determination of risperidone in tablets in the presence of its
determination of risperidone in tablets in the presence of its
determination of risperidone in tablets in the presence of its
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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 66 No. 5 pp. 461ñ470, 2009 ISSN 0001-6837<br />
Polish Pharmaceutical Society<br />
ANALYSIS<br />
DETERMINATION OF RISPERIDONE IN TABLETS IN THE PRESENCE OF<br />
ITS DEGRADATION PRODUCTS AND PLACEBO-DERIVED CONSTITUENTS<br />
ANNA MAåLANKA*, JAN KRZEK and ALEKSANDER PATRZA£EK<br />
Department <strong>of</strong> Inorganic and Analytical Chemistry, Jagiellonian University, Collegium Medicum,<br />
9 Medyczna St., 30-688 KrakÛw, Poland<br />
Abstract: New chromatographicñdensitometric assay was developed for identification and <strong>determ<strong>in</strong>ation</strong> <strong>of</strong><br />
<strong>risperidone</strong> <strong>in</strong> pharmaceutical formulations. Th<strong>in</strong>-layer chromatographic plates (TLC-F 254 ) as a stationary phase<br />
and n-butanolñacetic acidñwater (12:3:5 v/v/v) as a mobile phase were used for separation. Densitometric<br />
measurements were done for all constituents at λ = 280 nm. A decrease <strong>in</strong> stability <strong>of</strong> <strong>risperidone</strong> was observed<br />
<strong>in</strong> acidic, basic and antioxidant solutions. Degradation <strong>of</strong> active pharmaceutical <strong>in</strong>gredient was consistent with<br />
first-order k<strong>in</strong>etics and unrelated to <strong>the</strong> solution. This assay is specific for <strong>risperidone</strong>. No <strong>in</strong>terference <strong>of</strong> tablet<br />
orig<strong>in</strong> adjuvants and degradation products were observed. Moreover, high sensitivity, limit <strong>of</strong> detection (0.22<br />
µg/spot), limit <strong>of</strong> quantitation (0.67 µg/spot), recovery (98.2ñ100.82%), broad l<strong>in</strong>ear range (0.09 µg/spot to<br />
1.40 µg/spot) and accuracy (1.87% RSD) are characteristic tra<strong>its</strong> <strong>of</strong> <strong>the</strong> chromatographicñdensitometric assay.<br />
Keywords: pharmaceutical preparation analysis, TLC, densitometry, <strong>risperidone</strong><br />
Risperidone represents atypical neuroleptics<br />
and is classified on <strong>the</strong> basis <strong>of</strong> chemical constitution<br />
among neuroleptics that are derivatives <strong>of</strong> various<br />
heterocyclic systems with cyclic structure.<br />
Risperidone is a tetrahydropyrido[1,2-a]-pyrimid<strong>in</strong>-<br />
4-one derivative with 6-fluoro-3-(4-piperid<strong>in</strong>yl)-<br />
1,2-benzisoxazole group, as shown below (1):<br />
Risperidone ñ 4-[2-[4-(6-fluorobenzo[d]isoxazol-3-<br />
yl)-1-piperidyl]ethyl]-3-methyl-2,6-diazabicyclo[4.4.0]deca-1,3-dien-5-one<br />
Absorption assay by <strong>in</strong>frared spectrophotometry and<br />
liquid chromatography (LC) are recommended for<br />
estimation <strong>of</strong> <strong>the</strong> quality <strong>of</strong> <strong>risperidone</strong> by <strong>determ<strong>in</strong>ation</strong><br />
<strong>of</strong> substance accord<strong>in</strong>g to Polish<br />
Pharmacopoeia VII. High-performance liquid chromatography<br />
(HPLC) is used for purity analysis.<br />
However, a method <strong>of</strong> chloric(VII) acid titration<br />
with potentiometric <strong>determ<strong>in</strong>ation</strong> <strong>of</strong> end-po<strong>in</strong>t is<br />
used for quantitative analysis (2).<br />
Chromatographic methods, ma<strong>in</strong>ly LC and<br />
HPLC, have significant role <strong>in</strong> <strong>determ<strong>in</strong>ation</strong> <strong>of</strong><br />
<strong>risperidone</strong> and <strong>its</strong> impurities. HPLC, liquid chromatography<br />
coupled with mass spectrometry<br />
