto silica gel 60 F254 plates 

and the plate was developed in methanol/25% ammonia (14:1) to 8.0 cm. 

The analyte spot was identified by scanning in absorbance-reflectance 

mode at 200-400 nm and quantified by measuring the absorbance at 223 

nm. Calibration graph was linear for 2.4-12.2 μg spot -1 of dextromethor- 

phan with detection limit of 0.13 μg spot -1 . Recovery was 99.61-101.21%. 

Sodhi et al. (1997) studied simultaneous determination of 

dextromethorphane HBr by HPTLC. Ground tablet was dissolved in 

methanol, filtered and the filtrate made up to 25 ml with methanol. A 4.0 

ml portion was up to 1.0 ml of 4.0 mg ml -1 naproxen (internal standard) 

made up to 10 ml with methanol and 5 μL applied to a silica gel 60 F254 

HPTLC plate with CH2Cl2/acetone/methanol/triethylamine (70:40:5:2) as 

mobile phase. The plate was densitometrically scanned at 257 nm and the 

calibration graph was linear from 0.05-54 μg. RSD was 1.4% with 

recoveries of 98-102%. 

DiGregorio et al. (1999) studied quantification of dextromethorphan 

HBr in caplets, gelcaps, and tablets by HPTLC with UV absorption 

densitometry. Dextromethorphan HBr (DM) in solid and liquid gelcaps 

was analysed on silica gel 60 F254 GLP HPTLC plates (20 cm x 10 cm for 

CF analysis), cleaned with dichloromethane/methanol (1:1) and ethyl 

acetate/methanol/concentrated NH3 (17:1:2) for DM as mobile phase and

32 


detection at 225 nm for DM. Amount of DM in caplets and gelcaps from 

1.2 to 1.9 %and recovery of 99.4%. 

2.3. Literature Survey for Determination of Silymarin 

2.3.1. Spectrophotometric methods: 

Rajasekaran et al. (1997) studied the estimation of silymarin in 

pharmaceutical formulations; based on the reaction of silymarin with 

diazotized sulphanilic acid in alkaline medium at 460 nm. The chromogen 

formed is stable and beer’s law was obeyed in the concentration range of 

2.0- 10 mcg ml -1 . The probable mechanism of reaction is the coupling of 

diazo group in diazotised sulphanilic acid at 8 th position of flavone ring 

which imparts intense orange red color in alkaline medium 

Zarapkar et al. (2000) determined silymarin in pharmaceutical dosage 

forms; twenty tablets, containing the hepatoprotectant drug silymarin were 

accurately weighted and ground to a fine powder. Portion (125 mg) of 

powder were sonicated with 10 ml methanol in an ultrasonic bath until the 

sample had dissolved. The solution was diluted to 25 ml with more 

methanol and filtered (what man filter paper No.4). Portions (2.0 ml) of 

filtrate, or those of a standard solution of silymarin, were heated with 

2.0ml methanolic 1.0% dinitro phenyl hydrazine solution (containing 2.0% 

- 96% H2SO4) in a sealed flask at 60 ºC for one hour. A 2.0 ml portion of 

the reaction solution was mixed with 3.0 ml methanolic 10% tetramethyl 

ammonium hydroxide solution and the mixture was diluted to 100 ml with 

more methanol. After 30 min , absorbance of the solution was measured at 

490 nm (vs. a reagent blank). The calibration graph was linear from 8.0-28 

μg ml -1 of silymarin. The method was applied to the analysis of silymarin 

in a pharmaceutical formulation labeled as containing 70 mg of silymarin.

33 


The average recovery of silymarin was 100. 40% with an RSD (n = 6.0) of 

0.48%. The average recovery of standard amounts of silymarin added to 

drugs was 99.92%. 

Li et al. (2002) studied quantitation of flavonoids in silymarin loaded 

solid dispersion system; (30-60 mg) sample was dissolved in and diluted to 

50 ml with10 mM- NaOH, shaken ultrasonically for 3.0 min and filtered. 

A 1.0 ml portion of the filtrate was diluted to 10 ml with 10 mM-NaOH, 

and the flavovoids were determined by measuring the absoluted trough 

amplitude (H) of the first-derivative spectral peak of flavovoids at 338 nm. 

Calibration graph was linear from (4.0-20 mu ml -1 )of flavovoids. 

Recoveries were 98.93-102.1% RSD (n =3.0) was less than or equal to 

5.1%. 

A new simple and sensitive kinetic spectrophotometric method for the 

determination of silymarin in pure form and in pharmaceutical 

formulations was described by Rahman et al. (2004). The method is based 

on the oxidation of the drug with potassium permanganate at pH 7.0 0.2. 

The reaction is followed spectrophotometrically by measuring the decrease 

in the absorbance at 530 nm. The calibration graph is linear in the range of 

18-50 μg ml -1 The method has been successfully applied to the 

determination of silymarin in pharmaceutical formulations. 

2.3.2. Electrokinetic capillary methods 

Ding et al. (2000) developed a micellar electrokinetic capillary 

chromatographic (MECC) method for the separation and determination of 

silybin, isosilybin, silydianin and silycristin in Legalon capsules, Yiganling 

tablets and silymarin. A buffer solution containing 30 mM L -1 disodium 

tetraborate, 50 mM L -1 taurodeoxycholic acid sodium salt and 20 mM L -1

34 


β-cyclodextrin (pH 9.2) was found to be the most suitable electrolyte for 

this separation. The applied voltage was 20 kV and UV detection 

wavelength was 288 nm. Rutin was used as internal standard. The average 

recoveries of 99.9% for silycristin, 98.3% for silydianin, 99.0% for silybin 

and 98.2% for isosilybin were obtained. 

2.3.3. High-performance liquid chromatography (HPLC) Methods: 

A combination of two stereo selective assays was developed using 

column-switching HPLC with electrochemical detection for the 

determination of free (unconjugated) silibinin and RP-HPLC with UV 

detection for the measurement of total (free and conjugated) silibinin in 

human plasma by Rickling et al. (1995). After extraction of free silibinin 

and the internal standard hesperetin with diethyl ether the compounds were 

pre-separated on a RP-CN column. A cut fraction of eluate containing the 

analytes was then transferred to the RP-18 main column by means of a 

switching valve for final separation of the compounds. The limit of 

quantification with electrochemical detection for free silibinin was 0.25 ng 

ml -1 per diastereomer. For the determination of total silibinin diastereomers 

all conjugates were cleaved enzymatically using beta-glucuronidase/ 

arylsulfatase at pH 5.6 followed by extraction with diethyl ether of the pH 

8.5 alkalized solution. Separation of the diastereomers and of the internal 

standard naringenin was achieved on a RP-18 column. The limit of 

quantification with UV detection at 288 nm for total silibinin was 5 ng ml -1 

per diastereomer. 

Wang et al. (1998) studied the determination of the active flavonoids 

in silymarin, a sample (100 mg) was sonicated with 40 ml ethanol for 3 

min and diluted to 50 ml with more solvent. The active flavonoids,

35 


taxifolin, silydianin, silybins A and B, as well as isosilybins A and B in the 

solution were analysed by HPLC. On a 10 μm YWG C18 column (20 cm x 

4.6 mm i.d.), with methanol / H2O /0.5 M-potassium dihydrogen phosphate 

buffer adjusted to pH 4.0 with 0.2 M-H3PO4 (10:10:1) as mobile phase at 

0.9 ml min -1 and detection at 280 nm. Gradient HPLC was similarly 

conducted. The calibration graphs were linear from 0.016-2.68 g L -1 . The 

RSD were < 2.0%. 

Quaglia et al. (1999) studied determination of silymarin in the 

extract from the dried silybum marianum fruits by HPLC and HPCE. 

Powdered silybum marianum fruit was soxhlet extracted with petroleum 

ether and the solution was discarded . Extraction was repeated twice with 

methanol and the methanolic solutions were evaporated to dryness . The 

main active flavonoids silybine, isosilybine, silycristine, silydianine and 

taxifolin (TXF)usually expressed as silymarin content (with the exception 

of TXF) were determined by HPLC and HPCE .For HPLC, two different 

methods were used. The first was with use of a 5.0 m purospher C 18 

column (25 cm x 4.0 mm i.d.) with H2O acidified to pH 2.6 with 10 % 

H3PO4 / acetonitril (31:19) as mobile phase (1 ml min -1 ) and detection at 

289 nm. The second method was a 5.0 m lichrosphere C8 column (25 cm 

x 4.0 mm i.d.) and a gradient solvent system of H2O acidified to pH 2.3 

with 10% H3PO4 / acetonitril / methanol (details tabulated; 1.0 ml min -1 ) 

and detection at 289 nm. HPCE was performed in an uncoated capillary 

(43 cm x 50 m μm i.d.; 35 cm effective length) with 100 mM-borax 

adjusted to pH 9.0 with 100 mM-boric acid containing 15 % methanol and 

12 mM-dimethyl β-cyclodextrin as back ground electrolyte and detection 

at 200 nm. RSD for all the methods was < 2.25%.

36 


Ding et al. (2001) studied separation and determination of effective 

components in compound products of silymarin by HPLC, powdered 

sample (0.2 g) was sonicated with some methanol for 20 min and diluted 

with more solvent to 100 ml and filtered. A 1.0 ml portion of the filtrate 

was further diluted with methanol to 10 ml (solution A). Portion (10 μL) of 

solution A was analyzed for silybin, isosilybin, silydianin, and silycristin, 

including the diastereomers of silybin and isosilybin, by HPLC on a 5.0 m 

shim-pack VPODS column (15 cm x 4.6 cm i.d.) equipped with a guard 

column (1.0cm x 4.6 mm i.d.) of the same phase, operated at 40 ºC , with 

gradient elution (1.5 ml min -1 )with methanol (mobile phase A) and 

aqueous 10% dioxane (mobile phase B) and detection at 288 nm. The 

calibration graphs were linear from 84.6 ng to 1.398 µg. by standard 

addition methods, the recoveries were 97.9-101.7% with RSD of < 3.4%. 

2.3.4. Thin - layer chromatography (TLC) method: 

Wu and Wang (1989) studied determination of silybin [silymarin] in 

the peel of silybum marianum; powdered sample (3.5 g) was defatted with 

light petroleum (b.p.60 ºC to 90 ºC ) in asoxhlet apparatus, the residue was 

extracted with ethyl acetate, the extract was evaporated to dryness and the 

residue was dissolved in ethanol (100 ml). Apportion was applied to a 

silica gel G plat with CHCl3 : acetone : formic acid (9:2:1) as mobile 

phase. The plate was scanned at 335 nm (reference wave length at 400 

nm)for determination of silymarin. The calibration graph was rectilinear 

from 1.0 to 7.0 µg of silymarin. The mean recovery (n = 5.0) for 11 mg of 

silymarin was 96% with a coefficient of variation of 5.0%.

37 


2.4. Spectrophotometric Determination of Some Drugs Using 

Acid Dyes Technique: 

2.4.1. Structure of the studied acid dyes: 

HO 

Br 

Bromophenol blue Bromocresol purple 

M.W.=671.6 M.W.= 541.8 

M.F.=C18H10Br4O5S M.F.= C20H16Br2O5 

HO 

C 

H 3 

Bromocresol green Bromothymol blue 

M.W.=699.6 M.W.= 625.8 

M.F.=C20H14Br4O5S M.F.= C26H28Br2O5S 

HO 

Br 

Br 

Br 

O 

CH 3 

Br 

S O 

O 

O 

OH 

Br 

Br 

S O 

O 

CH 3 

OH 

Br 

C 

H 3 

HO 

CH 3 

Br 

Br 

CH 3 

O 

C 

H 3 

S O 

O 

C 

H 3 

O 

S O 

O 

OH 

Br 

CH 3 

CH 3 

OH 

Br

38 


2.4.2. Literature survey for spectrophotometric determination of some 

drugs using acid dyes technique: 

Lowry (1993) used bromothymol blue (BTB) for estimation of some 

quaternary ammonium compounds like benzalkonium chloride, 

benzenthionium chloride and chlorhexidine gluconate. 

Vladimirov et al. (1993 and 1995) described a simple colorimetric 

assay for the determination of molsidomine in pharmaceutical formulations 

using (BCG), the method is based on the formation of a coloured ion-pair 

complex at pH 2.8. The absorbance of the chloroformic phase was 

measured at 421 nm against a reagent blank. He also studied 

spectrophotometric determination of nizatidine in pharmaceutical 

formulations via the formation of ion-pair complex with BPB, at pH 3.25, 

the absorbance of the chloroformic extract was measured at 417 nm. 

Bromophenol blue (BPB) was utilized for the spectrophotometric 

determination of some β-odrenoceptor blocking agents by El-Gindy et al., 

(1993). 

El Ragehy et al. (1995) used (BTB) and (BPB) for the spectro- 

photometric determination of triamterene in pure form and in 

pharmaceutical preparations. The dyes form a choroform-soluble/ coloured 

ion-association complex with triamterene at pH 3.4 and 3.2 using BPB and 

BTB, respectively. The formed complex could be extracted and measured 

spectrophotometrically at 417 nm for both dyes. 

Bromocresol green (BCG) used for the determination of ondanestrone 

by Zamora and Calatayud (1996). The method is based on the formation 

of 1:1 ion-pair with BCG in the pH range 3.2-4.4 and then extracted into 

chloroform layer. The absorption spectra was measured at 420.8 nm.

39 


Amin (1997) described a simple and sensitive spectrophotometric 

method for the determination of some anthelminties that are used in 

veterinary medication, based on the formation of ion-pair complex with 

(BCP) and (BCG). Drugs analyzed are piperazine hexahydrate, tetramisole 

hydrochloride and metronidazole. The drugs were determined either in 

pure powdered forms or in pharmaceutical formulations using the standard 

addition technique. 

Issa et al. (1997) studied three spectrophotometric methods for the 

determination of ofloxacin and lomefloxacin hydrochloride, which are 

based on their extraction into chloroform as ion-pairs with (BPB), (BTB) 

and (BCP). The absorbance value of the organic layer were measured at 

410, 415 and 410 nm for BPB, BTB and BCP. 

Abdel-Gawad (1997) described a simple and sensitive 

spectrophotometric method for the assay of three piprazine derivatives: 

ketoconazole, piribedil and prazosin hydrochloride based upon the 

intereaction of the basic drug in dry chloroform with (BPB) in the same 

solvent to produce a stable yellow ion-pair complex which absorbs at 410 

nm. 

Saad et al. (1997) studied the spectrophotometric determination of the 

antitussive drug noscapine, based on the measurement of the absorbance 

of the organic soluble ion-association complex formed between the 

noscapine monocation and a bulky counter anion. (BCG) and (BTB) were 

used as the counter ions. 

A simple and sensitive spectrophotometric method for the 

determination of amlopidine besylate (ABD) in pure forms and tablets was 

described by Sridhar et al. (1997). This method is based on the formation

40 


of an ion association complex between the drug and an acidic dye, 

Bromothymol blue, which is extractable into chloroform and has an 

absorption maximum at 405 nm. 

Bsavaiah and Krishnamurthy (1998) studied a simple, accurate, 

rapid and sensitive spectrophotometric method for the assay of six 

phenothiazine derivatives in bulk and in their pharmaceutical preparations. 

The method based on the ion-pair complex reaction of phenothiazines with 

(BCG) in aqueous acidic buffer. 

Famotidine was determined spectrophotometrically by Abu Zuhri et 

al. (1999) by the formation of (1:1) ion-pair complex between the drug and 

each of bromocresol green (BCG)and bromothymol blue (BTB). The 

yellow ion-pair formed was quantitatively extracted into dichloromethane, 

and its absorption spectrum displayed an absorption band at 420 nm. 

El-Yazbi et al. (1999) described sensitive and accurate 

spectrophotometric method for the determination of benazepril hydro- 

chloride in its single and multi-component dosage forms. This method 

depends on the color formed by the reaction of the drug with (BCG). The 

yellow color of the resulting ion-pair complex was extracted with 

chloroform and measured at 412 nm. 

El Sherif (1999) studied spectrophotometric determination of 

enrofloxacin through the formation of an ion-pair complex with (BCP) in 

presence of phthalate buffer (pH 4.8 ±0.4). the yellow ion-pair is extracted 

with chloroform and the absorbance is measured at 410 nm. The method 

allows the determination of 4.0-48 µg ml -1 . 

Simple, sensitive and selective methods for the determination of 

trimethoprim (TMP) in pure form and in pharmaceutical formulations were

41 


described by El-Ansary et al. (1999). The methods are based on the 

reaction of TMP as л- electron donor with (BTB) and (BCG) as electron 

acceptors. The coloured products are quantified spectrophotometrically at 

their corresponding λ max values. 

Hassan (2000) studied a spectrophotometric method for the 

determination of ipratropium bromide by the formation of an ion-associate 

complex between the drug and an acidic dye, (BCG), which is extractable 

into chloroform and has an absorption maximum at 418 nm. 

Two simple, sensitive and accurate spectrophotometric methods for the 

determination of loperamide hydrochloride (lop. HCl) were described by 

El Sherif et al. (2000). The first method is based on the formation of ion- 

pair association complex (1:1) with bromothymol blue (BTB), 

bromophenol blue (BPB). The coloured products are extracted into 

chloroform, and measured spectrophotometrically at 414 (BTB) and 415 

(BPB). Beer’s law was obeyed in the ranges of 5–35 and 5–30 g ml −1 for 

BTB and BPB methods, respectively. 

Three simple and sensitive extractive spectrophotometric methods 

have been described by Rahman and Hejaz-Azmi (2000) for the assay of 

diltiazem hydrochloride either in pure form or in pharmaceutical 

formulations. The developed methods involve the formation of colored 

chloroform extractable ion-pair complexes of the drug with bromothymol 

blue (BTB), bromophenol blue (BPB) and bromocresol green (BCG) in 

acidic medium. The extracted complexes showed absorbance maxima at 

415 nm for all three methods. Beer's law is obeyed in the concentration 

ranges 2.5–20.0, 2.5–10.0 and 2.5–12.5 g ml-1 with BTB, BPB and BCG, 

respectively.

42 


El-Gindy et al. (2001) studied the determination of trazodone 

hydrochloride in pharmaceutical tablets. The spectrophotometric method 

was based on the formation of a yellow ion pair complex between the basic 

nitrogen of the drug and bromophenol blue at pH 3.4. The formed complex 

was extracted with chloroform and measured at 414 nm. 

A spectrophotometric method described by Marona and Schapoval 

(2001) for the determination of sparfloxacin in tablets. The procedure is 

based on the complexation of bromothymol blue 0.5% and sparfloxacin to 

form a compound of yellow colour with maximum absorption at 385 nm. 

Two simple, rapid and sensitive spetrophotometric methods have been 

developed by Gowda et al. (2001) for the assay of ceterizine 

hydrochloride (CTZH) in bulk drug and in pharmaceutical preparations. 

These methods are based on the formation of chloroform soluble 

complexes between CTZH with (BCP and BPB) in Walpole buffer at pH 

2.64 with an absorption maximum at 409 and 414 nm for BCP and BPB, 

respectively. 

