Simultaneous steroid measurement by isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS): comparison with routine analysis methods and reference intervals in normal subjects
Flaminia Fanelli, Ilaria Belluomo, Valentina Diana Di Lallo, Gaia Cuomo, Margherita Baccini, Valentina Vicennati, Alessandra Gambineri, Renato Pasquali & Uberto Pagotto
The development of a reliable and rapid method to simultaneously measure serum steroids represents a compelling challenge for modern endocrinology. Immunoassays are very sensitive methods, being widely employed in automated routine laboratories, lacking, however, in specificity due to cross-reactivity and matrix interference. Gas chromatographymass spectrometry (GC-MS) constitutes the reference method for quantitative analysis of steroids, but time-consuming sample pre-treatment prevents its wide application. We developed and validated an ID-LC-MS/MS method for simultaneous measurement of 8 serum steroids and compared it to currently employed immunoassays. After protein precipitation of 0.9 ml serum, SPE eluate was injected into a 2D-chromatographic system, purified in perfusion column and separated on a C8 column during 21 minutes gradient run. Each analyte undergoes atmospheric pressure chemical ionization before spectrometric revelation of two transitions, quantitative and qualitative, in multiple reaction monitoring mode by 4000Q-Trap triple quadrupole (Applied-Biosystems), ensuring high specificity. Accuracy was validated against GC-MS certified European sera. A comparison study, conducted on 200 samples, showed substantial agreement between ID-LC-MS/MS and Elecsys-E170 for measurement of cortisol, progesterone above 1 ng/ml and testosterone in males. Overestimation by a factor 2 for androstenedione and 3 for DHEA was shown by Immulite2000 and DSL9000, respectively, while disagreement was obtained between ID-LC-MS/MS and 17OHP-Bridge for 17OH-progesterone, and Elecsys-E170 for testosterone in females and progesterone below 1 ng/ml. Preliminary reference intervals were obtained for cortisol, corticosterone, 11desoxycortisol, androstenedione, testosterone, 17OH-progesterone, DHEA and progesterone in 134 male and 79 female healthy subjects. Participants had given informed consent and the study was approved by local ethics committee. Central 95th percentile was estimated for 18-69 y.o. male group, for 18-55 y.o. fertile females and for 41-70 y.o. females in menopause. In conclusion, our data highlighted the power of ID-LC-MS/MS in providing a simultaneous and reliable steroid profile and in documenting the limits shown by the routine methods runtime of 21 min/session.