Liquid chromatography-tandem mass spectrometry analysis of 17-hydroxyprogesterone in dried blood spots revealed matrix effect on immunoassay

• Published in: Analytical and bioanalytical chemistry Vol. 411; no. 2; pp. 395 - 402
• Main Authors: Han, Liqiao, Tavakoli, Norma P., Morrissey, Mark, Spink, David C., Cao, Zhimin (Tim)
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.01.2019; Springer; Springer Nature B.V
• Subjects: 17-alpha-Hydroxyprogesterone - blood; 17-alpha-Hydroxyprogesterone - chemistry; 17-hydroxyprogesterone; Adrenal glands; Analytical Chemistry; analytical specificity; Biochemistry; Blood; Blood tests; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, Liquid; Coefficient of variation; Cortisol; Food Science; Hormones; Humans; Hydroxyprogesterone; Hyperplasia; Immunoassay; Immunoassay - methods; Immunoassays; Infant, Newborn; Laboratory Medicine; Linearity; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Mathematical analysis; Medical screening; Methods; Molecular Structure; Monitoring/Environmental Analysis; Neonates; patients; Quantitation; Research Paper; Scientific imaging; screening; Sensitivity and Specificity; Serologic Tests; Spectroscopy; Tandem Mass Spectrometry; Newborn screening; 17-hydroxyprogesterone; Cortisol; Congenital adrenal hyperplasia; Mass spectrometry
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-018-1449-0

Immunoassays for measuring 17-hydroxyprogesterone (17-OHP) produce high rates of false positives that impact the identification of congenital adrenal hyperplasia (CAH) in neonates. A confirmatory test with high analytical specificity employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology is needed in newborn screening for CAH. 17-OHP and cortisol were extracted from dried blood spot (DBS) samples, resolved on a C18 column, and measured using tandem mass spectrometry. The results were compared with those determined using the AutoDELFIA immunoassay. The LC-MS/MS method had a limit of quantitation of 10.0 and 5.0 ng/mL for 17-OHP and cortisol, respectively. The method characteristics showed coefficient variation (%CV) ≤ 11.9% for both 17-OHP and cortisol, recoveries ranging from 83.1 to 101.5% for 17-OHP and from 95.1 to 102.8% for cortisol, and linearity with R 2  = 0.9994 for 17-OHP and R 2  = 0.9996 for cortisol, clinical sensitivity of 100.0% and a specificity of 96.4% as obtained by receiver operating characteristic analysis on 45 patient samples when 17-OHP > 39.1 ng/mL was selected as the cutoff value. Comparison between the LC-MS/MS and the AutoDELFIA immunoassay methods revealed a poor correlation for patient DBS samples ( R 2  = 0.6784); however, an excellent correlation was obtained for QC and proficiency test (PT) DBS samples ( R 2  = 0.9797). The LC-MS/MS method produced reliable results for 17-OHP and cortisol for the diagnosis of CAH. The AutoDELFIA immunoassay appears to be subject to matrix effects in the analysis for 17-OHP in DBS patient samples. The DBS samples of non-patient origin may not be suitable for assessing analytical accuracy of immunoassays.