LC-ESI-MS/MS method for the quantification of entecavir in human plasma and its application to bioequivalence study

Siddharth Institute of Pharmacy, Nalanda Educational Society, Guntur, Andhrapradesh, India. baluchalla
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (Impact Factor: 2.73). 02/2011; 879(11-12):769-76. DOI: 10.1016/j.jchromb.2011.02.023
Source: PubMed


Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used for a quantitative estimation of entecavir (EV) in human plasma using lamivudine (LM) as internal standard (IS). The method herein described is simple, sensitive, and specific. Chromatographic separation was performed on XBridge-C18, 4.6 mm × 50 mm, 5-μm column with an isocratic mobile phase composed of 10 mM ammonium hydrogen carbonate (pH 10.5):methanol (85:15 v/v), pumped at 0.3 ml/min. EV and LM were detected using proton adducts at m/z 278.1→152.1 and 230.2→112.0 in multiple reaction monitoring (MRM) positive mode. Solid phase extraction method was employed in the extraction of EV and LM from the biological matrix. This method was validated over a linear concentration range of 50.0-20000.0 pg/ml with a correlation coefficient (r) ≥0.9983. Intra and inter-day precision of EV was found within the range of 1.2-4.2 for EV and 4.4-4.5 for LM. EV was stable throughout three freeze/thaw cycles, bench top and postoperative studies. This method was successfully used in the analysis of plasma samples following oral administration of EV (0.5 mg) in 26 healthy human volunteers.

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Available from: Balasekhara Reddy Challa, May 22, 2014
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    • "To date, several analytical methods are available for the quantification of these drugs in human plasma but none of them is able to simultaneously quantify all the compounds in the same run [11] [12] [13] [14] [15] [16] [17] [18]. Moreover, the vast majority need a long process, expensive and cumbersome solid phase extractions (SPE) or salting-out processes [11] [19]. For these reasons, the aim of this work was to develop and validate a reliable, but still cheap and simple, chromatographic method for the quantification of these drugs in plasma samples from HBV+ patients, eligible for a routine use. "
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    ABSTRACT: Hepatitis B infection affects two billion people worldwide and 350 million of these are chronically infected. Chronic hepatitis B virus is one of the most important cause of mortality and morbidity worldwide. If it is left untreated, about one-third of affected people will develop progressive and possibly fatal liver disease, like hepatic cirrhosis and primary hepatocellular carcinoma. Currently, five nucleos(t)ide analogs are approved for the treatment of chronic HBV infection. They are: lamivudine, adefovir dipivoxil, telbivudine, entecavir and tenofovir disoproxil fumarate. In this work, we developed and validated an UPLC-Tandem mass spectrometry assay method capable of monitoring lamivudine, telbivudine, tenofovir and entecavir plasma concentrations. Both standards and quality controls (high, medium and low) were prepared in human plasma. Each sample was added with internal standard (5'amino-5'deoxy-thymidine) and then drugs were extracted through a protein precipitation protocol with acetonitrile+0.1% formic acid and then dried. The extracts were resuspended in water and then injected into the chromatographic system. The chromatographic separation was performed on an Acquity UPLC HSS T3 1.8μm 2.1×150mm column, with a gradient of water and acetonitrile, both added with formic acid (0.05%). Accuracy, intra-day and inter-day precision at quality controls levels fitted all FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 30 HBV+ patients with good results. This simple analytical method could represent a useful tool for the management of anti-HBV therapy. Copyright © 2015 Elsevier B.V. All rights reserved.
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    ABSTRACT: Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1ml, 30mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).
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    ABSTRACT: A direct spectrophotometric method for the determination of Entecavir is developed. Entecavir is a guanine analogue that is used in the treatment of hepatitis B. The absorbance of the pure drug was measured at wavelength 253 nm. The method was found linear from 2.5-40 μg mL-1 (r2, 0.999). The RSD was 1.1% and accuracy was 99%. The validation results and statistical data demonstrate that the method is reliable, simple, cost effective and reproducible and has an importance in quality assurance of Entecavir analysis.
    Full-text · Article · Dec 2012 · Journal- Chemical Society of Pakistan
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