DEVELOPMENT AND VALIDATION OF A SENSITIVE LCMS/MS METHOD DETERMINATION OF STAVUDINE, LAMIVUDINE AND NEVIRAPINE IN HUMAN DEVELOPMENT AND VALIDATION OF A SENSITIVE LCMS/MS METHOD DETERMINATION OF STAVUDINE, LAMIVUDINE AND NEVIRAPINE IN HUMAN PLASMA INTERNAT

Abstract: A simple and sensitive liquid chrom method for the determination of stavudine, lamivudine and nevirapine in human plasma was developed and validated to support the pharmacokinetic studies. Didanosine (for stavudine) and emtricitabine (for lamivudine and nevirapine) are used as internal standards and solid phase extraction was employed as the extraction technique with the sample volume of 300 µl. 2 mM ammonium acetate buffer and methanol in the ratio of 5:95 was used as mobile phase using C18 zorbax (4.6x150mm, 5µ) column for chromatographic separation. ESI/MSMS in the positive mode with the mass transitions (m/z) of 223.00/125.00 for stavudine, 228.10/133.90 for lamivudine, 264.90/181.90 for nevirapine, 234.90/134.90 for didanosine and 245.9 detection. The method linearity range was established as 5 to 1500 ng/ml for stavudine, 5 to 2000 ng/ml for lamivudine and 10 to 4000 ng/ml for nevirapine with the weighting factor of 1/X method was validated as per the regulatory guidelines and the results of the method validation were in compliance to the acceptable limits specified by the regulatory guidelines confirming the method readiness for the pharmacokinetic studies. A simple and sensitive liquid chromatography tandem mass spectrometry (LC method for the determination of stavudine, lamivudine and nevirapine in human plasma was developed and validated to support the pharmacokinetic studies. Didanosine (for stavudine) and ne and nevirapine) are used as internal standards and solid phase extraction was employed as the extraction technique with the sample volume of 300 µl. 2 mM ammonium acetate buffer and methanol in the ratio of 5:95 was used as mobile phase using C18 zorbax (4.6x150mm, 5µ) column for chromatographic separation. ESI/MSMS in the positive mode with the mass transitions (m/z) of 223.00/125.00 for stavudine, 228.10/133.90 for lamivudine, 264.90/181.90 for nevirapine, 234.90/134.90 for didanosine and 245.90/151.90 for emtricitabine were used for detection. The method linearity range was established as 5 to 1500 ng/ml for stavudine, 5 to 2000 ng/ml for lamivudine and 10 to 4000 ng/ml for nevirapine with the weighting factor of 1/X as per the regulatory guidelines and the results of the method validation were in compliance to the acceptable limits specified by the regulatory guidelines confirming the method readiness for the pharmacokinetic studies. hy tandem mass spectrometry (LCMS/MS) method for the determination of stavudine, lamivudine and nevirapine in human plasma was developed and validated to support the pharmacokinetic studies. Didanosine (for stavudine) and ne and nevirapine) are used as internal standards and solid phase extraction was employed as the extraction technique with the sample volume of 300 µl. 2 mM ammonium acetate buffer and methanol in the ratio of 5:95 was used as mobile phase using C18 zorbax eclipse (4.6x150mm, 5µ) column for chromatographic separation. ESI/MSMS in the positive mode with the mass transitions (m/z) of 223.00/125.00 for stavudine, 228.10/133.90 for lamivudine, 264.90/181.90 0/151.90 for emtricitabine were used for detection. The method linearity range was established as 5 to 1500 ng/ml for stavudine, 5 to 2000 ng/ml for lamivudine and 10 to 4000 ng/ml for nevirapine with the weighting factor of 1/X 2 . The as per the regulatory guidelines and the results of the method validation were in compliance to the acceptable limits specified by the regulatory guidelines confirming the method Keyword