Quantitative Analyses Of Didanosine And Stavudine In Human Plasma And Their Applications

OBJECTIVE: To develop two sensitive and specific liquid chromatographic-tandem mass spectrometric methods for respectively determination of didanosine and stavudine in human plasma respectively and to study the clinical pharmacokinetics of didanosine and stavudine.METHOD: To determine of didanosine in plasma by a liquid chromatographic-tandem mass spectrometric method, the samples were separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol - water - formic acid (40 : 60 : 0.2, v/v/v), after a simple protein precipitation using acetonitrile. The detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source. The mass spectrometer was operated in the positive ion detection mode. Quantification was performed using selected reaction monitoring of the transitions of m/z 237 to m/z 137 for didanosine and m/z 230 to m/z 112 for lamivudine (I.S.). To determine stavudine in plasma by LC/MS/MS, the plasma sample of stavudine was extracted from plasma using liquid-liquid extraction, and then separated on a Zorbax Extend C₁₈ column. The mobile phase consisted of methanol-water-1% ammonia (50 : 50 : 0.5, v/v/v). A Thermo Finnigan TSQ Ultra tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the negative ion mode. Selected reaction monitoring using the precursor to product ion combinations of m/z 223 to m/z 42 and m/z 241 to m/z 42 was performed to quantify stavudine and P-thymidine (I.S).RESULTS: The linear calibration curves of didanosine were obtained in the concentration range of 5.0 - 2000 ng·mL^-1. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range was less than 6%. Accuracy (RE) determined at three concentrations was within ± 4%. The linear calibration curves of stavudine were obtained inthe concentration range of 5.0 -1000 ng·mL^-1. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than...