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Liquid chromatography-tandem mass spectrometry analysis of 17-hydroxyprogesterone in dried blood spots revealed matrix effect on immunoassay
Abstract
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.
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“Cross-reactivity has been documented with 17-OH pregnenolone sulfate, a steroid originating in extremely high amounts from the fetal zone of the fetal adrenal (285), and 15β-hydroxylated compounds apparently generated by gut bacteria and resorbed through the entero-hepatic circulation (286).”
From Congenital Adrenal Hyperplasia—Current Insights in Pathophysiology, Diagnostics, and Management (2022) · cites this paper as [285]
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Congenital adrenal hyperplasia
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Steroid 21 Hydroxylase Deficiency Congenital Adrenal Hyperplasia
Nimkarn, S., Lin‐Su, K., & New, M. I. (2011) · Pediatric Clinics of North America · doi:10.1016/j.pcl.2011.07.012 · Cited by 64
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Congenital Adrenal Hyperplasia—Current Insights in Pathophysiology, Diagnostics, and Management
Claahsen - van der Grinten, H. L., Speiser, P. W., Ahmed, S. F., Arlt, W., Auchus, R. J., Falhammar, H., Flück, C. E., Guasti, L., Huebner, A., Kortmann, B. B. M., et al. (2022) · Endocrine Reviews · doi:10.1210/endrev/bnab016 · Cited by 556
Metadata
- Authors
- Han, L., Tavakoli, N. P., Morrissey, M., Spink, D. C., & Cao, Z. (.
- Affiliations
- Guangdong Provincial Hospital of Chinese Medicine (CN)
- New York State Department of Health (US)
- Corresponding author
- Cao, Zhimin (Tim)
- Venue
- Analytical and Bioanalytical Chemistry
- Published
- 2018-11-20
- Volume
- 411
- Issue
- 2
- Pages
- 395-402
- ISSN
- 1618-2642 (ISSN-L)
- 1618-2650
- OpenAlex
- W2901557701
- Cited by
- 16
- References
- 18
- Citations by year
- 2026: 2 · 2024: 4 · 2023: 3 · 2022: 1 · 2021: 3 · 2020: 1 · 2019: 2
- FWCI
- 0.53
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Analytical and Bioanalytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Multiple Sclerosis Society
- National Natural Science Foundation of China
- Topics
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Sexual Differentiation and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Keywords
- 17-hydroxyprogesterone, congenital adrenal hyperplasia, newborn screening, dried blood spots, matrix effects, immunoassay validation, cortisol, LC-MS/MS confirmation, false positives, proficiency testing
- MeSH terms
- Chromatography, Liquid, Humans, Immunoassay, Infant, Newborn, Sensitivity and Specificity, Serologic Tests, Molecular Structure, 17-alpha-Hydroxyprogesterone, Tandem Mass Spectrometry
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