ABSTRACT
A quantitative bioanalytical method involving chemical derivatization, solid phase extraction (SPE) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was developed for the determination of 4-fluorobenzyl chloride (4FBCl) in human plasma. 4FBCl is a volatile and reactive molecule that is very unstable in human plasma. In order to stabilize 4FBCl in plasma samples prior to storage, 4-dimethylaminopyridine (DMAP) was added, forming a stable quaternary amine salt derivative. A three-step weak cation-exchange SPE procedure was then employed to remove excess DMAP. The plasma extracts were analyzed by HPLC/MS/MS using a TurboIonspray interface and multiple reaction monitoring. Unlike 4FBCl, the quaternary amine derivative shows excellent sensitivity in electrospray mass spectrometry. The method was validated over a concentration range of 0.5-500 ng/mL using 45 microL of plasma. The maximum within-run and between-run precision observed in a three-run validation for quality control (QC) samples was 12.5 and 7.6%, respectively. The maximum percentage bias observed at all QC sample concentrations was 11.9%. The method has proven to be robust and compatible with high-throughput bioanalysis.
Subject(s)
Benzyl Compounds/blood , Benzyl Compounds/chemistry , Blood Chemical Analysis/methods , Chromatography, High Pressure Liquid/methods , Microchemistry/methods , Pyridines/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A relatively high-throughput high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method using a chiral derivatization reagent was developed for the quantitative determination of carvedilol enantiomers in human plasma. S-carvedilol and R-carvedilol are extracted from human plasma by protein precipitation using acetonitrile containing racemic [(2)H(5)]-carvedilol as an internal standard. Extracts are then derivatized with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate (GITC) and analysed using HPLC-MS/MS with a TurboIonspray (TIS) interface and selected reaction monitoring. Using 150microL of plasma, the method was validated over a concentration range of 0.2-200ng/mL. The maximum within-run precision observed in a three run quality control was 8.2% for S-carvedilol and 6.7% for R-carvedilol, respectively. The maximum percentage bias observed at all quality control sample concentrations was 9.4% for S-carvedilol and 11.6% for R-carvedilol, respectively. The HPLC-MS/MS method was also compared with a previously developed high-performance LC/fluorescence method by analysing 25 samples containing racemic carvedilol. Based on results obtained, these two methods were found to be equivalent. However, compared with LC/fluorescence method, HPLC-MS/MS method is more sensitive, uses less plasma, and also employs a less time-consuming sample preparation process.
Subject(s)
Carbazoles/blood , Carbazoles/chemistry , Propanolamines/blood , Propanolamines/chemistry , Carvedilol , Chromatography, High Pressure Liquid/methods , Humans , Mass Spectrometry/methods , StereoisomerismABSTRACT
A sensitive and selective chiral high performance liquid chromatographic method was developed for the direct determination of R- and S-warfarin enantiomers in human plasma. The method involved direct injection of human plasma onto a semipermeable surface (SPS) guard column, washing the proteins from the column with aqueous acetonitrile and back flushing the analytes onto a reversed phase ovomucoid silica HPLC column using switching valves. After separation, the analytes were simultaneously detected and quantitated with a fluorometer. The recoveries of R-warfarin from human plasma at 25 and 2500 ng/ml were 98.9% and 88.1%, respectively. The recoveries of S-warfarin at 25 and 2500 ng/ml were 105.4% and 93.9%, respectively. Using 100 microl of human plasma, the lower limit of quantification for both R- and S-warfarins was 25 ng/ml. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 25 to 2500 ng/ml for both enantiomers. Fluorescence chromatograms of drug-free human plasma showed no interfering peaks with retention times similar to those for R- and S-warfarins and the internal standard. Results from a 3-day validation study for both enantiomers demonstrated excellent precision (1.7-9.0%) and accuracy (97-109%) across the calibration range.