Derivative and Derivative Ratio Spectrophotometric Analysis of ...

Journal of the Chinese Chemical Society, 2008, 55, 971-978 971 

Derivative and Derivative Ratio Spectrophotometric Analysis of 

Antihypertensive Ternary Mixture of Amiloride Hydrochloride, 

Hydrochlorothiazide and Timolol Maleate 

Mohamed H. Abdel-Hay, Azza A. Gazy, Ekram M. Hassan and Tarek S. Belal* 

Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Alexandria, 

Elmessalah 21521, Alexandria, Egypt 

Two simple, rapid and reliable spectrophotometric methods are described for the resolution of the 

three-component mixture of amiloride hydrochloride (AMD), hydrochlorothiazide (HCT) and timolol 

maleate (TIM). The first method involves the use of derivative spectrophotometry with the zero-crossing 

technique where AMD was easily determined using its 0 Dand 1 D( = 6) amplitudes at 365 and 385 nm, 

respectively, while HCT and TIM were determined by measuring the 3 D( = 6) amplitude at 265 nm and 

the 1 D( = 8) amplitude at 315.4 nm, respectively. The second method involves the application of the ratio-spectra 

zero-crossing first and second derivative spectrophotometry where two points have been used 

for the quantification of each compound. For the determination of AMD, HCT was used as divisor and the 

1 DD ( =4)and 2 DD ( = 6) values at 299.4 and 311 nm, respectively, were plotted against AMD concentration; 

while - by using TIM as divisor - the 2 DD ( = 6) amplitudes at 264.2 and 290 nm were found 

to be proportional to HCT concentration. TIM was assayed in the mixture using its 1 DD ( =6)amplitudes 

at 289.8 nm (Divisor was AMD) and 314.8 nm (Divisor was HCT). Synthetic mixtures of different 

proportions and laboratory-made tablets were assayed by the proposed methods and the results revealed 

good accuracy and repeatability of the developed methods. 

Keywords: Amiloride hydrochloride; Hydrochlorothiazide; Timolol maleate; Derivative 

spectrophotometry; Derivative ratio spectrophotometry. 

INTRODUCTION 

Direct UV-spectrophotometric measurement is subjected 

to interference from co-formulated drugs, excipients 

and/or degradation products. Derivative spectrophotometry 

is an analytical technique of great utility for extracting 

quantitative information from spectra of unresolved bands. 

The zero-crossing method 1,2 has led to significant developments 

in the analysis of pharmaceutical compounds in 

multi-component mixtures. 3,4 

In 1990, Salinas et al. 5 developed the ratio spectra derivative 

spectrophotometry for the resolution of binary 

mixtures. In 1992, Berzas et al. 6 developed a method and 

discussed the theory for resolving ternary mixtures based 

on the use of first derivative of the ratio spectra of mixtures, 

followed by measurements at the zero crossing wavelengths 

of the first derivative of ratio spectra of single components. 

The ratio spectra zero crossing first derivative 

spectrophotometry was applied for the resolution of several 

ternary mixtures. 7,8 Also, higher derivative orders such 

as the 3 DD were applied for the determination of several 

ternary mixtures. 9,10 

Amiloride hydrochloride (AMD), chemically known 

as 3,5-diamino-N-carbamimidoyl-6-chloropyrazine-2- 

carboxamide hydrochloride dihydrate, is a weak diuretic 

which appears to act mainly on the distal renal tubules. It is 

described as potassium sparing since it increases the excretion 

of sodium and reduces the excretion of potassium. Hydrochlorothiazide 

(HCT), 6-chloro-3,4-dihydro-2H-1,2,4- 

benzothiadiazine-7-sulphonamide-1,1-dioxide, is a moderately 

potent diuretic. It exerts its effect by reducing the reabsorption 

of electrolytes from the renal tubules, thereby 

increasing the excretion of sodium and chloride ions, and 

consequently of water. Timolol maleate (TIM), (2S)-1- 

[(1,1-dimethylethyl)amino]-3-[[4-(morpholin-4-yl)-1,2,5- 

thiadiazol-3-yl]oxy]propan-2-ol (Z)-butenedioate, is a 

non-cardioselective -blocker used in the management of 

several cardio-vascular disorders. 11 Drug combinations of 

hydrochlorothiazide with -adrenergic blocking agents are 

* Corresponding author. Tel: +20-3-4871317; Fax: +20-3-4871351; E-mail: tbelaleg@yahoo.com

972 J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 Abdel-Hay et al. 

usually employed for the management of hypertension. 

