Standardized LC-MS/MS based steroid hormone profile-analysis

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• 作者：Therese Koal^a ; ^{therese.koal@biocrates.com} ; Diane Schmiederer^a ; Hai Pham-Tuan^a ; Cornelia Rö ; hring^a ; Manfred Rauh^b
• 关键词：Steroid hormones ; High performance liquid chromatography&ndash ; tandem mass spectrometry (HPLC&ndash ; MS/MS) ; Endocrinology ; Kit ; Standardization ; Metyrapone
• 刊名：The Journal of Steroid Biochemistry and Molecular Biology
• 出版年：2012
• 期刊代码：172_09600760
• 类别：med
• 出版时间：April, 2012
• 卷：129
• 期：3-5
• 页码：129-138
• 文件大小：1238 K

摘要

In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17尾-estradiol (E2), estrone (E1), etiocholanolone, 17伪-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R² > 0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8%and 8.2-18.6%for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5%and 109.3-128.2%for 11-deoxycorticosterone, inter-day 91.4-117.2%and 102.3-137.1%for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.