Development of a LC-MS/MS method to measure simultaneously 10 sexual steroids in Pediatric Endocrinology

AMBAO V; RODRÍGUEZ ME ; ALTUBE M; BALLERINI MG; BERGADÁ I; REY RA; ROPELATO MG

Florinópolis

Congreso; XXVIII Latin American Congress of Pediatric Endocrinology 2019 Annual Meeting of the Latin American Society for Pediatric Endocrinology (SLEP); 2019

Sociedad Latinoamericana de Endocrinología Pediátrica

Mass spectrometry (MS) allow the determination of a panel of steroids in small sample volume with superior sensibility and specificity than immunoassays, important advantages in pediatric samples. Objective: To develop LC-MS/MS method to measure concomitantly Cortisol (F), Δ4-Androstenedione (Δ4-A), Dehydroepiandrosterone (DHEA), Progesterone (P4), 17OH-Progesterone (17OHP4), Pregnenolone (P5), 17OH-Pregnenolone (17OHP5), Testosterone (T), 17 beta-Estradiol (E2) and Dihydrotestosterone (DHT) in pediatric serum samples.Material and Methods: An UHPLC-MS/MS system (Shimadzu-ABSciex QTRAP®6500) in Multiple Reaction Monitory mode (MRM) was used. Calibration curves covering pediatric ranges were used to test linearity. Isotopically labeled Internal Standards were used to calculate recovery. Precision and bias were evaluatedwith human serum quality controls (Biorad).Results: F, Δ4-A, DHEA, P4, 17OHP4, P5, 17OHP5, T, and E2 could be measured by APCI+ ionization; measurement of DHT resulted to be more sensitive by ESI+ ionization. Two MRM transitions per analyte (qualitative and quantitative), compound and source parameters were optimized. A 13-min (APCI) and 8-mingradient (ESI) allowing the separation without interferences were used. Sample preparation shown recoveries between 90 and 105%. All curves shown linearity within the concentration range studied (r>0.99). Lower limit of detection (LLD: signal/noise ratio=3) and lower limit of quantitation (LLQ: signal/noise=10) were: F: 0.013, 0.044 ug/dl, Δ4-A: 0.41, 1.37 ng/ml, DHEA: 0.37, 1.22 ng/ml, P4:0.025, 0.08 ng/ml, 17OHP4: 0.005, 0.018 ng/ml, P5: 0.23, 0.76 ng/ml, 17OHP5: 1.95, 6.49 ng/ml, T: 0.56, 1.87 ng/ml, E2: 20.5, 69 pg/ml, DHT: 0.13, 43 pg/ml, respectively. CV% lower than 5% were obtained in the reportable pediatric range for F, Δ4-A, T, DHEA, 17OHP4 and P4.Conclusions: A sensitive and selective clinical research LC-MS/MS method has been developed for concomitantly measurement of 10 sexual steroids in serum. Estradiol measurement performance must be improved in terms of sensitivity to be applied in pediatric setting.