Ramesh et al. (2001) described an extractive spectrophotometric 

method for the determination of Antiallergic drugs in pharmaceutical 

formulations using (BTB). This method based on the formation of ion- 

association complex between the antiallergic drugs and BTB. 

A highly sensitive color reaction has been developed by Liu et al. 

(2002) based on the fact that vitamin B1 reacted with a triphenylmethane 

acid dye such as bromothymol blue, bromophenol blue, bromocresol 

green, to form an ion-association complex in a weak-base aqueous solution 

in the presence of some solubilization agents e.g. polyvinyl alcohol, 

emulgent OP, Triton X-100 or Tween-20. The wavelengths of maximum

43 


absorbance of the ion-association complexes were between 420 and 450 

nm, and fading reaction appeared at the longer wavelength and the 

maximum fading wavelengths were between 550 and 620 nm. 

A simple, quick and sensitive spectrophotometric method was 

described by Mostafa et al. (2002) for the determination of enrofloxacin 

and Pefloxacin. The method is based on the reaction of these drugs with 

bromophenol blue (BPB) in buffered aqueous solution at pH 2.3–2.5 to 

give highly coloured complex species, extractable with chloroform. The 

coloured products are quantitated spectrophotometrically at 420 for BPB. 

Three simple, accurate and sensitive spectrophotometric methods were 

developed by Suslu (2002) for the determination of enoxacin. The 

methods based on the extraction of this drug into chloroform as ion pairs 

with sulphonphthalein dyes as bromophenol blue and bromocresol purple. 

The optimum conditions of the reactions were studied and optimized. The 

absorbance of yellow products was measured at 412 nm for enoxacin– 

bromophenol blue and 410 nm for enoxacin–bromocresol purple. Linearity 

ranges were found to be 2.0–20.0 μg ml -1 for enoxacin–bromophenol blue 

and 0.77–17.62 μg ml -1 for enoxacin–bromocresol purple. 

A direct, extraction-free spectrophotometric method has been 

developed by Abdine et al. (2002) for the determination of cinnarizine in 

pharmaceutical preparations. The method was based on an ion-pair 

formation between the drug and three acidic (sulphonphthalein) dyes; 

namely bromocresol green (BCG), bromocresol purple (BCP) and 

bromophenol blue (BPB) which induces an instantaneous bathochromic 

shift of the maximum in the drug spectrum.

44 


A spectrophotometric method was developed for the determination of 

etidocaine hydrochloride (EH) in injectable pharmaceutical preparation by 

Silva and Schapoval (2002). The proposal of this work was to develop a 

rapid, simple, inexpensive, precise and accurate visible spectrophotometric 

method. The method is based on the formation of the ion-pair complex by 

the EH reaction with bromocresol green in the pH value of 4.6 which after 

chloroform extraction gives a yellow color which changes in basic medium 

to blue color and exhibits a maximum absorbance at 625 nm. The 

calibration graph was linear over the range 2.0–6.0 g ml-1 EH calculated 

on the final yellow solution. 

Dinesh et al. (2002) studied an extractive spectrophotometric method 

for the determination of sildenafil citrate (SC). The method is based on the 

formation of an ion-association complex of (SC) with bromocresol green 

(BCG)in aqueous acidic buffer. The complex extractable to the chloroform 

phase, was quantitatively measured at 415 nm. Beer's law was obeyed in 

the SC concentration range 1.25-25 µg ml -1 with a limit of detection 0.16 

µg ml -1 . 

A simple and sensitive extractive spectrophotometric method in the 

visible region was described by Sadeghi et al. (2002) for the determination 

of diazepam in bulk sample and pharmaceutical formulations. The method 

is based on 1 : 1 ion-association complex formation with the acidic dye, 

bromocresol green at pH 3.5, which is extractable into chloroform from the 

aqueous phase. Spectrophotometric measurement was done at 410 nm. 

Beer's law is obeyed in a concentration range of 2–60 µg mL -1 for the 

investigated complex. 

A simple and accurate method was described by El-Yazbi et al. (2003) 

for the determination of nizatidine (NIZ) in pharmaceutical preparations.

45 


The method is based on the formation of an ion-pair complex between the 

drug and bromocresol purple with subsequent measurement of the 

developed color at 411 nm. 

New spectrophotometric procedures have been established by Erk 

(2003) for the assay of torvastatin either in bulk form or in pharmaceutical 

formulations. The procedures are based on the reaction between the 

examined drug and bromocresol green, or bromophenol blue producing an 

ion-pair complexes which can be measured at the optimum wavelengths. 

Simple, rapid, and extractive spectrophotometric methods were 

developed by Süslü (2003) for the determination of ofloxacin in bulk and 

pharmaceutical dosage form. These methods are based on the formation of 

yellow ion-pair complexes between the basic nitrogen of the drug and 

bromophenol blue and bromocresol purple as sulphonphthalein dyes in 

phthalate buffer pH 3.0 and pH 3.1, respectively. The formed complexes 

were extracted with chloroform and measured at 414 and 408 nm for 

ofloxacin–bromophenol blue and ofloxacin–bromocresol purple, 

respectively. 

Two sensitive and simple spectrophotometric methods for the 

estimation of reboxetine in pure form and in pharmaceutical formulations, 

were developed by Erk (2003). The methods are based upon the 

interaction of the basic drugs in dry chloroform with bromothymol blue 

(BTB) and bromocresol green (BCG) in the same solvent to produce a 

stable yellow ion-association complexes. The colored products are 

quantified spectro-photometrically at their corresponding max. Beer's law 

is obeyed in the range 16.0–34.0 µg mL -1 and 10.0–30.0 µg mL -1 for BTB 

and BCG, respectively.

46 


Spectrophotometric determination of Bisacodyl and Piribedil, was 

described by Abdel-Hay et al. (2004). The method is concerned with the 

reaction of the investigated drugs with three sulphonphthalein acid dyes, 

namely; bromocresol green (BCG), bromocresol purple (BCP) and 

bromophenol blue (BPB). The yellow ion-pair complexes formed show 

absorption spectra with maxima within the range from 400 to 415 nm. The 

stoichiometric ratio was found to be 11. 

Three simple, sensitive and accurate spectrophotometric methods have 

been developed by Rahman (2004) for the determination of nifedipine in 

pharmaceutical formulations. These methods are based on the formation of 

ion-pair complexes of the amino derivative of nifedipine with bromocresol 

green (BCG), bromophenol blue (BPB) and bromothymol blue (BTB) in 

acidic medium. The colored products are extracted with chloroform and 

measured spectrophotometrically at 415 nm (BCG, BPB and BTB).

47 


Aim of the work 

The aim of the present work is to develop spectrophotometric methods 

for determination of ketamine hydrochloride (analgesic and anaesthetic), 

dextromethorphan hydrobromide (cough suppressant) and silymarin 

(hepatoprotectant and antioxidant) using the acid dye reagents bromocresol 

green (BCG), bromocresol purple (BCP), bromothymol blue (BTB), 

bromophenol blue (BPB). The principle of these methods are based on 

allowing the reaction of drug with the dye at a selected acidic pH value. A 

highly colored ion-pair chromogen is formed which is extracted with an 

organic solvent to be measured spectrophotometrically at its λ max. We 

have stimulated this work toward the development of reliable 

spectroscopic procedures for the determination of micro amounts of 

ketamine hydrochloride, dextromethorphan hydrobromide and silymarin in 

pure forms, in pharmaceutical preparations, in biological samples (urine or 

serum) and any degradation products in the sample which are likely to 

occur at normal storage conditions.

3.1. Apparatus 

3. MATERIALS AND METHODS 

All the absorption spectral measurements were made using Kontron 

930 (UV-Visible) spectrophotometer (German) with scanning speed 200 

nm/min, and band width 1.0 nm equipped with 10 mm matched quartz 

cells. 

The pH values of universal buffer solutions were checked using an 

Orion research pH-meter model 601 A/digital ionalyzer. 

3.2. Materials 

3.2.1. Drugs 

The drugs under investigations are ketamine hydrochloride (KET), 

dextromethorphan hydrobromide (DEX) and silymarin (SIL). 

3.2.1.1. Ketamine hydrochloride 

Ketamine hydrochloride, (KET) was provided by Egyptian 

International Pharmaceutical Industries Company (EPICO). The purity of 

the sample was found to be not less than 99.0% on the dried basis 

according to British Pharmacopia method BP (1998). A stock solution of 

KET (5.0 10 -4 M) was prepared by dissolving 0.0137g of pure sample in 

least amount of bidistilled water in 100 ml measuring flask then diluted 

with bidistilled water to the mark. Further dilution was carried out with 

bidistilled water to obtain 100 μg ml -1 stock solution of the studied drug. 

3.2.1.1.A. Official method for ketamine hydrochloride (USP 23, 2000) 

Dissolve about 500 mg of ketamine hydrochloride accurately weighed, 

in 1.0 ml of formic acid, and add 50 ml of glacial acetic acid. Add 10 ml of

49 

____________________________________________ Materials and Methods 

mercuric acetate and one drop of crystal violet and titrate with 0.1 N 

perchloric acid to a blue-green end point. Perform a blank determination, 

and make any necessary correction. Each mL of 0.1 N perchloric acid is 

equivalent to 27.42 mg of C13H16ClNO.HCl of the drug. 

3.2.1.2. Dextromethorphan hydrobromide 

Dextromethorphan hydrobromide (DEX) was provided by EPICO and 

the purity of the sample was found to be 99.5% on the dried basis 

according to Unite State Pharmacopeia method USP (2002). A stock 

solution of DEX (5.0 x 10 -4 M) was prepared by dissolving 0.0187 g of 

pure sample in least amount of bidistilled water in 100 ml measuring flask 

then diluted with water to the mark. Further dilution was carried out with 

bidistilled water to obtain 100 μg ml -1 stock solution of the studied drug. 

3.2.1.2.A. Official method for dextromethorphan hydrobromide (BP, 

1998) 

Dissolve 0.3 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid and 

20 ml of alcohol. Titrate with 0.1 M sodium hydroxide, determining the 

end-point potentiometrically. Read the volume added between the two 

points of inflexion. Each 1.0 ml of 0.1 M sodium hydroxide is equivalent 

to 35.23 mg of C18H26BrNO of the drug. 

3.2.1.3. Silymarin 

Silymarin was kindly supplied by EPICO and the purity of the sample 

was found to be 99.7% on the dried basis according to British 

Pharmacopeia BP (1998) method. A stock solution of SIL (5.0 x 10 -4 M) 

was prepared by dissolving 0.0241 g of pure sample in the least amount of 

acetone transferred to 100 ml measuring flask then diluted with the same

50 


solvent to the mark. Further dilution was carried out with the same solvent 

to obtain 100 μg ml -1 stock solution of the studied drug. 

3.3. Reagents 

A stock solutions (5.0 10 -4 M) of bromocresol green (BCG), 

bromocresol purple (BCP), bromothymol blue (BTB) and bromophenol 

blue (BPB), (Merck) were prepared by dissolving an appropriate weight of 

the dye initially in 20 ml of acetone followed by dilution in 100 ml 

measuring flask by the same solvent to the mark. 

All of the solvents and chemicals used in this study were of analytical 

grade quality of the highest purity and all solutions were freshly prepared. 

(Chloroform, benzene, carbon tetrachloride, methylene chloride, acetone 

and hexane) were provided by (ADWIC). 

3.4. Market Samples 

1. Ketamar ampoules, manufactured by Amoun Pharmaceutical Industries 

Company, claimed to contain 50 mg/ml of ketamine hydrochloride per 

ampoule. 

2. Tussilar drops, Kahira Pharmaceutical and Chemical Industries, Cairo, 

claimed to contain 1.0 g/15 ml of dextromethorphan hydrobromide. 

3. Codiphan syrup manufactured by The Nile Company for 

Pharmaceuticals and Chemicals Industries, claimed to contain 15 

mg/5.0 ml dextromethorphan hydrobromide. 

4. Tussilar tablets, Kahira Pharmaceutical and Chemical Industries 

claimed to contain 10 mg of dextromethorphan hydrobromide per 

tablet.

51 


5. Hepamarin capsules, manufactured by Uni Pharma Company, claimed 

to contain 140 mg of silymarin per capsule. 

6. Legalex tablets, manufactured by Alexandria Company for Pharma- 

ceuticals, claimed to contain 70 mg of silymarin per tablet. 

7. Legalon tablets, manufactured by Cid company for Pharmaceutical 

Industries, Egypt, claimed to contain 70 mg of silymarin per tablet. 

3.5. Determination of the Molecular Structure: 

The spectrophotometric methods were used for the determination of 

the molecular structure and stability constants of the coloured ion pair 

complexes. 

3.5. A. The molar ratio method 

In the molar ratio method, the concentration of drug is kept constant 

(1.0 ml of 5.0 x10 -4 M) while the reagent is regularly varied (0.2 - 2.4 ml 

of 5.0 x 10 -4 M). The absorbance of the prepared solutions was measured at 

the λ max for each complex. The absorbance values were then plotted versus 

the molar ratio [reagent / drug]. The intersection of the straight lines 

obtained shows the molar ratio of the most stable complexes. 

3.5. B. The continuous variation method 

In the present work, the modification of Job’s continuous variation 

method is utilized for investigating the stoichiometric ratio of the reaction 

prouduct between drug and reagent. A series of solutions was prepared by 

mixing equimolar solutions of the drug and the reagent in different 

proportions (0.1-0.9 ml of 5.0 x 10 -4 M) while keeping the total molar 

concentration constant (1.0 ml of 5.0 x 10 -4 M). A plot of the absorbance of 

the solution measured at λ max versus the mole fraction of the drug

52 


manifests a maximum at the expected molar ratio of the most stable 

complexes. 

2.6. Spectrophotometric Determination of the Drugs Under 

Investigation Using Ion-Pair Complex Formation with Acid 

Dyes 


BCG. 

Aliquot portions of DEX, KET and SIL containing 10-130 μg ml -1 , 

(0.1 –1.4 ml) in case of DEX, (0.1-1.6 ml) in case of KET and (0.1- 1.5 

ml) in case of SIL were transferred into 10 ml measuring flask and mixed 

with 1.2, 1.0 ml and 1.2 ml of BCG (5.0 x 10 -4 M), followed by 2.0 ml pH 

3.0, 2.5 ml pH 4.0 and 1.5 ml pH 3.5 of universal buffer solution, for DEX, 

KET and SIL, respectively. The volume was completed to 10 ml with 

bidistilled water and the solution was transferred into a 25 ml separating 

funnel and the formed ion-pair was extracted with 5.0 ml of chloroform in 

case of DEX, KET and SIL by shaking for 3.0 min. The absorbance values 

of the extracted solutions were measured at 419 nm, 417 nm and 420 nm 

for DEX, KET and SIL, respectively, against extracted reagent blank 

prepared in the same manner at room temperature. A calibration graph for 

each drug was constructed from which the concentration of unknown 

samples could be deduced. 


BCP. 


(0.1 –1.3 ml) in case of DEX, (0.1-1.5 ml) in case of KET and (0.1- 1.6

53 



with 1.2 ml or 1.0 ml of BCP (5.0 10 -4 M), followed by 2.0 ml of 

universal buffer solution pH 4.0 or 3.0, for (DEX and SIL) or KET, 

respectively. The volume was completed to 10 ml with bidistilled water 

and the solution was transferred into a 25 ml separating funnel and the 

formed ion-pair was extracted with 5.0 ml of chloroform, by shaking for 

2.5 and 3.0 min for DEX and KET or with 5.0 ml of methylene chloride, 

by shaking for 3.0 min for SIL, respectively. The absorbance values of the 

extracted solution were measured at λmax 409 nm, 408 nm and 418 nm for 

DEX, KET and SIL, respectively, against extracted reagent blank prepared 

in the same manner at room temperature. A calibration graph for each drug 

was constructed from which the concentration of unknown samples could 

be deduced. 

3.6.3. Determination of the studied drugs in authentic powder 

using BTB. 


(0.1 –1.6 ml) in case of DEX, (0.1-1.4 ml) in case of KET and (0.1- 1.5 


with 1.0 or 1.2 ml of BTB (5.0 10 -4 M), followed by 1.5 ml pH 4.0 or 2.0 

ml pH 3.0 of universal buffer solution for DEX or (KET and SIL), 

respectively. The volume was completed to 10 ml with bidistilled water 

and the solution was transferred into a 25 ml separating funnel and the 

formed ion-pair was extracted with 5.0 ml of chloroform, by shaking for 

3.0 min for DEX, KET and SIL. The absorbance values of the extracted 

solutions were measured at 413 nm, 412 nm and 420 nm for DEX, KET 

and SIL, respectively against extracted reagent blank prepared in the same 

manner at room temperature. A calibration graph for each drug was

54 


constructed from which the concentration of unknown samples could be 

deduced. 

3.6.4. Determination of the studied drugs in authentic powder 

using BPB. 

Aliquot portions of DEX, KET and SILcontaining 10-130 μg ml -1 , (0.1 

–1.5 ml) in case of DEX, (0.1-1.4 ml) in case of KET and (0.1- 1.6 ml) in 

case of SIL were transferred into 10 ml measuring flask and mixed with 

1.0 and 1.1 ml of BPB (5.0 x 10 -4 M), followed by 2.0 ml buffer of pH 4.0 

for (DEX and SIL) or 1.5 ml buffer of pH 3.0 for KET, respectively. The 

volume was completed to 10 ml with bidistilled water and the solution was 

transferred into a 25 ml separating funnel and the formed ion-pair was 

extracted with 5.0 ml of chloroform, by shaking for 3.0 and 2.5 min in case 

of DEX and KET or with 5.0 ml of methylene chloride, by shaking for 3.0 

min for SIL, respectively. The absorbance of the extracted solution was 

measured at λmax 417 nm, 416 nm and 421 nm for DEX, KET and SIL, 

respectively, against the extracted reagent blanks prepared in the same 

manner at room temperature. A calibration graph for each drug was 

constructed from which the concentration of unknown samples could be 

deduced. 

3.7. Dosage Forms 

3.7.1. Determination of ketamine hydrochloride in ketamar ampoules 

Transfer an accurately measured volume of injection, equivalent to 

about 500 mg of KET to 100 ml measuring flask and shake well with 

bidistilled water, completed to a 100 ml with bidistilled water to obtain 

test solution of 100 μg ml -1 of KET. The general procedure described

55 


above were used for the determination of KET concentration in its dosage 

form. 

3.7.1.A. Official method for the determination of ketamine 

hydrochloride injection (USP 25, 2002) 

Transfer an accurately measured volume of injection, equivalent to 

about 500 mg of KET to 200 ml volumetric flask, dilute with water to 

volume, and mix. Transfer 20.0 ml of this solution to a 125 ml separating 

funnel, add 3.0 ml of 0.1 N sodium hydroxide, and extract with three 15 ml 

portions of chloroform. Collect the chloroform extracts in a second 125 ml 

separator, and extract with 30 ml portions of 0.1 N sulfuric acid, collecting 

the acid extracts in a 200 ml volumetric flask. Dilute with 0.1 N sulfuric 

acid (saturated with chloroform) to volume and mix. Concomitantly 

determine the absorbance of this solution and a Standard solution of USP 

KET in the same medium having a known concentration of about 250 μg 

ml -1 , in 1.0 cm cells at λ max 269 nm, with a suitable spectrophotometer 

using 0.1 N sulfuric acid (saturated with chloroform) as the blank. 

