title: Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.
abstract: An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.
authors.affiliation: Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, United States. jenny.y.zhang@Pfizer.com
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dawn
authors.initials: D
authors.lastname: Dufield
authors.forename: Jon
authors.initials: J
authors.lastname: Klover
authors.forename: Wenlin
authors.initials: W
authors.lastname: Li
authors.forename: Gabriella
authors.initials: G
authors.lastname: Szekely-Klepser
authors.forename: Christopher
authors.initials: C
authors.lastname: Lepsy
authors.forename: Nalini
authors.initials: N
authors.lastname: Sadagopan
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006152:Cyclic GMP / Q000097:blood* / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D006152:Cyclic GMP