Standardized LC-MS/MS based steroid hormone profile-analysis.
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), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-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 (R(2)>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.
BIOCRATES Life Sciences AG, Innrain 66, A-6020 Innsbruck, Austria. therese.koal@biocrates.com
Therese
T
Koal
Diane
D
Schmiederer
Hai
H
Pham-Tuan
Cornelia
C
Röhring
Manfred
M
Rauh
D000963:Antimetabolites / Q000494:pharmacology; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D005260:Female; D006728:Hormones / Q000097:blood*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods / Q000592:standards; D013256:Steroids / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
D000963:Antimetabolites; D006728:Hormones; D013256:Steroids; D008797:Metyrapone