Calculations: 

mg of Ketamine hydrochloride = (237.73 / 274.19) (2 C / V) (A u / A s) 

Where: 

237.73 and 274.19 are the molecular weights of ketamine and 

ketamine hydrochloride, respectively. 

C = the concentration of ketamine hydrochloride in the standard 

solution in μg ml -1 . 

V = the volume of injection taken in ml. 

Au = the absorbance of the solution from the injection.

56 


As = the absorbance of the standard solution. 


tablets 

Thoroughly powder and mix the contents of 10 tablets of the 

investigated drug and determine the average weight of each one. An 

accurately weighed amount of the powder equivalent to 10 mg of DEX 

was transferred to 100 ml measuring flask, shake well with warm distilled 

water for 5.0 min and completed to 100 ml with bidistilled water. Filter if 

necessary, and further dilution with bidistilled water was carried out to 

obtained test solution of 100 μg ml -1 of DEX. The general procedure 

described above was used for the determination of the drug concentration. 


drops 

Thoroughly the contents of one bottle of the investigated drug was 

transferred into a 25 ml beaker. An aliquot of the drops equivalent to 1.0 g 

of DEX was transferred to 100 ml measuring flask, shake well with warm 

distilled water for 5.0 min and completed to 100 ml with bidistilled water. 

Filter if necessary, and further dilution with bidistilled water was carried 

out to obtain test solution of 100 μg ml -1 of DEX. The general procedure 


3.7.4. Determination of dextromethorphan hydrobromide in codiphan 

syrup 

Thoroughly the contents of one bottle of the investigated drug was 

transferred into a 250 ml beaker. An aliquot of the syrup equivalent to 15 

mg of DEX was transferred to 100 ml measuring flask, shake well with

57 


warm distilled water for 5.0 min and completed to 100 ml with bidistilled 

water, filter if necessary, and further dilution with bidistilled water was 

carried out to obtain test solution of 100 μg ml -1 of DEX. The general 

procedure described above was used for the determination of the drug 

concentration. 

3.7.4.A. Official method for the determination of dextromethorphan 

hydrobromide syrup (USP 25, 2002) 

Pipet a volume of syrup equivalent to about 10 mg of DEX into a 100 

ml volumetric flask, dilute with water to volume, and mix. Calculate the 

quantity, in mg, of DEX in the volume of the syrup taken by the formula: 

in which: 

(370.33 / 352.32)(100 C)(r u / r s) 

370.33 and 352.32 are the molecular weights of dextromethorphan 

hydrobromide and anhydrous dextromethorphan hydrobromide, 

respectively. 

C = the concentration of dextromethorphan hydrobromide, on the 

anhydrous basis, in the standard solution in μg ml -1 . 

r u and r s are the peak responses obtained from the assay preparation 

and the standard preparation, respectively. 

3.7.5. Determination of silymarin in hepamarin capsules 

Thoroughly powder and mix the contents of 10 capsules of the 


accurately weighed amount of the powder equivalent to 140 mg of Sil was 

transferred to 100 ml measuring flask, shake well with warm distilled 

water for 5.0 min and completed to 100 ml with bidistilled water, filter if 

necessary, and further dilution with bidistilled water was carried out to

58 


obtained test solution of 100 μg ml -1 of SIL. The general procedure 


3.7.6. Determination of silymarin in legalex and legalon tablets 

Thoroughly powder and mix the contents of 10 tablets of the 


accurately weighed amount of the powder equivalent to 70 mg of Sil was 

transferred to 100 ml measuring flask, shake well with warm distilled 

water for 5.0 min and completed to 100 ml with bidistilled water. Filter if 

necessary, and further dilution with bidistilled water was carried out to 

obtained test solution of 100 μg ml -1 of SIL. The general procedure 


3.7.7. Determination of the studied drugs in urine samples 

In a 25 ml measuring flask, 5.0 ml urine aliquot of a healthy person 

was mixed with various concentrations of the investigated drugs then the 

optimum volume of the reagent solution was added and the solution was 

adjusted to the required pH values for each ion-pair formed, in sequence 

(drug-reagent-buffer) and the solutions were completed to the mark with 

bidistilled water, shaked well and left to stand for the optimum time in case 

of DEX, KET or SIL as described above for each reagent.

59 


3.7.8. Determination of ketamine hydrochloride in serum samples 

3.7.8.A. Determination of ketamine hydrochloride in serum samples 

(in vitro) 

In a 25 ml measuring flask, 2.0 ml serum aliquot of a healthy person 

was mixed with various concentrations of the investigated drug after 

deproteinization by 10 ml trichloroacetic acid solution, then the optimum 

volume of the reagent solution was added and the solution was adjusted to 

the required pH values for each ion-pair formed, in sequence (drug- 

reagent-buffer) and the solutions were completed to the mark with 

bidistilled water, shaked well and left to stand for the optimum time as 

described above for each reagent.

60 


3.7.8.B. Biochemical effect of KET injection on liver and kidney 

functions (in vivo) 

3.7.8.B. 1. Experimental animals: 

Adults male and female Swiss albino mice purchased from the unit of 

Egyptian Organization for Biological Products and Vaccines were used 

throughout this work. 

A total of 90 adult male and female Swiss albino mice weighing 20- 

25g were used in this experiment. The animals were randomly divided into 

five groups each contain 18 mice, divided as follow: 

Group I Normal control (N.C): Mice remained as normal control. 

Group II Normal treated with KET: Mice injected intrapentoreally (I.P.) 

by (5.0 μg/mouse) dose for 30-60 minutes. 

Group III Normal treated with KET: Mice injected intrapentoreally 

(I.P.) by (5.0 μg/mouse) dose for 3 hours. 

Group IV Normal treated with KET: Mice injected intrapentoreally 

(I.P.) by (10 μg/mouse) dose for 30-60 minutes. 

Group V Normal treated with KET: Mice injected intrapentoreally (I.P.) 

by (10 μg/mouse) dose for 3 hours. 

3.7.8.B.2. Collection of blood samples 

Because of the difficulties in obtaining large amounts of mouse blood 

serum for the assay, pooled blood samples from 3 mice, were collected in 

dry clean centrifuge tubes and allowed to clot at room temperature, then 

samples were centrifuged at 3000 r.p.m. for 10 minutes. Samples were 

kept at -20 ºC in sterile tubes in until assayed.

61 


Determination of serum transaminases (AST & ALT) activities: 

Transaminasas were determined according to Reitman and Frankel 

(1957). (Diamond Diagnostics Company). 

Principle: 

AST catalyses the following reaction: 

AST 

α-Ketoglutarate + L-aspartate L-glutamate + oxaloactate 

AST catalyses the following reaction: 

ALT 

α-Ketoglutarate + DL-alanine L-glutamate + pyruvate 

The evolved ketoacids react with 2, 4-dinitrophenylhydrazine giving 

the corresponding hydrazone derivatives (yellow) which changes to 

brownish color in an alkaline solution. 

Reagents: 

R1 (AST buffer substrate): 

Phosphate buffer pH 7.2 100 mmol/l 

α-Ketoglutarate 80 mmol/l 

L-aspartate 4.0 mmol/l 

R2 (Color reagent) 

2, 4-dinitrophenylhydrazine 4.0 mmol/l 

R1 (ALT buffer substrate): 

Phosphate buffer pH 7.2 100 mmol/l 

α-Ketoglutarate 80 mmol/l 

DL-alanine 4.0 mmol/l

62 


R2 (Color reagent) 

2, 4-dinitrophenylhydrazine 4.0 mmol/l 

Additional reagent 

Sodium hydroxide 0.4 N 

Procedure: 

Wavelength 546 nm (530-550) 

* Pipette into test tube: 

Sample 

R1(AST or ALT) 

Distilled water 

Blank Sample 

-------- 

0.5 ml 

100 μl 

100 μl 

0.5 ml 

------ 

** Mix, incubate for exactly 30 minutes at 37 ºC, then add: 

R2 0.5 ml 0.5 ml 

*** Mix, incubate for exactly 20 minutes at 20-25 ºC, then add: 

Na OH (0.4 N) 5.0 ml 5.0 ml 

Mix, and read the absorbance of the sample (A sample) after 5.0 

minutes against reagent blank. 

The colour intensity is stable for 60 minutes. 

Calculation was done using standard curves and results are shown in 

figures (A & B).

63 


Absorbance 

0.4 

0.3 

0.2 

0.1 

0 

0 20 40 60 80 100 120 140 160 

U/L 

Fig. (A): Standard curve of ALT. 

Absorbance 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

0 20 40 60 80 100 

U/L 

Fig. (B): Standard curve of AST.

64 


Determination of blood urea: 

Blood urea was determined according to the method of Patton and 

Crouch, (1977). 

Principle: 

Determination of urea according to the following reaction: 

urease 

Urea + H2O 2 NH3 + CO2 

In an alkaline medium, the ammonium ions react with the salicylate 

and hypochlorite to form a green coloured indophenol. 

Reagents: 

R1 Urea standard 50 mg/dl 

R2 Enzyme reagent 

Urease 

>5000 U/l 

R3 Buffer reagent 

Phosphate buffer pH 8 

100 mmol/l 

Sodium salicylate 

52 mmol/l 

Sodium nitroprusside 

2.5 mmol/l 

EDTA 

2.0 mmol/l 

R4 Alkaline reagent 

Sodium hydroxide 

80 mmol/l 

Procedure: 

Sodium hypochlorite 

4.0 mmol/l 

Wavelength 580 nm; 578nm 

Blank Standard Sample 

R3 (Buffer) 1.0 ml 1.0 ml 1.0 ml 

R2 (Urease) One drop One drop One drop

65 


R1 (Standard) ---- 10 μl ---- 

Sample --- ---- 10 μl 

Mix and incubate at least 3 minute at 37 ºC or 5 minute at 20-25 ºC. 

R4 

(alk. Reagent) 

200 μl 200 μl 200 μl 

Mix and incubate for 5 minutes at 37 ºC, or for 10 minutes at 20-25 ºC. 

Measure absorbance of sample (A s) and standard (A std) against reagent 

blank. 

Calculation: 

Where n = 50.0 mg/dl 

Results are shown in table C 

Determination of creatinine: 

Urea concentration = (A s / A std) x n 

Creatinene was determined according to the method of Folin, (1934). (BIO 

ADWIC Company). 

Principle: 

Creatinine reacts with alkaline picrate to form an amber yellow 

colour that is measured photometrically. 

Reagents: 

Regent 1 Picric acid 36 mmol / L 

Reagent 2 (Standard) Creatinine 20 mg / L 

Reagent 3 Sodium hydroxide 1.5 N

66 


Procedure: 

Wavelength 530 nm (510 – 550 nm) 

Zero adjustment Reagent Blank 

Pipette into Centrifuge tube: 

Serum 0.5 ml 

Reagent 1: 1.5 ml 

Mix well, centrifuge for 10 minutes. 

Supernatant 

or dil. Urine 

Blank Standard Test 

--- --- 1000 μl 

Reagent 1 750 μl 750 μl --- 

Regent 2 

(Standard) 

--- 250 μl --- 

Dist. Water 250 μl --- --- 

Reagent 3 50 μl 50 μl 50 μl 

Mix well, wait exactly for 20 min. at room temperature, then measure 

the absorbance of sample (As) and that of the standard (Astd) against blank. 

Calculation: 

Where 

Creatinine mg/dl = (A s / A std) x n 

n = 2 (for serum plasma)

67 


Determination of Ket by the proposed methods in serum samples: 

Deproteinizatin was achieved by adding 20 ml of trichloroacetic acid 

solution after centrifugation, then apply the same procedures for 

determination of KET for each reagent. In 10 ml measuring flask, 100 μL 

serum was mixed with the optimum volume of the reagent solution and 

the solution was adjusted to the required pH values for each ion-pair 

formed, in sequence (drug-reagent-buffer). The solutions were completed 

to the mark with bidistilled water, shaked well and left to stand for the 

optimum time as described above for each reagent. 

STATISTICAL ANALYSIS: 

The data obtained in this study were statistically analyzed according to 

the analysis described by Miller (1993). 

1- Arithmetic mean: 

where x = sum 

x 

X = 

n 

n = number of values or observations. 

2- Standard deviation: 

S.D. = 

x 

2 

x 

 

 

n 

n 1 

where x 2 = sum of the square of observations. 

(x) 2 = square of the sum of observations. 

2

68 


3- Standard error: 

S.E. = 

S.D. 

where n = square root of the number of values. 

4- Student's t-test: 

t = 

x 

n 

2 2 

SE SE 

1 

1 

x 

where x1 = mean of the first set of observations. 

x2 = mean of the second set of observations. 

The result of t-value is then checked on student's t-table to find out 

the significance level (P-value) Miller et al. (1993). 

The difference was considered significant only at P 0.05). 

Table (B) showed no significant changes in serum aspartate transferase 

level for all groups of mice (P > 0.05). 

Table (C) showed high significant changes in blood urea for groups (II 

2 

and IV) of mice. On the other hand no significant change was 

noticed in any other group of mice. 

Table (D) showed high significant changes in serum creatinine for groups 

(II and IV) of mice. On the other hand no significant change 

was noticed in any other group of mice. 

2

4. RESULTS AND DISCUSSION 

4. Determination of the Studied Drugs by Ion-Pair Complex 

Formation with Acid Dyes: 

4.1. Absorption spectra of the studied drugs with BCG: 

In order to investigate the optimum reaction conditions for the colour 

intensity development of ion–pair complex formed between the studied 

drugs and BCG (5.0 x 10 -4 M). The optimum wavelength corresponding to 

each ion–pair complex of the drugs with BCG was at 419, 417 and 420 nm 

in case of DEX, KET and SIL, respectively as shown in Fig. (1). 

The effect of different experimental variables were studied and 

recorded below. 

4.1.1. Effect of pH 

The effect of pH on the colour intensity of ion-pair complex formed 

between the studied drugs and BCG was investigated. DEX, KET and SIL 

were allowed to react with BCG (5.0 x 10 -4 M) in aqueous universal buffer 

of various pH values (2.0 – 7.0). The formed ion-pair was extracted with 

chloroform to measure the absorbance value at λmax. The highest 

absorbance value was obtained at pH = 3.0 for DEX, pH = 4.0 for KET 

and pH = 3.5 for SIL which were selected for the ion-pairs formation (Fig. 

2.). Furthermore, the amount of buffer added found to be 2.0 ml for DEX, 

KET and SIL was used (Fig. 3). 

4.1.2. Effect of time 

The effect of time required for complete color development of the 

formed ion-pair between the studied drugs and BCG (5.0 x 10 -4 M) was 

investigated. The reactants were allowed to stand and shaked for different

70 

____________________________________________ Results and Discussion 

time intervals. It was observed that shaking for 3.0 min is quite sufficient 

to obtain a maximum color intensity, before extraction of the drugs. The 

formed ion-pair of each drug was extracted with chloroform, the optimum 

shaking time before extraction is 3.0 min. as shown in Fig. (4). The 

intensity of the extracted ion-pair was found to be stable over the 

temperature range 20-40 ºC. Hence room temperature, (25 ±1 ºC), was 

used. The formed ion-pairs were found to be stable for more than 6 hours. 

4.1.3. Effect of the extracting solvent 

The polarity of the solvent affects both extraction efficiency and 

absorbance intensity. The results obtained using different extraction 

solvents (benzene, chloroform, carbon tetrachloride, hexane and methylene 

chloride), applying BCG reagent on the studied drugs indicated that 

chloroform is the best solvent for extraction of the ion-pairs formed. This 

solvent was selected due to its slightly higher sensitivity and considerably 

lower extraction ability of the reagent blank. Complete extraction was 

attained by extraction with 5.0 ml of chloroform for at a time. 

4.1.4. Effect of reagent concentration 

Various concentrations of BCG were added to a fixed concentration of 

the studied drugs. The obtained results indicated that the absorbance was 

increased with increasing reagent volume till 1.0 ml in case of KET and 

1.2 ml of BCG (5.0 x 10 -4 M) in case of DEX and SIL solutions as shown 

in Fig. (5). These concentrations of reagent were found to be sufficient for 

the production of maximum and reproducible color intensity. Higher 

concentration of reagent indicate a slightly decrease in the absorbance and 

color intensity of the formed ion-pair.

71 


4.1.5. Molecular ratio of the complexes 

The stoichiometry of the ion-pair complexes was established by the 

molar ratio and continuous variation methods using both variable reagent 

BCG (5.0 x 10 -4 M) and drugs concentrations. The results showed that the 

stoichiometric ratio of the complex was equimolar (1 : 1) (reagent : drug) 

and the shape of the resulting curves indicated in Figs.(6, 7). 

Consequently, a large excess of reagent must be always used to enhance 

the formation of the complex. 

4.1.6. Sequence of addition 

Different sequences were used to achieve maximum color 

development. The sequence of (drug-BCG-buffer) gave the best sequent 

before extraction process, for the highest color intensity and the least time 

for developing maximum absorbance, all other sequences needed longer 

times and gave lower intensity. 

4.1.7. Suggested mechanism 

Acid dye technique is an ion-pair mechanism in which ion-pair formed 

between negative ion produced from ionization of BCG which convert into 

BCG sodium salt in the buffer and positive ion of the drugs. The ion-pair 

formed exhibits maximum absorbance at λmax 419, 417 nm and 420 nm 

for DEX, KET and SIL as shown in Fig. (8). 

4.1.8. Interference 

No interference (less than 3.0% in absorbance is considered non- 

interference) from the presence of additives and excipients that are usually 

present in pharmaceutical formulations was observed in the determination 

of KET, DEX and SIL with BCG. Also there were no interference from

72 


common degradation products resulted from oxidation of the studied drug, 

which are likely to occur at normal storage conditions. 

4.1.9. Evaluation of the stability constants of the ion-pair complexes 

Spectrophotometric methods can be applied for the determination of 

the stability constant of the ion-pair complexes. Generally, the 

spectrophotometric methods that are usually applied to establish the 

stoichiometry of the complexes can also be used for the determination of 

their stability constants of the concerned ion-pair complexes. The stability 

constants were calculated using the spectrophotometric data of the molar 

ratio and continuous variation methods applying the Harvey and Manning 

method, using the following equation: 

Where: 

Kn = 

A/Am 

(1 

A/Am) 

n 1 

A, is the absorbance at reagent concentration CR. 

Am, is the absorbance at full colour development. 

n, is the stoichiometric ratio of the complex. 

Kn, is the stability constant. 

