Home Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents
Article
Licensed
Unlicensed Requires Authentication

Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents

  • Ashley Di Meo , Mehrdad Yazdanpanah , Victoria Higgins , Matthew Nichols , Mary Kathryn Bohn , Agnes Tan , Shazina Zainab , Lusia Sepiashvili and Khosrow Adeli EMAIL logo
Published/Copyright: April 11, 2023
Become an author with De Gruyter Brill
Submit Manuscript Author Information
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 61 Issue 10

Abstract

Objectives

Monitoring estradiol (E2) is important for determining the onset of pubertal development as well as in the evaluation of girls with precocious puberty. However, E2 measurement remains an analytical challenge in children, who have lower circulating levels. We developed and evaluated a simple and sensitive LC-MS/MS procedure for serum E2 quantification in pediatric populations and established age- and sex-specific pediatric reference intervals.

Methods

Residual patient serum samples were used to evaluate the analytical performance of our in-house LC-MS/MS E2 assay. The evaluation included accuracy, precision, linearity, functional sensitivity (LLoQ), and method comparison. Age- and sex-specific pediatric E2 reference intervals were also established from a cohort of 405 healthy children (birth to 18 years) recruited with informed consent. Age- and sex-specific differences were assessed, and outliers were removed. Reference intervals were established using the robust method.

Results

The assay imprecision was <5.3 %. Assay linearity ranged from 13.7 to 1923.3 pmol/L. The LLoQ corresponding to a CV of 20 % was determined to be 8.9 pmol/L. Bland-Altman analysis revealed a mean bias of 29.3 pmol/L or 9.1 % between our LC-MS/MS E2 assay and an external reference laboratory measuring E2 by LC-MS/MS.

Conclusions

Our LC-MS/MS E2 assay shows acceptable accuracy, precision, functional sensitivity (LLoQ), and linearity for E2 quantification. Our LC-MS/MS E2 assay also showed good agreement with an external reference laboratory measuring E2 by LC-MS/MS. In addition, using CALIPER samples, we established robust age- and sex-specific pediatric E2 reference intervals to improve accuracy of test result interpretation and clinical decision making.

Keywords: estradiol; immunoassay; LC-MS/MS; pediatric; reference intervals
Corresponding author: Khosrow Adeli, PhD, FCACB, DABCC, FAACC, Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada; Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada; and SickKids Research Institute, 686 Bay Street, Toronto, ON M5G 0A4, Canada, Phone: 416 813 8682, E-mail:
Ashley Di Meo and Mehrdad Yazdanpanah contributed equally to this work.

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The local Institutional Review Board deemed the study exempt from review.

References

1. Bay, K, Andersson, AM, Skakkebaek, NE. Estradiol levels in prepubertal boys and girls--analytical challenges. Int J Androl 2004;27:266–73. https://doi.org/10.1111/j.1365-2605.2004.00487.x.Search in Google Scholar PubMed

2. Ferreira, MS, Arruda, AMM, Pepi, GT, Martho, AC, Maximiano, PM, Ricci, L, et al.. High sensitivity method validated to quantify estradiol in human plasma by LC-MS/MS. J Chromatogr, B: Anal Technol Biomed Life Sci 2017;1064:109–14. https://doi.org/10.1016/j.jchromb.2017.09.005.Search in Google Scholar PubMed

3. Pauwels, S, Antonio, L, Jans, I, Lintermans, A, Neven, P, Claessens, F, et al.. Sensitive routine liquid chromatography-tandem mass spectrometry method for serum estradiol and estrone without derivatization. Anal Bioanal Chem 2013;405:8569–77. https://doi.org/10.1007/s00216-013-7259-5.Search in Google Scholar PubMed

4. Albin, AK, Niklasson, A, Westgren, U, Norjavaara, E. Estradiol and pubertal growth in girls. Horm Res Paediatr 2012;78:218–25. https://doi.org/10.1159/000343076.Search in Google Scholar PubMed

5. Guo, T, Gu, J, Soldin, OP, Singh, RJ, Soldin, SJ. Rapid measurement of estrogens and their metabolites in human serum by liquid chromatography-tandem mass spectrometry without derivatization. Clin Biochem 2008;41:736–41. https://doi.org/10.1016/j.clinbiochem.2008.02.009.Search in Google Scholar PubMed PubMed Central