(LC/MS) and nuclear magnetic resonance (NMR)<br />
were used for identification and structural analyses<br />
<strong>of</strong> impurities and degradation products <strong>of</strong> <strong>risperidone</strong><br />
(3-5). Determ<strong>in</strong>ations <strong>of</strong> <strong>risperidone</strong> and 9-<br />
hydroxy<strong>risperidone</strong>, an active metabolite, <strong>in</strong> biological<br />
material were accomplished us<strong>in</strong>g LC/MS<br />
method (6ñ15). Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> and <strong>its</strong><br />
degradation products <strong>in</strong> <strong>tablets</strong> (16, 17), and biologic<br />
material (18ñ23) were performed us<strong>in</strong>g HPLC<br />
method coupled with mass spectrometry or spectrophotometric<br />
detection. TLC 60-F 254 plates and<br />
acetonitrileñmethanolñ1-propanolñtriethanolam<strong>in</strong>e<br />
(8.5:1.2:0.6:0.2 v/v/v/v) mobile phase were used for<br />
separation <strong>of</strong> degradation products <strong>of</strong> <strong>risperidone</strong>.<br />
Densitometric measurements were used for detection<br />
under UV light at λ = 280 nm (17). Capillary<br />
electrophoresis (CE) was also used as a separation<br />
method for <strong>determ<strong>in</strong>ation</strong> <strong>of</strong> <strong>risperidone</strong> (24, 25).<br />
Polarography (26), chemilum<strong>in</strong>escence (27) and<br />
colorimetry (28) were used amongst o<strong>the</strong>r analytical<br />
* Correspond<strong>in</strong>g author: e-mail: amasl@cm-uj.krakow.pl<br />
461
462 ANNA MAåLANKA et al.<br />
methods for <strong>the</strong> <strong>determ<strong>in</strong>ation</strong> <strong>of</strong> <strong>risperidone</strong> <strong>in</strong><br />
<strong>tablets</strong> and biological material.<br />
This research paper deals with <strong>the</strong> development<br />
<strong>of</strong> new chromatographicñdensitometric assay<br />
for identification and <strong>determ<strong>in</strong>ation</strong> <strong>of</strong> <strong>risperidone</strong> <strong>in</strong><br />
<strong>tablets</strong> and <strong>its</strong> degradation products that can occur <strong>in</strong><br />
acidic and basic solutions, as well as <strong>in</strong> <strong>the</strong> <strong>presence</strong><br />
<strong>of</strong> oxidiz<strong>in</strong>g or reduc<strong>in</strong>g agents.<br />
EXPERIMENTAL<br />
Apparatus<br />
(a) TLC ñ Densitometer ñ Scanner 3 equipped<br />
<strong>in</strong> Cats 4.0x s<strong>of</strong>tware, L<strong>in</strong>omat IV (Camag, Muttenz,<br />
Switzerland); (b) TLC alum<strong>in</strong>um plates covered by<br />
silica gel (20 ◊ 20 cm, Merck, Germany) that were cut<br />
<strong>in</strong>to 10 ◊ 10 cm plates; (c) Chromatographic chamber<br />
18 ◊ 9 ◊ 18 cm (Sigma Aldrich); (d) Balance scale,<br />
WPA 60/C model, 0.1 mg accuracy (Radwag,<br />
Poland); (e) Bench centrifuge, MPW-223e model<br />
(MPW Med. Instruments, Poland).<br />
Reagents<br />
Methanol (POCH, Gliwice, Poland), 1-butanol<br />
(Chempur, Piekary ålπskie, Poland), acetic acid<br />
(Chempur, Piekary ålπskie, Poland), all analytical<br />
grade.<br />
Reference substances and formulations<br />
A substance that meets requirements <strong>of</strong> Polish<br />
Pharmacopoeia VI and European Pharmacopoeia<br />
Monographs was used dur<strong>in</strong>g studies: Risperidone<br />
CRS ñ LGC Promochem Sp.z o.o, Poland; coated<br />
<strong>tablets</strong> <strong>of</strong> Risperdal (4 mg) manufactured by<br />
Janssen-Cilag, France, placebo: lactose, corn starch,<br />
microcrystall<strong>in</strong>e cellulose, hydroxypropyl methyl<br />
cellulose, magnesium stearate, anhydrous colloidal<br />
silica, sodium lauryl sulfate.<br />
Reference solutions<br />
Amount <strong>of</strong> 5.6 mg <strong>of</strong> comparative substance<br />