C 

n 

R 

n 

2

73 


4.1.10. Validity of Beer ' s law 

Calibration graphs were constructed using standard solutions of KET, 

DEX and SIL. Under the optimum conditions, a linear relationship existed 

between the absorbance and concentration of the drugs over the 

concentration range listed in (Table. 1). The correlation coefficient, slopes, 

intercepts, standard deviation of slopes and standard deviation of intercepts 

of the calibration data for Ket, Dex and Sil are calculated. The 

reproducibility of the method was determined by running six replicate 

samples, each contain 8.0 μg ml -1 of drug in case of Ket, Dex and Sil. At 

these concentrations, the relative standard deviation was found to be ≤ 

1.205 % (Table. 1). For more accurate results, Ringbom optimum 

concentration range was determined by plotting log [C] in μg ml -1 against 

percent transmittance. The linear portion of the S-shaped curve gave 

accurate range of analysis as shown in Fig. (10) and The mean molar 

absorpitivity, Sandell sensitivity, detection and quantification limits are 

calculated and recorded in (Table. 1). Representation curves on the validity 

of Beer ' s law for BCG-ion pairs is shown in Fig. (9). 

4.1.11. Accuracy and precision 

In order to determine the accuracy and precision of the proposed 

methods, solutions containing six different concentration of Ket, Dex and 

Sil were prepared and analyzed in quintuplicate. The analytical results 

obtained from these investigations are summarized in (Table. 2). The 

relative standard deviations and the percentage range of error at 95% 

confidence level were calculated. The results can be considered to be 

satisfactory, at least for the level of concentrations examined.

74 



using BCG 

In a 25 ml-volume measuring flask, 5.0 ml urine aliquot of a healthy 

person was spiked with various concentrations of the investigated drugs. 

Deproteinization was achieved by adding 20 ml of trichloroacetic acid 

solution then 1.2 and 1.0 ml of BCG (5.0 x 10 -4 M) were added for (DEX 

or SIL) and KET, respectively. The solution was adjusted to the required 

pH values 3.0, 4.0 and 3.5 for each ion-pair in case of DEX, KET and SIL, 

in sequence (drug-BCG-buffer). The solutions were completed to the mark 

with bidistilled water, shaked well and left to stand for 3.0 min. The 

absorbance was measured following the general procedure described 

above. The relative standard deviation (RSD), recovery and confidence 

limits of the added drugs are computed and recorded as shown in (Tables 

3, 4). 


samples (in vitro) using BCG 

To a sample of serum (2.0 ml) appropriate amount of KET was added. 


solution, after centrifugation, 1.0 ml of the supernatant solution was mixed 

with 1.0 ml of BCG (5.0 x 10 -4 M). The solution was adjusted to the 

required pH value 4.0. The solution was completed to the mark with 

bidistilled water, shaked well and left to stand for 3.0 min. The absorbance 

was measured following the general procedure described above. No 

interference was observed from the serum components which remain after 

deproteinization. The relative standard deviation (RSD), recovery and 

confidence limits of the added drug are computed and recorded as shown 

in Table (5-A).

75 


4.1.13.B. Determination of ketamine hydrochloride in serum samples 

(in vivo) using BCG 

After extraction of the serum from the treated mice by the different 

doses of KET (5.0, 10 and 15 μg/mouse). To a sample of serum (1.00 ml) 

deproteinizatin was achieved by adding 10 ml of trichloroacetic acid 


with 1.0 ml of BCG (5.0 x 10 -4 M). The solution was adjusted to the 



was measured following the general procedure described above. There is 

an interference was observed due to ketamin , s anaesthetic action which 

terminated by redistribution from CNS peripheral tissues and hepatic 

biotransformation to an metabolite norketamine. Other metabolic pathways 

include hydroxylation of the cyclohexone ring and conjugation with 

glucuronic acid. A part of KET was excreted in the urine as metabolites 

and affect on urea and creatinine concentrations. The relative standard 

deviation (RSD), recovery and confidence limits of the added drug are 

computed and recorded as shown in Table (5-B).

76 


4.1.14. Analytical applications 

The validity of the proposed procedures are tested to determine KET, 

DEX and SIL in pharmaceutical preparations manufactured in the local 

company as mentioned before. The concentration of the studied drugs in 

dosage forms was calculated from the appropriate calibration graphs using 

standard addition method. There was no shift in the absorption maximum 

due to the presence of other constituents on the dosage forms. The results 

are compared with those obtained by applying the official methods. The 

results obtained were compared statistically by the Student ' s t-test and 

variance ratio F-value with those obtained using the official methods on 

the sample of the same batch. The student ' s t-test values obtained at 95% 

confidence level and five degree of freedom did not exceed the theoretical 

tabulated value indicating no significant difference between the methods 

compared. The F-values also showed that there is no significant difference 

between precision of the proposed and the official method Table (6). The 

accuracy of the proposed method when applied to pharmaceutical 

preparations is evaluated by applying standard addition method in which 

variable amounts of the drugs (KET, DEX and SIL) were added to the 

previously analyzed portion of pharmaceutical preparations. The results 

are recorded in (Tables. 7 and 8) confirming that the proposed method is 

not liable to interference by fillers usually formulated with the drugs (KET, 

DEX and SIL). The proposed methods are sensitive, therefore they could 

be used easily for the routine analysis in pure form and in there 

pharmaceutical preparations.

77 


Absorbance 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

1 

3 

2 

350 375 400 425 450 475 500 525 550 

Wavelength, nm 

1 = DEX 

2 = KET 

3 = SIL 

Fig. (1): Absorption spectra of studied drugs using BCG (5.0 x 10 -4 M) at 

the optimum conditions.

78 


Absorbance 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

0 1 2 3 4 5 6 7 8 

Fig. (2): Effect of pH on the absorbance of the studied drugs using (5.0 x 

Absorbance 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

10 -4 M) BCG. 

pH 

DEX 

KET 

0 1 2 3 

ml add of buffer 

4 5 6 

SIL 

DEX 

KET 

SIL 

Fig. (3): Effect of ml added of buffer on the absorbance of the studied 

drugs using (5.0 x 10 -4 M) BCG.

79 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

KE 

T 

DE 

X 

SIL 

0 1 2 3 

time (min) 

4 5 6 7 

Fig. (4): Effect of shaking time on the absorbance of the studied drugs 

absorbance 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

solution using (5.0 x 10 -4 M) BCG. 

0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 

ml add of (5.0 x10 -4 M) BCP 

SIL 

DEX 

Ket 

Fig. (5): Effect of reagent concentration on the absorbance of the studied 

drugs solution using (5.0 x 10 -4 M) BCG.

80 


Absorbance 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

DEX 

SIL 

KET 

0 0.4 0.8 1.2 1.6 2 2.4 2.8 

Reagent / drug 

Fig. (6): Molar ratio for BCG-Drugs (5.0 x 10 -4 M) under consideration. 

Absorbance 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

DEX 

KET 

SIL 

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 

mole fraction of drug 

Fig. (7): Continuous variation using (5.0 x 10 -4 M) BCG reagent with (5.0 

10 -4 M) of the drugs under consideration.

81 


Fig (8): Proposed mechanism of the reaction between ketamine hydro- 

HO 

Br 

Br 

chloride and bromocresol green salt. 

CH 3 

Br 

O 

S 

O 

O 

CH 3 

OH 

Br 

HO 

Br 

Br 

CH 3 

Br 

SO 3 H 

Bromocresol green (quinoid ring) 

(lactoid ring) 

CH 3 

HO 

CH 3 

O 

Br 

Br 

HO 

Br 

CH 3 

Br 

Br 

CH 3 

O 

Br 

SO 3 Na 

NH 

+ 

Br 

Br 

CH3 SO3Na O 

Cl 

.HCl 

Ketamine hydrochloride Bromocresol green sodium salt 

pH = 4.0 

O 

Cl 

CH 3 

NH 

+ - 

Br 

Br 

HO 

Ketamine hydrochloride- BCG ion-pair complex 

Br 

CH 3 

SO 3 

CH 3 

O 

Br 

CH 3 

+ NaCl 

O 

Br

82 


Absorbance 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

DEX 

KET 

0 5 10 15 20 25 30 35 

[D] μg/ml 

Fig. (9): Application of Beer ' s law for the studied drugs using (5.0 x 10 -4 

T % 

M) BCG. 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 

Log C μg/ml 

Fig. (10): Ringbom plotting for the studied drugs solution using (5.0 x 10 -4 

M) BCG. 

SIL 

KET 

طخ 

DEX 

SIL

83 


Table (1): Analytical data and characteristics of coloured product, 

pH 

λ max (nm) 

precision and accuracy of the studied drugs using BCG. 

Parameters 

Stability constant 

Beer's Law Limits (μg ml -1 ) 

Ringbom Limits (μg ml -1 ) 

Regression equation (A*): 

Slope (b) 

Intercept (a) 

Molar Absorpitivity (ξ) x10 4 

(L mol -1 cm -1 ) 

Sandell , s Sensitivity (ng cm -2 ) 

Standard deviation (SD)** % 

Correlation coefficient (r) 

Detection limit (μg ml -1 ) 

Quantification limit (μg ml -1 ) 

RSD % 

RE % 

KET 

4.0 

417 

4.36 

1.0-14 

1.5-12 

0.097 

-0.0203 

2.604 

10.529 

0.6793 

0.9995 

0.0195 

0.065 

0.9357 

0.9821 

Bromocresol green (BCG) 

DEX 

3.0 

419 

4.47 

1.0-21 

2.0-18.5 

0.0809 

0.0034 

3.00 

12.34 

0.7203 

0.9999 

0.0486 

0.162 

1.108 

1.163 

3.5 

420 

3.95 

SIL 

2.0-29 

3.0-27.5 

0.0269 

0.0023 

1.37 

35.21 

0.2651 

0.9994 

0.0336 

0.112 

1.205 

1.265 

* : A = a + b C, where A is the absorbance unit, a is the intercept, b is the 

slope and C is the concentration of drug in μg ml -1 . 

** : Six-replicate samples (concentration of 8.0 μg of drug per ml).

84 


Table (2): Evaluation of the accuracy and precision of the proposed 

Drugs 

Ketamine 

hydrochloride 

method using BCG. 

Taken 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD a 

(%) 

RE 

(%) 

Confidence b 

limits 

3.0 2.99 99.68 1.4451 1.5162 2.99±0.0453 

6.0 6.003 100.06 0.9283 0.9740 6.003±0.0585 

9.0 8.99 99.90 0.6728 0.7059 8.99±0.0635 

Dextromethorphan 

hydrobromide 3.0 3.007 100.23 1.0917 1.1454 3.007±0.0344 

Silymarin 

6.0 5.969 99.48 0.7679 0.8057 5.969±0.0481 

9.0 9.001 100.01 0.8283 0.8691 9.001±0.0782 

5.0 4.973 99.46 1.0124 1.0622 4.973±0.0528 

10 10.02 100.2 0.7307 0.7666 10.02±0.0768 

15 14.98 99.86 0.5164 0.5418 14.98±0.0812 

a Relative standard deviation for six determinations. 

b 95% confidence limits and five degrees of freedom.

85 



method for investigated KET and DEX using BCG in spiked 

urine samples. 

Dosage Forms Added 

(μg ml -1 ) 

Ketamar 

ampoules 

(50 mg / ml) 

Codiphan syrup 

(15 mg / 5.0 ml) 

Tussilar drops 

(1.0 g / 15 ml) 

Tussilar tablet 

(10 mg per tab) 

Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD a 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.48 99.2 1.2903 2.48±0.0336 

5.0 5.01 100.2 0.9594 5.01±0.0504 

7.5 7.51 100.13 0.8247 7.51±0.0650 

10 9.97 99.7 0.9218 9.97±0.0964 

- - - - - 

2.5 2.505 100.2 1.2345 2.505±0.0324 

5.0 4.98 99.6 0.8356 4.98±0.0437 

7.5 7.49 99.86 0.7485 7.49±0.0588 

10 10.05 100.5 0.6857 10.05±0.0723 

- - - - - 

2.5 2.485 99.4 1.1241 2.485±0.0293 

5.0 5.027 100.54 1.0472 5.027±0.0552 

7.5 7.513 100.17 0.8927 7.513±0.0704 

10 10.06 100.6 0.9035 10.06±0.0954 

- - - - - 

2.5 2.495 99.8 1.3273 2.495±0.0347 

5.0 5.048 100.96 0.9512 5.048±0.0503 

7.5 7.504 100.05 0.6792 7.504±0.0535 

10 9.985 99.85 0.8426 9.985±0.0883 



86 



Dosage Forms 

method for investigated SIL using BCG in spiked urine 

samples. 

Hepamarin capsules 

(140 mg per cap.) 

Legalex tablets 

(70 mg per tab.) 

Legalon tablets 


Added 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD a 

(%) 

Confidence b 

limits 

- - - - - 

3.5 3.486 99.60 0.8411 3.486±0.0308 

7.0 7.018 100.25 0.9514 7.018±0.0701 

10.5 10.497 99.97 0.7692 10.497±0.0847 

15.0 14.985 99.90 0.6248 14.985±0.09823 

- - - - - 

3.5 3.504 100.12 1.1656 3.504±0.0429 

7.0 6.989 99.84 0.5761 6.989±0.0422 

10.5 10.489 99.89 0.4806 10.489±0.0529 

15.0 15.05 100.33 0.5075 15.05±0.0801 

- - - - - 

3.5 3.495 99.85 0.9438 3.495±0.0346 

7.0 7.015 100.21 0.7715 7.015±0.0568 

10.5 10.502 100.01 0.5654 10.502±0.0623 

15.0 14.992 99.94 0.4274 14.992±0.0672 



87 


Table (5-A): Evaluation of the accuracy and precision of the proposed 

Dosage form 

Ketamar ampoule 

(50 mg / ml) 

method for investigated KET using BCG in spiked serum 

samples, (in vitro). 

Table (5-B): Evaluation of the accuracy and precision of the proposed 

method for investigated KET using BCG in serum Samples, 

(in vivo). 

Add 

(μg ml -1 ) 

2.5 

5.0 

7.5 

10 

Found 

(μg ml -1 ) 

2.47 

4.89 

7.31 

9.91 

Recovery 

% 

98.80 

97.80 

97.47 

99.10 


b 95% confidence limits and five degrees of freedom. 

RSD a 

% 

1.1552 

0.9528 

0.8541 

0.7624 

Confidence b 

limit 

2.482±0.030 

4.96±0.049 

7.45±0.066 

9.91±0.0793 

Found 

(μg ml 

Recovery 

% % limit 

-1 Add 

(μg ml ) 

-1 Dosage form 

) 

Ketamar ampoule 5.0 4.042 80.84 0.8504 4.042±0.036 

(50 mg / ml) 10 7.595 75.95 0.7006 7.595±0.056 

15 

11.769 

78.46 

RSD a 

0.6585 

Confidence b 

11.769±0.081

88 


Table (6): Evaluation of the accuracy and precision of the proposed and 

Dosage 

forms 

Ketamar ampoules 

(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 



Hepamarin 

capsules 







in it ' s pharmaceutical forms using BCG. 

Official method Proposed method 

Taken 

mg 

50 

15 

1000 

10 

140 

70 

70 

Found* 

mg 

49.612 

14.77 

979.2 

9.853 

138.18 

70.02 

69.63 

*: Average of six determinations. 

Recovery Taken Found* Recovery t ** 

(%) mg mg (%) Value 

99.224 

98.47 

97.92 

98.53 

98.70 

100.03 

99.47 

50 

15 

1000 

10 

140 

70 

70 

49.38 

14.84 

982.2 

9.904 

138.8 

69.72 

69.45 

98.76 

98.93 

98.22 

99.04 

99.14 

99.60 

99.21 

1.187 

1.007 

0.697 

1.259 

0.916 

0.879 

0.558 

F ** 

test 

2.146 

1.142 

1.888 

1.829 

2.223 

1.851 

2.388 

**: Theoretical values for t- and F- values for five degree of freedom and 

95% confidence limits are 2.57 and 5.05, respectively.

89 


Table (7): Determination of the studied drugs (KET and DEX) in it ' s 

Dosage forms 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 




method using BCG. 


Taken 

(μg ml -1 Added 

) (μg ml -1 Found* 

) (μg ml -1 Recovery 

) (%) 

3.0 0.0 3.01 100.33 

2.0 4.98 99.6 

4.0 7.015 100.21 

6.0 8.99 99.88 

8.0 11.05 100.41 

3.0 0.0 2.98 99.33 

2.0 5.015 100.3 

4.0 6.97 99.57 

6.0 9.02 100.22 

8.0 10.97 99.72 

3.0 0.0 2.96 98.66 

2.0 4.985 99.70 

4.0 6.975 99.64 

6.0 9.025 100.27 

8.0 10.95 99.54 

3.0 0.0 3.01 100.33 

2.0 5.03 100.6 

4.0 6.99 99.85 

6.0 9.035 100.38 

8.0 11.04 100.36

90 


Table (8): Determination of SIL in it ' s pharmaceutical dosage forms 

applying the standard addition method using BCG. 

Dosage forms 







Taken 

(μg ml -1 ) 


Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

5.0 0.0 5.01 100.2 

2.5 7.485 99.8 

5.0 10.025 100.25 

7.5 12.497 99.976 

10 14.96 99.73 

5.0 0.0 4.95 99.00 

2.5 7.51 100.13 

5.0 9.96 99.6 

7.5 12.506 100.04 

10 15.045 100.3 

5.0 0.0 5.023 100.46 

2.5 7.495 99.93 

5.0 10.06 100.6 

7.5 12.495 99.96 

10 14.99 99.93

91 


4.2. Absorption spectra of the studied drugs with BCP 


intensity development of ion-pair complex formed between the studied 

drugs and BCP (5.0 x 10 -4 M). The optimum wavelength corresponding to 

each ion-pair complex is at 408, 409 and 418 nm in case of KET, DEX and 

SIL, respectively as shown in Fig. (11). 

below. 

The effect of different experimental variables was studied and recorded 



between the studied drugs and BCP was investigated. DEX, KET and SIL 

were allowed to react with BCP (5.0 x 10 -4 M) in aqueous universal buffer 

solutions of various pH values (2.0–8.0). The formed ion-pair was 

extracted with chloroform or methylene chloride to measure the 

absorbance value at λmax. The highest absorbance value was obtained at pH 

= 3.0 and pH = 4.0 in case of KET and (DEX or SIL), respectively which 

selected for ion-pairs formation (Fig. 12). Furthermore, the amount of 

buffer added was examined and found to be 2.0 ml in case of DEX, KET 

and SIL, respectively was used as shown in (Fig. 13). 


The effect of time required for complete colour development of the 

formed ion-pair between the studied drugs and BCP (5.0 x 10 -4 M) was 

investigated. The reactants were allowed to stand and shaked for different 

time intervals. It was observed that shaking for 2.5 min in case of DEX and 

3.0 min. in case of (KET or SIL) are quite sufficient to obtain a maximum 

colour intensity, before extraction of the drug. The formed ion-pair of each

92 


drug was extracted with chloroform, the optimum shaking time before 

extraction is shown in (Fig. 14). The formed ion-pairs were found to be 

stable for more than 8.0 hours. 




solvents (benzene, chloroform, carbon tetrachloride, hexane, and 

methylene chloride), applying BCP reagent on the studied drugs indicated 

that chloroform is the best solvent for extraction of the ion-pairs formed in 

case of KET and DEX and methylene chloride is the best solvent for 

extraction of the ion-pair formed in case of SIL. Those solvents were 

selected due to their slightly higher sensitivity and considerably lower 

extraction of the reagent itself. Complete extraction was attained by 

extraction with 5.0 ml of the solvent for one time. 