Amiloride is useful for the prevention of hypokalemia induced 

by hydrochlorothiazide especially during prolonged 

treatment. 11 

The three drugs are official in both: the BP 12 and the 

USP. 13 Also binary mixtures of HCT with either AMD or 

TIM are official and are assayed by HPLC methods. 13 Binary 

and ternary mixtures containing AMD and HCT have 

been analyzed by several reported methods such as capillary 

zone electrophoresis, 14 spectrophotometry, 15 derivative 

spectrophotometry, 16 differential pulse polarography 

with partial least squares, 17 derivative ratio spectrophotometry, 

18 and HPLC. 18-20 HCT and TIM have been simultaneously 

assayed in their binary mixture using several spectrophotometric 

methods including flow-injection, 21 orthogonal 

function 22 and derivative and derivative-ratio 

spectrophotometry. 23 

The combination of the three studied drugs has been 

recently assayed using several chemometric methods 24 including 

classical least squares (CLS), partial least squares 

(PLS) and principal component regression (PCR). 

This work describes the application of the zero crossing 

derivative and the ratio spectra zero crossing first 

( 1 DD) and second ( 2 DD) derivative spectrophotometry for 

the resolution of AMD-HCT-TIM ternary mixture. The 

proposed methods are rapid, simple, accurate and do not require 

solving equations or working with additional sophisticated 

software. In addition, the methods are direct and do 

not need separation or pretreatment. 

EXPERIMENTAL 

Apparatus 

All spectrophotometric measurements were performed 

using a Perkin-Elmer, Lambda EZ201 UV-VIS 

spectrophotometer with matched 1-cm quartz cuvettes. The 

instrument is connected to a Panasonic impact dot matrix 

printer KX-P3626. 

Materials 

All chemicals and solvents used throughout the study 

were of analytical grade. Authentic samples of AMD, HCT 

and TIM were kindly donated by Pharco Pharmaceuticals 

Co., Alexandria, Egypt and were used as received. 

Laboratory-made tablets containing 25 mg hydrochlorothiazide, 

10 mg timolol maleate and 2.5 mg amiloride 

hydrochloride per tablet were prepared as documented 

in several drug indexes 25,26 for Moducren (Merck Sharp & 

Dohme). 

Preparation of Stock Solutions 

Stock solutions of AMD 200 g/mL, HCT 400 g/mL 

and TIM 400 g/mL were prepared in methanol and stored 

refrigerated at 4 C. 

Calibration Graphs 

Derivative Method 

The working standard solutions were prepared by dilution 

of the stock solutions of AMD, HCT and TIM with 

0.1 M sodium hydroxide solution to reach the concentration 

ranges mentioned in Table 1. The absorbance, 1 Dand 

3 D spectra were recorded against 0.1 M sodium hydroxide 

solution. The absolute values of the A max and 1 D( =6) 

amplitudes at 365 and 385 nm, respectively (for AMD), the 

3 D( = 6) amplitudes at 265 nm (for HCT) and the 1 D( 

= 8) amplitudes at 315.4 nm (for TIM) were plotted against 

the corresponding concentrations. 

Derivative Ratio Method 

The working standard solutions were prepared by dilution 

of AMD, HCT and TIM stock solutions with 0.1 M 

sodium hydroxide solution to reach the concentration ranges 

mentioned in Table 1. The absorption spectra were recorded 

against 0.1 M sodium hydroxide solution and stored. 

