INTRODUCTION:

Trihexyphenidyl Hydrochloride, (1RS)-1-Cyclohexyl-1-phenyl-3-(piperidin-1-yl) propan-1-ol hydrochloride is white, crystalline powder, slightly soluble in water, soluble in chloroform and methanol, sparingly soluble in methylene chloride¹. The drug is used as an anticholinergic agent and anti Parkinson agent². Its mode of action is preventing the effects of ACh by blocking its binding to muscarinic cholinergic receptors at neuroeffector sites on smooth muscle, cardiac muscle, and gland cells; in peripheral ganglia; and in the central nervous system^2,3.

Literature review shows various methods for the estimation of Trihexyphenidyl HCl includes radioimmune assay (RIA)⁴, HPTLC⁵, HPLC^6,7, LC-ESI-MS⁸, Capillary electrophorosis⁹, Gas chromatography^10-16, Gravimetry¹⁷, Argentometric¹⁸ and Polarographic¹⁹ methods, but no UV estimation method was found. So the aim of our study is to estimate Trihexyphenidyl HCl as API and in pharmaceutical dosage forms.

MATERIAL AND METHOD:

Shimadzu model 1601 UV-Visible double beam spectrophotometer with matched quartz cells (1cm) was used. Digital pH meter model 335 (systronic) was used to adjust the pH of the buffer.

All the chemicals used were of analytical grade. Standard Trihexyphenedyl hydrochloride was provided by Oasis lab, Ahmedabad and API was manufactured by Padmawati chemicals.

Market formulations Lahexy (La Pharma) and Bexole (Intas) were purchased from the local market.

Preparation of stock solution:

Accurately weighted 20 mg of trihexyphenidyl hydrochloride was transferred to 200 ml volumetric flask, 50ml of water was added and sonicated for 10 minutes, volume was made with water (100 mg/ml). 10 ml of above solution was pipette out and diluted it to 50 ml with water.

50 mg of bromocresol green powder was transferred in 100 ml volumetric flask, 0.72 ml of 0.1 M sodium hydroxide solution was added, 20 ml of ethanol was added, after solution was effected volume was make up with water¹⁶(7.1 X 10^-4 M).

13.6 gm. of sodium acetate trihydrate was transferred in 1000 ml volumetric flask, 6 ml of glacial acetic acid was added, volume was make up with water¹⁶.

Preparation of calibration curve:

Aliquots of standard solution of trihexyphenidyl hydrochloride (20 mg/ml) like (1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9ml) was diluted to 10 ml to prepare solution of concentration (2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml). 5 ml of each solution was taken in a

separator and 5 ml acetate buffer (pH 5.0) was added, 5 ml of bromocresol green solution was added and 10 ml of chloroform was added and separator was shake for 5 minutes. Chloroform layer transferred in a test tube. Absorbance of the solution was measure at 410 nm. Against blank preparation and calibration curve was plotted. The Beer’s Law was linear in concentration range of 2mg/ml to 18 mg/ml at 410 nm.

From the calibration curve data, the linear regression equation and correlation coefficient for complex was found to be y = 0.0571(±0.00019)x -0.02(±0.0010) and r = 0.9991 (0.00013), where ‘y’ is absorbance and ‘x’ is the concentration of drug solution respectively.

Figure 1: Chemical structure of Trihexyphenidyl Hydrochloride

VALIDATION OF THE PROPOSED METHOD:

Specificity:

For specificity determination, excipients was mixed with standard drug such as 50 % (600 mg) lactose, 7 % (48 mg) magnesium stearate, and 15 % (180 mg) starch were added, then after filter these excipients and any change in absorbances are observed. Interference found was 0.13 % which is less then 0.5% limit.

Precision:

Method was validated in terms of repeatability, interday and intraday and % RSD found was 0.16, 0.20-0.41 and 0.17-0.19 respectively which was found to be in the limit of 2.

Accuracy:

Results of analysis, obtained in three experiments with API and different tablet dosage forms, had good agreement with the labeled amount of the drug. In three different 50 ml volumetric flasks, 5 ml of the pre-analyzed tablet solution (100 mg/ml) was taken and it was labeled as f1, f2, f3. and 3 ml, 5 ml, 7 ml of standard solution (100mg/ml) added respectively, volume was made up to 50 ml with water, 25 ml from each flask was diluted to 50 ml. % recovery was found 98.80 – 99.55 which was in limit 98.00 – 102.00

Linearity :

Linearity was assessed under section 5.3.7. Linearity rang found to be 2-18 µg/ml. The correlation coefficient was 0.9991 which adhere good linearity between above rang. The slop was found to be 0.0571 and intercept was found to be -0.02 which was close to zero intercept.

Figure 2: UV Spectra of chloroform extracted Trihexyphenidyl HCl - bromocresolgreen complex using acetate buffer. (410 nm)

Range :

Working range, linearity range, and Target concentration was found to be 0.35-18, 2-18 and 10 mg/ml. Target range was found to be 8,10 and 12 mg/ml.

LOD and LOQ:

Limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.11 and 0.35 µg/ml respectively.

ESTIMATION OF TIRHEXYPHENIDYL HYDROCHLORIDE:

As an API:

20 mg of the sample was taken in a 200 ml volumetric flask. 50 ml of water was added and sonicated for 10 minutes. 5 ml of above solution was diluted to 50 ml. 5 ml of this solution was taken in a separator, 5 ml of acetate buffer was added, 5 ml of bromocresol green was added and 10 ml of chloroform was added and shaken for 5 minutes. Chloroform layer was transferred to test tube. Absorbance was measured at 410 nm.

