Design of a stable isotope dilution gas chromatography/mass spectrometric assay for cAMP: Comparison with standard protein-binding and radioimmunoassay methods

Abstract

A stable isotope dilution assay is presented in which picomole quantities of cAMP can be determined with high precision and selectivity using gas chromatography and electron impact mass spectrometry with multiple ion detection techniques. Using synthetic [2,8- 2H 2,6- 15N]-cAMP as the internal standard, suitable specificity was obtained by monitoring the (MCH 3) + fragment ions of the trimethylsilyl derivatives of cAMP and the internal standard at m z 530 and m z 533, respectively. The sensitivity of the assay as judged from the lower limit of detection of the mass spectrometer was 3.0 pmol. Rat liver and human urine cAMP levels were assayed using gas chromatography/mass spectrometry and were compared with levels determined by protein-binding assays and radioimmunoassays for the same samples. The intraassay coefficients of variation of the gas chromatography/mass spectrometry assay were 5.3% for the rat liver sample (cAMP level 832 pmol/g) and 6.0% for the urine sample (cAMP level 2.50 μmol/liter). Comparison of the levels of cAMP determined by the three assay methods showed correlation to within 10% variation.