Department of Physical Pharmacy and Pharmacokinetics, Karol Marcinkowski University of Medical Sciences in Poznań, 6 Swiecickiego Street, 60-781 Poznań, Poland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Feb 1;878(3-4):283-9. doi: 10.1016/j.jchromb.2009.11.016. Epub 2009 Nov 26.
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.
11β-羟类固醇脱氢酶同工酶 2（11β-HSD2）负责将皮质醇（F）转化为无活性的皮质酮（E）。其活性的紊乱可导致高血压。为了估计 11β-HSD2 的活性，除了 F 和 E 之外，还应该测定它们的四氢化物（THF、THE）以及差向四氢化物（allo-THF、allo-THE）代谢物。本研究描述了一种用于定量测定血浆和尿液（总游离和游离）内源性糖皮质激素（GCs）及其代谢物的 HPLC-FLD 方法。在 pH7.4 下用二氯甲烷提取后，用 9-蒽基腈将 GCs（F、E、THF、allo-THF、THE、allo-THE 和内标——泼尼松龙）衍生化，并使用 C（18）柱通过 SPE 进行纯化。使用β-葡萄糖醛酸酶进行结合类固醇的酶水解。研究了有机碱对类固醇 9-AN 衍生化的影响。在存在 10.0%三乙胺（TEA）和 0.1%奎宁啶（Q）混合物的情况下，获得了衍生化的最佳产率。在 Chromolith RP-18e 整体柱上完成了色谱分离。该方法经过验证。F、E 和 THF 的校准曲线在 5.0-1000.0ngmL（-1）范围内呈线性，allo-THF 和 THE+allo-THE 的校准曲线在 10.0-1000.0ngmL（-1）范围内呈线性。所有分析物的检测限（S/N=3:1）为 3.0ngmL（-1）。GCs 的回收率超过 90%。该方法精确、准确，日内和日间精密度分别为 3.0-12.1%和 9.2-14.0%，准确度范围为 0.2-15.1%。该方法用于估计血浆和尿液中的内源性 GCs。F 和 E 的血浆水平分别在 133.0-174.5ngmL（-1）和 17.4-35.9ngmL（-1）范围内。游离尿类固醇的水平分别在 12.0-54.1μg/24h（UFF）和 37.8-76.2μg/24h（UFE）范围内。（THF+allo-THF）/（THE+allo-THE）的比值为 1.01-1.23。所得结果证实了所提出的方法在评估人类 11β-HSD2 活性方面的实用性。
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