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A strategy for high‐throughput analysis of levosimendan and its metabolites in human plasma samples using sequential negative and positive ionization liquid chromatography/tandem mass spectrometric detection
Author(s) -
Zhang Jun,
Gage Eric M.,
Ji Qin C.,
ElShourbagy Tawakol A.
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3046
Subject(s) - chemistry , chromatography , ammonium acetate , levosimendan , bioanalysis , liquid chromatography–mass spectrometry , sample preparation , high performance liquid chromatography , chemical ionization , acetonitrile , atmospheric pressure chemical ionization , elution , mass spectrometry , selected reaction monitoring , tandem mass spectrometry , ionization , medicine , heart failure , ion , organic chemistry
Levosimendan (Simdax®) is an approved drug in approximately 40 countries and currently in phase III clinical studies in the USA and Europe. An accurate, high‐throughput and rugged assay is critical to support these clinical trials. Due to the mechanism of drug metabolism, the drug and its active metabolites often have significant differences in their chemical properties. In order to achieve high assay throughput and low sample volumes, a single bioanalytical assay for the drug and its metabolites is preferred. However, this need may prevent the optimization of both high‐performance liquid chromatography (HPLC) and mass spectrometric ionization conditions. The chemical properties of levosimendan are significantly different from those of its two active metabolites, OR‐1855 and OR‐1896. Here, we present a novel strategy for high‐throughput analysis of levosimendan and its metabolites. A 96‐well liquid/liquid extraction procedure was developed for sample preparation. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) system with two separate mobile phases, shared backwash solvent and conditioning solvent, was developed to perform sequential LC separation for levosimendan and the metabolites. Levosimendan was eluted by 5 mM ammonium acetate in 33.3% acetonitrile and detected using negative ionization mode MS/MS monitoring. The metabolites were eluted by 5 mM ammonium acetate and 0.2% acetic acid in 20% acetonitrile and detected with positive ionization mode MS/MS monitoring. The method has been demonstrated to have excellent precision and accuracy, with high assay ruggedness during method validation and clinical sample analysis. The linear dynamic ranges were approximately 200–50 000 pg/mL for levosimendan and approximately 500–130 000 pg/mL for both metabolites. The coefficient of determination (r 2 ) for all analytes was greater than 0.9985. The intra‐assay %CVs for QC samples were from 0.9% to 2.0% for levosimendan, 0.9% to 3.2% for OR‐1855, and 0.4% to 4.9% for OR‐1896. The inter‐assay %CVs for QC samples were from 1.2% to 1.8% for levosimendan, 1.3% to 2.7% for OR‐1855, and 1.4% to 3.4% for OR‐1896. The mean % biases for QC samples were from 1.5% to 5.5% for levosimendan, −1.4% to 2.6% for OR‐1855, and −0.3% to 4.5% for OR‐1896. By using a single extraction approach coupled with sequential LC/MS/MS analysis for levosimendan and its metabolites, the assay maintained high throughput and low sample volume usage. Copyright © 2007 John Wiley & Sons, Ltd.