Determination of Pyridostigmine in Human Plasma by High-Performance Liquid Chromatography

An easy to perform, specific, reproducible and sensitive high performance liquid chromatographic (HPLC) method to measure pyridostigmine concentration in human plasma was developed and validated. Sample clean-up consists of ion-pair extraction into dichloromethane in the presence of neostigmine as internal standard, followed by back extraction into an aqueous phase. Mean recovery of 110% (with a standard deviation of 10%) was determined for concentrations of 5 - 100 ng/ml. Chromatography on a 125·4 mm CN-propyl column using a mobile phase composed of 10% acetonitrile in 3.5×10 −4 M NaH 2 PO 4 and UV detection at 270 nm, yields clean chromatograms without any interferences from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection (LD) of 3 ng/ml, with %CV (precision) and bias (accuracy) ≥ 10% for concentrations in the range of 0-100 ng/ml. The method is being used in human pharmacokinetic studies of oral dosage forms of pyridostigmine.