Various concentrations of BCP were added to a fixed concentration of 


increased with increasing reagent concentration till 1.0 ml in case of KET, 

1.2 ml in case of DEX and SIL of BCP (5.0 x 10 -4 M) solutions, 

respectively as shown in Fig. (15). These concentrations of reagent were 

found to be sufficient for the production of maximum and reproducible 

colour intensity. Higher concentration of reagent slightly decrease the 

absorbance and colour intensity of the formed ion-pairs. 



molar ratio and continuous variation methods using both variable reagent

93 


BCP (5.0 x 10 -4 M) and KET, DEX and SIL, concentrations. The results 

showed that the stoichiometric ratio of the complex is (1 : 1) (reagent : 

drug) and the shape of the resulting curves indicated that the complex is 

labile, as shown in Figs. ( 16, 17). Consequently, a large excess of reagent 

must be always used to enhance the formation of the complex. 


Different sequences were used to achieve maximum colour 

development. The sequence of (drug-BCP-buffer) gave the best sequent 

before extraction process, for the highest colour intensity and the least time 

for developing maximum absorbance, all other sequences needed longer 

times and gave lower intensity. 



between negative ion produced from ionization of BCP which convert into 

BCP sodium salt in the buffer and positive ion of the drugs. The ion-pair 

formed exhibits maximum absorbance at λmax 409 nm, 408 nm and 418 

nm for DEX, KET and SIL, respectively as shown in Fig. (18). 





of KET, DEX and SIL with BCP. Also there were no interference from 


which are likely to occur at normal storage conditions.

94 






concentration range listed in Table (9). The correlation coefficient, slopes, 

intercepts, standard deviation of slopes and standard deviation of intercepts 

of the calibration data for KET, DEX and SIL are calculated. The 

reproducibility of the method was determined by running six replicate 

samples, each contain 8.0 μg ml -1 of drug in case of KET, DEX and SIL. 

At these concentration, the relative standard deviation was found to be ≤ 

1.126 % (Table. 9). For more accurate results, Ringbom optimum 

concentration range was determined by plotting log [C] in μg ml -1 against 

percent transmittance. The linear portion of the S-shaped curve gave 

accurate range of analysis as shown in Fig. (20) and the mean molar 


calculated and recorded in (Table. 9). Representation curves on the validity 

of Beer ' s law for BCP-ion pairs is shown in (Fig. 19). 



methods, solutions containing six different concentration of KET, DEX 

and SIL were prepared and analyzed in quintuplicate. The analytical 

results obtained from these investigations are summarized in (Table. 10). 

The relative standard deviations and the percentage range of error at 95% 



95 



using BCP 




solution, then 1.2 and 1.0 ml of BCP (5.0 x 10 -4 M) in case of (DEX or 

SIL) and KET were added. The solution was adjusted to the required pH 

values 4.0 and 3.0 for each ion-pair in case of (DEX or SIL) and KET, in 

sequence (drug-BCP-buffer). The solutions were completed to the mark 

with bidistilled water, shaked well and left to stand for 2.5 min. in case of 

DEX and 3.0 min. in case of (KET or SIL). The absorbance was measured 

following the general procedure described above. The relative standard 


computed and recorded as shown in Table ( 11, 12). 

4.2.12.A. Determination of ketamine hydrochloride in serum samples 

(in vitro) using BCP 



solution. After centrifugation, 1.0 ml of the supernatant solution was 

mixed with 1.2 ml of BCP (5.0 x 10 -4 M). The solution was adjusted to the 







in Table (13-A).

96 


4. 2. 12. B. Determination of ketamine hydrochloride in serum samples 

(in vivo) using BCP 



deproteinization was achieved by adding 10 ml of trichloroacetic acid 

solution (240 g l -1 ). After centrifugation, 1.0 ml of the supernatant solution 

was mixed with 1.2 ml of BCP (5.0 x 10 -4 M). The solution was adjusted to 

the required pH value 3.0. The solution was completed to the mark with 







glucuronic acid. A part of KET was excreted in the urine as metabolites 

and affect on urea and creatinine concentrations. The relative standard 


computed and recorded as shown in Table (13-B). 




company as mentioned before. The concentration of the studied drug in 

dosage forms was calculated from the appropriate calibration graph using 


due to the presence of other constituent on the dosage forms. The results 


results obtained were compared statistically by the student's t-test and

97 



the sample of the same batch. The student's t-test values obtained at 95% 




between precision of the proposed and the official method Table (14). The 





are recorded in Tables (15 and 16) confirming that the proposed method is 



be used easily for the routine analysis of pure form and its pharmaceutical 

preparations.

98 


Absorbance 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

1 

2 

3 

1 = DEX 

2 = KET 

3 = SIL 

325 350 375 400 425 450 475 500 525 550 


Fig. (11): Absorption spectra of the studied drugs using BCP (5.0 10 -4 

M) at the optimum conditions.

99 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

DEX 

KET 

SIL 

0 1 2 3 4 5 6 7 8 9 

pH 

Fig. (12): Effect of pH on the absorbance of the studied drugs using (5.0 

Absorbance 

10 -4 M) BCP. 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

DEX 

KET 

SIL 

0 1 2 3 


4 5 6 

Fig. (13): Effect of ml added of buffer on the absorbance of the studied 

drugs using (5.0 10 -4 M) BCP.

100 


Absorbance 

2.5 

2 

1.5 

1 

0.5 

0 

Ket 

DEX 

SIL 

0 1 2 3 4 5 6 7 

Time (min) 


absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

solution using (5.0 10 -4 M) BCP. 

0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 

ml add of (5.0 x10 -4 M) BCP 

SIL 

DEX 

Ket 


drugs solution using (5.0 10 -4 M) BCP.

101 


Absorbance 

2.5 

2 

1.5 

1 

0.5 

0 

KET 

DEX 

SIL 

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 

Reagent/drug 

Fig. (16): Molar ratio for BCP-Drugs (5.0 10 -4 M) under consideration. 

Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

KET 

DEX 

SIL 

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 


Fig. (17): Continuous variation using (5.0 10 -4 M) BCP reagent with 

(5.0 10 -4 M) of the drugs under consideration.

102 


Fig (18): Proposed mechanism of the reaction between dextromethorphan 

HO 

Br 

CH 3 

hydrobromide and bromocresol purple sodium salt. 

CH 3 

O 

S 

O 

O 

OH 

Br 

HO 

Br 

CH 3 

CH 3 

SO 3 H 

Bromocresol purple (quinoid ring) 


H 3 C 

O 

.HBr 

+ 

HO 

Br 

O 

Br 

CH 3 

HO 

Br 

CH 3 

CH 3 

SO 3 Na 

+ - 

HO 

Br 

CH 3 

SO 3 

CH 3 

O 

Br 

O 

Br 

CH 3 

SO 3 Na 

Dextromethorphan hydrobromide Bromocresol purple sodium salt 

H 3 C 

O 

H 

H 

N 

CH 3 

N 

CH 3 

pH = 4.0 

Dextromethorphan hydrobromide-BCP ion-pair Complex 

+ NaBr 

O 

Br

103 


Absorbance 

2 

1.6 

1.2 

0.8 

0.4 

0 

KET 

DEX 

SIL 

0 5 10 15 20 25 

[D] μg/ml 

Fig. (19): Application of Beer ' s law for the studied drugs using the 

optimum volume of (5.0 x 10 -4 M) BCP. 

T % 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

0 0.4 0.8 1.2 1.6 2 

Log C μg/ml 

Fig. (20): Ringbom plotting for the studied drugs solution using (5.0 10 -4 

M) BCP. 

DEX 

KET 

SIL

104 



precision and accuracy of the studied drugs using BCP. 

Parameters 

pH 

λ max (nm) 





Slope (b) 

Intercept (a) 


(L mol -1 cm -1 ) 






RSD % 

RE % 

KET 

3.0 

408 

4.42 

1.0-15 

1.2-14 

0.0976 

0.0037 

2.72 

10.08 

0.736 

0.9991 

0.0366 

0.121 

0.9316 

0.9778 

Bromocresol purple (BCP) 

DEX 

4.0 

409 

4.29 

1.8-22 

2.0-20.5 

0.0838 

0.0113 

3.18 

11.64 

0.584 

0.9993 

0.04 

0.1342 

0.8343 

0.8757 

SIL 

4.0 

418 

4.62 

1.0-24 

2.5-23 

0.0319 

0.0009 

1.20 

40.20 

0.214 

0.9987 

0.0444 

0.148 

1.126 

1.182 




105 



method using BCP. 

Drugs Taken 

(μg ml -1 Found 

) (μg ml -1 Recovery RSD 

) (%) 

a 

RE Confidence 

(%) (%) 

b 

limits 

Ketamine 

hydrochloride 

3.0 2.97 99.00 1.0765 1.1295 2.97±0.0335 

6.0 6.016 100.26 0.9325 0.9784 6.014±0.0588 


hydrobromide 

Silymarin 

9.0 8.985 99.83 0.7958 0.8350 8.985±0.0750 

3.0 3.017 100.56 0.9821 1.0304 3.017±0.0311 

6.0 5.98 99.66 0.8062 0.8459 5.98±0.0506 

9.0 9.024 100.26 0.7683 0.8061 9.024±0.0727 

5.0 4.94 98.8 1.1547 1.2115 4.94±0.0477 

10 10.01 100.1 0.7682 0.8060 10.01±0.0646 

15 14.965 99.76 0.6201 0.6506 14.965±0.078 



106 



method for investigated KET and DEX using BCP in urine 

samples. 


(μg ml -1 ) 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 



Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.501 100.04 1.2453 2.501±0.0327 

5.0 4.975 99.5 0.9207 4.975±0.0481 

7.5 7.502 100.02 0.8582 7.502±0.0675 

10 9.985 99.85 0.7238 9.985±0.0758 

- - - - - 

2.5 2.487 99.48 1.4081 2.487±0.0367 

5.0 5.015 100.3 0.9256 5.015±0.0487 

7.5 7.486 99.81 0.7548 7.486±0.0593 

10 10.019 100.19 0.6584 10.019±0.0692 

- - - - - 


2.5 2.468 98.72 0.7648 4.936±0.0396 

5.0 4.99 99.8 0.5637 9.98±0.059 

7.5 7.495 99.93 0.4419 14.989±0.0695 

10 9.992 99.92 0.3891 19.944±0.0814 

- - - - - 

2.5 2.5025 100.1 1.0485 2.502±0.0275 

5.0 4.99 99.8 0.8305 4.99±0.0435 

7.5 7.505 100.6 0.7683 7.505±0.0605 

10 9.987 99.87 0.6785 9.987±0.0711 


107 



Dosage Forms 







method for investigated SIL using BCP in urine samples. 

Added 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.475 99.00 1.1472 2.475±0.0298 

5.0 4.945 98.9 0.9528 4.945±0.0494 

7.5 7.485 99.8 0.6219 7.485±0.0488 

10 9.972 99.72 0.4821 9.972±0.0504 

- - - - - 

2.5 2.502 100.08 1.2167 2.502±0.0319 

5.0 4.968 99.36 1.0434 4.968±0.0544 

7.5 7.465 99.53 0.8642 7.465±0.0677 

10 9.935 99.35 0.4673 9.935±0.0487 

- - - - - 

2.5 2.483 99.32 1.3208 2.483±0.0344 

5.0 4.96 99.2 1.0927 4.96±0.0569 

7.5 7.482 99.76 0.8792 7.482±0.069 

10 9.87 98.7 0.7134 9.87±0.0739 



108 



Dosage form 

Ketamar ampoule (50 

mg / ml) 

method for investigated KET using BCP in serum samples, 

(in vitro). 

Add 

(μg ml -1 ) 

2.5 

5.0 

7.5 

10 

Found 

(μg ml -1 ) 

2.43 

4.90 

7.40 

9.77 

Recovery 

% 

97.20 

98.00 

98.67 

97.70 

RSD 

% 

1.1249 

0.8792 

0.7364 

0.6805 

Confidence b 

limit 

2.43±0.029 

4.90±0.045 

7.40±0.057 

9.77±0.070 


method for investigated KET using BCP in serum samples, 

(in vivo). 

Dosage form 

Ketamar ampoule (50 

mg / ml) 

Add 

(μg ml -1 ) 

5.0 

10 

15 

Found 

(μg ml -1 ) 

3.87 

8.04 

11.09 

Recovery 

% 

77.40 

80.40 

73.93 



RSD a 

% 

0.644 

0.845 

0.689 

Confidence b 

limit 

3.87±0.026 

8.04±0.071 

11.09±0.080

109 



Dosage forms 


(50 mg / ml) 


(15 mg / 5.0 ml) 

Tussilar-drops 

(1.0 g / 15 ml) 










in it ' s pharmaceutical forms using BCP. 


Taken Found* Recovery Taken Found* Recovery 

mg mg (%) 

mg mg (%) 

50 

15 

1000 

10 

140 

70 

70 

49.612 

14.77 

979.2 

9.953 

138.18 

70.02 

69.63 


99.224 

98.47 

97.92 

99.53 

98.70 

100.03 

99.47 

50 

15 

1000 

10 

140 

70 

70 

49.45 

14.87 

983 

10.01 

137.7 

69.73 

69.34 

98.90 

99.13 

98.30 

100.1 

98.35 

99.614 

99.057 

t ** 

Value 

0.834 

1.604 

0.869 

1.466 

0.698 

0.849 

0.855 

**: Theoretical values for t- and F- values for five degree of freedom and 

95% confidence limits are 2.57 and 5.05, respectively. 

F ** 

test 

2.227 

1.946 

1.718 

2.136 

1.787 

1.846 

1.906

110 




method using BCP. 

Dosage forms Taken 

(μg ml -1 ) 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 




Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

3.0 0.0 3.015 100.5 

2.0 4.982 99.64 

4.0 7.02 100.28 

6.0 8.975 99.72 

8.0 11.03 100.27 

3.0 0.0 2.99 99.66 

2.0 4.85 97.00 

4.0 6.95 99.28 

6.0 9.014 100.15 

8.0 10.935 99.4 

3.0 0.0 3.02 100.66 

2.0 4.98 99.60 

4.0 7.01 100.14 

6.0 8.93 99.22 

8.0 10.87 98.81 

3.0 0.0 2.94 98.00 

2.0 5.03 100.6 

4.0 6.97 99.57 

6.0 9.025 100.27 

8.0 11.04 100.36

111 



applying the standard addition method using BCP. 


(μg ml -1 ) 








Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

5.0 0.0 4.97 99.40 

2.5 7.051 100.01 

5.0 9.985 99.85 

7.5 12.502 100.016 

10 14.975 99.83 

5.0 0.0 5.01 100.20 

2.5 7.47 99.60 

5.0 9.86 98.60 

7.5 12.48 99.84 

10 14.92 99.46 

5.0 0.0 4.94 98.80 

2.5 7.51 100.13 

5.0 10.02 100.2 

7.5 12.46 99.68 

10 15.018 100.12

112 


4.3. Absorption spectra of the studied drugs with BTB 



drugs and BTB (5.0 x 10 -4 M), The optimum wavelength corresponding to 

each ion–pair complex is at 412, 413 and 420 nm in case of KET, DEX 

and SIL, as shown in Fig. (21). 

The effect of different experimental variables was studied and 

recorded below. 



between the studied drugs and BTB were investigated. DEX, KET and SIL 

was allowed to react with BTB (5.0 x 10 -4 M) in aqueous universal buffer 


chloroform to measure the absorbance value at λmax. The highest 

absorbance value was obtained at pH = 4.0 and pH = 3.0 in case of DEX 

and (KET or SIL), respectively which selected for ion-pairs formation. 

(Fig. 22). Furthermore, the amount of buffer added was examined and 

found to be 1.5 and 2.0 ml in case of DEX and (KET or SIL), respectively 

as shown in (Fig. 23). 



formed ion-pair between the studied drugs and BTB (5.0 x 10 -4 M) was 

investigated. The reactants were allowed to stand and shaked for different 

time intervals. It was observed that shaking for 3.0 min is quite sufficient 

to obtain a maximum colour intensity, before extraction in case of DEX, 

KET and SIL. The formed ion-pair of each drug was extracted with

113 


chloroform, the optimum shaking time before extraction is 3.0 min. as 

shown in (Fig. 24). The intensity of the extracted ion-pairs was found to 

be stable over the temperature range 20-40 ºC. Hence room temperature, 

(25 ±1 ºC), was used. The formed ion-pairs were found to be stable for 

more than 10 hours. 





methylene chloride), applying BTB reagent on the studied drugs indicated 

that chloroform is the best solvent for extraction of the ion-pairs formed. 

This solvent was selected due to its slightly higher sensitivity and 

considerably lower extraction ability of the reagent blank. Complete 

extraction was attained by extraction with 5.0 ml of chloroform for one 

time. 


Various concentrations of BTB were added to a fixed concentration of 


increased with increasing reagent volume up to 1.0 ml in case of DEX and 

1.2 ml of BTB (5.0 x 10 -4 M) in case of KET and SIL solutions as shown 

in (Fig. 25). These concentrations of reagent were found to be sufficient 

for the production of maximum and reproducible colour intensity. Higher 


colour intensity of the formed ion-pair.

114 



The stoichiometry of the ion-pair complex was established by the 

molar ratio and continuous variation methods using both variable reagent 

BTB (5.0 x 10 -4 M) and KET, DEX and SIL, concentrations. The results 



labile, as shown in (Figs. 26, 27). Consequently, a large excess of reagent 




development. The sequence of (drug-BTB-buffer) gave the best sequent 

before extraction process, for the highest colour intensity and the least 

time for developing maximum absorbance, all other sequences needed 

longer times and gave lower intensity. 



between negative ion produced from ionization of BTB which convert into 

BTB sodium salt in the buffer and positive ion of the drugs. The ion-pair 


for DEX, KET and SIL as shown in (Fig. 28). 





of KET, DEX and SIL with BTB. Also there were no interference from

115 



which are likely to occur at normal storage conditions. 





concentration range listed in (Table. 17). The correlation coefficient, 

slopes, intercepts, standard deviation of slopes and standard deviation of 

intercepts of the calibration data for KET, DEX and SIL are calculated. 

The reproducibility of the method was determined by running six replicate 

samples, each contain 8.0 μg ml -1 of drug in case of KET, DEX and SIL. 

At these concentrations, the relative standard deviation was found to be ≤ 

0.892 % as recorded in (Table. 17). For more accurate results, Ringbom 

optimum concentration range was determined by plotting log [C] in μg ml - 

1 

against percent transmittance. The linear portion of the S-shaped curve 

gave accurate range of analysis as shown in (Fig. 30), and the results are 

recorded in (Table. 17). The mean molar absorpitivity, Sandell sensitivity, 

detection and quantification limits are calculated and recorded in (Table. 

