A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the determination of endogenous cyclic nucleotides in rat brain

A robust high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3 ',5 '-cyclic adenosine -C-13(5) monophosphate (C-13(5)-cAMP) and 3 ',5 '-cyclic guanosine-C-13 N-15(2) monophosphate ((C5N2)-C-13-cGMP) were used as surrogate analytes to measure endogenous 3 ',5 '-cyclic adenosine monophosphate (cAMP) and 3 ',5 '-cyclic guanosine monophosphate (cGMP). Pre weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC-MS/MS. The calibration curves for both analytes ranged from 5 to 2000 ng/g and showed excellent linearity (r(2) > 0.996). Relative surrogate analyte-to-analyte LC-MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for C-13(5)-CAMP and (C15N2)-C-13-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for (13)C(5)cAMP and (13)C(15)N(2-)cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies. (C) 2017 The Authors. Published by Elsevier B.V.