The stored spectra were divided (amplitude at each wavelength) 

by the spectrum of a standard solution of 3 g/mL 

HCT(forAMD)and4g/mL TIM (for HCT). The first and 

the second derivatives of the ratio spectra were obtained. 

The absolute values of the 1 D( =4)and 2 D( =6)amplitudes 

at 299.4 and 311 nm, respectively (for AMD) and 

the 2 D( = 6) amplitudes at 264.2 and 290 nm (for HCT) 

were plotted against the corresponding concentrations. 

For the determination of TIM, the stored spectra were 

divided (amplitude at each wavelength) by the spectrum of 

a standard solution of either 2 g/mL AMD or 3 g/mL 

HCT, the first derivative of the ratio spectra were obtained 

and the absolute values of the 1 D( = 6) amplitudes at 

289.8 and 314.8 nm, respectively, were plotted against the 

corresponding concentrations. 

Determination of AMD-HCT-TIM in Tablets 

A total of 20 tablets (Laboratory-made tablets containing25mgHCT,10mgTIMand2.5mgAMDpertablet 

in addition to lactose, starch, talc and magnesium stearate 

as tablet fillers) were massed and finely powdered. To an 

accurately weighed quantity of the powder equivalent to 

the average weight per tablet, 60 mL methanol were added, 

stirred for 10 minutes then filtered into a 100-mL volumetric 

flask. The residue was washed with two 10 mL portions 

of methanol and washings were added to the filtrate and di-

Analysis of Amiloride, Hydrochlorothiazide and Timolol J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 973 

Table 1. Analytical parameters for the determination of AMD, HCT and TIM by the proposed methods 

Linearity Regression Equation 

a 

b 

Compound Wavelength (nm) Range Intercept Slope Corr. S a S b 

(g/mL) (a) (b) Coeff. (r) 

AMD 

S y/x 

c 

LOD d 

(g/mL) 

LOQ e 

(g/mL) 

A 365 3-15 0.003 0.0640 0.99991 0.0043 4.93 10 -4 0.0042 0.22 0.67 

Zero-Crossing Derivative: 

D 385 1-15 0.004 0.1129 0.99992 0.0054 8.08 10 -4 0.0051 0.16 0.49 

Derivative Ratio: 

DD 299.4 (HCT) * 1-15 -0.050 2.7905 0.99995 0.1049 0.0192 0.0857 0.12 0.36 

2 DD 311 (HCT) * 1-15 -0.034 2.4549 0.99997 0.0715 0.0131 0.0584 0.10 0.30 

HCT 

TIM 


D 265 2-25 -0.004 0.0866 0.99992 0.0083 6.23 10 -4 0.0079 0.32 0.97 


DD 264.2 (TIM) * 2-25 -0.039 1.3633 0.99995 0.1016 0.0093 0.0830 0.25 0.76 

2 DD 290 (TIM) * 2-25 -0.051 1.2986 0.99996 0.0884 0.0081 0.0721 0.23 0.70 


1 D 315.4 3-20 -0.002 0.0394 0.99992 0.0038 2.83 10 -4 0.0036 0.32 0.97 


1 DD 289.8 (AMD) * 1-20 -0.053 2.2286 0.99997 0.1353 0.0124 0.1104 0.20 0.61 

1 DD 314.8 (HCT) * 1-20 0.045 1.8875 0.99999 0.0581 0.0053 0.0474 0.10 0.30 

* The compound used as divisor 

a Standard deviation of the intercept 

b Standard deviation of the slope 

c Standard deviation of residuals 

d Limit of detection 

e Limit of quantification 

luted to volume with methanol. Aliquots of the tablet solution 

(prepared in methanol) were diluted with 0.1 M sodium 

hydroxide solution to obtain final concentrations 

within the ranges mentioned in Table (1) and then treated as 

under the procedures for derivative and derivative-ratio 

methods. 

