Development of high performance liquid chromatography ropivacaine serum concentration determination method

Local anesthetics dates back to 18th century, and many different regional anesthetics methods have been developed since then. Nowadays local anesthesia is widely used to reduce pain during or after surgery, specially after knee surgery which is one of most painful. Nevertheless, It is known that if local anesthesia drugs gets into blood circulation, they can potentially cause life threatening complications, one such drug is ropivacaine. Therefore, therapeutic drug monitoring can help assess patients safety. The objective of this work is to develop HPLC (high performance liquid chromatography) method to determine ropivacaine serum levels of patients during and after surgery. The patients received ropivacaine via N. Femoralis block before knee surgery or via infiltration anesthesia during the surgery. The blood samples were taken from patients using EDTA anticoagulated vacutainer, before anesthesia, 10, 30, 60 and 120 min. after. The serum was obtained via centrifugation and 0.5 ml were taken for analysis. Prior to analysis, the centrifuged samples were kept at – 80 ℃. Ropivacaine was extracted using diethyl ether after basification with KOH. Afterwards, the ether layer was evaporated, and the dry residue redissolved in HPLC mobile phase. HPLC analysis was done on Thermo Ultimate 3000 HPLC-UV system equipped with ascentis C18 column. The calibration was done by spiking pooled, blank patient plasma with diluted ropivacaine hydrochloride. The analytical method is capable to determine ropivacaine serum concentration starting from approximately 0.05 up to 5 µg/ml with excellent linearity coefficient R2 = 0.999. Ropivacaine plasma concentrations ranges from undetected up to 1.2 µg/ml, the peak concentration is reached after approximately 1-2 hours. The current analytical method provides valuable information on patient ropivacaine serum concentration levels. The currently gathered data, comparing prementioned ropivacaine administration methods, suggest that ropivacaine serum levels do not surpass venous toxicity threshold of 2.2 µg/ml described by literature.