INTRODUCTION:

Palonosetron hydrochloride is an antiemetic and antinauseant agent. It is a serotonin subtype 3 (5-HT₃) receptor antagonist with a strong binding affinity for this receptor. Chemically, palonosetron hydrochloride¹ is: (3aS)-2-[(S)-1-Azabicyclo [2.2.2]oct-3-yl]-2,3,3a,4,5,6-hexahydro-1-oxo-1Hbenz[de]isoquinoline hydrochloride. The empirical formula is C₁₉H₂₄N₂O.HCl, with a molecular weight of 332.87. Palonosetron hydrochloride^2-10 is a white to off-white crystalline powder. It is freely soluble in water, soluble in propylene glycol, and slightly soluble in ethanol and 2-propanol.Palonosetron hydrochloride injection is a sterile, clear, colorless, non-pyrogenic, isotonic, buffered solution for intravenous administration.

Each 5 mL vial of palonosetron hydrochloride injection contains 0.25 mg palonosetron base as hydrochloride, 207.5 mg mannitol, disodium edetate and citrate buffer in water for intravenous administration.

The pH of the solution is 4.5 to 5.5. Palonosetron hydrochloride exists as a single isomer and has the following structural formula:

Fig 1: Structure of Palonosetron Hydrochloride

EXPERIMENTAL:

Materials and Methods:

Palonosetron Hydrochloride was obtained as a gift sample from M/s. Vishnu Chemicals Ltd, Hyderabad. Acetonitrile, Potassium Dihydrogen Orthophosphate and water used were of HPLC grade (Qualigens). Commercially available Palonosetron Hydrochloride Injectables (Aloxi 0.25® Injectables) were procured from local market.

Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Shimadzu LC 2010 dual λ detector equipped with automatic injector with injection volume 20 µl. The HPLC system was equipped with LC solution Software.

HPLC Conditions:

The contents of the mobile phase were mixture of buffer 0.03M Potassium Dihydrogen Orthophosphate in water and pH adjusted to 3.20 with Orthophosphoric acid and acetonitrile in the gradient program was used (shown in table-IV). 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 1.0 mL/min. The run time was set at 30.0 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 242 nm.

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 10 mg of Palonosetron Hydrochloride in 10 mL volumetric flask and dissolved in diluent (Acetonitrile and Water:50:50), sonicated for about 15 min and then made up to 10 mL with diluent get 1000 mcg/mL standard stock solution.

Working Standard solution:

0.25mL of the above stock solution was taken with micropipette in 10 mL volumetric flask and thereafter made up to 10 mL with diluent (Acetonitrile and Water: 50:50) to get a concentration of 25mcg/mL.

Preparation of Sample solution:

Twenty Injectables (Aloxi 0.25® Injectables) were taken and transfer the liquid into a 100 mL volumetric falsk. A sample of the 5mL equivalent to 250mcg of the active ingredient, add 10 mL of diluent to get working sample solution of 25mcg/mL and then filtered through a 0.45 μm membrane filter.

Linearity:

Aliquots of standard Palonosetron Hydrochloride 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 Palonosetron Hydrochloride are in the range of 5-30 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 242 nm and a Calibration graph was obtained by plotting peak area versus concentration of Palonosetron Hydrochloride (Fig 3).

The plot of peak area of each sample against respective concentration of Palonosetron Hydrochloride was found to be linear in the range of 5–30 mcg/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=3644.3x-3644.7. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in table I.

Assay:

20 µL of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 10.9 minutes. The amount of drug present per parentaral 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 Palonosetron Hydrochloride, known amount of standard was added to the preanalysed 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.

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Palonosetron Hydrochloride. The parameters studied to evaluate the suitability of the system are given in table III.

Table I: Linear Regression Data for Calibration curves:

Drug

Palonosetron Hydrochloride

Concentration range (mcg/mL)

Slope (m)

Intercept (b)

Correlation coefficient

% RSD

5-30

3644.3

-3644.7

0.9999

0.55

Table II: Results of HPLC Assay and Recovery studies:

Sample

Amount claim

(mg/ Injectable)

% found by the proposed method

% Recovery*

0.25

99.72

99.50

99.66

99.36

99.21

99.33

*Average of three different concentration levels.