hydroxide solution are shown in Fig. 1. They exhibited 

broad bands of UV absorption with maxima at 365, 272 and 

RESULTS AND DISCUSSION 

Both acidic (0.1 M hydrochloric acid solution) and alkaline 

(0.1 M sodium hydroxide solution) media were tried 

for the resolution of the ternary mixture and the best results 

for analytical purposes (best zero crossings resolving the 

mixture) were obtained using 0.1 M sodium hydroxide solution 

as a medium for both derivative and derivative ratio 

measurements. Solutions were stable in 0.1 M sodium hydroxide 

medium at ambient temperature for several hours 

and therefore the amplitudes of the peaks used for quantitative 

measurement remained unchanged. 

Derivative Method 

The absorption (zero-order) UV spectra of AMD, 

HCT and TIM over the range: 215-420 nm in 0.1 M sodium 

Fig. 1. Absorption spectra of 4 g/mL AMD (——), 

10 g/mL HCT (– – –) and 8 g/mL TIM (…) in 

0.1 M sodium hydroxide solution.


295 nm for AMD, HCT and TIM, respectively. 

Although being the minor component in this mixture, 

AMD could be easily determined because of its peak at 365 

nm at which the other two components show no interference 

(Fig. 1), therefore, conventional A max method was applied 

for the determination of AMD in this ternary mixture. 

To improve the sensitivity of AMD determination in the 

mixture, the measurement of the first ( 1 D) derivative signal 

at 385 nm ( = 6) was carried out where the other two 

components give zero readings. 

For the determination of HCT, the first ( 1 D) and the 

second ( 2 D) derivative orders did not solve the problem of 

the extensive spectral overlap, while the 3 D spectrum ( = 

6) enabled the determination of HCT at 265 nm where 

AMD and TIM exhibited no interference. Fig. 2 shows the 

3 D spectra of several serial concentrations of HCT. 

TIM suffered in this mixture from being the least absorbing 

component (A (1%,1 cm) = 645, 530 and 210 for 

AMD, HCT and TIM, respectively, at their maxima mentioned 

above) as well as from the extensive spectral overlap. 

Several trials were made in order to find an iso-differential 

point suitable for the selective determination of TIM, 

and it was successfully determined through its 1 D spectrum 

at 315.4 nm and = 8 nm (zero crossing for both AMD 

and HCT). 

Fig. 2. Third derivative spectra ( = 6 nm) of 4, 8, 12, 

16 and 20 g/mL HCT (——), 2 g/mL AMD 

(…) and 8 g/mL TIM (– – –) in 0.1 M sodium 

hydroxide solution. 

Derivative Ratio Method 

The absorption spectra of AMD, HCT and TIM in 0.1 

M sodium hydroxide solution are strongly overlapped over 

the range of 215-350 nm (Fig. 1). This spectral overlapping 

was sufficient enough to demonstrate the resolving power 

of the derivative ratio technique. This approach represents 

the integration and simultaneous use of the traditional zerocrossing 

technique and the Salinas method. 5 One of the advantages 

of using higher derivatives is the possibility of selecting 

more suitable wavelengths from a larger range of 

possibilities; therefore, both the 1 DD and 2 DD modes were 

applied for the resolution of this mixture. 

The influence of the different parameters was studied 

to optimize the signal of the derivative ratio spectra; i.e., 

to give good selectivity and higher sensitivity in the determination. 

Medium scan speed (200 nm/min.) was chosen 

throughout the work. 

A study was carried out to test for the effect of the divisor 

concentration on the calibration graphs. The divisor 

concentration can be modified and different calibration 

graphs are then obtained. When the concentration of divisor 

is increased or decreased, the resulting derivative values 

are proportionately decreased or increased, respectively, 

although the maxima and minima remain at the same 

wavelengths. For all subsequent measurements, spectra of 

2 g/mL AMD, 3 g/mL HCT and 4 g/mL TIM were used 

as standard divisors. These assured the best compromise in 

terms of sensitivity, repeatability and signal to noise ratio. 

