LC/MS/MS High Sensitivity Bioanalytical Method: Entecavir in Human Plasma

Hepatitis B virus (HBV) is a deoxyribonucleic acid (DNA) virus that causes both acute and chronic infection of the liver in human. Entecavir used as an oral antiviral drug is one of the FDA approved drugs for treatment of HBV infection. Entecavir is a reverse transcriptase inhibitor which prevents the hepatitis B virus from multiplying and reduces the amount of virus in the body by acting like a deoxyguanosine analogue that inhibit the activities of HBV polymerase with reverse transcription, DNA replication and transcription in the viral replication process. Bioanalyses of entecavir for pharmacokinetics and bioequivalence studies have been reported, which are carried out mostly on LC/MS/MS with liquid-liquid extraction (LLE) or solid phase extraction (SPE) method for high sensitivity and selectivity. The reliability of a bioassay depends on the performance of LC/MS/MS system employed and the method of sample pre-treatment. With rapid progress of interface and triple quadrupole mass spectrometry techniques, simpler sample pre-treatment without SPE may be adopted to achieve not only reliable quantitative results, but also higher throughput and lower running cost. Here we describe a simple, fast and high sensitivity bioanalytical method for quantitative determination of entecavir in human plasma on Shimadzu LCMS-8060 system with a heated ESI and coupled with UHPLC. The high sensitivity and robust interface design of the system allow the use of protein-precipitation only in plasma pre-treatment and achieve a LLOQ of 40 pg/mL in plasma or 10 pg/mL in the injection solution after pre-treatment of plasma sample with 1uL injection volume.