A high-performance liquid chromatography electrospray tandem mass spectrometry analysis of cortisol and metabolites in placental perfusate

A reversed-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry assay (HPLC-ESI-MS/MS) was developed to quantitate cortisol, cortisone, 20 alpha- and beta-dihydrocortisol, 20 alpha- and beta-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyze perfusate from the isolated human placental lobule for cortisol and its metabolites. Analytes were prepared from the perfusion medium using C18 solid-phase extraction cartridges. The internal standard for the analyses was 6 alpha-methylprednisolone. Chromatography was performed on a Novapak C18 column at ambient temperature using 53% methanol and 47% 10 mM ammonium formate buffer (pH 4.0) as mobile phase, at a flow rate of 80 mu L/min. A PE-SCIEX API III triple quadrupole instrument was used for mass spectrometric detection. An ionspray (pneumatically assisted electrospray) interface was used in negative and positive ionization mode. The assay was linear over the range 100-2000 mu g/L for each analyte. The instrumental limit of detection was 50 pg. Assay imprecision at 400 and 800 mu g/L was less than or equal to 10% (total coefficient of variation). Accuracy ranged between 83.2% for 20 beta-dihydrocortisone to 102.6% for cortisone. Recovery of 1000 mu g/L analyte ranged from 91.3% for cortisone to 109.7% for tetrahydrocortisol. (C) 1997 Academic Press.