SIMULTANEOUS DETERMINATION OF GLUCOCORTICOIDS IN PLASMA OR URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH PRECOLUMN FLUOROMETRIC DERIVATIZATION BY 9-ANTHROYL NITRILE

A new method for simultaneous determination of glucocorticoids (GCs) i n plasma or urine by high-performance liquid chromatography (HPLC) wit h fluorimetric detection has been developed, Following extraction with ethyl acetate using a reversed-phase disposable cartridge, the six GC s [cortisol (F), cortisone (E), prednisolone (PL), prednisone (PN), 6 beta-hydroxycortisol (6 beta-OHF) and 6 beta-hydroxyprednisolone (6 be ta-OHP)] and an internal standard (6 beta-hydroxycotortisone) were der ivatized by treatment with 9-anthroyl nitrile (9-AN) in a mixture of b asic catalysts (triethylamine and quinuclidine) to give the fluorescen t esters through the 21-hydroxyl group. The GC derivatives so obtained were then cleaned by a straight-phase disposable cartridge and chroma tographed on a straight-phase column with an isocratic HPLC technique. The fluorescence derivatives of the GCs, including the internal stand ard, were separated as clear single peaks and no interfering peaks wer e observed on the chromatograms. The lower limits of detection for F, E, PL and PN in plasma or urine were 0.1 ng/ml and those for 6 beta-OH F and 6 beta-OHP in plasma or urine were 0.5 ng/ml, at a signal-to-noi se ratio of 3. The analytical recovery of known amounts of the GCs add ed to plasma or urine were almost 100%. This method can be applied to the determination of plasma or urinary F in renal transplant patients who received PL and can be applied for other metabolic investigations in relation to the change in blood pressure via 11 beta-hydroxysteroid dehydrogenase or in hepatic metabolizing via CYP3A4. (C) 1998 Elsevie r Science B.V.