The influence of for plotting the first and second 

derivatives of the ratio spectra was tested to obtain the optimum 

wavelength interval. The value affects both shape 

and position of peaks of the analyzed compound as well as 

the position of the zero-crossing points of the other compound 

in the mixture. In some cases, this effect is dramatic 

in that only a single value can be successful while others 

give poor resolution; therefore, we can say that the effect 

of divisor concentration is mainly a matter of sensitivity, 

while the effect of is mainly a matter of resolution. In 

the mixture under analysis, = 6 nm was the optimum for 

all determinations except for the measurement of AMD at 

1 DD 299.4 where = 4 nm was applied. 

For each compound, 2 points can be successfully 

used for its specific determination where the other compounds 

show no interference. The ratio spectra of different 

AMD standards at increasing concentrations in 0.1 M sodium 

hydroxide solution were obtained by dividing each 

with the spectrum of 3 g/mL HCT in the same solvent. The


first ( = 4) derivative was plotted from the smoothed ratio 

spectra. As illustrated in Fig. 3, AMD can be determined 

by measuring the 1 DD amplitude at 299.4 nm where there is 

no contribution from TIM. Similarly, the second ( =6) 

derivative was plotted from the smoothed ratio spectra and 

AMD can be determined by measuring the 2 DD amplitude 

at 311 nm. For the determination of HCT, a similar procedure 

was followed. The ratio spectra of different standards 

of HCT were obtained by dividing each with the spectrum 

of 4 g/mL TIM in sodium hydroxide solution then smoothing. 

The first derivative spectra didn’t provide suitable zerocrossings 

for HCT determination, while the second derivative 

( = 6) permitted its determination at 264.2 and 290 

nm where the corresponding values of AMD were equal to 

zero (Fig. 4). 

For the determination of TIM, two pathways could be 

followed. The first pathway involved measuring the first 

derivative amplitudes ( = 6) of the smoothed ratio spectra 

at 289.8 nm (zero-crossing point for HCT), using 2 g/ 

mL AMD in 0.1 M sodium hydroxide solution as divisor. 

The first derivative curves of TIM are illustrated in Fig. 5. 

The second pathway for the determination of TIM involved 

the use of 3 g/mL HCT in 0.1 M sodium hydroxide solution 

as divisor. The resultant ratio spectra were smoothed 

then differentiated with respect to wavelength at =6 

nm. The first derivative amplitude at 314.8 nm, which corresponds 

to a zero-crossing of AMD, is proportional to 

TIM concentration. 

Statistical Analysis of Results 

Concentration Ranges and Calibration Graphs 

A critical evaluation of the proposed derivative and 

derivative-ratio methods was performed by statistical anal- 

Fig. 4. Second derivative of ratio spectra ( =6nm) 

of 4, 8, 12 and 16 g/mL HCT and 3 g/mL 

AMD in 0.1 M sodium hydroxide solution. 

Fig. 3. First derivative of ratio spectra ( =4nm)of 

2, 4, 6 and 8 g/mL AMD and 5 g/mL TIM in 

0.1 M sodium hydroxide solution. 

Fig. 5. First derivative of ratio spectra ( =6nm)of 

4, 8, 12 and 16 g/mL TIM and 8 g/mL HCT 

in 0.1 M sodium hydroxide solution.


Table 2. Precision and accuracy for the determination of AMD/HCT/TIM synthetic mixtures by zero-crossing 

derivative spectrophotometry 

Nominal Value 

(X SD) a (g/mL) RSD (%) b E r (%) c 

(g/mL) AMD AMD AMD 

HCT TIM HCT TIM HCT TIM 

AMD HCT TIM A 1 365 D 1 385 A365 D 1 385 A365 D 385 

8 8 8 8.02 0.04 7.99 0.03 07.94 0.06 8.02 0.07 0.50 0.38 0.76 0.87 0.25 -0.13 -0.75 0.25 