was dissolved <strong>in</strong> 5.0 mL <strong>of</strong> methanol and mixed.<br />
Solutions at <strong>the</strong> follow<strong>in</strong>g concentrations: 0.560<br />
mg/mL, 0.280 mg/mL, 0.140 mg/mL, 0.070 mg/mL,<br />
0.035 mg/mL and 0.0175 mg/mL were obta<strong>in</strong>ed by<br />
dilution <strong>of</strong> stock solution with methanol.<br />
Solutions for stability analysis<br />
Coated <strong>tablets</strong> <strong>of</strong> Risperdal formulation were<br />
used after remov<strong>in</strong>g a coat<strong>in</strong>g by water wett<strong>in</strong>g.<br />
Powdered tablet mass correspond<strong>in</strong>g to 32.0 mg <strong>of</strong><br />
<strong>risperidone</strong> was extracted with 16.0 mL <strong>of</strong> methanol<br />
followed by shak<strong>in</strong>g for 15 m<strong>in</strong>. Suspension was<br />
centrifuged at 3000 rpm and supernatant was collected.<br />
Each time, <strong>the</strong> volume <strong>of</strong> 1.5 mL <strong>of</strong> solution<br />
was evaporated to dryness.<br />
Acidic solution<br />
A volume <strong>of</strong> 4 mL 0.5 mol/L HCl was added to<br />
dried residue. The solution was poured <strong>in</strong>to 0.5-mL<br />
glass vials. The solutions were heated at 100 O C,<br />
simultaneously samples for fur<strong>the</strong>r studies were collected<br />
after 1, 2, 3 and 5 h. After cool<strong>in</strong>g down, <strong>the</strong><br />
collected solutions were diluted immediately with<br />
methanol at a ratio <strong>of</strong> 1:1.<br />
Basic solution<br />
Volumes <strong>of</strong> 1 mL methanol and 3 mL <strong>of</strong> 0.5<br />
mol/L NaOH were added to dried residue. Dissolved<br />
solution was poured <strong>in</strong>to 0.5-ml glass vials.<br />
Solutions were heated at 100 O C and simultaneously<br />
samples for fur<strong>the</strong>r studies were collected after 1, 2,<br />
3 and 5 h. After cool<strong>in</strong>g down, <strong>the</strong> solutions were<br />
diluted immediately with methanol at a ratio <strong>of</strong> 1:1.<br />
Oxidant solution<br />
A volume <strong>of</strong> 4-mL 0.9 mol/L H 2 O 2 ñmethanol<br />
solution was added to <strong>the</strong> dried residue. The solution<br />
was left at room temperature (RT). Samples for fur<strong>the</strong>r<br />
studies were collected after 0.5, 1, 2 and 5 h.<br />
Reductant solution<br />
A volume <strong>of</strong> 4-mL waterñmethanol 0.1 mol/L<br />
Na 2 S 2 O 3 solution was added to dried sample. The<br />
solution was left at RT. Samples for fur<strong>the</strong>r studies<br />
were collected after 1, 2, 7 h and 14 days.<br />
Pharmaceutical agent solution for <strong>risperidone</strong> <strong>determ<strong>in</strong>ation</strong><br />
Powdered tablet mass correspond<strong>in</strong>g to 4.0 mg<br />
<strong>of</strong> <strong>risperidone</strong> was extracted with 4.0 mL <strong>of</strong><br />
methanol, shaken for 15 m<strong>in</strong> and centrifuged.<br />
Supernatant was diluted 10 times with methanol.<br />
Chromatographic analysis<br />
Volumes <strong>of</strong> 5 mL <strong>of</strong> reference solution and<br />
degradation solutions were applied to chromatographic<br />
plates (TLC, 10 ◊ 10 cm) us<strong>in</strong>g an applicator.<br />
Chromatograms were developed as far as 9.5<br />
cm, us<strong>in</strong>g mobile phase: n-butanolñacetic<br />
acidñwater (12:3:5 v/v/v), dried at RT and recorded<br />
under UV light. Densitometric record<strong>in</strong>g was done<br />
at λ = 280 nm. This wavelength was chosen on <strong>the</strong><br />
basis <strong>of</strong> directly recorded spectrum on chromatogram.<br />
Retardation factor value (R F ~ 0.49) and<br />
UV absorption spectrum recorded directly from<br />
chromatogram were used for identification <strong>of</strong><br />
<strong>risperidone</strong>.
Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> <strong>tablets</strong> <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong>... 463<br />
Table 1. Parameters <strong>of</strong> separation.<br />
Constitues TLC plates HPTLC plates<br />
(1) : (2) R F(1) R F(2) α R s R F(1) R F(2) α R s<br />
Risperidone : peak 2<br />
solution HCl<br />
~0.49 ~0.79 4.00 4.21 ~0.53 ~0.78 3.18 4.09<br />
peak 2 : Risperidone<br />
solution NaOH<br />
~0.43 ~0.48 1.22 0.70 ~0.44 ~0.51 1.32 0.91<br />
Risperidone : peak 4<br />
solution NaOH<br />
~0.48 ~0.72 2.77 3.43 ~0.51 ~0.70 2.23 3.22<br />
Risperidone : peak 2<br />
solution H 2 O 2<br />
~0.48 ~0.50 1.08 0.63 ~0.53 ~0.56 1.13 0.60<br />
R F ñ retardation factor, a ñ separation factor, R s ñ resolution factor<br />
R s ñ resolution factor, R s = 2 ◊ (distance between <strong>the</strong> centers <strong>of</strong> two adjacent spots)/(sum <strong>of</strong> <strong>the</strong> widths <strong>of</strong> <strong>the</strong> two spots <strong>in</strong> <strong>the</strong> direction<br />
<strong>of</strong> development)<br />
Table 2. Percentage content <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong> degradation products <strong>in</strong> solutions: NaOH, HCl, H 2 O 2 and Na 2 S 2 O 3 . K<strong>in</strong>etics<br />
parameters <strong>of</strong> degradation reaction <strong>in</strong> <strong>in</strong>dividual solutions.<br />
0.5 mol/L NaOH 0.5 mol/L HCl 0.9 mol/L H 2 O 2 0.1 mol/L Na 2 S 2 O 3<br />
Content <strong>of</strong> Content <strong>of</strong> Content <strong>of</strong> Content <strong>of</strong><br />
Time <strong>risperidone</strong> Time <strong>risperidone</strong> Time <strong>risperidone</strong> Time <strong>risperidone</strong><br />
[%] [%] [%] [%]<br />
1 h 0.073 1 h 0.074 0.5 h 0.031 1 h 0.150<br />
2 h 0.066 2 h 0.072 1 h 0.028 2 days 0.150<br />
3 h 0.062 3 h 0.069 2 h 0.027 7 days 0.150<br />
5 h 0.055 5 h 0.065 5 h 0.020 14 days 0.150<br />
k = 0.0692 h -1 ± 0.0267 h -1 k = 0.0334 h -1 ± 0.0062 h -1 k = 0.0926 h -1 ± 0.0396 h -1<br />
t 0.1 = 1.52 h t 0.1 = 3.15 h t 0.1 = 1.37 h ó-<br />
t 0.5 = 10.01 h t 0.5 = 20.75 h t 0.5 = 7.48 h<br />
k ñ reaction rate constant, t 0.1 ñ degradation time <strong>of</strong> 10% substrate, t 0.5 ñ degradation time <strong>of</strong> 50% substrate<br />
K<strong>in</strong>etics estimation<br />
Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> was done by <strong>the</strong><br />
use <strong>of</strong> k<strong>in</strong>etics parameters (29).<br />
Assay validation<br />
Assay validation was done by <strong>determ<strong>in</strong>ation</strong> <strong>of</strong><br />
specificity, accuracy, l<strong>in</strong>earity, limit <strong>of</strong> detection<br />
and quantification, stability, and robustness (30).<br />
Specificity<br />
Assay specificity was determ<strong>in</strong>ed by compar<strong>in</strong>g<br />
peak areas, R F values and absorption spectra recorded<br />
for reference solution <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong> placebo.<br />
Model mixtures conta<strong>in</strong><strong>in</strong>g lactose, starch corn,<br />
microcrystall<strong>in</strong>e cellulose, hydroxypropyl methyl<br />
cellulose, magnesium stearate, anhydrous colloidal<br />
silica and sodium lauryl sulfate were prepared (Fig.<br />
1). Placebo was added to <strong>the</strong>se model mixtures.<br />
Accuracy<br />
Assay accuracy was determ<strong>in</strong>ed by estimat<strong>in</strong>g<br />
recovery percentage (%E) for chosen constituents.<br />
Known amount <strong>of</strong> reference substances (80% to 120%<br />
declared content) were added to model solutions already<br />
conta<strong>in</strong><strong>in</strong>g placebo constituents. Recovery <strong>in</strong> percentage<br />
value was calculated on <strong>the</strong> basis <strong>of</strong> determ<strong>in</strong>ed content<br />
<strong>of</strong> <strong>in</strong>dividual constituents to weighed amount (Table 3).<br />
Precision<br />
Consistency <strong>of</strong> experimentally determ<strong>in</strong>ed results<br />
