Simple rapid method for quantification of antiretrovirals by liquid chromatography—tandem mass-spectrometry

Objectives: The aim of the current study was to develop a simple, fast and universal method for quantification of any combination of all currently marketed anti-HIV drugs in human plasma, using a LC-tandem mass spectrometer (API-2000, SCIEX, Toronto, Canada). Methods: 80 μL plasma were spiked with internal standard (cimetidine), and protein precipitated with 200 μL acetonitrile. The sample was centrifuged and 30 μL aliquot was injected onto the HPLC column, where it underwent an online extraction with ammonium acetate. After that the automatic switching valve was activated, changing the mobile phase to methanol and thereby eluting the analytes into the tandem mass spectrometer. Stavudine, AZT and efavirenz were analyzed in the negative MS/MS mode, while all other drugs were analyzed in the positive mode. The high selectivity of a tandem mass analyzer allows determination of any combination of the drugs within a 4.5 min run. Results: Between-day precision was below 10% for all analytes at the concentrations tested. Accuracy ranged between 95% and 105% (n = 20). The method was linear over the measuring ranges of all analytes. Within-run precision gave a CV < 7% for all analytes. Good correlation with other analytical methods was observed. Conclusions: The simplicity, universality and high throughput of the method make it suitable for application in a clinical laboratory. The method has been implemented in our laboratory for routine use.