HPLC method for determination of fluorescence derivatives of cortisol, cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids.
11Beta-hydroxysteroid dehydrogenase isoform 2 (11beta-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11beta-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard--prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C(18) cartridges. The enzymatic hydrolysis of conjugated steroids was provided using beta-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0-1000.0 ng mL(-1), for allo-THF and THE + allo-THE 10.0-1000.0 ng mL(-1). LOD (S/N=3:1) for all analytes amounted 3.0 ng mL(-1). Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0-12.1% and 9.2-14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0-174.5 ng mL(-1) and 17.4-35.9 ng mL(-1), respectively. Free urinary steroids were in the ranges: 12.0-54.1 microg/24 h (UFF) and 37.8-76.2 microg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11beta-HSD2 activity in man.
Główka
Franciszek K
FK
Department of Physical Pharmacy and Pharmacokinetics, Karol Marcinkowski University of Medical Sciences in Poznań, 6 Swiecickiego Street, 60-781 Poznań, Poland. glowka@ump.edu.pl
Kosicka
Katarzyna
K
Karaźniewicz-Łada
Marta
M
D001826:Body Fluids / Q000737:chemistry*; D002021:Buffers; D002138:Calibration; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003348:Cortisone / Q000032:analysis* / Q000097:blood / Q000652:urine; D005453:Fluorescence; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis* / Q000097:blood / Q000652:urine; D006868:Hydrolysis; D057230:Limit of Detection; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D013760:Tetrahydrocortisol / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood / Q000652:urine; D013761:Tetrahydrocortisone / Q000032:analysis* / Q000097:blood / Q000652:urine; D013997:Time Factors
D002021:Buffers; C006461:allotetrahydrocortisol; D013761:Tetrahydrocortisone; D013760:Tetrahydrocortisol; D005966:Glucuronidase; D003348:Cortisone; D006854:Hydrocortisone