was checked on <strong>the</strong> model solutions prepared by dissolv<strong>in</strong>g<br />
<strong>the</strong> substance <strong>in</strong> methanol. Five <strong>determ<strong>in</strong>ation</strong>s<br />
were done for each model solution (Table 3).<br />
Indirect precision<br />
Determ<strong>in</strong>ation was done for <strong>the</strong> model solutions<br />
by two analytical chemists <strong>in</strong> two different<br />
days. In each case, five <strong>determ<strong>in</strong>ation</strong>s were done<br />
for each model solution (Table 3).<br />
L<strong>in</strong>earity<br />
L<strong>in</strong>earity was determ<strong>in</strong>ed as <strong>the</strong> relationship <strong>of</strong><br />
peak areas (p [mm 2 ]) to mass [mg/spot]. Five meas-
464 ANNA MAåLANKA et al.<br />
Figure 1. Densitogram <strong>of</strong> methanol solution <strong>of</strong> placebo constituents <strong>of</strong> pharmaceutical formulation after extraction <strong>of</strong> 1.0 g <strong>of</strong> powdered<br />
mass with 10.0 mL <strong>of</strong> methanol.<br />
Fig. 2. Densitogram <strong>of</strong> methanol extract <strong>of</strong> <strong>risperidone</strong> from pharmaceutical formulation.<br />
urements were done for concentrations 0.018<br />
mg/mL to 0.280 mg/mL, apply<strong>in</strong>g 5.0 mL <strong>of</strong> each<br />
solution. The measurement <strong>of</strong> l<strong>in</strong>earity <strong>of</strong> <strong>risperidone</strong><br />
was shown as a straight l<strong>in</strong>e equation with <strong>the</strong><br />
content <strong>of</strong> correlation coefficient (r): p = 4134.3 ◊ m<br />
+ 742.9, r = 0.99657.<br />
Limit <strong>of</strong> detection (LOD) and limit <strong>of</strong> quantitation<br />
(LOQ)<br />
Us<strong>in</strong>g standard deviation and slope <strong>of</strong> a<br />
straight l<strong>in</strong>e coefficient, <strong>the</strong> values <strong>of</strong> LOD and LOQ<br />
were determ<strong>in</strong>ed us<strong>in</strong>g <strong>the</strong> follow<strong>in</strong>g equations:<br />
LOD = 3.3 ◊ S y /a, LOQ = 10 ◊ S y /a, where: S y ñ<br />
error <strong>of</strong> estimation, a ñ slope <strong>of</strong> a straight l<strong>in</strong>e coefficient<br />
(Table 3).<br />
Stability<br />
Methanol extract was prepared from<br />
Risperdal <strong>tablets</strong> at concentration 1.0 mg/mL <strong>of</strong><br />
<strong>risperidone</strong>. The methanol extract was stored at RT<br />
and also at 5 O C for 7 days. Determ<strong>in</strong>ation <strong>of</strong> <strong>the</strong><br />
content was done ex tempore (without pretreat-
Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> <strong>tablets</strong> <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong>... 465<br />
Fig. 3. Absorption spectrum <strong>of</strong> <strong>risperidone</strong> recorded directly from chromatogram<br />
Fig. 4. Densitogram <strong>of</strong> <strong>risperidone</strong> (peak 1) and <strong>its</strong> degradation product (peak 2) <strong>in</strong> acidic solution.<br />
ment) and after 24 h, 48 h and a whole week<br />
aga<strong>in</strong>st reference solutions prepared directly before<br />
<strong>determ<strong>in</strong>ation</strong> (Table 4).<br />
Robustness<br />
The impact <strong>of</strong> replacement <strong>of</strong> TLC 60-F 254<br />
plates with HPTLC-F 254 plates covered by silica 60<br />
was studied. Volume <strong>of</strong> 5 µL <strong>of</strong> each solution after<br />
hydrolysis <strong>in</strong> HCl and NaOH solutions were<br />
applied on both types <strong>of</strong> plates. Then, <strong>the</strong> plates<br />
have undergone chromatographic analysis.<br />
Separation and resolution factors were calculated<br />
for two adjacent peaks on <strong>the</strong> basis <strong>of</strong> developed<br />
densitograms (Table 1).<br />
Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> pharmaceutical<br />
formulation<br />
Usability <strong>of</strong> this assay was proved on pharmaceutical<br />
formulation. The results are presented <strong>in</strong><br />
Table 2.