2 20 8 —— 2.00 0.02 19.81 0.16 8.14 0.05 — 1.00 0.81 0.61 — 0.00 -0.95 1.75 

2 20 4 —— 2.01 0.02 19.73 0.13 4.08 0.03 — 1.00 0.66 0.74 — 0.50 -1.35 2.00 

8 4 4 7.99 0.04 8.03 0.04 03.96 0.05 3.99 0.03 0.50 0.50 1.26 0.75 -0.13 0.38 -1.00 -0.25 

4 4 8 4.00 0.03 3.99 0.02 04.05 0.02 8.02 0.08 0.75 0.50 0.49 1.00 0.00 -0.25 1.25 0.25 

a Mean SD for three determinations 

b % relative standard deviation 

c % relative error 

N.B. In some mixtures, AMD was determined using the 1 D mode only because its content was 2 g/mL which was 

lower than the linearity range of the A max method. 

Table 3. Precision and accuracy for the determination of AMD/HCT/TIM synthetic mixtures 

by derivative ratio spectrophotometry 

Nominal Value 

(g/mL) 

AMD HCT TIM 

(X SD) a for AMD 

(g/mL) 

RSD (%) b 

E r (%) c 

1 DD 299.4 2 DD 311 1 DD 299.4 2 DD 311 1 DD 299.4 2 DD 311 

8 8 8 7.97 0.04 8.02 0.06 0.50 0.75 -0.38- 0.25 

2 20 8 2.02 0.02 1.99 0.02 0.99 1.01 1.00 -0.50- 

2 20 4 1.99 0.02 1.99 0.02 1.01 1.01 -0.50- -0.50- 

8 4 4 8.02 0.07 7.90 0.05 0.87 0.63 0.25 -1.25- 

4 4 8 4.03 0.04 4.00 0.04 0.99 1.00 0.75 0.00 

Nominal Value 

(g/mL) 

AMD HCT TIM 

(X SD) a for HCT 

(g/mL) 

RSD (%) b 

E r (%) c 

2 DD 264.2 2 DD 290 2 DD 264.2 2 DD 290 2 DD 264.2 2 DD 290 

8 8 8 8.01 0.05 8.08 0.07 0.62 0.87 0.13 1.00 

2 20 8 19.93 0.13 19.81 0.14 0.65 0.71 -0.35- -0.95- 

2 20 4 20.01 0.14 19.93 0.11 0.70 0.55 0.05 -0.35- 

8 4 4 3.97 0.03 3.98 0.03 0.76 0.75 -0.75- -0.50- 

4 4 8 3.98 0.03 4.01 0.03 0.75 0.75 -0.50- 0.25 

Nominal Value 

(g/mL) 

AMD HCT TIM 

(X SD) a for TIM 

(g/mL) 

RSD (%) b 

E r (%) c 

1 DD 289.8 1 DD 314.8 1 DD 289.8 1 DD 314.8 1 DD 289.8 1 DD 314.8 

8 8 8 8.03 0.07 7.95 0.05 0.87 0.63 0.38 -0.63- 

2 20 8 7.94 0.07 8.01 0.07 0.88 0.87 -0.75- 0.13 

2 20 4 4.04 0.05 4.03 0.04 1.24 0.99 1.00 0.75 

8 4 4 3.96 0.03 3.95 0.04 0.76 1.01 -1.00- -1.25- 

4 4 8 7.97 0.07 8.00 0.06 0.88 0.75 -0.38- 0.00 

a Mean SD for three determinations 

b % relative standard deviation 

c % relative error


ysis of the experimental data. The linearity ranges and regression 

equations calculated with the methods described 

above are assembled in Table 1, together with other statistical 

data. The linearity of the calibration graphs and the adherence 

of the system to Beer’s law were validated by the 

high values of the correlation coefficients and the small intercepts. 

Other important statistical parameters such as the 

standard error of the estimate (S y/x ), the standard deviation 

of the slope (S b ) and the intercept (S a ) are also given in 

Table 1. 

