Determination of lamivudine/stavudine/efavirenz in human serum using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch

Abstract

A high-performance liquid chromatography/tandem mass spectrometry (LC-MS-MS) method with ionization polarity switch was developed and validated in human serum for the determination of a lamivudine (3TC)/stavudine (d4T)/efavirenz combination HIV therapy. Solid phase extraction (SPE) was used to extract these anti-HIV drugs and internal standard aprobarbital. A gradient mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer with pH adjusted to 4.5 using glacial acetic acid was utilized to separate these drugs on a hexylsilane column (150 x 2.0 mm i.d.). The total run time between injections was 18 min. The precursor and major product ions of these drugs were monitored on a triple quadrupole mass spectrometer in the multiple reactions monitoring (MRM) mode. Ionization polarity was switched in the middle of the LC run allowing these anti-HIV drugs with different physicochemical properties to be detected simultaneously. The effect of ion suppression from human serum was studied and no interference with the analysis was noted. The method was validated over the range of 1.1-540 ng/mL for 3TC, 12.5-6228 ng/mL for d4T and 1.0-519 ng/mL for efavirenz. The method was shown to be accurate, with intra-day and inter-day accuracy less than 14.0% and precise, with intra-day and inter-day precision less than 13.1%. The extraction recoveries of all analytes were higher than 90%.

Validation of new methods

“…In the first approach, a solution of the analyte is constantly infused into the eluent from the column via postcolumn tee connection using a syringe pump. The continuous post-column infusion leads to a constant signal in the detector, unless compounds that elute from the column suppress or enhance ionisation, which would lead to a decreased or increased detector response, respectively [32][33][34][35][36][40][41][42]. A comprehensive strategy for the second approach was recently published by Matuszewski et al [19].…”

Rapid and Simultaneous Determination of Efavirenz, 8-Hydroxyefavirenz, and 8,14-Dihydroxyefavirenz Using LC–MS–MS in Human Plasma and Application to Pharmacokinetics in Healthy Volunteers

“…4. The total run time per sample of this assay was within 3.0 min, which is shorter than the reported methods [20,22,[29][30][31][32][33][34][35]. In addition, the present method was sensitive and accurate to measure efavirenz and its hydroxy metabolites, simultaneously.…”

Determination of salivary efavirenz by liquid chromatography coupled with tandem mass spectrometry

“…Several analytical methods have been published for the analysis of EFV in plasma, either alone or in combination with other drugs, using HPLC-UV [14][15][16][17] and LC-MS/MS [18][19][20][21][22][23] techniques. Determination of EFV levels in human hair [7] and reports of determining nevirapine [8,10] and indinavir [24] from saliva have been published, but this study is the first validated LC-MS/MS method for the assay of EFV in saliva to be reported.…”