6. Ankarberg-Lindgren, C, Dahlgren, J, Andersson, MX. High-sensitivity quantification of serum androstenedione, testosterone, dihydrotestosterone, estrone and estradiol by gas chromatography-tandem mass spectrometry with sex- and puberty-specific reference intervals. J Steroid Biochem Mol Biol 2018;183:116–24. https://doi.org/10.1016/j.jsbmb.2018.06.005.Search in Google Scholar PubMed

7. Wang, Q, Mesaros, C, Blair, IA. Ultra-high sensitivity analysis of estrogens for special populations in serum and plasma by liquid chromatography-mass spectrometry: assay considerations and suggested practices. J Steroid Biochem Mol Biol 2016;162:70–9. https://doi.org/10.1016/j.jsbmb.2016.01.002.Search in Google Scholar PubMed PubMed Central

8. Ankarberg-Lindgren, C, Norjavaara, E. A purification step prior to commercial sensitive immunoassay is necessary to achieve clinical usefulness when quantifying serum 17beta-estradiol in prepubertal children. Eur J Endocrinol 2008;158:117–24. https://doi.org/10.1530/eje-07-0403.Search in Google Scholar

9. Rosner, W, Hankinson, SE, Sluss, PM, Vesper, HW, Wierman, ME. Challenges to the measurement of estradiol: an endocrine society position statement. J Clin Endocrinol Metab 2013;98:1376–87. https://doi.org/10.1210/jc.2012-3780.Search in Google Scholar PubMed PubMed Central

10. Ankarberg-Lindgren, C, Elfving, M, Wikland, KA, Norjavaara, E. Nocturnal application of transdermal estradiol patches produces levels of estradiol that mimic those seen at the onset of spontaneous puberty in girls. J Clin Endocrinol Metab 2001;86:3039–44. https://doi.org/10.1210/jcem.86.7.7667.Search in Google Scholar PubMed

11. Ketha, H, Girtman, A, Singh, RJ. Estradiol assays--the path ahead. Steroids 2015;99:39–44. https://doi.org/10.1016/j.steroids.2014.08.009.Search in Google Scholar PubMed

12. Mejia-Otero, JD, White, P, Lopez, X. Effectiveness of puberty suppression with gonadotropin-releasing hormone agonists in transgender youth. Transgender Health 2021;6:31–5. https://doi.org/10.1089/trgh.2020.0007.Search in Google Scholar PubMed PubMed Central

13. Kushnir, MM, Rockwood, AL, Bergquist, J, Varshavsky, M, Roberts, WL, Yue, B, et al.. High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol. Am J Clin Pathol 2008;129:530–9. https://doi.org/10.1309/lc03bhq5xjpjyekg.Search in Google Scholar PubMed

14. Rahhal, SN, Fuqua, JS, Lee, PA. The impact of assay sensitivity in the assessment of diseases and disorders in children. Steroids 2008;73:1322–7. https://doi.org/10.1016/j.steroids.2008.04.014.Search in Google Scholar PubMed

15. Ankarberg, C, Norjavaara, E. Diurnal rhythm of testosterone secretion before and throughout puberty in healthy girls: correlation with 17beta-estradiol and dehydroepiandrosterone sulfate. J Clin Endocrinol Metab 1999;84:975–84. https://doi.org/10.1210/jc.84.3.975.Search in Google Scholar

16. Ankarberg-Lindgren, C, Norjavaara, E. Changes of diurnal rhythm and levels of total and free testosterone secretion from pre to late puberty in boys: testis size of 3 mL is a transition stage to puberty. Eur J Endocrinol 2004;151:747–57. https://doi.org/10.1530/eje.0.1510747.Search in Google Scholar PubMed

17. Bohn, MK, Wilson, S, Hall, A, Adeli, K. Pediatric reference interval verification for endocrine and fertility hormone assays on the Abbott Alinity system. Clin Chem Lab Med 2021;59:1680–7. https://doi.org/10.1515/cclm-2021-0337.Search in Google Scholar PubMed

18. Wooding, KM, Hankin, JA, Johnson, CA, Chosich, JD, Baek, SW, Bradford, AP, et al.. Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization. Steroids 2015;96:89–94. https://doi.org/10.1016/j.steroids.2015.01.007.Search in Google Scholar PubMed PubMed Central

19. Fiers, T, Casetta, B, Bernaert, B, Vandersypt, E, Debock, M, Kaufman, JM. Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization. J Chromatogr, B: Anal Technol Biomed Life Sci 2012;893–894:57–62. https://doi.org/10.1016/j.jchromb.2012.02.034.Search in Google Scholar PubMed

