title: 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.
abstract: 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 (100 × 4.6 mm, 5 µm) 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 μL 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 223.0 → 72.0 and 306.0 → 140.0 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 (r ≥ 0.998). 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.
authors.affiliation: Department of Pharmacy, Guangdong Pharmaceutical University, No. 280 Outer Ring East Road, Guangzhou Higher Education Mega Center, Guangdong, 510006, People's Republic of China; Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai, 200433, People's Republic of China; Shanghai Key Laboratory for Pharmaceutical Metabolite Research, No. 325 Guohe Road, Shanghai, 200433, People's Republic of China.
authors.forename: Di
authors.initials: D
authors.lastname: Cao
authors.forename: Wenxue
authors.initials: W
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Zhao
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Ye
authors.forename: Fanlu
authors.initials: F
authors.lastname: Sun
authors.forename: Jinying
authors.initials: J
authors.lastname: Li
authors.forename: Fenyun
authors.initials: F
authors.lastname: Song
authors.forename: Guorong
authors.initials: G
authors.lastname: Fan
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D004355:Drug Stability; D016014:Linear Models; D009388:Neostigmine / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009388:Neostigmine
keywords: Beagle dogs; LC-MS/MS; Sirocco 96-well filtration plates; neostigmine; pharmacokinetic study