466 ANNA MAåLANKA et al.<br />
Fig. 5. Densitogram <strong>of</strong> <strong>risperidone</strong> (peak 3) and <strong>its</strong> degradation product (peak: 1, 2 and 4) <strong>in</strong> basic solution.<br />
Fig. 6. Densitogram <strong>of</strong> <strong>risperidone</strong> (peak 1) and <strong>its</strong> degradation product (peak: 2) <strong>in</strong> hydrogen peroxide solution.<br />
RESULTS AND DISCUSSION<br />
Accord<strong>in</strong>g to assumption made <strong>in</strong> this paper,<br />
new chromatographicñdensitometric assay allows<br />
for simultaneous identification and quantity analysis<br />
<strong>of</strong> <strong>risperidone</strong> <strong>in</strong> pharmaceutical formulations <strong>in</strong> <strong>the</strong><br />
<strong>presence</strong> <strong>of</strong> degradation products occurr<strong>in</strong>g <strong>in</strong><br />
acidic, basic and hydrogen peroxide solutions.<br />
Good results <strong>of</strong> chromatographic separation<br />
were obta<strong>in</strong>ed by us<strong>in</strong>g n-butanolñacetic acidñwater<br />
(12:3:5 v/v/v) mobile phase and TLC-F 254 plates<br />
covered with silica gel 60. Peak <strong>of</strong> <strong>risperidone</strong> is<br />
symmetrical and well-shaped under develop<strong>in</strong>g conditions.<br />
It allows for easy identification and quality<br />
analysis (Fig. 1, 2).<br />
Characteristic absorption spectrum recorded<br />
directly from chromatograms can be useful for identity<br />
studies. Moreover, this spectrum allows to<br />
choose analytical wavelength at λ = 280 nm (Fig. 3).<br />
Risperidone-orig<strong>in</strong> peaks (R F ~ 0.49) always<br />
occur close to degradation products on recorded<br />
chromatograms for studied and comparative solutions
Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> <strong>tablets</strong> <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong>... 467<br />
Fig. 7. Densitogram <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> sodium thiosulfate(VI) solution.<br />
Fig. 8. Logarithmic dependence <strong>of</strong> <strong>risperidone</strong> concentration to degradation time <strong>in</strong> HCl, NaOH and hydrogen peroxide solutions.<br />
Regressionequations: 1: log c = 0.0145 ◊ t ñ 1.1178, <strong>in</strong> acidic solution; 2: log c = 0.0297 ◊ t ñ 1.1129, <strong>in</strong> basic solution; 3: log c = 0.0402<br />
◊ t ñ 1.4968, <strong>in</strong> hydrogen peroxide solution.<br />
<strong>in</strong> established conditions. It could be assumed that<br />
this assay is specific aga<strong>in</strong>st tested constituent and it<br />
guarantees that <strong>the</strong> obta<strong>in</strong>ed results are reliable.<br />
Exemplary densitograms <strong>of</strong> degradation products<br />
<strong>of</strong> <strong>risperidone</strong> are presented <strong>in</strong> Figures 4ñ7. The<br />
peaks <strong>of</strong> products degradation orig<strong>in</strong> that differ with<br />
retention coefficient values, which depend on solution<br />
<strong>of</strong> <strong>the</strong>ir orig<strong>in</strong>, are observed on chromatographic<br />
densitograms <strong>in</strong> acidic, basic and hydrogen peroxide<br />
solutions.<br />
Separation <strong>of</strong> <strong>risperidone</strong> from <strong>its</strong> degradation<br />
products was satisfactory under studied conditions<br />
(Table 1). No significant differences were observed<br />
when stationary phase was changed by replacement<br />
<strong>of</strong> TLC with HPTLC plates.<br />
The <strong>presence</strong> <strong>of</strong> one degradation product <strong>of</strong><br />
