Brief communication (Original). Validation of a high-performance liquid chromatography method for the determination of pyridostigmine in plasma

Background: Pyridostigmine is a reversible acetylcholinesterase inhibitor that is used in military medicine as a prophylactic agent against intoxication by nerve agents and in the treatment of myasthenia.

Objective: We developed and validated a high-performance liquid chromatography (HPLC) method for the quantification of pyridostigmine in plasma samples.

Methods: Pyridostigmine was isolated from plasma using one-step protein precipitation by chloroform. The separation was performed on an analytical C18 column (250 × 4.6 mm; 4 μm particle size). The detector wavelength was set at 270 nm. The mobile phase was a mixture of acetonitrile and aqueous solution (15:85 v/v) of sodium-1-hepatene sulfonate and triethylamine, adjusted to pH 3.0 at a flow rate of 1.5 ml min^-1. Hydrochlorothiazide was used as internal standard.

Results: The recovery of drug from plasma samples was above 90%. Using the current method, pyridostigmine (r_t = 7.3) and hydrochlorothiazide (r_t = 6.4) peaks were well resolved. The calibration curve was linear over the concentration range of 15-60 ng ml^-1. The inter- and intraday assay coefficients of variation were found to be less than 8%.

Conclusion: The currently described procedure could be used as a simple, rapid and sensitive tool in bioavailability and bioequivalency investigations for the quantification of pyridostigmine in human plasma samples.