title: Development and validation of a sample stabilization strategy and a UPLC-MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF).
abstract: A UPLC-MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core-Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC-MS/MS. This UPLC-MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9±0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC-MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025-5 ng/mL for ACh and 0.05-10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9-12.3% CV and -10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0-16.0% CV and -5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.
authors.affiliation: Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Groton Laboratories, Groton, CT 06340, United States. jenny.y.zhang@pfizer.com
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Zhang
authors.forename: F David
authors.initials: FD
authors.lastname: Tingley
authors.forename: Elaine
authors.initials: E
authors.lastname: Tseng
authors.forename: Max
authors.initials: M
authors.lastname: Tella
authors.forename: Xin
authors.initials: X
authors.lastname: Yang
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Groeber
authors.forename: Jianhua
authors.initials: J
authors.lastname: Liu
authors.forename: Wenlin
authors.initials: W
authors.lastname: Li
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schmidt
authors.forename: Rick
authors.initials: R
authors.lastname: Steenwyk
mesh_terms: D000109:Acetylcholine / Q000134:cerebrospinal fluid* / Q000378:metabolism / Q000493:pharmacokinetics; D000818:Animals; D001572:Benzofurans / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000494:pharmacology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000077265:Donepezil; D004355:Drug Stability; D006632:Histamine / Q000134:cerebrospinal fluid* / Q000378:metabolism / Q000493:pharmacokinetics; D057927:Hydrophobic and Hydrophilic Interactions; D007093:Imidazoles / Q000134:cerebrospinal fluid* / Q000493:pharmacokinetics; D007189:Indans / Q000494:pharmacology; D008297:Male; D008761:Methylhistamines / Q000134:cerebrospinal fluid / Q000493:pharmacokinetics; D010880:Piperidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001572:Benzofurans; D002800:Cholinesterase Inhibitors; D007093:Imidazoles; D007189:Indans; D008761:Methylhistamines; D010880:Piperidines; C406662:prucalopride; C002667:methylimidazoleacetic acid; D006632:Histamine; D000077265:Donepezil; C020404:tele-methylhistamine; D000109:Acetylcholine