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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 49; no. 4; pp. 1027 - 1033
• Main Authors: Zhang, Duxi, Fu, Yunlin, Gale, Jane P., Aubry, Anne F., Arnold, Mark E.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2009; Elsevier
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Antiviral Agents - blood; Antiviral drug; Biological and medical sciences; Chromatography, High Pressure Liquid; Drug Contamination; Entecavir; Freezing; Fundamental and applied biological sciences. Psychology; General pharmacology; Guanine - analogs & derivatives; Guanine - blood; Hepatitis B virus; High-pH mobile phase; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; LC–MS/MS; Mass Spectrometry; Medical sciences; pg/mL; Pharmacology. Drug treatments; Quality Control; Reference Standards; Solid Phase Extraction; Specimen Handling; Spectrometry, Mass, Electrospray Ionization; Solid phase extraction; Antiviral drug; LC–MS/MS; pg/mL; High-pH mobile phase; Entecavir; Human; Biological fluid; HPLC chromatography; Determination; Mobile phase; Chemical enrichment; Blood plasma; LC-MS/MS; Sample preparation; Mass spectrometry MS/MS; pH; Nucleoside analog; Antiviral; Quantitative analysis
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2009.02.003

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 (∼80%), however, the large volume of plasma used did result in a considerable matrix effect (∼0.45) which was well compensated by the analog internal standard. The method was applied to sample analysis of a Phase I clinical study.