Detection and Quantification Limits 

In accordance with the recommendations of ICH, 27 

the limit of detection, LOD = 3.3 /s, where is the standard 

deviation of the intercept of the regression line and s is 

the sensitivity, namely the slope of the calibration graph. 

On the other hand, the limit of quantification, LOQ, is defined 

as 10 /s. Detection and quantification limits of the 

three drugs using the proposed methods were calculated 

and are presented in Table 1. The LOD values were verified 

and they were found experimentally detectable. 

Precision and Accuracy 

The applicability of the developed methods was tested 

by the analysis of AMD, HCT and TIM in several synthetic 

mixtures of different proportions. Good accuracy, expressed 

as percentage relative error (E r %), and high precision, expressed 

as percentage relative standard deviation (RSD%), 

were obtained. The results, summarized in Tables 2 and 3, 

show that the E r (%) and RSD(%) values do not exceed 2% 

which demonstrates the accuracy and repeatability of the 

developed methods. 

Assay of Tablets 

Due to the unavailability of the commercial tablets in 

the Egyptian market, laboratory-made tablets were pre- 

Table 4. Assay of AMD, HCT and TIM in tablets using the proposed methods 

Derivative Ratio 

AMD A 1 365 D 385 1 DD 2 299.4 DD 311 

% Recovery SD* 99.68 1.44 100.13 1.01 99.60 1.67 100.79 1.39 

RSD (%) 1.45 1.01 1.68 1.38 

E r (%) -0.32 0.13 -0.40 0.79 

ANOVA (single factor): F = 0.767 (Critical F = 3.239) 

HCT 

3 D 265 


DD 2 264.2 DD 290 

% Recovery SD* 99.58 1.43 100.48 0.78 99.27 0.83 

RSD (%) 1.44 0.78 0.84 

E r (%) -0.42 0.48 -0.73 


TIM 

1 D 315.4 


DD 1 289.8 DD 314.8 

% Recovery SD* 101.30 0.76 99.75 0.97 100.93 1.16 

RSD (%) 0.75 0.97 1.15 

E r (%) 1.30 -0.25 0.93 


ANOVA (Two-factor with replication) 

Source of Variation SS df MS F P-value F (critical) 

Component 05.873 02 2.937 2.216 0.124 3.259 

Methods 01.542 02 0.771 0.582 0.564 3.259 

Interaction 12.503 04 3.126 2.358 0.072 2.634 

Within 47.716 36 1.325 

Total 67.634 44 

* Mean % recovery SD for five determinations.


pared and analyzed by the proposed derivative and derivative-ratio 

spectrophotometric methods. No interference 

from commonly encountered tablet excipients such as talc, 

starch, lactose and magnesium stearate was observed in the 

analyses. The results, shown in Table 4, agree with nominal 

content showing reasonable precision and accuracy. This is 

evident from the low RSD(%) and E r (%) values. For each 

component, the results obtained by the proposed methods 

were statistically compared using the single factor analysis 

of variance (One-way ANOVA) test. 28 The calculated F- 

value did not exceed the critical value for any of the three 

drugs, indicating that there is no significant difference between 

the proposed methods. 

Furthermore, the two factor (two-way) ANOVA test 28 

was applied for the overall comparison concerning the two 

factors: the analyzed component and the applied method, 

using the percentage recoveries of the analytes from tablets 

as the statistical population. As shown in Table 4, the obtained 

F-values did not exceed the critical values for either 

the component or the method factors. 

CONCLUSION 

Derivative and derivative-ratio spectrophotometry 

were successfully adopted for the simultaneous quantification 

of a ternary mixture of amiloride hydrochloride, hydrochlorothiazide 

and timolol maleate. Compared with the 

previously published chemometric methods, 24 the proposed 

methods are simple, direct and do not require any additional 

software programs. Also, they are accurate, precise 

and time saving and therefore they can be considered alternative 

tools for the routine analysis of commercial tablets 

containing the three drugs with minimum sample preparation. 

Received November 9, 2007. 

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