<strong>risperidone</strong> <strong>in</strong> a solution <strong>of</strong> 0.5-mol/L acetic acid (R F<br />
~ 0.79) was observed toge<strong>the</strong>r with <strong>the</strong> peak <strong>of</strong><br />
tested constituent (R F ~ 0.49) under develop<strong>in</strong>g<br />
conditions (Fig. 4). Three degradation products
468 ANNA MAåLANKA et al.<br />
Table 3. Assay validation parameters.<br />
Validation parameters Value <strong>of</strong> validation parameter Statistical estimation<br />
LOD<br />
[mg/spot]<br />
0.222 ó-<br />
LOQ<br />
[mg/spot]<br />
0.672 ó-<br />
95,96 x ñ = 98.27<br />
Recovery 97,81 S x = 2.332<br />
80 % 101,29 t 95% = ±2.89<br />
[%] 96,27 RSD = 2.37<br />
100,03<br />
101,89<br />
Recovery 99,76 x ñ = 99.60<br />
100 % 98,40 S x = 1.761<br />
[%] 97,41 t 95% = ±2.18<br />
100,55 RSD = 1.76<br />
101,63<br />
Recovery 100,58 x ñ = 100.82<br />
120 % 101,30 S x = 1.162<br />
[%] 101,70 t 95% = ±1.44<br />
98,90 RSD = 0.01<br />
4,10<br />
4,02 x ñ = 4.00<br />
Precision 3,92 S x = 0.075<br />
[mg/4 mL] 3,93 t 95% = ±0.093<br />
4,03 RSD = 1.87<br />
3,97<br />
Indirect precision 3,91 x ñ = 4.01<br />
[mg/4 mL] 3,96 S x = 0.100<br />
Analytical chemist I 4,11 t 95% = ±0.125<br />
4,14 RSD = 2.50<br />
3,91<br />
Indirect precision 4,12 x ñ = 4.01<br />
[mg/4 mL] 4,08 S x = 0.103<br />
Analytical chemist I 3,90 t 95% = ±0.128<br />
4,07 RSD = 2.56<br />
Regression equation p = 3729.9 ◊ m + 212.1<br />
Correlation coefficient r = 0.99411 ó-<br />
R F ñ retardation factor; x ñ ñ mean value; S x ñ standard deviation, t 95% ñ confidence <strong>in</strong>terval for 95% probability; RSD ñ relative standard<br />
deviation; p ñ peak surface area [mm 2 ]; m ñ mass [mg/spot]; r ñ correlation coefficient<br />
(R F ~ 0.40, R F ~ 0.48 and R F ~ 0.72) were observed<br />
<strong>in</strong> a solution <strong>of</strong> 0.5 mol/L sodium hydroxide (Fig. 5).<br />
Degradation product with R F ~ 0.50 was detected <strong>in</strong><br />
<strong>the</strong> <strong>presence</strong> <strong>of</strong> hydrogen peroxide (Fig. 6). No<br />
<strong>in</strong>fluence <strong>of</strong> sodium thiosulfate(VI) was observed on<br />
robustness <strong>of</strong> <strong>risperidone</strong> (Fig. 7).<br />
In conclusion, <strong>the</strong> solution, where <strong>the</strong> stability<br />
<strong>of</strong> <strong>risperidone</strong> was studied, has an impact on type<br />
and number <strong>of</strong> degradation products that differ with<br />
<strong>the</strong> values <strong>of</strong> retardation factor. It has to be highlighted<br />
that <strong>the</strong> impact <strong>of</strong> degradation process on<br />
method specificity was not observed.<br />
Interest<strong>in</strong>g conclusions can be made on <strong>the</strong><br />
basis <strong>of</strong> concentrations change <strong>of</strong> <strong>risperidone</strong> to time<br />
<strong>of</strong> measurement. Degradation process is <strong>in</strong>dependent<br />
on conditions but is consistent with k<strong>in</strong>etics <strong>of</strong><br />
<strong>the</strong> first-order reaction (Fig. 8).<br />
Reaction rate constant (k) demonstrates various<br />
values <strong>in</strong> different solutions. Degradation is <strong>the</strong><br />
fastest <strong>in</strong> hydrogen peroxide solution (k = 0.0926 h -1 ),<br />
followed by basic solution (k = 0.0692 h -1 ) and <strong>the</strong><br />
slowest <strong>in</strong> acidic solution (k = 0.0334 h -1 ).<br />
Risperidone ma<strong>in</strong>ta<strong>in</strong>s <strong>its</strong> stability dur<strong>in</strong>g 14 days <strong>of</strong><br />
storage <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong> reduc<strong>in</strong>g agent.
