Analysis of bupivacaine enantiomers in plasma as total and unbound concentrations using LC-MS/MS: Application in a pharmacokinetic study of a parturient with placental transfer

Abstract

Bupivacaine, a drug used in obstetric anesthesia and analgesia, is commercially available as a racemic mixture of the R-bupivacaine and S-bupivacaine enantiomers, which show differences in pharmacokinetics, efficacy and toxicity. Changes in bupivacaine plasma protein binding is of clinical relevance considering its high protein binding (approximately 95%) and its classification as an intermediate hepatic extraction ratio drug (E = 0.38). Furthermore, the plasma protein binding of bupivacaine is also of clinical relevance considering that pregnancy is a physiological condition associated with reduced plasma albumin concentration. Also, different pathological conditions, such as pre-eclampsia, can reduce the maternal plasma protein concentrations and consequently increase the bupivacaine placental transfer. This report describes the development and validation of analytical methods for the sequential analysis of the total and unbound concentrations of bupivacaine enantiomers in human plasma using liquid chromatography coupled to mass spectrometry (LC–MS/MS) with a sensitivity compatible with application in pharmacokinetic studies including placental transfer. Aliquots of 200 μL of plasma or plasma ultra-filtrate were extracted with n-hexane in alkaline medium after the deproteinization of the matrix with acetonitrile and water. The separation of bupivacaine enantiomers was obtained on a Chirex® 3020 chiral stationary phase column using as a mobile phase a mixture of 95% n-hexane:ethanol (80:20, v/v) at a flow rate of 0.8 mL/min. The lower limit of quantification was 0.25 ng of each enantiomer/mL of plasma as the total concentration and 0.125 ng of each enantiomer/mL of plasma as the unbound concentration. The methods were applied to study the pharmacokinetics of bupivacaine enantiomers after the administration of 2.5 mg of 0.5% racemic bupivacaine hydrochloride with 1:200,000 epinephrine via the epidural route to an HIV-positive parturient woman undergoing antiretroviral treatment. The parturient showed lower AUC0−∞ (25.42 vs. 30.57 ng.h/mL) and higher volume of distribution (841.96 vs 655.05 L) and total clearance (98.34 vs 81.79 L/h) for the R-bupivacaine enantiomer. The pharmacokinetics of bupivacaine were enantioselective displaying a lower plasma proportion of the enantiomer R-bupivacaine (AUC(R)/(S) ratio equal to 0.83). The placental transfer was approximately 60% for both bupivacaine enantiomers. The unbound fraction (Fu) for the R-bupivacaine enantiomer was higher (10.84%) than the eutomer S-bupivacaine (6.29%).