Analytical and Bioanalytical Chemistry

, Volume 411, Issue 2, pp 395–402 | Cite as

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

  • Liqiao Han
  • Norma P. Tavakoli
  • Mark Morrissey
  • David C. Spink
  • Zhimin (Tim) CaoEmail author
Research Paper


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.


Newborn screening Congenital adrenal hyperplasia 17-hydroxyprogesterone Cortisol Mass spectrometry 







Congenital adrenal hyperplasia


Centers for Disease Control and Prevention


Dried blood spot


Liquid chromatography-tandem mass spectrometry


Limit of detection


Limit of quantitation


New York State Newborn Screening


Proficiency test


Receiver operating characteristic



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.

Compliance with ethical standards

Conflict of interest

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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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Liqiao Han
    • 1
  • Norma P. Tavakoli
    • 2
  • Mark Morrissey
    • 2
  • David C. Spink
    • 2
  • Zhimin (Tim) Cao
    • 2
    • 3
    Email author
  1. 1.Department of Laboratory MedicineThe Second Affiliated Hospital of Guangzhou University of Chinese MedicineGuangzhouChina
  2. 2.New York State Department of HealthWadsworth CenterAlbanyUSA
  3. 3.College of Arts and SciencesUniversity at Albany, State University of New YorkAlbanyUSA

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