17). Representation curves on the validity of Beer ' s law for BTB-ion pairs 

is shown in (Fig. 29). 




and SIL were prepared and analyzed in quintuplicate. The analytical 

results obtained from these investigations are summarized in (Table. 18). 

The percent standard deviations and the percentage range of error at 95 %

116 



satisfactory, at least for the level of concentrations examined. 

4.3.11. Determination of the studied drugs in urine spiked samples 

using BTB 




solution then 1.0 and 1.2 ml of BTB (5.0 x 10 -4 M) were added for DEX 

and (KET or SIL), respectively. The solution was adjusted to the required 

pH values 4.0 and 3.0 for each ion-pair in case of DEX and (KET or SIL), 

in sequence (drug-BTB-buffer). The solutions were completed to the mark 

with bidistilled water, shaked well and left to stand for 3.0 min. The 

absorbance was measured following the general procedure described 

above. The relative standard deviation (RSD), recovery and confidence 

limits of the added drugs are computed and recorded as shown in (Tables. 

19, 20). 


samples in vitro using BTB 

To a sample of serum (2.00 ml) appropriate amount of KET was 

added. Deproteinization was achieved by adding 20 ml of trichloroacetic 

acid solution, after centrifugation, 1.0 ml of the supernatant solution was 

mixed with 1.2 ml of BTB (5.0 x 10 -4 M). The solution was adjusted to the 




interference was observed from the serum components which remain after

117 




in (Table 21-A). 

4.3.12.B. Determination of ketamine hydrochloride in serum samples 

in vivo using BTB 



appropriate amount of KET was added. Deproteinization was achieved by 

adding 20 ml of trichloroacetic acid solution, after centrifugation, 1.0 ml of 

the supernatant solution was mixed with 1.2 ml of BTB (5.0 x 10 -4 M). The 

solution was adjusted to the required pH = 3.0. The solution was 

completed to the mark with bidistilled water, shaked well and left to stand 

for 3.0 min. The absorbance was measured following the general 

procedure described above. There is an interference was observed due to 

ketamin , s anaesthetic action which terminated by redistribution from CNS 

peripheral tissues and hepatic biotransformation to an metabolite 

norketamine. Other metabolic pathways include hydroxylation of the 

cyclohexone ring and conjugation with glucuronic acid. A part of ketamine 

was excreted in the urine as metabolites and affect on urea and creatinine 

concentrations. The relative standard deviation (RSD), recovery and 


in Table (21-B).

118 












the sample of the same batch. The student ' s t-test values obtained at 95 % 




between precision of the proposed and the official method (Table. 22). The 





are recorded in (Tables. 23 and 24) confirming that the proposed method is 



be used easily for the routine analysis in pure form and in there 


119 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

2 

1 

3 

340 365 390 415 440 465 490 515 540 


1 = DEX 

2 = KET 

3 = SIL 

Fig. (21): Absorption spectra of the studied drugs using BTB (5.0 10 -4 


120 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

DEX 

KET 

0 1 2 3 4 5 

pH 

6 7 8 9 10 

Fig. (22): Effect of pH on the absorbance of 8.0 μg ml -1 of the studied 

Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

drugs using (5.0 10 -4 M) BTB. 

0 1 2 3 4 5 6 


SIL 

KET 

DEX 

SIL 

Fig. (23): Effect of ml added of buffer on the absorbance of 8.0 μg ml -1 of 

the studied drugs using (5.0 10 -4 M) BTB.

121 



Absorbance 

Absorbance 

2 

1.6 

1.2 

0.8 

0.4 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

0 

0 1 2 3 4 5 6 7 

Time (min) 

solution using (5.0 10 -4 M) BTB. 

DEX 

KET 

SIL 

KET 

DEX 

SIL 

0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 

ml add of reagent 


drugs solution using (5.0 10 -4 M) BTB.

122 


Absorbance 

2.4 

2 

1.6 

1.2 

0.8 

0.4 

0 

DE 

X 

KET 

0 0.4 0.8 1.2 

Reagent/drug 

1.6 2 2.4 2.8 

Fig. (26): Molar ratio for BTB-Drugs (5.0 x 10 -4 M) under consideration. 

Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 


EX 

KET 

SIL 

Fig. (27): Continuous variation using (5.0 x 10 -4 M) BTB reagent with (5.0 


123 


Fig. (28): Proposed mechanism of the reaction between ketamine 

hydrochloride and bromothymol blue sodium salt. 

HO 

Br 

C 3 H 7 

C 3 H 7 

OH 

HO 

C 3 H 7 

C 3 H 7 

CH3 O 

S 

O 

Br 

CH3 O 

Br 

CH3 Br 

CH3 SO3H Br 

CH3 O 

HO 

C 3 H 7 

C 3 H 7 

SO 3 Na 

Bromothymol blue (quinoid ring) Bromothymol blue 

(lactoid ring) sodium salt 

O 

Cl 

CH 3 

NH 

.HCl 

+ 

HO 

Br 

C 3 H 7 

C 3 H 7 

SO 3 Na 

Ketamine hydrochloride Bromothymol blue sodium salt 

pH = 3.0 

O 

Cl 

CH 3 

NH 

SO 3 

CH 3 

+ - 

C3H7 C3H7 HO 

Br 

CH 3 

CH 3 

CH 3 

Ketamine hydrochloride- BTB ion-pair complex 

O 

Br 

O 

Br 

CH 3 

+ NaCl 

O 

Br

124 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

0 5 10 

[D] μg/ml 

15 20 25 


optimum volume of (5.0 10 -4 M) BTB. 

100 

90 

80 

70 

60 

50 

T % 

40 

30 

20 

10 

0 

KET 

SIL 

DEX 

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 

log C µg/ml 


M) BTB. 

KET 

DEX 

SIL 

ط

125 



precision and accuracy of the studied drugs using BTB. 

pH 

λ max (nm) 

Parameters 





Slope (b) 

Intercept (a) 


(L mol -1 cm -1 ) 






RSD % 

RE % 

KET 

3.0 

412 

4.58 

1.0-15 

1.4-14 

0.1168 

0.0009 

3.208 

8.547 

0.714 

0.999 

0.021 

0.071 

0.776 

0.814 

Bromothymol blue (BTB) 

DEX 

4.0 

413 

3.99 

1.0-19 

1.5-17.5 

0.0653 

-0.0021 

2.36 

15.69 

0.455 

0.9994 

0.0426 

0.142 

0.892 

0.936 

3.0 

420 

4.37 

SIL 

2.0-23.5 

3.0-22 

0.0255 

0.0006 

1.24 

38.9 

0.152 

0.9995 

0.0825 

0.275 

0.76 

0.798 




126 



Drugs 

Ketamine 

hydrochloride 


hydrobromide 

Silymarin 

method using BTB. 

Taken 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD a 

(%) 



RE 

(%) 

Confidence b 

limits 

3.0 3.02 100.66 1.2241 1.2843 3.02±0.0388 

6.0 5.99 99.83 0.8969 0.9410 5.99±0.0564 

9.0 9.024 100.26 0.6794 0.7128 9.024±0.0643 

3.0 2.98 99.33 1.1809 1.2390 2.98±0.0369 

6.0 6.03 100.5 0.9215 0.9668 6.03±0.0583 

9.0 8.99 99.88 0.6085 0.6384 8.99±0.0574 

5.0 4.96 99.2 1.0265 1.0770 4.96±0.0534 

10 9.87 98.7 0.7227 0.7583 9.87±0.0748 

15 14.94 99.60 0.5118 0.5370 14.94±0.0802

127 



Dosage Forms 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 



method for investigated KET and DEX using BTB in spiked 


Added 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 



RSD a 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.504 100.16 1.2462 2.504±0.0327 

5.0 5.025 100.5 1.0436 5.025±0.055 

7.5 7.48 99.73 0.8287 7.48±0.065 

10 10.01 100.1 0.5646 10.01±0.0593 

- - - - - 

2.5 2.483 99.32 1.6737 2.483±0.0436 

5.0 4.99 99.8 1.1258 4.99±0.0589 

7.5 7.495 99.93 0.9357 7.495±0.0736 

10 9.992 99.92 0.5216 9.992±0.0547 

- - - - - 

5.0 4.98 99.6 1.3085 4.98±0.0684 

10 9.92 99.2 0.7158 9.92±0.0745 

15 15.003 100.02 0.5215 15.003±0.0821 

20 19.94 99.7 0.4424 19.94±0.0926 

- - - - - 

2.5 2.505 100.2 2.0173 2.505±0.053 

5.0 5.015 100.3 1.0644 5.015±0.056 

7.5 7.497 99.96 0.5172 7.497±0.0407 

10 10.03 100.3 0.3266 10.03±0.0344

128 



Dosage Forms 

method for investigated SIL using BTB in spiked urine 

samples. 







Added 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 

RSD a 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.495 99.8 1.1723 2.495±0.0307 

5.0 4.96 99.2 0.9505 4.96±0.0495 

7.5 7.483 99.77 0.8254 7.483±0.0648 

10 9.974 99.74 0.7172 9.974±0.0751 

- - - - - 

2.5 2.486 99.44 1.1348 2.486±0.0296 

5.0 5.01 100.2 0.8748 5.01±0.0460 

7.5 7.492 99.89 0.7095 7.492±0.0558 

10 10.026 100.26 0.4925 10.026±0.0518 

- - - - - 

2.5 2.492 99.68 1.3715 2.492±0.0359 

5.0 4.976 99.52 0.9692 4.976±0.0506 

7.5 7.486 100.18 0.7546 7.486±0.0593 

10 9.99 99.9 0.3547 9.99±0.0372 



129 



Dosage form 


(50 mg / ml) 

method for investigated KET using BTB in spiked serum 



Dosage form 


(50 mg / ml) 

Add 

(μg ml -1 ) 

method for investigated KET using BTB in serum 

samples, (in vivo). 

Add 

(μg ml -1 ) 

5.0 

10 

15 

2.5 

5.0 

7.5 

10 

Found 

(μg ml -1 ) 

2.46 

4.89 

7.30 

9.88 

Found 

(μg ml -1 ) 

3.992 

8.15 

11.487 

Recovery 

% 

98.40 

97.80 

97.33 

98.80 

Recovery 

% 

79.84 

81.50 

76.58 



RSD a 

% 

1.478 

0.9271 

0.6805 

0.4326 

RSD a 

% 

0.7465 

0.7059 

0.6895 

Confidence b 

limit 

2.46±0.038 

4.89±0.048 

7.30±0.053 

9.88±0.045 

Confidence b 

limit 

3.992±0.031 

8.15±0.060 

11.487±0.083

130 



Dosage forms 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 










in it ' s pharmaceutical forms using BTB. 


Taken Found* Recovery Taken Found* Recovery t ** 

mg mg (%) mg mg (%) Value 

50 

15 

1000 

10 

140 

70 

70 

49.612 

14.77 

979.2 

9.953 

138.18 

70.02 

69.63 

* Average of six determinations. 

99.224 

98.47 

97.92 

99.53 

98.70 

100.03 

99.47 

50 

15 

1000 

10 

140 

70 

70 

49.79 

14.92 

982. 

9.98 

137.62 

69.6 

69.28 

99.58 

99.46 

98.25 

99.8 

98.37 

99.428 

98.971 

0.948 

0.962 

0.783 

0.706 

0.827 

1.217 

1.002 

** Theoretical values for t- and F- values for five degree of freedom and 


F ** 

test 

2.832 

1.979 

2.143 

2.376 

2.052 

1.742 

1.610

131 



Dosage forms 


(50 mg / ml) 

Codiphen syrup 

(15 mg / 5.0 ml) 


(1.0 g /15 ml) 




technique using BTB. 

Taken 

(μg ml -1 ) 


Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

3.0 0.0 3.01 100.33 

2.0 4.99 99.8 

4.0 6.98 99.71 

6.0 9.02 100.22 

8.0 11.015 100.13 

3.0 0.0 2.975 99.18 

2.0 4.985 99.7 

4.0 7.03 100.42 

6.0 8.995 99.94 

8.0 10.982 99.83 

3.0 0.0 2.991 99.7 

2.0 5.024 100.48 

4.0 6.97 99.57 

6.0 8.98 99.77 

8.0 11.02 100.18 

3.0 0.0 3.018 100.6 

2.0 5.04 100.8 

4.0 6.975 99.64 

6.0 8.981 99.78 

8.0 11.05 100.45

132 



Dosage forms 







applying the standard addition technique using BTB. 

Taken 

(μg ml -1 ) 


Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

5.0 0.0 4.96 99.20 

2.5 7.47 99.60 

5.0 10.05 100.5 

7.5 12.45 99.60 

10 14.98 99.86 

5.0 0.0 4.97 99.40 

2.5 7.502 100.02 

5.0 9.975 99.75 

7.5 12.49 99.92 

10 14.97 99.80 

5.0 0.0 4.95 99.00 

2.5 7.482 99.76 

5.0 9.99 99.90 

7.5 12.501 100.00 

10 14.95 99.66

133 


4.4. Absorption Spectra of the Studied Drugs with BPB 



drugs and BPB (5.0 x 10 -4 M), The optimum wavelength corresponding to 

each ion–pair complex is at 416 nm in case of KET, 417 nm in case of 

DEX, and 421 nm in case of SIL, as shown in (Fig. 31). 

The effect of different experimental variables was studied and recorded 

below. 



between the studied drugs and BTB was investigated, DEX, KET and SIL 

were allowed to react with BPB (5.0 x 10 -4 M) in aqueous universal buffer 


chloroform or methylene chloride to measure the absor-bance value at λmax. 

The highest absorbance value was obtained at pH = 3.0 and 4.0 for KET 

and (DEX or SIL), respectively which selected for ion-pair formation. 

(Fig. 32). Furthermore, the amount of universal buffer added was 

examined and found to be 1.5 ml and 2.0 ml for KET and (DEX or SIL) as 

shown in (Fig. 33). 



formed ion-pair between the studied drugs and BPB (5.0 x 10 -4 M) was 

investigated. It was observed that shaking for 3.0 min in case of (SIL or 

DEX), whereas in case of KET shaking for 2.5 min, respectively are quite 

sufficient to obtain a maximum colour intensity, before extraction as 

shown in (Fig. 34). The formed ion-pair of each drug was extracted with 

chloroform in case of KET and DEX or with methylene chloride in case of

134 


SIL. The intensity of the extracted ion-pairs was found to be stable over 

the temperature range 20-40ºC. Hence room temperature, (25 ±1ºC), was 

used. The formed ion-pairs were found to be stable for more than 10 hours. 



absorbance intensity. The results obtained using different extracted 


methylene chloride), applying BPB reagent on the studied drugs under 

consideration indicated that chloroform is the best solvent for extraction of 

the ion-pairs formed in case of KET or DEX and methylene chloride is the 

best solvent for extraction of the ion-pair formed in case of SIL. Those 

solvents were selected due to their slightly higher sensitivity and 

considerably lower extraction of the reagent itself. Complete extraction 

was attained by extraction with 5.0 ml of the solvent for one time. 


Various concentrations of BPB were added to a fixed concentration of 


increased with increasing reagent volume up to 1.0 ml and 1.1 ml for 

(DEX or SIL) and KET of BPB (5.0 x 10 -4 M) respectively as shown in 

(Fig. 35). These concentrations of reagent were found to be sufficient for 

the production of maximum and reproducible colour intensity. Higher 


colour intensity of the formed ion-pairs. 



molar ratio and continuous variation methods using both variable reagent

135 


BPB (5.0 x 10 -4 M) and KET, DEX and SIL, concentrations. The results 



labile, as shown in (Figs. 36, 37). Consequently, a large excess of reagent 




development. The sequence of (drug-BPB-buffer) gave the best sequent 

before extraction process, for the highest colour intensity and the least 

time for developing maximum absorbance, all other sequences needed 

longer times and gave lower intensity. 



between negative ion produced from ionization of BPB which convert into 

BPB sodium salt in the buffer and positive ion of the drugs. The ion-pair 


for DEX, KET and SIL as shown in (Fig. 38). 





of KET, DEX and SIL with BPB. Also there were no interference from 


which are likely to occur at normal storage conditions.

136 



Calibration graphs were constructed using standard solutions of 

KET, DEX and SIL. Under the optimum conditions, a linear relationship 

existed between the absorbance and concentration of the drugs in the 

concentration range listed in (Table. 25). The correlation coefficient, 

slopes, intercepts, standard deviation of slopes and standard deviation of 

intercepts of the calibration data for KET, DEX and SIL are calculated. 

The reproducibility of the method was determined by running six replicate 

samples, each contain 8.0 μg ml -1 of drug in case of KET, DEX and SIL. At 

these concentrations, the relative standard deviation was found to be ≤ 

1.017% as recorded in (Table. 25). For more accurate results, Ringbom 

optimum concentration range was determined by plotting log [C] in 

μg ml -1 against percent transmittance. The linear portion of the S-shaped 

curve gave accurate range of analysis as shown in (Fig. 40), and the results 

are recorded in (Table. 25). The mean molar absorpitivity, Sandell 

sensitivity, detection and quantification limits are calculated and recorded 

in (Table. 25). Representation curves on the validity of Beer ' s law for BPB- 

ion pairs is shown in (Fig. 39). 




and SIL were prepared and analysed in quintuplicate. The analytical results 

obtained from these investigations are summarized in (Table. 26). The 

percent standard deviations and the percentage range of error at 95% 



137 



using BPB 




solution, then 1.0 and 1.1 ml of BPB (5.0 x 10 -4 M) in case of (DEX or 

SIL) and KET, respectively were added. The solution was adjusted to the 

required pH values 4.0 and 3.0 for each ion-pair in case of (DEX or SIL) 

and KET, in sequence (drug-BPB-buffer). The solutions were completed to 

the mark with bidistilled water, shaked well and left to stand for 3.0 and 

2.5 min for (DEX or SIL) and KET, respectively. The absorbance was 

measured following the general procedure described above. The relative 

standard deviation (RSD), recovery and confidence limits of the added 

drug are computed and recorded as shown in (Table. 27, 28). 

4.4.12.A. Determination of KET in spiked serum samples in vitro using 

BPB 




with 1.1 ml of BPB (5.0 x 10 -4 M). The solution was adjusted to the 







in (Table. 29-A).

138 


4.4.12.B. Determination of KET in serum samples in vivo using BPB 



deproteinization was achieved by adding 10 ml of trichloroacetic acid 


with 1.1 ml of BPB (5.0 x 10 -4 M). The solution was adjusted to the 








glucuronic acid. A part of ketamine was excreted in the urine as 

metabolites and affect on urea and creatinine concentrations. The relative 

standard deviation (RSD), recovery and confidence limits of the added 

drug are computed and recorded as shown in Table (29-B). 










variance ratio F-value with those obtained using the official methods on

139 


the sample of the same batch. The student ' s t-test values obtained at 95% 




between precision of the proposed and the official method (Table. 30). 

