Gu X Q, Fryirs B, Mather L E
Department of Anaesthesia and Pain Management, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW, Australia.
J Chromatogr B Biomed Sci Appl. 1998 Nov 20;719(1-2):135-40. doi: 10.1016/s0378-4347(98)00380-6.
Chiral separation of rac-bupivacaine extracted from blood was achieved with similar limits of detection but using a much simpler sample preparation than reported previously. The simple one-step sample preparation devised was highly robust and efficient and allowed a very high throughput of samples. The high-performance liquid chromatography (HPLC) conditions used gave baseline separation of the enantiomers with high sensitivity. R-(+)-bupivacaine and S-(-)-bupivacaine blood concentrations were determined using a chiral stationary phase (AGP, ChromTech) with diode array detection at 220 nm; this wavelength produced a stable baseline allowing semi-automated analysis. Sample preparation involved addition of internal standard (diphenhydramine), basification of blood, extraction with n-hexane, concentration of the extract to dryness and reconstitution in 0.002 M phosphoric acid. At rac-bupivacaine concentrations of 0.5, 5 and 50 microg/ml in blood, assay accuracy as estimated by coefficients of variation (C.V.s), were 3.3, 1.4, and 1.6%, respectively, for R-(+)-bupivacaine and 3.7, 2.0 and 1.5%, respectively, for S-(-)-bupivacaine. Using 0.6-ml samples, the estimated limits of detection for R-(+)-bupivacaine and S-(-)-bupivacaine were both 15 ng/ml of blood. Calibration curves (n=188) were linear from 0.1 to 50 microg/ml with all correlation coefficients being greater than 0.99. This semi-automated method was applied to studies involving whole body pharmacokinetics with intravenous doses ranging from 12.5 to 350 mg and regional myocardial pharmacokinetics with coronary arterial doses ranging from 2.5 to 12.5 mg. These studies generated approximately 12000 blood samples.
从血液中提取的消旋布比卡因的手性分离得以实现，检测限相似，但使用的样品制备方法比之前报道的要简单得多。所设计的简单一步法样品制备具有高度的稳健性和高效性，且允许非常高的样品通量。所使用的高效液相色谱（HPLC）条件实现了对映体的基线分离且灵敏度高。使用手性固定相（AGP，ChromTech）在220 nm处进行二极管阵列检测来测定R-(+)-布比卡因和S-(-)-布比卡因的血药浓度；该波长产生稳定的基线，允许进行半自动分析。样品制备包括加入内标（苯海拉明）、将血液碱化、用正己烷萃取、将萃取物浓缩至干并在0.002 M磷酸中复溶。在血液中消旋布比卡因浓度为0.5、5和50μg/ml时，以变异系数（C.V.s）估计的R-(+)-布比卡因的测定准确度分别为3.3%、1.4%和1.6%，S-(-)-布比卡因分别为3.7%、2.0%和1.5%。使用0.6 ml样品时，R-(+)-布比卡因和S-(-)-布比卡因的估计检测限均为15 ng/ml血液。校准曲线（n = 188）在0.1至50μg/ml范围内呈线性，所有相关系数均大于0.99。这种半自动方法应用于涉及静脉注射剂量为12.5至350 mg的全身药代动力学研究以及冠状动脉注射剂量为2.5至12.5 mg的局部心肌药代动力学研究。这些研究共产生了约12000份血样。