20. CLSI. Defining, establishing, and verifying reference intervals in the clinical laboratory; approved guidelines – third edition CLSI document C28-A3. Wayne, PA: Clinical and Laboratory Standards Institute (CLSI); 2008.Search in Google Scholar

21. Colantonio, DA, Kyriakopoulou, L, Chan, MK, Daly, CH, Brinc, D, Venner, AA, et al.. Closing the gaps in pediatric laboratory reference intervals: a CALIPER database of 40 biochemical markers in a healthy and multiethnic population of children. Clin Chem 2012;58:854–68. https://doi.org/10.1373/clinchem.2011.177741.Search in Google Scholar PubMed

22. Tukey, JW. Exploratory data analysis. Boston: Addison-Wesley; 1977.Search in Google Scholar

23. Hubert, M, Veeken, SVD. Outlier detection for skewed data. J Chemometr 2008;3–4:235–46. https://doi.org/10.1002/cem.1123.Search in Google Scholar

24. Horn, PS, Pesce, AJ, Copeland, BE. A robust approach to reference interval estimation and evaluation. Clin Chem 1998;44:622–31. https://doi.org/10.1093/clinchem/44.3.622.Search in Google Scholar

25. Zhou, H, Wang, Y, Gatcombe, M, Farris, J, Botelho, JC, Caudill, SP, et al.. Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 2017;409:5943–54. https://doi.org/10.1007/s00216-017-0529-x.Search in Google Scholar PubMed PubMed Central

26. van Winden, LJ, Kok, M, Acda, M, Dezentje, V, Linn, S, Shi, RZ, et al.. Simultaneous analysis of E1 and E2 by LC-MS/MS in healthy volunteers: estimation of reference intervals and comparison with a conventional E2 immunoassay. J Chromatogr, B: Anal Technol Biomed Life Sci 2021;1178:122563. https://doi.org/10.1016/j.jchromb.2021.122563.Search in Google Scholar PubMed

27. Yi, X, Leung, EKY, Bridgman, R, Koo, S, Yeo, KJ. High-sensitivity micro LC-MS/MS assay for serum estradiol without derivatization. J Appl Lab Med 2016;1:14–24. https://doi.org/10.1373/jalm.2016.020362.Search in Google Scholar PubMed

28. Bailey, D, Colantonio, D, Kyriakopoulou, L, Cohen, AH, Chan, MK, Armbruster, D, et al.. Marked biological variance in endocrine and biochemical markers in childhood: establishment of pediatric reference intervals using healthy community children from the CALIPER cohort. Clin Chem 2013;59:1393–405. https://doi.org/10.1373/clinchem.2013.204222.Search in Google Scholar PubMed

29. Karbasy, K, Lin, DC, Stoianov, A, Chan, MK, Bevilacqua, V, Chen, Y, et al.. Pediatric reference value distributions and covariate-stratified reference intervals for 29 endocrine and special chemistry biomarkers on the Beckman Coulter Immunoassay Systems: a CALIPER study of healthy community children. Clin Chem Lab Med 2016;54:643–57. https://doi.org/10.1515/cclm-2015-0558.Search in Google Scholar PubMed

30. Higgins, V, Fung, AWS, Chan, MK, Macri, J, Adeli, K. Pediatric reference intervals for 29 Ortho VITROS 5600 immunoassays using the CALIPER cohort of healthy children and adolescents. Clin Chem Lab Med 2018;56:327–40. https://doi.org/10.1515/cclm-2017-0349.Search in Google Scholar PubMed

31. Bohn, MK, Horn, P, League, D, Steele, P, Hall, A, Adeli, K. Pediatric reference intervals for endocrine markers and fertility hormones in healthy children and adolescents on the Siemens Healthineers Atellica immunoassay system. Clin Chem Lab Med 2021;59:1421–30. https://doi.org/10.1515/cclm-2021-0050.Search in Google Scholar PubMed

32. Frederiksen, H, Johannsen, TH, Andersen, SE, Albrethsen, J, Landersoe, SK, Petersen, JH, et al.. Sex-specific estrogen levels and reference intervals from infancy to late adulthood determined by LC-MS/MS. J Clin Endocrinol Metab 2020;105:754–68. https://doi.org/10.1210/clinem/dgz196.Search in Google Scholar PubMed PubMed Central

