Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults |
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Authors: | Fanelli Flaminia Belluomo Ilaria Di Lallo Valentina D Cuomo Gaia De Iasio Rosaria Baccini Margherita Casadio Elena Casetta Bruno Vicennati Valentina Gambineri Alessandra Grossi Gabriele Pasquali Renato Pagotto Uberto |
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Affiliation: | a Endocrinology Unit and Centre of Applied Biomedical Research, Department of Clinical Medicine, S.Orsola-Malpighi Hospital, Alma Mater Studiorum, University of Bologna, Italy b Central Laboratory, S.Orsola-Malpighi Hospital, Bologna, Italy c AB Sciex, 20052 Monza, Italy |
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Abstract: | BackgroundThe simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.Methods0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.ResultsOf the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone > 1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone < 1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.ConclusionOur ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations. |
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Keywords: | ID-LC-MS/MS, isotopic dilution-liquid chromatography-tandem mass spectrometry DOC, deoxycorticosterone DHEA, dehydroepiandrosterone IS, internal standard APCI, atmospheric pressure chemical ionization MRM, multiple reaction monitoring GC-MS, gas chromatography-mass spectrometry DHEA-S, DHEA-sulphate BSA, bovine serum albumin HPLC, high pressure liquid chromatography SPE, solid phase extraction QC, quality control BMI, body mass index CAD, collision activated dissociation CUR, curtain gas LLOQ, lower limit of quantification S/N, signal to noise ratio LOD, limit of detection IR, ion ratio IQR, interquartile range CI, confidence interval MW, molecular weight RT, retention time DP, declustering potential CE, collision energy CXP, cell exit potential Sy/x, standard deviation of residuals M, males F, females pre-M, pre-menopausal females post-M, post-menopausal females s.d., standard deviation |
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