Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults.
BACKGROUND
The 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.


METHODS
0.9 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.


RESULTS
Of 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.


CONCLUSIONS
Our 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.
Centre of Applied Biomedical Research, Department of Clinical Medicine, S.Orsola-Malpighi Hospital, Alma Mater Studiorum, University of Bologna, Bologna, Italy.
Flaminia
F
Fanelli
Ilaria
I
Belluomo
Valentina D
VD
Di Lallo
Gaia
G
Cuomo
Rosaria
R
De Iasio
Margherita
M
Baccini
Elena
E
Casadio
Bruno
B
Casetta
Valentina
V
Vicennati
Alessandra
A
Gambineri
Gabriele
G
Grossi
Renato
R
Pasquali
Uberto
U
Pagotto
D000328:Adult; D000735:Androstenedione / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D007118:Immunoassay / Q000379:methods; D007201:Indicator Dilution Techniques; D008297:Male; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D011374:Progesterone / Q000097:blood; D012016:Reference Values; D012680:Sensitivity and Specificity; D013256:Steroids / Q000097:blood*; D013739:Testosterone / Q000097:blood
D013256:Steroids; D013739:Testosterone; D000735:Androstenedione; D011374:Progesterone; D006854:Hydrocortisone