Abstract
A stereoselective, sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MSMS) method for the determination of R-acenocoumarol and S-acenocoumarol in human plasma was developed and validated at IPRC bioanalytical labs. The procedure involved solid phase extraction of both enantiomers and their corresponding internal standard. The chromatographic separation was accomplished employing a chiral column and proper mobile phase. Detection was carried out using Waters Micromass® Quattro Premier mass spectrometer in multiple reaction monitoring (MRM) mode using turbo ion spray with negative ionization. The method was validated over a linear range of 0.40 - 40.00 ng/ml for R-acenocoumarol and 0.20 - 20.00 ng/ml for the S-acenocoumarol. Method validation covered different parameters such as linearity, accuracy, precision and stability. The method was successfully applied for the determination of R and S-acenocoumarol in plasma samples of 28 healthy subjects who participated in a pharmacokinetics study.
Highlights
Acenocoumarol, 4-hydroxy-3-[1-(4-nitrophenyl)-3-oxobutyl]-2H-1-benzopyran-2-one (Figure 1), is a vitamin K antagonists that belongs to the group of most frequently used drugs worldwide [1]-[3]
Several analytical techniques have been published for the analysis of acenocoumarol in different matrices, e.g., racemic determination by high performance liquid chromatography (HPLC-UV) in plasma [5] or plasma and urine [6]
Thijssen et al reported in 2001 [7] the use of HPLC-UV stereoselective quantification of the R and Sacenocoumarol in human plasma after derivatization with N-carbobenzoyl-Lproline
Summary
Acenocoumarol, 4-hydroxy-3-[1-(4-nitrophenyl)-3-oxobutyl]-2H-1-benzopyran-2-one (Figure 1), is a vitamin K antagonists that belongs to the group of most frequently used drugs worldwide [1]-[3]. Several analytical techniques have been published for the analysis of acenocoumarol in different matrices, e.g., racemic determination by high performance liquid chromatography (HPLC-UV) in plasma [5] or plasma and urine [6]. One of the reasons is due to the absence of well-documented specific and sensitive validated chiral method for the determination of R- and S-acenocoumarol after oral administration of the drug in a well controlled clinical/pharmacokinetics study. The main aim of our work was to develop and validate a highly specific and sensitive stereoselective liquid chromatographic-mass spectrometric method for the determination of R- and S-acenocoumarol in human plasma, as well, its application for the enantioselective pharmacokinetics evaluation of both enantiomers after oral dosing in human healthy subjects. Six different batches of plasma were obtained from healthy blood donors who were proved to be HIV, hepatitis B & C negative
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