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

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, 13C10,15N5-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 (R2 = 0.197, P = 0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5–20 ng/mL. The LC–MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.