Table III Validation Summary:

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor

Retention time in minutes

% Area

10247

1.14

10.9

99.98

LOD (ng/mL)

LOQ (ng/mL)

2.5

7.5

Table IV: Gradient Program in HPLC method:

Time in mins	Buffer	Acetonotrile
0.01	80	20
5	80	20
12	30	70
20	30	70
25	80	20
30	80	20

Fig 2: Typical Chromatogram of Palonosetron Hydrochloride by HPLC

Fig 3: Calibration curve of the Palonosetron Hydrochloride by RP-HPLC.

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

The limit of detection (LOD) and limit of quantification (LOQ) for Palonosetron Hydrochloride were found to be 2.5 ng/mL and 7.5 ng/mL respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ. From the typical chromatogram of Palonosetron Hydrochloride as shown in fig 2, it was found that the retention time was 10.9 min. A mixture of buffer 0.03M Potassium Dihydrogen Orthophosphate in water and pH adjusted to 3.20 with Orthophosphoric acid and acetonitrile in the gradient program was used (shown in table-IV) 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 (r²=0.9999) was observed between the concentration range of 5-30 mcg/mL. Low values of standard deviation are indicative of the high precision of the method. The assay of Palonosetron Hydrochloride injectables was found to be 99.6%. From the recovery studies it was found that about 99.3% of Palonosetron Hydrochloride 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 injectables. 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 parental dosage forms of Palonosetron Hydrochloride within a short analysis time.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Vishnu chemicals Limited, Hyderabad for the supply of as a gift sample Palonosetron Hydrochloride and to the Management, Vishnu Chemicals Limited, Hyderabad, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. Martindale-The Complete Drug Reference, 36, 1759, (2009)

2. R.C. Reynolds, Prokinetic agents: a key in the future of gastroenterology, Gastroenterol. Clin. N.A. 18 (1989) 437–457.

3. A. De Leon, Palonosetron (Aloxi): a second generation 5-HT3 receptor antagonist for chemotherapy-induced nausea and vomiting, Proc. Bayl Univ. Med. Cent. 19 (2006) 413–416.

4. G. Stacher, Palonosetron (Helsinn), Curr. Opin. Investig. Drugs 3 (2002) 1502–1507.

5. B.A. Kowalczyk, N.H. Dyson, Hydrogenation of a chiral 1H-benz[de]isoquinolin-1-one and an equilibration using palladium catalyst, Org. Process Res. Dev. 1 (1997) 117–120.

6. R.D. Clark, A.B. Miller, J. Berger, D.B. Repke, K.K. Weinhardt, B.A. Kowalczyk, R.M.Eglen, D.W. Bonhaus, C.H. Lee, 2-(Quinuclidin-3-yl)pyrido[4 3-b]indol-1-ones and isoquinolin-1-ones. Potent conformationally restricted 5-HT3 receptor antagonists, J. Med. Chem. 36 (1993) 2645–2657.

7. R. Stoltz, J.C. Cyong, A. Shah, S. Parisi, Pharmacokinetic and safety evaluation of Palonosetron, a 5-hydroxytryptamine-3 receptor antagonist, in U.S. and Japanese Healthy subjects, J. Clin. Pharmacol. 44 (2004) 520–531.

8. T.L. Hunt, S.C. Gallegher, M. Cullen, A.K. Shah, Evaluation of safety and pharmacokinetics of consecutive multiple-day dosing of Palonosetron in healthysubjects, J. Clin. Pharmacol. 44 (2005) 589–596.

9. L. Ding, Y. Chen, L. Yang, A. Wen, Determination of Palonosetron in human plasma by liquid chromatography–electrospray ionization-mass spectrometry, J. Pharm. Biomed. Anal. 44 (2007) 575–580.

10. W. Zhang, F. Feng, Sensitive and selective LC-MS-MS assay for the quantiﬁcation of Palonosetron in human plasma and its application to a pharmacokinetic study, Chromatographia 68 (2008) 193–199.

Received on 14.05.2012 Accepted on 26.05.2012

Asian J. Pharm. Tech. 2(2): April-June 2012; Page 77-79