Development And Validation Of A Rapid And High-Sensitivity Liquid Chromatography-Tandem Mass Spectrometry Assay For The Determination Of Neostigmine In Small-Volume Beagle Dog Plasma And Its Application To A Pharmacokinetic Study

A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100x4.6 mm, 5 mu m) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 L into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.072.0 and 306.0140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs. Copyright (c) 2013 John Wiley & Sons, Ltd.