Bristol-Myers Squibb, Research and Development, Analytical and Bioanalytical Development, Route 206 & Province Line Road, Princeton, NJ 08543, USA.
J Pharm Biomed Anal. 2012 Nov;70:574-9. doi: 10.1016/j.jpba.2012.05.026. Epub 2012 May 30.
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.
卡维地洛被广泛用于治疗高血压、心力衰竭和心肌梗死后左心室功能障碍。本研究开发并验证了一种灵敏且可靠的液相色谱-串联质谱（LC-MS/MS）分析方法，可用于可靠且高效地分析人血浆中卡维地洛（R）-和（S）-对映异构体及其具有药理活性的 4'-羟苯基代谢物。采用 96 孔板格式的固相萃取（SLE）进行血浆提取后，用 2,3,4,6-四-O-乙酰基-β-D-吡喃葡萄糖基异硫氰酸酯（GITC）衍生提取样品。通过在 ACQUITY UPLC HSS T3 分析柱上进行梯度洗脱实现色谱分离。通过实施色谱和质谱技术的组合，最大限度地减少了几种潜在干扰的同重异构体代谢物对 4'-羟苯基代谢物（R）-和（S）-对映异构体定量的影响。衍生化分析物和稳定标记的内标通过正离子电喷雾串联质谱检测。该方法在（R）-和（S）-卡维地洛浓度范围为 0.200-100ng/mL，（R）-和（S）-4'-羟苯基卡维地洛浓度范围为 0.0200-10.0ng/mL 进行了验证。在验证过程中，所有分析物的日内和日间精密度的重复性质控样品的精度值均在 11.9%以内。所有分析物的平均质控准确度值均在名义值的±9.4%以内。分析物回收率高（>76%），内标归一化基质效应最小。四种分析物在室温下至少 24 h、-20°C 下 89 天和-70°C 下以及至少五个冻融循环后，在人血浆中稳定。验证后的分析方法成功应用于人血浆中卡维地洛及其具有药理活性的 4'-羟苯基代谢物的（R）-和（S）-对映异构体的定量分析，支持人体药代动力学研究。
