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

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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 R2 = 0.9994 for 17-OHP and R2 = 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 (R2 = 0.6784); however, an excellent correlation was obtained for QC and proficiency test (PT) DBS samples (R2 = 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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Abbreviations

17-OHP:

17-Hydroxyprogesterone

ACN:

Acetonitrile

CAH:

Congenital adrenal hyperplasia

CDC:

Centers for Disease Control and Prevention

DBS:

Dried blood spot

LC-MS/MS:

Liquid chromatography-tandem mass spectrometry

LoD:

Limit of detection

LoQ:

Limit of quantitation

NYS-NBS:

New York State Newborn Screening

PT:

Proficiency test

ROC:

Receiver operating characteristic

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Acknowledgements

We thank the National Natural Science Foundation of China (grant no. 81572088 and 81601861) for supporting Liqiao Han in performing the study at Wadsworth Center, New York State Department of Health.

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  1. Liqiao Han and Zhimin (Tim) Cao contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China

    Liqiao Han

  2. New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY, USA

    Norma P. Tavakoli, Mark Morrissey, David C. Spink & Zhimin (Tim) Cao

  3. College of Arts and Sciences, University at Albany, State University of New York, Albany, NY, USA

    Zhimin (Tim) Cao

Authors
  1. Liqiao Han
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  2. Norma P. Tavakoli
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  5. Zhimin (Tim) Cao
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Correspondence to Zhimin (Tim) Cao.

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The authors declare that they have no conflict of interest.

Informed consent

Consent was obtained from the participants, who voluntarily donated whole blood for preparation of the control and calibrators materials. All other DBS samples were de-identified for patient information prior to receipt for analysis of the study. This study was approved by the Institutional Review Board of the New York State Department of Health.

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Use of trade names and commercial sources is for identification only and does not constitute endorsement by the Wadsworth Center of the New York State Department of Health.

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Han, L., Tavakoli, N.P., Morrissey, M. et al. Liquid chromatography-tandem mass spectrometry analysis of 17-hydroxyprogesterone in dried blood spots revealed matrix effect on immunoassay. Anal Bioanal Chem 411, 395–402 (2019). https://doi.org/10.1007/s00216-018-1449-0

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  • DOI: https://doi.org/10.1007/s00216-018-1449-0

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