Determination of Genotoxic Impurities N-nitrosamine's in Efavirenz Drug Substance Using Rp-Hplc Technique

Abstract

Aim: The main purpose of the research is to  develop a effective, sensitive, economical and simple reverse phase HPLC method for the determination of genotoxic N-Nitrosamine's impurities in Efavirenz drug substance. Study Design: HPLC based quantification studies Place and Duration of Study: Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh between April 2022 and August 2022. Methodology: Determination of genotoxic N-Nitrosamine's in Efavirenz drug substance. N-Nitrosamine's impurities were determined by RP-HPLC method using Zorbax SB C18 (150x4.6mm, 3.5µ) column as stationary phase. Column temperature maintained 25°C, injection volume 20 µL, flow rate 1.0 mL/min and sample cooler temperature 5°C and run time was 15 minutes.  The mixture of 0.1% formic acid buffer and methanol in the ratio of 50:950 (v/v) was used as mobile phase. wavelength 240 nm. respectively. Results:  There is no interference of blank at N-Nitrosamine's impurities peaks. The elution order and the retention times of impurities and Efavirenz obtained from individual standard preparations and mixed standard preparations are comparable. The limit of detection (LOD) and limit of quantitation (LOQ) for NDMA 0.03 & 0.09 µg/mL, NDEA 0.02 & 0.06 µg/mL  and NDIPA 0.03 & 0.09 µg/mL respectively. The linearity results for N-Nitrosamine's impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.Calibration curve was plotted and correlation co-efficient for impurities found to be 1.000, 1.000 and 1.000 respectively. The accuracy studies were shown as %recovery for N-Nitrosamine's impurities at specification level. The limit of % recovered shown is in the range of 80 and 120% and the results obtained were found to be within the limits. Hence the method was found to be accurate. The technique has been validated in accordance with ICH rules, and all validation parameters meet the acceptance criteria of the ICH Q2 specification. Conclusion:  According to ICH guidelines, an RP-HPLC method that can assess genotoxic Nitrosoamines in Efavirenz at trace level concentration has been established. The method's specificity, precision, linearity, and accuracy ensured its efficacy. As a result, the technique is appropriate for the goals it was designed to achieve and can be successfully used for release testing of efavirenz into the market.