Cholinesterase inhibition based determination of pancuronium bromide in biological samples

Pancuronium bromide (PCBr) inhibition effect on enzyme cholinesterase from pooled human serum (Che, EC 3.1.1.8 acylcholine acylhydrolase) was used for development of a spectrophotometric kinetic method for PCBr determination in human serum and urine. Optimal conditions for the basic and inhibitor reactions were established: pH=7.7 and substrate concentration c(benzoylcholine chloride)=1.33 mmol/L. Kinetic parameters were also determined: Michaelis-Menten's constant K(M)=0.40 mmol/L, maximal reaction rate V(max)=52.2 micromol/L min, inhibition constant K(i)=0,56 micromol/L and IC(50)=1.31 micromol/L. Linear dependence between the reaction rate and inhibitor concentration exists in PCBr concentration range 8.20-68.25 nmol/L, which corresponds to the real sample concentrations from 0.328 to 2.730 micromol/L. The method detection and quantification limits were 2.01 nmol/L and 6.67 nmol/L, respectively. Precision of the method was tested for three pancuronium concentrations (10.70, 29.35 and 51.25 nmol/L). Relative standard deviation (RSD) was in the range 0.15-7.45%. Accuracy was examined by standard addition method. Influence of the substances usually present in serum and urine on the reaction rate was tested. The developed method was applied for PCBr content determination in serum model samples, urine model samples and in urine taken during surgery. The method has good sensitivity, accuracy, precision and it is suitable for clinical practice.