Determ<strong>in</strong>ation <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> <strong>tablets</strong> <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong>... 469<br />
Table 4. Stability <strong>of</strong> methanol extract <strong>of</strong> <strong>risperidone</strong> from <strong>tablets</strong>.<br />
Content <strong>of</strong> stored solution at 23 O C [µg]<br />
Content <strong>of</strong> stored solution at 5 O C [µg]<br />
Ex tempore After 1 day After 2 days After 7 days After 1 day After 2 days After 7 days<br />
1.10 1.15 1.20 1.17 1.15 1.15 1.13<br />
Table 5. Determ<strong>in</strong>ation results <strong>of</strong> <strong>the</strong> content <strong>of</strong> <strong>risperidone</strong> <strong>in</strong> <strong>the</strong> pharmaceutical formulation studied.<br />
Formulation Determ<strong>in</strong>ed content Statistical<br />
(declared content) [mg/tablet] estimation<br />
4.10 x ñ = 4.00<br />
Risperdal 4.02 S x = 0.075<br />
3.92 t 0,95 = ±0.093<br />
(4 mg/tablet) 3.93 RSD = 1.87%<br />
4.03 E = 0.00%<br />
x ñ ñ mean value; S x ñ standard deviation, t 95% ñ confidence <strong>in</strong>terval for 95% probability; RSD ñ relative standard deviation; E ñ relative<br />
error<br />
A suggestion can be made on <strong>the</strong> basis <strong>of</strong> <strong>the</strong><br />
calculated values t 0.1 and t 0.5 that <strong>the</strong> smallest impact<br />
on stability <strong>of</strong> <strong>risperidone</strong> has <strong>the</strong> reduc<strong>in</strong>g agent and<br />
<strong>the</strong> highest one has hydrogen peroxide (Table 2).<br />
This new chromatographicñdensitometric<br />
method has high sensitivity (LOD = 0.222 mg, LOQ<br />
= 0.672 µg). A recovery <strong>of</strong> tested substance was<br />
98.27%, 99.60% and 100.82% for <strong>the</strong> levels <strong>of</strong>:<br />
80%, 100% and 120%, respectively. L<strong>in</strong>earity range<br />
was with<strong>in</strong> 0.087 mg/spot to 1.400 µg/spot. This<br />
method is very accurate (RSD = 1.87%). No impact<br />
on <strong>the</strong> results and values was observed dur<strong>in</strong>g test<strong>in</strong>g<br />
this method by two different analytical chemists<br />
(RSD = 2.50% and 2.56%) (Table 3).<br />
Methanol extracts <strong>of</strong> <strong>risperidone</strong> made from<br />
pharmaceutical formulation have ma<strong>in</strong>ta<strong>in</strong>ed <strong>the</strong>ir<br />
stability for 7 days dur<strong>in</strong>g storage <strong>in</strong> 5 O C or RT<br />
(Table 4).<br />
This assay is also specific aga<strong>in</strong>st placebo constituents,<br />
as well as degradation products (Fig. 1).<br />
Quantity analysis <strong>of</strong> <strong>risperidone</strong> was done <strong>in</strong> pharmaceutical<br />
formulation (Table 5). Developed assay<br />
has satisfactory accuracy and precision. The determ<strong>in</strong>ed<br />
content <strong>of</strong> pharmaceutical formulation was<br />
like declared. Relative error is 0.00%.<br />
CONCLUSION<br />
Developed chromatographicñdensitometric<br />
assay allows for fast and direct <strong>determ<strong>in</strong>ation</strong> <strong>of</strong><br />
<strong>risperidone</strong> <strong>in</strong> <strong>the</strong> <strong>presence</strong> <strong>of</strong> constituents from<br />
placebo formulation and degradation products.<br />
This assay is very sensitive and has broad l<strong>in</strong>earity<br />
range, good accuracy and precision. It can be<br />
used for quality evaluation <strong>of</strong> pharmaceutical formulations<br />
dur<strong>in</strong>g manufactur<strong>in</strong>g and distribution, as<br />
well as <strong>the</strong>ir stability.<br />
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Received: 23. 03. 2009