INTRODUCTION:

Risperidone is chemically, 3-[2-[4-(6-fluoro-1, 2-benzisoxozol-3-yl)-1 piperidinyl 1] ethyl]-6, 7, 8, 9,-tetrahydro-2-methyl-4H-pyrido [1, 2-a] pyrimidin-4-one¹. It is indicated for the treatment of acute and chronic schizophrenic psychoses, and other psychotic conditions². Trihexyphenidyl, 1-cyclohexyl-1-phenyl-3-(1-piperidyl) propane-1-ol, is an antiparkinson drug of the antimuscarinic class of agents and is chemically a tertiary amine³. The drug is available as the hydrochloride salt. Form the literature survey; it was found that there are many analytical methods reported for Risperidone and Trihexyphenidyl hydrochloride either individually by chemiluminescence⁴, HPLC⁵, Polarography⁶or in combination with other drugs by LC-MS/MS^7-10, and HPLC¹¹ methods.

However no method is reported for simultaneous estimation of these two drugs in tablet dosage form by HPLC. Hence the present work was attempted to develop accurate, simple and sensitive method for simultaneous estimation of Risperidone and Trihexyphenidyl hydrochloride in tablet dosage form.

MATERIALS AND METHODS:

Reagents and chemicals:

HPLC grade acetonitrile and methanol were procured from Merck Co., Mumbai. Ortho phosphoric acid and triethylamine AR grade were procured from SD fine chemicals. Water obtained from milli-Q RO water system. Risperidone and Trihexyphenidyl hydrochloride standards used in this study were gifted by Ranbaxy, Mumbai. The tablets used in this study were SIZODON PLUS (Sun pharmaceutical industries Ltd) labeled to contain 3mg of Risperidone and 2mg of Trihexyphenidyl hydrochloride.

Chromatographic conditions:

A Liquid chromatographic separation was performed on Shimadzu LC-20 AT isocratic Liquid chromatographic system, equipped with Rheodyne injector model 7725 with 20µL fixed loop. UV-Visible detector SPD-20A was employed and separation achieved on C₁₈ Phenomenex Gemini (250mm X4.6mm, i.d., 5µ) column using a mixture of 0.3% orthophosphoric acid buffer, acetonitrile and methanol (50:30:20% v/v/v) adjusted to pH 3with triethylamine as mobile phase. The elution was carried out at the flow rate of 1mL/minute. Detection was made at 220m at ambient temperature and obtained data were analyzed by Class-VP software.

Preparation of standard stock solution:

Accurately weighed 30mg of Risperidone and 20mg of Trihexyphenidyl hydrochloride were transferred into a

Table-1. Assay of tablets

Drug name	Label Claim mg/tab	MeanPeak Area		*Amount found ± SD (mg/tab)**	%Label claim ± SD
Drug name	Label Claim mg/tab	Standard	Sample	*Amount found ± SD (mg/tab)**	%Label claim ± SD
Risperidone	3mg	6448.46	6451.31	3.015±0.021	100.50 ±0.01
Trihexypenidyl hydrochloride	2mg	2118.98	2087.15	2.012±0.036	100.60±0.05

Table-2. System suitability parameters

S. No.	PARAMETERS	OBTAINED VALUES
S. No.	PARAMETERS	Riperidone	Trihexypenidyl hydrochloride
1.	Theoretical plates (N)	6521	2215
2.	Tailing factor (T)	1.351	1.201
3.	Retention time (min)	2.685	5.912
4.	% RSD of Peak Retention Time	0.306%	0.214%

Fig:1 Typical standard chromatogram of Riperidone and Trihexyphnidyl hydrochloride.

50mLvolumetric flask, diluted with little amount of mobile phase and the contents were shaken thoroughly to dissolve and finally make up the volume to 50mL with the mobile phase. 5mL of the above solution is transferred to a 25mL volumetric flask; dilute it to 25mL with mobile phase.

Preparation of working standard solution:

Working standard solution were prepared by diluting to 10mL of the stock solution to 100mL with mobile phase to achieve the concentration of 12.1µg/mL of Risperidone and 8.2 µg/mL of Trihexyphenidyl hydrochloride.

Estimation of drug in commercial tablet formulation:

For the estimation of drug in tablet formulation, twenty tablets were weighed and their average weight was determined. The tablets were then finely powdered. Accurately weighed tablet powder equivalent to 30mg of Risperidone and 20mg of Trihexyphenidyl was transferred to a 50mL volumetric flask. Little amount of mobile phase was added and the contents were shaken well to dissolve and sonicated for 15minutes. The volume was made up to 50mL with mobile phase and filtered through syringe membrane filter. 5mL of the above solution is taken and diluted to 25mL with mobile phase. Further this solution was diluted suitably to get the concentration of 12. µg/mL of Risperidone and 8.2 µg/mL of Trihexyphenidyl hydrochloride. Both the standard and sample preparations were injected separately and peak area responses were recorded. The percentage label claim was calculated and given in table-1.

