INTRODUCTION:

Aprepitant is a novel antiemetic agent used in cancer chemotherapy; with a chemical name-5-([(2R,3S)-2-((R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy)-3-(4-fluoro phenyl) morpholino]methyl)-1H-1,2,4-triazol-3(2H)-one. It’s molecular weight is 534.427 g/mol with molecular formula C₂₃H₂₁F₇N₄O_3. Literature survey reveals many Chromatographic methods^{1, 2} for the determination of Aprepitant and its metabolites in biological fluids and enantimeric separation. So far, no assay procedure has been reported for the estimation of Aprepitant from pharmaceutical dosage form. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Aprepitant in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Aprepitant in bulk drug samples and in pharmaceutical dosage forms.

Structure of Aprepitant

EXPERIMENTAL:

Aprepitant was obtained as a gift sample from Hetero Drugs Ltd, Hyderabad. Ammonium Acetate and Formic acid were of analytical grade, and supplied by M/s S.D.Fine Chem Limited, Mumbai. Methanol and water used were of HPLC grade (Qualigens). Commercially available Aprepitant Capsules (Aprecap 80, 120 mg, Glenmark) were procured from local market.

Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Waters separation 2996, PDA detector module equipped with automatic injector with injection volume 20 µl, and 2693 pump. A RP C-18 X-Terra column (250x4.6 mm i.d; particle size 5 μm) was used. The HPLC system was equipped with Empower Software.

Table I: Linear Regression Data for Calibration curves.

Drug

Aprepitant

Concentration range (µg/ml)

Slope (m)

Intercept (b)

Correlation coefficient

% RSD

14-168

267157.4

169105

0.9999

0.44

HPLC Conditions:

The contents of the mobile phase were 0.01 M Ammonium Acetate containing 0.1% Formic acid and Methanol in the ratio of 35:65 v/v in the ratio of 15:85 v/v. They were filtered before use through a 0.45 μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.5 ml/min. The run time was set at 25 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 205 nm.

Table II: Results of HPLC assay and Recovery studies

Sample	Amount claim [mg/ Capsules]	% found by the proposed method.	% Recovery
1.	80	99.85	101.77
2.	80	100.25	100.92
3.	80	99.82	100.37

*Average of three different concentration levels.

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 70 mg of Aprepitant in 100 ml volumetric flask containing 30 ml of mobile phase, sonicated for about 15 min and then made up to 100 ml with diluent [HPLC Grade water: Methanol 20: 80] to get 700 ppm/ml standard stock solution.

Working Standard solution:

10 ml of the above stock solution was taken in 50 ml volumetric flask and thereafter made up to 50 ml with mobile phase to get a concentration of 140ppm/ml.

Preparation of Sample solution:

Twenty Capsules (Aprecap, Glenmark) were weighed, and drug powder is collected. A sample of the capsule powder, equivalent to 70 mg of the active ingredient, was mixed with 25 ml of diluent [HPLC Grade water: Methanol 20: 80]. The mixture was allowed to stand for 1 hr with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 μm membrane filter, followed by adding mobile phase to obtain a stock solution of 700 µg/ml. Transfer 10ml of this solution to a 50 ml volumetric flask and made up to sufficient volume with mobile phase to give an concentration of 140 μg/ml.

Linearity:

Aliquots of standard Aprepitant stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Aprepitant are in the range of 14-168 μg/ml. Each of these drug solutions (20 μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 205 nm and a Calibration graph was obtained by plotting peak area versus concentration of Aprepitant (Fig 2). The plot of peak area of each sample against respective concentration of Aprepitant was found to be linear in the range of 14–168 µg/ml with correlation coefficient of 0.9999. Linear regression least square fit data obtained from the measurements are given in table I. The respective linear regression equation being

Y=267157.4x+169105. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table I.

Fig 1: Typical Chromatogram of Aprepitant by HPLC:

Assay:

20 µl of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 13.73 minutes. The amount of drug present per Capsules was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table II.

Recovery Studies:

Accuracy was determined by recovery studies of Aprepitant, known amount of standard was added to the preanalyzed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in Table II. The study was done at three different concentration levels.

Table III Validation Summary

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor.

Retention time in minutes.

18427.75

1.11

13.612

LOD (µg/ml)

LOQ (µg/ml)

0.28

0.84

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Aprepitant. Parameters that were studied to evaluate the suitability of the system are given in Table III.

Fig 2: Calibration curve of Aprepitant by RP-HPLC.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection (LOD) and limit of quantification (LOQ) for Aprepitant were found to be 0.28µg/ml and 0.84µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ.

From the typical chromatogram of Aprepitant as shown in fig 1, it was found that the retention time was 13.73 min. A mixture of 0.01 M Ammonium Acetate containing 0.1% Formic acid and Methanol in the ratio of 35:65 v/v in the ratio of 15:85 v/v was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r=0.9999) was observed between the concentration range of 14-168 µg/ml. Low values of standard deviation are indicative of the high precision of the method. The assay of Aprepitant Capsuless was found to be 99.85%. From the recovery studies it was found that about 102.4 % of Aprepitant was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the Capsuless. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible. Thus, the developed method can be easily used for the routine quality control of bulk and Capsules dosage form of Aprepitant within a short analysis time.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Hetero Drugs, Hyderabad for the supply of as a gift sample Aprepitant and to the Management, Sree Chaitanya Institute of Pharmaceutical Sciences, LMD.Colony, Karimnagar-505527.AP, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. Chavez-Eng C. M; Constanzer M. L ; Matuszewski B. K.^, Journal of pharmaceutical and biomedical analysis 2004, vol. 35,[5] pp. 1213-1229.

2. P. Radhakrishnanand, D. V. Subba Rao, K. V. Surendranath, D. Subrahmanyam and V. Himabindu; Chromatographia, 2008, 68[7-8], pp 669-673.

Received on 16.04.2009 Modified on 10.05.2009

Research J. Pharm. and Tech.2(2): April.-June.2009,;Page 412-414