A modification of the existing spectrophotometric kinetic method for the determination of pancuronium bromide (PCBr), based on pooled human serum cholinesterase (ChE, EC 3.1.1.8 acylcholine acylhydrolase) inhibition, was developed. Butyrylthiocholine iodide (concentration 1.667 mmol/L) was used as substrate and determination was performed at pH 7.6. Essential basic kinetic parameters were also determined: Michaelis-Menten's constant KM=0.33 mmol/L, maximal reaction rate Vmax=42.29 micromol/L min, inhibition constant KI=0.34 micromol/L, and IC50=0.235 micromol/L. Linear dependence between the reaction rate and the inhibitor concentration exists in PCBr concentration range 8.29-265.28 nmol/L, which corresponds to the real sample concentrations from 0.166 to 5.306 micromol/L. The method detection limit was established to be 1.86 nmol/L and the quantification limit was 6.18 nmol/L. Precision of the method was tested for three pancuronium concentrations (16.58, 99.48, and 198.96 nmol/L). The relative standard deviation (RSD) was in the range 0.78-5.13%. Accuracy was examined by the standard addition method. The influence of substances usually present in serum and urine on the reaction rate was determined. The method developed was applied for PCBr determination in spiked serum and urine samples and in the urine taken during surgery. The method was proven to have good sensitivity, accuracy, and precision and can be considered suitable for clinical practice.

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