VALIDATION OF THE METHOD:¹²

System suitability:

For system suitability, six replicates of standard solutions were injected and parameters studied were number of theoretical plates, peak area, resolution, retention time and tailing factor. The relevant data is shown in table -2.

Accuracy:

The accuracy of the experiment was established using recovery technique i.e. by external standard addition method. The result of recovery analysis is presented in table -3. The result of recovery was well within the acceptable limit.

Precision:

The validation of the proposed method was verified by system precision and method precision. The system precision was evaluated by measuring the peak area responses of Risperidone and Trihexyphenidyl hydrochloride for five replicate injections of the standard solutions. The method precision was determined by quantifying the sample solutions as per the proposed method, which yielded quite concurrent results, indicating reliability of the method. The value of SD and RSD were within the prescribed limit of 2% showing high precision of the method.

Linearity and range:

During linearity study, it was observed that the absorbance values of Risperidone and Trihexyphenidyl hydrochloride in the marketed formulation were linear in the concentration range of 80-120% of the test concentration with R² close to one for this method of analysis.

Table.3. Summary of validation parameters

Parameters	Data
Parameters	Riperidone	Trihexypenidyl hydrochloride
Linearity range	4.8-19.2 µg/mL	3.2-12.8 µg/mL
Correlation coefficient	1.000	0.9991
Limit of detection	50 ng/mL	20 µg/mL
Limit of quantitation	0.5µg/mL	0.2µg/mL
%Recovery (n=6)	99.0 to 100.0%	100.2 to 101.51%
Precision (%RSD) System Precision Method precision	0.699% 0.375%	1.090% 0.338%
Robustness (%RSD)	0.747%	0.394%
Ruggedness (%RSD)	0.773	0.624

Robustness and Ruggedness:

Robustness of the method is determined by analyzing the sample in duplicate with varying the method conditions i.e., very small changes in flow rate, showed there were no marked changes in chromatographic behavior and content of the drug, as evident from the low value of RSD indicating the method is robust. The method was also confirmed by ruggedness study, analyzing the product day to day, analyst to analyst and instrument to instrument. The results are shown in table-4, proved that the method was reproducible.

RESULTS AND DISCUSSION:

Estimation of Risperidone and Trihexyphenidyl hydrochloride in tablet dosage form by RP-HPLC method was carried out using optimized chromatographic conditions. The typical chromatogram of Risperidone and Trihexyphenidyl hydrochloride is shown in figure-1. The retention time for Risperidone and Trihexyphenidyl hydrochloride were found to be around were 2.685±0.012 and 5.912±0.030 minutes. The resolution value of more than 2 indicates satisfactory results in quantitative work and the high resolution value obtained indicate the complete separation of the drugs. The linearity was studied in the concentration range from 4.8 µg/mL to 19.2 µg/mL for Risperidone and 3.2 µg/mL to 12.8 µg/mL for Trihexyphenidyl hydrochloride. The regression co-efficient (R²) value for Risperidone and trihexyphenidyl hydrochloride was found to be 1.0 and 0.9991, respectively. The mean recovery for Risperidone was 99.0 to 100.0% and 100.2 to 101.51% for Trihexyphenidyl hydrochloride, which is largely within the 90-110% range that is considered acceptable and it reveals that the method is accurate. The validation of the proposed method was verified by system precision and method precision. The %RSD was found to be less than 2 for both drug indicates the proposed method is precise. The specificity of the method was confirmed by injecting the placebo and observed that there was no interference due to placebo.

The data for ruggedness of Risperidone and Trihexyphenidyl hydrochloride were found to be within the acceptance limit. Different validation parameters for the proposed HPLC method for determining Risperidone and Trihexyphenidyl hydrochloride were summarized in table-3. The results of analysis show that the amounts of drugs were in good agreement with the label claim of the formulation.

CONCLUSION:

The proposed method is simple, accurate, cost effective, less time consuming and the statistical analysis proved that the method is reproducible and efficient for the simultaneous estimation of Risperidone and Trihexyphenidyl hydrochloride as bulk drugs and in combined pharmaceutical dosage forms without any interference from the excipients. The developed method could be conveniently adopted for routine analysis in quality control laboratories.

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Received on 25.12.2009 Modified on 19.01.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 549-551