A sensitive method for the determination of entecavir at picogram per milliliter level in human plasma by solid phase extraction and high-pH LC-MS/MS.
Entecavir is a guanine nucleoside analogue used in the treatment of hepatitis B virus (HBV) infection. In this paper, we describe an LC-MS/MS method that was developed and validated for the quantitation of entecavir in human EDTA plasma with both high sensitivity (lower limit of quantitation (LLOQ) of 5 pg/mL) and a wide concentration range (5000-fold) intended for low dose ascending clinical studies. High enrichment was achieved by taking advantage of the excellent loading capacity and reproducibility of Oasis HLB 96-well solid phase extraction plate, which allowed 1 mL of plasma samples to be processed in two equal sequential loading steps. Lobucavir, a structural analogue, was used as the internal standard. A filtration step following the reconstitution proved to be vital for the method robustness. The analyte and internal standard were separated on an Xterra MS C18 column with a gradient elution and high-pH mobile phases. Analytes were detected by positive ion electrospray tandem mass spectrometry. The high-pH mobile phase provided both excellent analyte on-column retention and peak shape, leading to the desired sensitivity. Validation results show good intra-assay (12.3%CV) and inter-assay (3.1%CV) precisions, and good assay accuracy (+/-7.6%Dev). Recovery was high (approximately 80%), however, the large volume of plasma used did result in a considerable matrix effect (approximately 0.45) which was well compensated by the analog internal standard. The method was applied to sample analysis of a Phase I clinical study.
Zhang
Duxi
D
Bristol-Myers Squibb Company, Research & Development, Bioanalytical Sciences, Princeton, NJ 08543, USA. duxi.zhang@bms.com
Fu
Yunlin
Y
Gale
Jane P
JP
Aubry
Anne F
AF
Arnold
Mark E
ME
D000998:Antiviral Agents / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D005615:Freezing; D006147:Guanine / Q000031:analogs & derivatives* / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D011786:Quality Control; D012015:Reference Standards; D052616:Solid Phase Extraction; D013048:Specimen Handling; D021241:Spectrometry, Mass, Electrospray Ionization
D000998:Antiviral Agents; D007202:Indicators and Reagents; C413685:entecavir; D006147:Guanine