33. Bae, YJ, Zeidler, R, Baber, R, Vogel, M, Wirkner, K, Loeffler, M, et al.. Reference intervals of nine steroid hormones over the life-span analyzed by LC-MS/MS: effect of age, gender, puberty, and oral contraceptives. J Steroid Biochem Mol Biol 2019;193:105409. https://doi.org/10.1016/j.jsbmb.2019.105409.Search in Google Scholar PubMed

34. Dowis, J, Woroniecki, W, French, D. Development and validation of a LC-MS/MS assay for quantification of serum estradiol using calibrators with values assigned by the CDC reference measurement procedure. Clin Chim Acta 2019;492:45–9. https://doi.org/10.1016/j.cca.2019.02.003.Search in Google Scholar PubMed

35. Wang, Q, Rangiah, K, Mesaros, C, Snyder, NW, Vachani, A, Song, H, et al.. Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography-tandem mass spectrometry. Steroids 2015;96:140–52. https://doi.org/10.1016/j.steroids.2015.01.014.Search in Google Scholar PubMed PubMed Central

36. Madsen, A, Almas, B, Bruserud, IS, Oehme, NHB, Nielsen, CS, Roelants, M, et al.. Reference curves for pediatric endocrinology: leveraging biomarker Z-scores for clinical classifications. J Clin Endocrinol Metab 2022;107:2004–15. https://doi.org/10.1210/clinem/dgac155.Search in Google Scholar PubMed PubMed Central

37. Norjavaara, E, Ankarberg, C, Albertsson-Wikland, K. Diurnal rhythm of 17 beta-estradiol secretion throughout pubertal development in healthy girls: evaluation by a sensitive radioimmunoassay. J Clin Endocrinol Metab 1996;81:4095–102. https://doi.org/10.1210/jcem.81.11.8923866.Search in Google Scholar PubMed

38. Mitamura, R, Yano, K, Suzuki, N, Ito, Y, Makita, Y, Okuno, A. Diurnal rhythms of luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol secretion before the onset of female puberty in short children. J Clin Endocrinol Metab 2000;85:1074–80. https://doi.org/10.1210/jcem.85.3.6445.Search in Google Scholar PubMed

39. Ankarberg-Lindgren, C, Norjavaara, E. Twenty-four hours secretion pattern of serum estradiol in healthy prepubertal and pubertal boys as determined by a validated ultra-sensitive extraction RIA. BMC Endocr Disord 2008;8:10. https://doi.org/10.1186/1472-6823-8-10.Search in Google Scholar PubMed PubMed Central

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2022-1231).

Received: 2022-12-01
Accepted: 2023-03-28
Published Online: 2023-04-11
Published in Print: 2023-09-26

© 2023 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Laboratory Medicine: from just testing to saving lives
  4. Reviews
  5. Serum biomarkers of remodeling in severe asthma with fixed airway obstruction and the potential role of KL-6
  6. Safety monitoring of drug-induced muscle injury and rhabdomyolysis: a biomarker-guided approach for clinical practice and drug trials
  7. Mini Review
  8. Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications
  9. Opinion Paper
  10. Recommendation for the design of stability studies on clinical specimens
  11. General Clinical Chemistry and Laboratory Medicine
  12. Assessment of WHO 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays
  13. veRification: an R Shiny application for laboratory method verification and validation
  14. Impact of storage temperature and time before analysis on electrolytes (Na+, K+, Ca2+), lactate, glucose, blood gases (pH, pO2, pCO2), tHb, O2Hb, COHb and MetHb results
  15. The stability of blood gases and CO-oximetry under slushed ice and room temperature conditions
  16. Elevated levels of renal function tests conferred increased risks of developing various pregnancy complications and adverse perinatal outcomes: insights from a population-based cohort study
  17. Poor comparability of plasma renin activity measurement in determining patient samples: the status quo and recommendations for harmonization
  18. Salivary cortisol and cortisone in diagnosis of Cushing’s syndrome – a comparison of six different analytical methods
  19. Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application
  20. Analytical evaluation of a GAD65 antibodies chemiluminescence immunoassay for CSF in neurological syndromes
  21. Reference Values and Biological Variations
  22. Evaluation of low-density lipoprotein cholesterol equations by cross-platform assessment of accuracy-based EQA data against SI-traceable reference value
  23. Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents
  24. Cancer Diagnostics
  25. Practical delta check limits for tumour markers in different clinical settings
  26. Comparison between free β subunit of human chorionic gonadotropin (hCG) and total hCG assays in adults with testicular cancer
  27. Hematology and Coagulation
  28. Value of monocyte distribution width for predicting severe cholecystitis: a retrospective cohort study
  29. Performance of digital morphology analyzer Medica EasyCell assistant
  30. Validation of non-invasive point of care blood content analysis using the TensorTip™ MTX device: a method comparison study
  31. Infectious Diseases
  32. Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration
  33. Letters to the Editor
  34. The first case of VEXAS syndrome in Austria
  35. Acetylcholine receptor and muscle-specific tyrosine kinase antibodies detection: is it time for a change?
  36. Performance of the 2009 CKDEPI, 2021 CKDEPI, and EKFC equations among high-risk patients in Denmark
  37. Biotin interference in immunoassays: water under the bridge?
  38. The new synthetic benzimidazole opioid etonitazepipne: an emerging fatal harm and a challenge for laboratory medicine
  39. Unexplained increase of serum carcinoembryonic antigen: don’t forget the thyroid!
  40. Falsely elevated cortisol serum levels in preterm infants due to use of immunoassay
  41. Misdiagnosis of Hb Bart’s disease: prenatal screening and diagnosis of thalassemia in special population
https://doi.org/10.1515/cclm-2022-1231
Keywords for this article
estradiol; immunoassay; LC-MS/MS; pediatric; reference intervals

