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-t andem mass spectrometry assay (HPLC-ESI-MS/MS) was developed to quanti tate cortisol, cortisone, 20 alpha- and beta-dihydrocortisol, 20 alpha - and beta-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortiso ne. The technique was used to analyze perfusate from the isolated huma n placental lobule for cortisol and its metabolites. Analytes were pre pared from the perfusion medium using C18 solid-phase extraction cartr idges. The internal standard for the analyses was 6 alpha-methylpredni solone. Chromatography was performed on a Novapak C18 column at ambien t 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 A PI III triple quadrupole instrument was used for mass spectrometric de tection. An ionspray (pneumatically assisted electrospray) interface w as used in negative and positive ionization mode. The assay was linear over the range 100-2000 mu g/L for each analyte. The instrumental lim it 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 cortis one. Recovery of 1000 mu g/L analyte ranged from 91.3% for cortisone t o 109.7% for tetrahydrocortisol. (C) 1997 Academic Press.