The accuracy of the proposed method when applied to pharmaceutical 

preparations is evaluated by applying standard addition method. In which 


previously analysed portion of pharmaceutical preparations. The results are 

recorded in (Tables. 31 and 32) confirming that the proposed method is not 

liable to interference by fillers usually formulated with the drugs (KET, 


be used easily for the routine analysis in pure form and there 


140 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

2 

3 

1 

1 = DEX 

2 = KET 

3 = SIL 

350 375 400 425 450 


475 500 525 550 

Fig. (31): Absorption spectra of the studied drugs using BPB (5.0 10 -4 


141 


Absorbance 

2.4 

2 

1.6 

1.2 

0.8 

0.4 

0 

0 1 2 3 4 5 6 7 8 

pH 

Fig. (32): Effect of pH on the absorbance of 8.0 μg ml -1 of the studied 

Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

drugs using (5.0 10 -4 M) BPB. 

0 1 2 3 

ml add of pH 

4 5 6 

DE 

X 

KE 

T 

DEX 

KET 

SIL 

Fig. (33): Effect of ml added of buffer on the absorbance of 8.0 μg ml -1 of 

the studied drugs using (5.0 x 10 -4 M) BPB.

142 


Absorbance 

2.5 

2 

1.5 

1 

0.5 

0 

KET 

DEX 

SIL 

0 1 2 3 4 5 6 7 

Time (min) 


Absorbance 

2 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

solution using (5.0 10 -4 M) BPB. 

DEX 

SIL 

KET 

0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 

ml add of BPB (5.0 x 10 -4 M) 


drugs solution using (5.0 10 -4 M) BPB.

143 


Absorbance 

2.5 

2 

1.5 

1 

0.5 

0 

KET 

DEX 

SIL 

0 0.4 0.8 1.2 1.6 2 2.4 2.8 

Reagent/drug 

Fig. (36): Molar ratio for BPB-Drugs (5.0 x 10 -4 M) under consideration. 

Absorbance 

2.5 

2 

1.5 

1 

0.5 

0 

KET 

DEX 

SIL 

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 


Fig. (37): Continuous variation using (5.0 10 -4 M) BPB reagent with (5.0 


144 


Fig. (28): Proposed mechanism of the reaction between ketamine 

HO 

Br 

Br 

hydrochloride and bromophenol blue sodium salt. 

Br 

O 

S 

O 

O 

OH 

Br 

HO 

Br 

Br 

Br 

SO 3 H 

O 

Br 

HO 

Br 

Br 

Br 

SO 3 Na 

Bromophenol blue (quinoid ring) Bromophenol blue sodium salt 


H 3C 

O 

.HBr 

+ 

HO 

Br 

Br 

SO 3 

Br 

SO 3Na 

Dextromethorphan hydrobromide Bromophenol blue sodium salt 

H 3C 

O 

H 

H 

N 

CH 3 

N 

CH 3 

pH = 4.0 

+ - 

Br 

Br 

HO 

Dextromethorphan hydrobromide-BPB Complex 

Br 

O 

Br 

O 

Br 

O 

Br 

+ NaBr

145 


Absorbance 

2.4 

2 

1.6 

1.2 

0.8 

0.4 

0 

KET 

DEX 

SIL 

0 4 8 12 16 20 24 28 

[D] µg/ml 


T% 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

optimum volume of (5.0 10 -4 M) BPB. 

0 0.2 0.4 0.6 0.8 

LogC µg/ml 

1 1.2 1.4 1.6 


M) BPB. 

DEX 

SIL 

KET

146 



precision and accuracy of the studied drugs using BPB. 

pH 

λ max (nm) 

Parameters 


Beer's Law Limits (μg ml-1) 

Ringbom Limits (μg ml-1) 


Slope (b) 

Intercept (a) 


(L mol -1 cm -1 ) 






RSD % 

RE % 

KET 

3.0 

416 

4.58 

1.0-14.5 

2.0-13.5 

0.1158 

0.0042 

3.20 

8.568 

0.677 

0.9998 

0.015 

0.05 

0.744 

0.781 

Bromophenol blue (BPB) 

DEX 

4.0 

417 

4.15 

1.0-20 

2.5-19 

0.1053 

0.0045 

3.92 

9.449 

0.824 

0.9997 

0.0381 

0.127 

1.017 

1.067 

5.0 

421 

4.48 

SIL 

2.0-27 

3.0-26 

0.0276 

0.0046 

1.356 

35.575 

0.191 

0.9958 

0.0645 

0.215 

0.849 

0.891 




147 



method using BPB. 

Drugs Taken 

(μg ml -1 Found 

) (μg ml -1 Recovery 

RE 

) (%) (%) (%) limits 

Ketamine 

hydrochloride 

3.0 3.02 100.66 1.2892 1.3526 3.02±0.0408 

6.0 5.96 99.33 1.0765 1.1295 5.96±0.0673 


hydrobromide 

RSD a 

Confidence b 

9.0 9.01 100.11 0.5399 0.5665 9.01±0.051 

3.0 2.975 99.16 1.1472 1.2036 2.975±0.0358 

6.0 6.03 100.50 0.9528 0.9997 6.03±0.0603 

9.0 8.983 99.81 0.5425 0.5692 8.983±0.0511 

Silymarin 5.0 4.99 99.80 0.9637 1.0111 4.99±0.0505 

10 9.95 99.50 0.5881 0.6170 9.95±0.0614 

15 14.992 99.94 0.4722 0.4954 14.99±0.0743 



148 



method for investigated KET and DEX using BPB in spiked 



(μg ml -1 ) 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g /15 ml) 



Found 

(μg ml -1 ) 

Recovery 

(%) 



RSD a 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.504 100.16 1.3930 2.504±0.0366 

5.0 4.985 99.70 1.1462 4.985±0.0599 

7.5 7.475 99.66 0.8167 7.475±0.0641 

10 10.02 100.2 0.5343 10.02±0.0562 

- - - - - 

2.5 2.487 99.48 1.2553 2.487±0.0328 

5.0 4.96 99.20 0.9079 4.96±0.0472 

7.5 7.47 99.60 0.6466 7.47±0.0507 

10 9.99 99.90 0.4912 9.99±0.0515 

- - - - - 

5.0 4.984 99.68 1.1835 4.984±0.0619 

10 9.985 99.85 0.7352 9.985±0.0770 

15 14.85 99.00 0.4632 14.85±0.0722 

20 19.95 99.75 0.3836 19.95±0.0803 

- - - - - 

2.5 2.502 100.08 1.5162 2.502±0.0398 

5.0 4.95 99.00 1.0473 4.95±0.0544 

7.5 7.504 100.05 0.6335 7.504±0.0499 

10 9.975 99.75 0.7129 9.975±0.0746

149 



Dosage Forms 







method for investigated SIL using BPB in spiked urine 

samples. 

Added 

(μg ml -1 ) 

Found 

(μg ml -1 ) 

Recovery 

(%) 



RSD a 

(%) 

Confidence b 

limits 

- - - - - 

2.5 2.501 100.04 1.0129 2.501±0.0266 

5.0 4.975 99.50 1.1577 4.975±0.0604 

7.5 7.483 99.77 0.8522 7.483±0.0669 

10 9.965 99.65 0.7823 9.965±0.0818 

- - - - - 

2.5 2.485 99.40 1.7621 2.485±0.0459 

5.0 4.96 99.20 0.9786 4.96±0.0509 

7.5 7.51 100.13 0.6388 7.51±0.0503 

10 10.04 100.40 0.3405 10.04±0.0359 

- - - - - 

2.5 2.52 100.80 1.0159 2.52±0.0269 

5.0 5.03 100.60 0.8634 5.03±0.0456 

7.5 7.505 100.06 0.6399 7.505±0.0504 

10 9.99 99.90 0.4709 9.99±0.0494

150 



Dosage form 

Ketamar 

ampoule (50 mg / 

ml) 

method for investigated KET using BPB in spiked serum 


Add 

(μg ml -1 ) 

2.5 

5.0 

7.5 

10 

Found 

(μg ml -1 ) 

2.44 

4.91 

7.43 

9.87 

Recovery 

% 

97.60 

98.20 

99.07 

98.70 

RSD a 

% 

1.07 

0.7807 

0.6635 

0.5632 

Confidence b 

limit 

2.44±0.027 

4.91±0.040 

7.43±0.052 

9.87±0.058 


Dosage form 


(50 mg / ml) 

method for investigated KET using BPB in serum samples, 

(in vivo). 

Add 

(μg ml -1 ) 

5.0 

10 

15 

Found 

(μg ml -1 ) 

4.02 

7.982 

11.758 

Recovery 

% 

80.40 

79.82 

78.39 


RSD a 

% 

0.702 

0.815 

0.565 


Confidence b 

limit 

4.020±0.030 

7.982±0.068 

11.758±0.070

151 




in it ' s pharmaceutical forms using BPB. 

Dosage forms Official method Proposed method 

Taken Found* Recovery Taken Found* Recovery 

mg mg (%) mg mg (%) 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g / 15 ml) 



Hepamarin 

capsules 






50 

15 

1000 

10 

140 

70 

70 

49.612 

14.77 

979.2 

9.953 

138.18 

70.02 

69.63 

* Average of six determinations. 

99.224 

98.47 

97.92 

99.53 

98.70 

100.03 

99.47 

50 

15 

1000 

10 

140 

70 

70 

49.88 

14.86 

984 

9.92 

137.6 

69.75 

69.52 

99.76 

99.07 

98.40 

99.20 

98.28 

99.64 

99.31 

t ** 

Value 

1.407 

1.432 

1.112 

0.828 

0.845 

0.807 

0.335 

** Theoretical values for t- and F- values for five degree of freedom and 


F ** 

test 

2.554 

1.803 

1.852 

2.009 

1.878 

1.995 

2.032

152 



Dosage forms 


(50 mg / ml) 


(15 mg / 5.0 ml) 


(1.0 g /15 ml) 




method using BPB. 

Taken 

(μg ml -1 ) 


Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

3.0 0.0 2.98 99.33 

2.0 5.02 100.40 

4.0 7.01 100.14 

6.0 9.02 100.22 

8.0 11.04 100.36 

3.0 0.0 2.96 98.66 

2.0 4.99 99.80 

4.0 6.95 99.28 

6.0 9.04 100.44 

8.0 11.02 100.18 

2.0 0.0 2.01 100.50 

2.0 3.97 99.25 

4.0 5.99 99.83 

6.0 8.01 100.12 

8.0 9.985 99.85 

4.0 0.0 4.03 100.75 

2.0 6.01 100.16 

4.0 7.99 99.87 

6.0 10.02 100.20 

8.0 11.98 99.83

153 



applying the standard addition method using BPB. 


(μg ml -1 ) 








Added 

(μg ml -1 ) 

Found* 

(μg ml -1 ) 

Recovery 

(%) 

5.0 0.0 4.97 99.40 

2.5 7.48 99.73 

5.0 10.01 100.1 

7.5 12.46 99.68 

10 14.99 99.93 

5.0 0.0 4.95 99.00 

2.5 7.502 100.02 

5.0 9.985 99.85 

7.5 12.504 100.03 

10 15.01 100.06 

5.0 0.0 5.015 100.30 

2.5 7.465 99.53 

5.0 9.975 99.75 

7.5 12.482 99.85 

10 15.03 100.2

154 


Results of Biochemical Studies: 

Table A: Serum Alanine Transferase Level (ALT) (U/l) 

No. of 

EXP. 

G I 

(N-C) 

1 41.32 

2 43.06 

3 39.86 

4 40.21 

5 38.93 

6 42.05 

Means 40.91 

±S.D 1.52 

G II 

(5.0 μg after 

1 h) 

39.65 

38.73 

40.27 

37.68 

38.76 

41.31 

39.40 

1.29 

G III 


3 h) 

38.95 

40.15 

38.64 

39.55 

41.25 

39.22 

39.63 

G IV 

(10 μg after 

1 h) 

41.25 

42.33 

43.22 

40.16 

42.88 

41.68 

41.92 

0.95 1.13 

G V 

(10 μg after 

3 h) 

38.50 

39.75 

40.23 

39.86 

37.24 

41.03 

39.44 

1.35 

±S.E 0.62 0.53 0.39 0.46 0.55 

%change No significant change 

Table B. Serum Aspartate Transferase Level (AST) (U/l) 

No. of EXP. 

G I 

(N-C) 

G II 


1 h) 

G III 


3 h) 

G IV 

(10 μg after 

1 h) 

G V 

(10 μg after 

3 h) 

1 175.30 186.5 187.00 191.30 179.48 

2 182.60 189.50 172.54 185.40 190.24 

3 186.40 179.80 177.30 189.63 186.29 

4 183.40 183.20 178.60 193.46 180.43 

5 179.50 191.60 169.70 190.45 187.61 

6 185.60 184.60 176.50 182.46 185.34 

Means 182.13 185.87 176.94 188.78 184.90 

±S.D 4.14 4.29 5.93 4.08 4.18 

±S.E 1.69 1.75 2.42 1.66 1.71 

%change No significant change

155 


Table C: Blood Urea (mg /dl) 

No. of EXP. 

G I 

(N-C) 

1 21.65 

2 22.34 

3 20.97 

4 19.35 

5 23.14 

6 18.64 

Means 21.02 

±S.D 1.74 

G II 


1 h) 

18.35 

17.64 

14.65 

13.54 

15.46 

12.64 

15.38 

2.25 

G III 


3 h) 

21.68 

23.58 

24.35 

19.56 

24.15 

20.81 

22.36 

1.97 

G IV 

(10 μg after 

1 h) 

11.34 

13.26 

12.35 

14.65 

10.95 

15.32 

12.89 

1.76 

G V 

(10 μg after 

3 h) 

19.67 

23.54 

18.76 

21.30 

26.03 

17.65 

21.16 

3.15 

±S.E 0.71 0.92 0.80 0.72 1.29 

P value V.H.S N.S V.H.S N.S 

%Change -26.83% -6.37 -38.68 -0.66 

Table D: Serum Creatinine (mg/dl) 

No. of 

EXP. 

G I 

(N-C) 

1 0.99 

2 0.96 

3 0.87 

4 0.94 

5 0.90 

6 0.91 

G II 


1 h) 

0.33 

0.32 

0.29 

0.35 

0.37 

0.28 

G III 


3 h) 

0.88 

0.89 

0.85 

0.87 

0.88 

0.90 

G IV 

(10 μg after 

1 h) 

0.31 

0.32 

0.26 

0.30 

0.28 

0.27 

G V 

(10 μg after 

3 h) 

0.97 

0.96 

0.87 

0.90 

0.90 

0.91 

Means 0.93 0.33 0.88 0.29 0.92 

±S.D 0.044 0.038 0.017 0.024 0.04 

±S.E 0.018 0.015 0.007 0.097 0.016 

P value V.H.S N.S V.H.S N.S 

%Change -35.38 -5.38 -68.8 -1.08

156 


Aminotransferase are found in both cytosol and mitochondria. The 

aspartate transferase (AST) (Cytoplasmic and mitochondrial) is present in 

great concentration in cardiac muscle, liver skeletal muscle and kidney, 

while alanine transferase (ALT) (Solely cytoplasmic) is mainly present in 

the liver. 

The mean value of serum alanine transferase (ALT) activity in normal 

mice was found to be 40.91±1.52 R. F. Units/ml (Table. A). this results in 

agreement with of Evett and Harrison (1983) who reported an average of 

40 ± 14 R. F. Units/ml of serum ALT activity in mice. The treatment of the 

normal mice with KET using (doses 5.0 μg and 10 μg after one or three 

hours I.P.) showed non significant changes in serum ALT compared with 

control group (Group I N.C.). 

The mean value of AST serum level for normal control mice was found 

to be 182.13 ± 4.14 R. F. Units/ml (Table. B) which is near that found and 

reported as 153 ± 79 R. F. Units/ml of serum ALT activity in mice by 

Evett and Harrison (1983). The treatment of the normal mice with KET 

using (doses 5.0 μg and 10 μg after one or three hours I.P.) showed no 

significant changes in serum ALT compared with control group (Group I 

N.C.). 

The mean value of urea for normal control mice was found to be 21.02 

± 1.74 mg/dl (Table. C) which is in accordance with that found and 

reported as 20.7 ± 5.1 mg/dl by Evett and Harrison (1983). The treatment 

of the normal mice with KET using (doses 5.0 μg and 10 μg after one 

hour, I.P.) showed a very high significant decrease in urea concentration 

which reaches 15.38 

± 2.25 mg/dl and 12.89 ± 1.76 mg/dl (Groups II and 

IV), respectively compared with control group (Group I N.C.). This 

decrease in urea levels, below those observed may occur in mice with

157 


severe hepatic disease where circulating ammonia is inadequately 

converted to urea by the remaining functional hepatic tissue. In this 

instance, decrease urea level would be accompanied by increases in blood 

ammonia levels, while the treatment of the normal mice with KET using 

(doses 5.0 μg and 10 μg after three hours, I.P.) showed no significant 

changes in urea concentration which reaches 22.36 ± 1.97 mg/dl and 21.16 

± 3.15 mg/dl (Groups III and V), respectively compared with control group 

(Group I N.C.) 

The mean value of creatinine for normal control mice was found to be 

0.93 ± 0.044 mg/dl (Table. D) which is in agreement with that found and 

reported as 0.50 ± 0.56 mg/dl by Evett and Harrison (1983). The 

treatment of the normal mice with KET using (doses 5.0 μg and 10 μg after 

one hour, I.P.) showed a significant decrease of creatinine concentration 

which reaches 0.33 ± 0.038 mg/dl and 0.29 ± 0.024 mg/dl (Groups II and 

IV), respectively compared with control group (Group I N.C.). The 

implications of decrease in creatinine are similar to those previously cited 

for urea so that determination of serum creatinine offers no real 

interpretative advantage. While The treatment of the normal mice with 

KET using (doses 5.0 μg and 10 μg after three hours, I.P.) showed no 

significant changes in creatinine concentration which reaches 0.88 ± 0.017 

mg % and 0.92 ± 0.04 mg % (Groups III and V), respectively compared 

with control group (Group I N.C.).

SUMMARY AND CONCLUSION 

The present work illustrates practical application for micro- 

determination of some drugs using acid dye reagents and comprises the 

following: 

The introduction which include two parts: the first part gives an idea 

about the importance of drug analysis and the drugs under consideration, a 

discussion about the definitions, actions, chemical structures and chemical 

names, characters of the studied drugs ketamine hydrochloride, 

dextromethorphan hydrobromide and silymarin are given. The second part 

gives a literature survey of the previous studies for the analysis of the 

studied drugs including spectrophotometric, ultra-violet spectrophoto- 

metric, capillary electrophoresis, high-performance liquid chromatography, 

electroanalytical and other chromatographic methods. Chemical structures 

and chemical names and a literature survey of the acid dyes used 

bromocresol green (BCG), bromocresol purple (BCP), bromothymol blue 

(BTB) and bromophenol blue (BPB). 

The experimental part which includes apparatus used for 

measurements and procedures for preparations of the drug solutions and 

reagents. It also contains the proposed spectrophotometric methods for 

determination of the studied drugs in pure forms, biological fluids (urine) 

and in dosage forms which analysed. Also, it contains pharmacopoeial and 

official methods for analysis of the studied drugs. Also, it contains 

biochemical studies and analytical application for determination of 

ketamine hydrochloride in serum (in vitro and in vivo). 