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Laboratory Medicine: from just testing to saving lives
  4. Reviews
  5. Serum biomarkers of remodeling in severe asthma with fixed airway obstruction and the potential role of KL-6
  6. Safety monitoring of drug-induced muscle injury and rhabdomyolysis: a biomarker-guided approach for clinical practice and drug trials
  7. Mini Review
  8. Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications
  9. Opinion Paper
  10. Recommendation for the design of stability studies on clinical specimens
  11. General Clinical Chemistry and Laboratory Medicine
  12. Assessment of WHO 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays
  13. veRification: an R Shiny application for laboratory method verification and validation
  14. Impact of storage temperature and time before analysis on electrolytes (Na+, K+, Ca2+), lactate, glucose, blood gases (pH, pO2, pCO2), tHb, O2Hb, COHb and MetHb results
  15. The stability of blood gases and CO-oximetry under slushed ice and room temperature conditions
  16. Elevated levels of renal function tests conferred increased risks of developing various pregnancy complications and adverse perinatal outcomes: insights from a population-based cohort study
  17. Poor comparability of plasma renin activity measurement in determining patient samples: the status quo and recommendations for harmonization
  18. Salivary cortisol and cortisone in diagnosis of Cushing’s syndrome – a comparison of six different analytical methods
  19. Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application
  20. Analytical evaluation of a GAD65 antibodies chemiluminescence immunoassay for CSF in neurological syndromes
  21. Reference Values and Biological Variations
  22. Evaluation of low-density lipoprotein cholesterol equations by cross-platform assessment of accuracy-based EQA data against SI-traceable reference value
  23. Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents
  24. Cancer Diagnostics
  25. Practical delta check limits for tumour markers in different clinical settings
  26. Comparison between free β subunit of human chorionic gonadotropin (hCG) and total hCG assays in adults with testicular cancer
  27. Hematology and Coagulation
  28. Value of monocyte distribution width for predicting severe cholecystitis: a retrospective cohort study
  29. Performance of digital morphology analyzer Medica EasyCell assistant
  30. Validation of non-invasive point of care blood content analysis using the TensorTip™ MTX device: a method comparison study
  31. Infectious Diseases
  32. Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration
  33. Letters to the Editor
  34. The first case of VEXAS syndrome in Austria
  35. Acetylcholine receptor and muscle-specific tyrosine kinase antibodies detection: is it time for a change?
  36. Performance of the 2009 CKDEPI, 2021 CKDEPI, and EKFC equations among high-risk patients in Denmark
  37. Biotin interference in immunoassays: water under the bridge?
  38. The new synthetic benzimidazole opioid etonitazepipne: an emerging fatal harm and a challenge for laboratory medicine
  39. Unexplained increase of serum carcinoembryonic antigen: don’t forget the thyroid!
  40. Falsely elevated cortisol serum levels in preterm infants due to use of immunoassay
  41. Misdiagnosis of Hb Bart’s disease: prenatal screening and diagnosis of thalassemia in special population
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2025 De Gruyter Brill
Downloaded on 11.9.2025 from https://www.degruyterbrill.com/document/doi/10.1515/cclm-2022-1231/html
Scroll to top button