Liquid chromatography tandem mass spectrometry method for the quantification of total and free ropivacaine in human plasma

• Fast e robust method for the simultaneous quantification of both free and total ropivacaine. • Fast and reproducible UHPLC separation, quantification by positive ESI and MRM. • Adoption of stable-isotope-linked internal standard. • Thorough evaluation of method ruggedness in terms on matrix effect and recovery. • Full method validation and application of real human samples. Ropivacaine, a local anaesthetic with a convenient pharmacokinetic/pharmacodynamic profile, deserved attention for several applications. Despite a good safety profile, this drug can still be associated with systemic toxicity, due to erroneous site of injection or due too fast absorption or low volume of distribution. Moreover, ropivacaine is normally bound to α1-acid glycoprotein, and low concentrations of this transport protein can lead to higher-than-normal free concentrations. Since the description of total and free concentrations of Ropivacaine is pivotal to understand the probability of local anaesthetic systemic toxicity (LAST) and the underlying reasons, in this work we developed and validated a simple method for their quantification in human plasma. Plasma samples underwent protein precipitation for the determination of total Ropivacaine concentrations and an unltrafiltration process with Centrifree® filters, to isolate the free concentration. Reverse phase separation was obtained with a gradient run of water and acetonitrile:methanol 60:40, both added with 0.1 % formic acid, on a Kinetex® biphenyl 2.1 × 100 mm, 2.6 um column. Quantification was performed in ESI+ MRM mode and internal standardization with Stable-Isotope-Linked Ropivacaine. The method was validated following EMA ICH guidelines and showed satisfactory performance in terms of accuracy and precision (both bias and coefficient of variation) lower than 10 %, linearity, matrix effect, recovery, sensitivity and specificity. Ultrafiltration was not associated with drug loss due to adsorption to the molecular filters. Finally, the method was applied on samples from patients enrolled in a clinical study, confirming its clinical suitability for the analysis of Ropivacaine in human plasma.