Simultaneous Quantification of Lamivudine, Zidovudine and Related Impurities in Fixed Oral Dosage Combination Using RP-HPLC with DAD detection

A simple and fast isocratic Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed and validated for the simultaneous determination of Lamivudine, zidovudine and their related impurities in tablets. The method consists of a mobile phase combination of Acetonitrile (HPLC grade) and Buffer (0.0680 g of Potassium Dihydrogen Orthophosphate, 0.3 ml of Triethylamine, pH adjusted to 8.0 with Orthophosphoric acid to a final volume preparation of 100 ml) in the ratio 10:90. Phenomenex Luna 5-µm C18 (2)-250 x 4.6-mm, 5-µm) was used as the stationary phase. The column oven was set to a temperature of 30±1oC. Quantification was achieved with a DAD detector set at 270 nm. Resolution was achieved at a short run time of 25 minutes. Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine and Zidovudine related impurity B eluted at 3.749±0.004, 4.862±0.013, 15.332±0.064, 21.201±0.076 and 23.682±0.117 respectively. Relative retention times (RRT) for lamivudine unknown related impurities with respect to Zidovudine were 0.15, 0.17, 0.30 and 0.59. RRT for Zidovudine unknown related impurities with respect to Zidovudine were 0.39 and 0.63.  The method was found to be specific, robust, accurate and precise for the estimation of Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine and Zidovudine related impurity B in fixed oral dosage tablets over the concentration ranges of 0.0204 mg/mL-0.0088 mg/mL, 0.0962 mg/mL-0.7699 mg/mL, 0.1929 mg/mL-1.5410 mg/mL and 0.0088 mg/mL-0.024 mg/mL respectively. The Correlation Coefficient (r2) for Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine and Zidovudine related impurity B were greater than 0.998. The LOD were found to be between 1.9x10-4 mg/mL to 2.69 x10-4 mg/mL.  The proposed method is precise, specific, accurate and robust for the simultaneous estimation of Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine, Zidovudine related impurity B and other related impurities in dosage forms. Keywords: Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine, Zidovudine related impurity B, Lamivudine related impurities, Zidovudine related impurities RP-HPLC, Validation.

Relative Response Factor for Lamivudine and Zidovudine Related substances by RP-HPLC with DAD detection

A study was conducted to establish the Relative Response Factors for Zidovudine related impurity C, Lamivudine salicylic acid and Zidovudine related impurity B.  A simple and fast isocratic Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method was used for the simultaneous determination of the related impurities. The method consists of a mobile phase combination of Acetonitrile (HPLC grade) and Buffer (0.0680 g of Potassium Dihydrogen Orthophosphate, 0.3 ml of Triethylamine, pH adjusted to 8.0 with Orthophosphoric acid to a final volume preparation of 100 ml) in the ratio 10:90 using Phenomenex Luna 5-µm C18 (2)-250 x 4.6-mm, 5-µm) as a stationary phase, flow rate of 1.0 mL/min with detection at 270 nm. The RRF for Zidovudine related impurity C, Lamivudine salicylic acid and Zidovudine related impurity B were 2.07, 0.13 and 1.28 respectively.   Results obtained for quantification of the related substance in lamivudine and zidovudine single dose oral solid dosage form using the RRF and know standards shows no significant difference at 95 % confidence interval. The RRF can therefore be used for the quantification of know related impurities in lamivudine zidovudine oral dosage form using the stated chromatographic conditions