Stability-indicating HPLC determination of gemcitabine in pharmaceutical formulations.

Rahul Singh,Ashok K Shakya,Rajashri Naik,Naeem Shalan

doi:10.1155/2015/862592

Abstract

A simple, sensitive, inexpensive, and rapid stability indicating high performance liquid chromatographic method has been developed for determination of gemcitabine in injectable dosage forms using theophylline as internal standard. Chromatographic separation was achieved on a Phenomenex Luna C-18 column (250 mm × 4.6 mm; 5μ) with a mobile phase consisting of 90% water and 10% acetonitrile (pH 7.00 ± 0.05). The signals of gemcitabine and theophylline were recorded at 275 nm. Calibration curves were linear in the concentration range of 0.5–50 μg/mL. The correlation coefficient was 0.999 or higher. The limit of detection and limit of quantitation were 0.1498 and 0.4541 μg/mL, respectively. The inter- and intraday precision were less than 2%. Accuracy of the method ranged from 100.2% to 100.4%. Stability studies indicate that the drug was stable to sunlight and UV light. The drug gives 6 different hydrolytic products under alkaline stress and 3 in acidic condition. Aqueous and oxidative stress conditions also degrade the drug. Degradation was higher in the alkaline condition compared to other stress conditions. The robustness of the methods was evaluated using design of experiments. Validation reveals that the proposed method is specific, accurate, precise, reliable, robust, reproducible, and suitable for the quantitative analysis.

Highlights

Gemcitabine hydrochloride (Figure 1), (4-amino-1-[(2R, 4R, 5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl) oxolan2-yl] pyrimidin-2-one) is a β-difluoronucleoside, purine antimetabolite
During optimization gemcitabine hydrochloride and internal standard were injected into various mobile phases of water : methanol or water : acetonitrile (90 : 10, 80 : 20, 70 : 30, 60 : 40, and 50 : 50, pH 5, 6, or 7) and the retention time, tailing factor along with resolution factor, was recorded
The absorption maximum of the drug at 275 nm was selected for detection (Figure 2), as there was no interference from excipients present in drug or baseline disturbance

Summary

Introduction

Gemcitabine hydrochloride (Figure 1), (4-amino-1-[(2R, 4R, 5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl) oxolan2-yl] pyrimidin-2-one) is a β-difluoronucleoside, purine antimetabolite. The drug is an antitumor agent, employed extensively against several human malignancies like ovarian, lung, pancreatic, bladder, urothelial, and breast cancer. It is currently marketed as a lyophilized powder. A few stability indicating that HPLC methods [3, 11, 12] have been reported, which provides variable level of degradation of gemcitabine. The validated stability indicating method which can separate the hydrolytic degraded product of gemcitabine. To the best of our knowledge none of the HPLC method reported the oxidative degraded product of gemcitabine. The present investigation describes a simple, rapid, accurate, precise, robust stability indicating RP-HPLC method for the determination of gemcitabine for dosage forms.

Experimental

Preparation of Stock and Standards Solutions

Analytical Method Validation

Results and Discussion

Conclusion