A validated enantioselective LC-MS/MS assay for the simultaneous determination of carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma: application to a clinical pharmacokinetic study.
Carvedilol is widely prescribed for the treatment of hypertension, heart failure and left ventricular dysfunction following myocardial infarction. A sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable and efficient bioanalysis of the (R)- and (S)-enantiomers of carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma. Following plasma extraction using supported liquid extraction (SLE) in a 96-well plate format, extracted samples were derivatized with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC). Chromatographic separation was achieved by gradient elution on an ACQUITY UPLC HSS T3 analytical column. The impact of several potentially interfering isobaric metabolites on the quantification of the 4'-hydroxyphenyl metabolite (R)- and (S)-enantiomers was minimized by implementation of a combination of chromatographic and mass spectrometric techniques. Derivatized analytes and stable-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The assay was validated over concentration ranges of 0.200-100 ng/mL for (R)- and (S)-carvedilol and 0.0200-10.0 ng/mL for (R)- and (S)-4'-hydroxyphenyl carvedilol. Intra- and inter-assay precision values for replicate quality control samples were within 11.9% for all analytes during the assay validation. Mean quality control accuracy values were within ±9.4% of nominal values for all analytes. Assay recoveries were high (>76%) and internal standard normalized matrix effects were minimal. The four analytes were stable in human plasma for at least 24 h at room temperature, 89 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of the (R)- and (S)-enantiomers of both carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma in support of a human pharmacokinetic study.
Bristol-Myers Squibb, Research and Development, Analytical and Bioanalytical Development, Route 206 & Province Line Road, Princeton, NJ 08543, USA. michael.furlong@bms.com
Michael T
MT
Furlong
Bing
B
He
William
W
Mylott
Song
S
Zhao
Thomas
T
Mariannino
Jim
J
Shen
Bruce
B
Stouffer
D000284:Administration, Oral; D018674:Adrenergic Antagonists / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics*; D001711:Biotransformation; D002138:Calibration; D002227:Carbazoles / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics*; D000077261:Carvedilol; D056148:Chromatography, Reverse-Phase / Q000592:standards; D004355:Drug Stability; D006801:Humans; D006900:Hydroxylation; D007536:Isomerism; D017879:Isothiocyanates / Q000737:chemistry; D057230:Limit of Detection; D016014:Linear Models; D015394:Molecular Structure; D011412:Propanolamines / Q000008:administration & dosage / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000592:standards; D013696:Temperature
D018674:Adrenergic Antagonists; D002227:Carbazoles; D017879:Isothiocyanates; D011412:Propanolamines; D000077261:Carvedilol; C041943:2,3,4,6-tetra-O-acetylglucopyranosylisothiocyanate