The results and discussion which include the spectrophotometric 

procedures for the determination of the studied drugs using acid dyes

159 

__________________________________________ Summary and Conclusion 

reagents bromocresol green (BCG), bromocresol purple (BCP), 

bromothymol blue (BTB) and bromophenol blue (BPB). the proposed 

methods are based on coloured ion-pair complex formation between the 

acid dyes and drugs which is extracted with an organic solvents, 

(chloroform, benzene, carbon tetrachloride, hexane, and methylene 

chloride) and determination of the concentration by measuring the 

absorbance of the extracted complex against reagent blank prepared by the 

same way. The following experimental variables are investigated. 

1 - Effect of pH. 

2 – Effect of time. 

3 – Effect of the extracting solvent. 

4 – Effect of reagent concentration. 

5 – Molecular ratio of the complex. 

6 – Sequence of addition. 

7 – Suggested mechanism. 

8 – Evaluation of the stability constants of the ion-pair complexes. 

Using BCG: 

Beer's law is obeyed in the concentration ranges 1.0-14, 1.0-21 and 

2.0-29 μg ml -1 for Ket, Dex and Sil, respectively. For more accurate results, 

Ringbom optimum concentration ranges are determined. Molar 


calculated. The stoichiometric ratios of the studied drugs with BCG are 

established using the molar ratio and continuous variation methods and 

found to be 1 : 1 for the drugs under consideration with BCG. In order to 

determine the accuracy and precision of the proposed methods, solution

160 


containing three different concentrations of the studied drugs are prepared 

and analysed in six replicates. The recovery, the relative standard 

deviation, the relative error and confidence limits are calculated. The 

proposed methods can successfully applied to determine the pure form of 

the studied drugs. Also the proposed methods are successfully applied to 

determine the studied drugs in their dosage forms. The results obtained are 

compared statistically by Student's t- test and variance ratio F-test with the 

official methods at 95 % confidence level. The results showed that the t- 

and F- value are less than the critical value indicating that there is no 

significant difference between the proposed and official methods. Thus the 

proposed spectrophotometric methods can applied in determination of the 

studied drugs in pure and in dosage forms. Also the studied drugs are 

determined in biological samples (urine samples) (in vitro) and the results 

showed that no interferences between the studied drugs and urine 

components after deproteinization. Also ket is determined in serum after 

injection Ket doses (5.0, 10 and 15 μg) into the mice and the results 

showed that the recovery ≥ 75.95 %, this indicated that there is 

interference due to anaesthetic action and a part of ket was excreted in the 

urine as metabolites and affect on urea and creatinine concentrations. 

Using BCP: 





calculated. The stoichiometric ratios of the studied drugs with BCP are 


found to be 1 : 1 for the drugs under consideration with BCP. In order to

161 


determine the accuracy and precision of the proposed methods, solution 













determined in biological fluids (urine samples) (in vitro) and the results 


components after deproteinization. Also ketamine hydrochloride is 

determined in serum after injection Ket doses (5.0, 10 and 15 μg) into the 

mice and the results showed that the recovery ≥ 73.93 %, this indicated 

that there is interference due to anaesthetic action and a part of ket was 

excreted in the urine as metabolites and affect on urea and creatinine 

concentrations. 

Using BTB: 


2.0-23.5 μg ml -1 for Ket, Dex and Sil, respectively. For more accurate 

results, Ringbom optimum concentration ranges are determined. Molar 


calculated. The stoichiometric ratios of the studied drugs with BTB are

162 



found to be 1 : 1 for the drugs under consideration with BTB. In order to 
















components after deproteinization. Also ket is determined in serum after 

injection Ket doses (5.0, 10 and 15 μg) into the mice and the results 

showed that the recovery ≥ 76.58 %, this indicated that there is 

interference due to anaesthetic action and a part of ket was excreted in the 

urine as metabolites and affect on urea and creatinine concentrations. 

Using BPB: 

Beer's law is obeyed in the concentration ranges 1.0-14.5, 1.0-20 and 



absorpitivity, Sandell sensitivity, detection and quantification limits are

163 


calculated. The stoichiometric ratios of the studied drugs with BPB are 


found to be 1 : 1 for the drugs under consideration with BPB. In order to 
















components after deproteinization. Also Ket is determined in serum (in 

vivo) after injection Ket doses (5.0, 10 and 15 μg) into the mice and the 

results showed that the recovery ≥ 78.39 %, this indicated that there is 

interference due to anaesthetic action and a part of Ket was excreted in the 

urine as metabolites and affect on urea and creatinine concentrations.

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ﻲﺑرﻌﻟا صﺧﻠﻣﻟا 

نﻳـــﻳﻌﺗﻟ ﺔـــﺛﻳدﺣﻟا قرـــطﻟا عرـــﺳأو قدأو ﻝﻬـــﺳأ نـــﻣ ﺎﻬﺗﺎﻘﺗـــﺷﻣ و ﺔﻳﺋوـــﺿ فـــﻳطﻟا قرـــطﻟا رـــﺑﺗﻌﺗ 

مادﺧﺗـﺳا مﺗـﻳ ثـﻳﺣ . ﺎـﻬﻠﻠﺣﺗ ﺞﺗاوـﻧ ﻊـﻣ وأ طﻳﻟﺎـﺧﻣ ﻰـﻓ 

، درـﻔﻧﻣ ءاوـﺳ ﺔﻳﺋاودﻟا تارﺿﺣﺗﺳﻣﻟا ﻝﻳﻠﺣﺗو 

ﺎـــﻬﺗﻗد و ﺎﻬﺗﻋرـــﺳﻟ ارـــظﻧ ﺔـــﻳودﻷا ﻊﻳﻧـــﺻﺗ تﺎﻛرـــﺷ ﻊـــﻳﻣﺟ ﻰـــﻓ ﺎـــﻳﻟﺎﺣ ﺔﻳﺋوـــﺿ فـــﻳطﻟا قرـــطﻟا ﻩذـــﻫ 

ﺔطﻳـﺳﺑ قرـط دﺎـﺟﻳإ ثـﺣﺑﻟا اذـﻫ نﻣ فدﻬﻟا و . ﺔﺿﻔﺧﻧﻣﻟا ﺔﻳﺋاودﻟا تازﻳﻛرﺗﻟا ﻰﻓ ﺔﺻﺎﺧ ﺔﻳﻫﺎﻧﺗﻣﻟا 

نﺎﻓروﺛﻳﻣورﺗـﺳﻛد 

- 

دـﻳروﻠﻛوردﻳﻫ نﻳﻣﺎـﺗﻳﻛ) 

ﺔـﻳودﻷا ضﻌﺑـﻟ ﻰﺋﺎـﻳﻣﻳﻛﻟا ﻝـﻳﻠﺣﺗﻠﻟ ﺔـﺳﺎﺳﺣ و ﺔـﻘﻳﻗد و 

ﺔـﻳﺋاودﻟا تارـﺿﺣﺗﺳﻣﻟا ﻰـﻠﻋ قرــطﻟا ﻩذـﻫ قـﻳﺑطﺗ و ﺔـﻳﻘﻧﻟا ةروــﺻﻟا ﻰـﻓ ( نﻳرﺎﻣﻳﻠﻳـﺳ – دـﻳﻣورﺑوردﻳﻫ 

. ﺔﻳـﺿﻣﺣﻟا تﺎﻐﺑـﺻﻟا ضـﻌﺑ مادﺧﺗـﺳﺎﺑ .( ﻝوـﺑﻟا و مدـﻟا) 

ﺔـﻳوﻳﺣﻟا تﺎـﻧﻳﻌﻟا ضـﻌﺑ و تﺎـﺑﻛرﻣﻟا ﻩذـﻬﻟ 

: ثﺣﺑﻟا نﻣﺿﺗﻳو 

مﻳــﺳﻘﺗﻟا و ﺔــﻳودﻷا ﻝــﻣﻋ ﺔــﻳﻔﻳﻛ و ﺔــﻳودﻷا ﻩذــﻫ نــﻋ ةرــﺻﺗﺧﻣ ةذــﺑﻧ ﻰــﻠﻋ ىوــﺗﺣﺗ ﻲــﺗﻟا ﺔــﻣدﻘﻣﻟا 

ﺢــﺳﻣ ﻰــﻟإ ﺔﻓﺎــﺿﻹﺎﺑ ﺎﻬــﺻﺋﺎﺻﺧ كﻟذــﻛ و ﻲــﺑﻳﻛرﺗﻟا ءﺎــﻧﺑﻟاو ﻲﺋﺎــﻳﻣﻳﻛﻟا مــﺳﻻا ﺎــﺿﻳأو ﺎــﻬﻟ ﻲﺋﺎــﻳﻣﻳﻛﻟا 

ةذــﺑﻧ كﻟذــﻛو ﺎــﻬﻟ ﺔــﻳﺋاودﻟا تارــﺿﺣﺗﺳﻣﻟا و ﺔــﻳﻘﻧﻟا ةروـــﺻﻟا ﻰــﻓ ﺎــﻬﻠﻳﻠﺣﺗﻟ ﺔﻣدﺧﺗــﺳﻣﻟا قرــطﻠﻟ ﻝﻣﺎــﺷ 

. ﺔﻳودﻷا ضﻌﺑ ﻝﻳﻠﺣﺗ ﻰﻓ ﺔﻳﺿﻣﺣﻟا تﺎﻐﺑﺻﻟا مادﺧﺗﺳا نﻋ ةرﺻﺗﺧﻣ 

ﻝـﻳﻟﺎﺣﻣﻟا رﻳـﺿﺣﺗﻟ ﺔﻣدﺧﺗﺳﻣﻟا قرطﻟاو سﺎﻳﻘﻠﻟ ﺔﻣدﺧﺗﺳﻣﻟا ةزﻬﺟﻷا ﻝﻣﺷﻳ ﻲﻠﻣﻌﻟا ءزﺟﻟا ﺎﺿﻳأو 

ةروـﺻﻟا ﻲـﻓ ﺔـﻳودﻷا ﻩذـﻫ نﻳـﻳﻌﺗﻟ ﺔـﺣرﺗﻘﻣﻟا قرطﻟا نﻋ ﻰﻓاو 

حرﺷ ﻰطﻌﻳ كﻟذﻛو ﺔﻳودﻷاو فﺷاوﻛﻟاو 

ﺔــﻳودﻷا ﻩذــﻫ نﻳــﻳﻌﺗﻟ ﺔــﺻﺎﺧﻟا قرــطﻟا ضرــﻋ ﻊــﻣ ﺎــﻬﺑ ﺔــﺻﺎﺧﻟا ﺔﻳﻧﻻدﻳــﺻﻟا تارــﺿﺣﺗﺳﻣﻟا و ﺔــﻳﻘﻧﻟا 

ةدﻣﺗﻌﻣﻟا ﺔﻳروﺗﺳدﻟا قرطﻟا وأ ﺔﻳودﻷا تﺎﻛرﺷ ﻰﻓ ﺔﻘﺑطﻣﻟا ءاوﺳ 

نﻳﻣﺎــــﺗﻳﻛ) 

ﺔــــﺳا ردﻟا دــــﻳﻗ ﺔــــﻳودﻸﻟ ﻰــــﻔﻳطﻟا رﻳدــــﻘﺗﻟا طــــطﺧ ﺔــــﺷﻗﺎﻧﻣﻟاو ﺞﺋﺎــــﺗﻧﻟا نﻣــــﺿﺗﺗ كﻟذــــﻛو 

مادﺧﺗﺳﺎﺑ ( نﻳرﺎﻣﻳﻠﻳﺳ 

– 

دﻳﻣورﺑوردﻳﻫ نﺎﻓروﺛﻳﻣورﺗﺳﻛد 

- 

دﻳروﻠﻛوردﻳﻫ 

(Bromocresol green, Bromocresol purple, Bromothymol blue and 

Bromophenol blue) 

بـﺳﺎﻧﻣ يوﺿﻋ بﻳذﻣ ﻰﻓ ﻪﺟارﺧﺗﺳاو نوﻠﻣ ﻰﺋﺎﻧﺛ بﻛارﺗﻣ نﻳوﻛﺗ ﻰﻠﻋ دﻣﺗﻌﺗ ﺔﺣرﺗﻘﻣﻟا قرطﻟا 

ﻝــﻣاوﻌﻟا ضــﻌﺑ رﻳﺛﺄــﺗ ﺔــﺳارد مــﺗ دــﻗو . ﻰﺟوــﻣ ﻝوــط بــﺳﻧا دــﻧﻋ ﺎــﻬﻟ ﻰﺋوــﺿﻟا صﺎــﺻﺗﻣﻻا 

سﺎــﻳﻗو 

: ﻰﻫو رﻳدﻘﺗﻠﻟ ﺔﻳﺑﻳرﺟﺗﻟا فورظﻟا نﺳﺣأ طﺎﺑﻧﺗﺳﻻ 

ﺔﻳﺿﻣﺎﺣﻟا ﺔﺟرد رﻳﺛﺄﺗ - 

نﻣزﻟا رﻳﺛﺄﺗ 

-

2 

___________________________________________________________ ﻰﺑرﻌﻟا صﺧﻠﻣﻟا 

مﺎــــﻣﺗﻻ ىوــــﺿﻋ بﻳذــــﻣ بــــﺳﻧﺄﻛ دــــﻳروﻠﻛ نﻳــــﻠﻳﺛﻳﻣﻟا وا مروــــﻓوروﻠﻛﻟا رﺎــــﻳﺗﺧاو تﺎﺑﻳذــــﻣﻟا رﻳﺛﺄــــﺗ - 

نﺎﻓروﺛﻳﻣورﺗــــﺳﻛد 

- 

1 

20-1 

2 

، 

، 

15-1 

، 

23.5 

19 

- 

، 

-1 

2 

، 

- 

دــــﻳروﻠﻛوردﻳﻫ نﻳﻣﺎــــﺗﻳﻛ) 

نــــﻣ ﻝــــﻛﻟ نوــــﻠﻣﻟا 

ﻰﺋﺎــــﻧﺛﻟا بــــﻛارﺗﻣﻟا صﻼﺧﺗــــﺳا 

.( نﻳرﺎﻣﻳﻠﻳﺳﻟاو دﻳﻣورﺑوردﻳﻫ 

15-1 

، 

24 

22-1 

- 

،14- 

2 

، 

، 

ﺔﻐﺑﺻﻟا زﻳﻛرﺗ رﻳﺛﺄﺗ - 

بﻛارﺗﻣﻟا ﺎﻬﻧﻣ نوﻛﺗﻳ ﻰﺗﻟا بﺳﻧﻟا نﻳﻳﻌﺗ - 

بﺋاوﺷﻟا رﻳﺛﺄﺗ - 

1 ىزﻳﻛرﺗﻟا ىدﻣﻟا ﻰﻓ ﺔﺣرﺗﻘﻣﻟا قرطﻟا قﻳﺑطﺗ مﺗ دﻗو 

21-1 

، دﻳروﻠﻛوردﻳﻫ نﻳﻣﺎﺗﻳﻛﻠﻟ 

كﻟذو ﻰﻠﻠﻣ/ 

مارﺟورﻛﻳﻣ 14.5 

29 

- 

2 

و دﻳﻣورﺑوردﻳﻫ نﺎﻓروﺛﻳﻣورﺗﺳﻛدﻠﻟ كﻟذو ﻰﻠﻠﻣ/ 

مارﺟورﻛﻳﻣ 

نﻣ ﻼﻛ ﻊﻣ نﻳرﺎﻣﻳﻠﻳﺳﻠﻟ كﻟذو ﻰﻠﻠﻣ/ 

مارﺟورﻛﻳﻣ 27- 

(Bromocresol green, Bromocresol purple, Bromothymol blue and 

Bromophenol blue) 

دـﻳﻗ ﺔـﻳودﻷا نـﻣ ﺔـﻔﻠﺗﺧﻣ تازـﻳﻛرﺗ ثﻼـﺛ ﻝـﻳﻠﺣﺗ مـﺗ ، ﺔـﺣرﺗﻘﻣﻟا قرــطﻟا ﺔـﻗد بﺎـﺳﺣﻟو بـﻳﺗرﺗﻟﺎﺑ 

ةروـــﺻﻟا ﻰـــﻓ 

. ﺄطﺧﻟا ﺔﺑﺳﻧو ىرﺎﻳﻌﻣﻟا فارﺣﻧﻻا ﺔﺑﺳﻧ 

، 

، ﺔﻗدﻟا بﺎﺳﺣ مﺗو ﺔﻳﻟﺎﺗﺗﻣ تارﻣ ﺔﺗﺳ ﺔﺳاردﻟا 

ﺔـــﻳﻘﻧﻟا ةروـــﺻﻟا ﻰـــﻓ ﺔـــﺳاردﻟا دـــﻳﻗ ﺔـــﻳودﻷا رﻳدـــﻘﺗﻟ ﺔـــﺣرﺗﻘﻣﻟا قرـــطﻟا قـــﻳﺑطﺗ مـــﺗو 

رـﻬظﺗو ةدـﻣﺗﻌﻣﻟا ﺔﻳروﺗـﺳدﻟا قرـطﻟا ﻊـﻣ ﺎﻳﺋﺎـﺻﺣإ ﺞﺋﺎـﺗﻧﻟا ﺔﻧرﺎﻘﻣ مﺗو ﺔﻳوﻳﺣﻟا تﺎﻧﻳﻌﻟا 

و ﺔﻳﻧﻻدﻳﺻﻟا 

ﻪـﻧأ ﺢـﺿﺗﻳ كﻟذـﺑو ةدﻣﺗﻌﻣﻟا ﺔﻳروﺗﺳدﻟا قرطﻟاو ﺔﺣرﺗﻘﻣﻟا قرطﻟا نﻳﺑ ﺢﺿاو قرﻓ دﺟوﻳ ﻻ ﻪﻧا ﺞﺋﺎﺗﻧﻟا 

ةروـــﺻﻟا كﻟذـــﻛو ﺔـــﻳﻘﻧﻟا ةروـــﺻﻟا ﻰـــﻓ ﺔـــﺳاردﻟا دـــﻳﻗ ﺔـــﻳودﻷا نﻳـــﻳﻌﺗﻟ ﺔـــﺣرﺗﻘﻣﻟا قرـــطﻟا قـــﻳﺑطﺗ نـــﻛﻣﻳ 

. 

ﺔﻳﻧﻻدﻳﺻﻟا

ةينلاديصلا تابكرملا ضعب 

ةيويحلاو 

مادختساب 

نم ةمدقم ةلاسر 

ملاس ةدوج حوتفلاوبأ نميأ 

" 

ءايميك" 

( ءايميكلا) 

مولع 

ىلع لوصحلل 

مولعلا 

ءايميكلا مسق 

مولعلا ةيلك 

قيزاقزلا ةعماج 

2005 

سويرولاكب 

ىف ريتسجاملا ةجرد 

ةيليلحت تاسارد

Ayman Abou El-Fetouh Gouda

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