Development of a rapid UPLC–PDA method for quantification of lidocaine in human serum

Abstract A simple and rapid ultra-performance liquid chromatography (UPLC) method was developed to quantify lidocaine in human serum, using procainamide as the internal standard (IS). Serum samples were subjected to liquid–liquid extraction (LLE) with diethyl ether under alkaline conditions and analysed using a Waters ACQUITY H-Class UPLC system equipped with a C18 column (100 × 2.1 mm, 1.7 µm). Isocratic separation was performed with acetonitrile and phosphate buffer (12:88, v/v; KH 2 PO 4 containing sulfuric acid and triethylamine) at approximately pH 3.0 and a flow rate of 0.40 mL min −1 . The total run time was 4 min, with mean retention times of 0.77 ± 0.008 min for procainamide and 2.23 ± 0.02 min for lidocaine. Photodiode-array (PDA) detection was set at 277 nm for the IS and switched to 210 nm at 2 min for lidocaine. Calibration was evaluated over 50–5,000 ng mL −1 using 1/x 2 -weighted linear regression, selected from the back-calculated performance across the calibration range. The lower limit of quantification (LLOQ) was 50 ng mL −1 , with a mean measured concentration of 52.59 ± 0.59 ng mL −1 , an RSD of 1.11%, a +5.18% bias, and an accuracy of 105.18%. Intra-day precision ranged from 1.4 to 7.0% RSD, with bias from −14.0 to +20.9%. Inter-day precision ranged from 23.0 to 59.1% RSD, with bias from −19.4 to +27.4%. The method showed adequate selectivity and good within-day repeatability, but greater between-day variability and deviations in accuracy were observed at selected concentrations. Further assessment of recovery, carryover, stability, matrix effect, and dilution integrity is required before the method can be considered fully validated for regulated bioanalytical applications.