RESULT AND DISCUSSION:

Concentration was found to be 99.85 % of trihexyphenidyl HCl. This concentration found within pharmacopoieal rang (99.00 -101.00).

In Tablets Dosage Form:

Twenty tablets (of same respective batch number) of two pharmaceutical companies were taken and average weight was determined.

These tablets were powdered and triturated well. Quantity of powder equivalent to 20 mg of tirhexyphenidyl hydrochloride was transferred to 200 ml volumetric flask, and mixed with 50 ml of water and

Table 1: Result recovery study using Bexole (Intas)

Conc. Selected (µg/ml )	Conc. found before spiking (µg/ml )	Conc. of std added (µg/ml )	Conc. found after spiking (µg/ml )	% recovery
5	4.96	3	7.93	99.44
5	4.96	5	9.90	98.80
5	4.96	7	11.92	99.42

Table 2: Recovery study using La hexy (La Pharma)

Conc. selected (µg/ml )	Conc. found before spiking (µg/ml )	Conc. of std added (µg/ml )	Conc. found after spiking (µg/ml )	% recovery
5	4.94	3	7.91	99.00
5	4.94	5	9.90	99.20
5	4.94	7	11.86	98.85

Table 3: Optical and Regression parameters

l _max	410 nm
Beers’ law limit (µg/ml)	2 – 18
Molar absorptivity (1 mol^-1 cm^-1)	5.6 X 10⁴
Sandell’s sensitivity	1.84 X 10^-2
Regression equation	y = 0.0571x – 0.02
Slope	0.0571
Intercept	-0.02
Correlation coefficient	0.9996

Table 4: Estimation of table dosage form and comparison with HPLC method

S. No.	Brand name	Label claim	*mg/ tablet	% of Trihexyphenidyl HCl
S. No.	Brand name	Label claim	*mg/ tablet	By UV- VIS spectroscopy	By HPLC
1	Bexole (Intas)	2 mg / tab	2.02	101.0 %	100.45 %
2	La Hexy (La Pharma)	2 mg / tab	2.01	100.5 %	100.30 %

* Mean of three analyses.

Table 5: Comparison of estimation of trihexyphenidyl HCl by two analyst

	Brand	Label claim	Mg/table	% of drug	Mean ± SD	RDS
Analyst 1	A	2 mg / tab	2.02	101.0 %	100.83 % ± 0.288	0.286
			2.01	100.5 %
			2.02	101 %
Analyst 2	A	2 mg / tab	2.00	100.0 %	100.5 % ± 0.5	0.497
			2.02	101.0 %
			2.01	100.5 %

Table 6: Summary of validation data

Specificity		% interference 0.13
Precision	Repeatability	RSD = 0.16
	Intraday	RSD = 0.20 – 0.41
	Interday	RSD = 0.17 – 0.19
Accuracy		% recovery = 98.80 – 99.55
Linearity		2-18 µg/ml
Range	Linear Range	0.35 – 18 µg/ml
	Working Range	8 – 12 µg/ml
	Target Range	10 µg/ml
LOD		0.11 µg/ml
LOQ		0.35 µg/ml

solution was sonicated for 10 minutes there after volume was made up to 200 ml with water. Solution was filtered through Whatmann filter paper No. 41. From the filtrate, 5 ml was transferred to 50 ml volumetric flasks and volume was made up to 50 ml with water. 5 ml of this solution is

taken in the separator, 5 ml of acetate buffer added, 5 ml of bromocresol green solution

CONCLUSION:

In present work method was developed and validated for estimation of Trihexyphenidyl Hcl as API and in tablet dosage form by spectrophotometry.

The developed method is accurate, economical, precise, sensitive, and reproducible. This method is use for determination of trihexyphenidyl HCl in routine analysis as an API and in tablet dosage form. In this method ion pair complex is form between trihexyphenidyl HCl and bromocresol green which gives l_max410 nm. Limit of quantitation at 410 nm was found to be 0.35 µg/ml and limit of detection 0.11 µg/ml. Beers’ law was found to follow in the range 2 µg/ml to 18 µg/ml. The method shows good repeatability that is demonstrated by RSD of lower than 0.16%. The RSD (%) for Interday determination found to be 0.17, 0.18, and 0.19.and intraday determination found to be 0.20, 0.29, and 0.41. The RSD was found to within acceptance limit (<2.0%), so it was concluded that there was no significant difference for the assay, which was tested within day and between days. Hence, method at selected wavelength was found to be precise.

Specificity studies show good agreement with results, indicating that the excipients did not interfere with the analysis. The recovery studies were carried out at three levels 80, 100 and 120%. Good recoveries were obtained with %RSD lower than 2%. The mean % recovery varies from 98.80 to 99.44% and it was within the desirable confidence interval of 98-102%, it can be said that the proposed method is accurate.

It is reveal from this study that the methods are simple, precise, specific and accurate. The newly developed methods can be used for routine analysis for the estimation of trihexyphenidyl HCl in bulk and tablet dosage form in pharmaceutical industry.

Figure 3: Probable Structure of Complex Formed

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Received on 06.12.2008 Modified on 11.01.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 104-107