Sensitive simultaneous quantitation of testosterone and estradiol in serum by LC–MS/MS without derivatization and comparison with the CDC HoSt program

Ryan C. Schofield, Damodara R. Mendu, Lakshmi V. Ramanathan, Melissa S. Pessin, Dean C. Carlow

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC–MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. Methods Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes. Results The lower limits of quantitation (LLOQs) of E2 and Te were 5 pg/mL and 1 ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5–600 pg/mL and 1–1,170 ng/dL for Te. Assay accuracy was determined both by comparison with a LC–MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N = 10) and between-day (N = 20) CVs at concentrations spanning the AMR were less than 7% for both analytes. Conclusion We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC–MS/MS without chemical derivatization and using commercially available calibrators.

Original languageEnglish (US)
Pages (from-to)70-76
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1048
DOIs
StatePublished - Mar 24 2017

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Centers for Disease Control and Prevention (U.S.)
Testosterone
Estradiol
Assays
Serum
Endocrinology
Pediatrics
Hexanes
Immunoassay
Mass spectrometry
Mass Spectrometry
Estrogens
Negative ions
Positive ions
Ions

Keywords

  • CDC HoSt
  • Estradiol
  • Liquid chromatography
  • Mass spectrometry
  • Method correlation
  • NIST SRM 971
  • Testosterone

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

Cite this

Sensitive simultaneous quantitation of testosterone and estradiol in serum by LC–MS/MS without derivatization and comparison with the CDC HoSt program. / Schofield, Ryan C.; Mendu, Damodara R.; Ramanathan, Lakshmi V.; Pessin, Melissa S.; Carlow, Dean C.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 1048, 24.03.2017, p. 70-76.

Research output: Contribution to journalArticle

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abstract = "Background Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC–MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. Methods Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes. Results The lower limits of quantitation (LLOQs) of E2 and Te were 5 pg/mL and 1 ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5–600 pg/mL and 1–1,170 ng/dL for Te. Assay accuracy was determined both by comparison with a LC–MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N = 10) and between-day (N = 20) CVs at concentrations spanning the AMR were less than 7{\%} for both analytes. Conclusion We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC–MS/MS without chemical derivatization and using commercially available calibrators.",
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N2 - Background Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC–MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. Methods Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes. Results The lower limits of quantitation (LLOQs) of E2 and Te were 5 pg/mL and 1 ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5–600 pg/mL and 1–1,170 ng/dL for Te. Assay accuracy was determined both by comparison with a LC–MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N = 10) and between-day (N = 20) CVs at concentrations spanning the AMR were less than 7% for both analytes. Conclusion We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC–MS/MS without chemical derivatization and using commercially available calibrators.

AB - Background Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC–MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. Methods Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18 column and mass spectrometry was performed in both positive and negative ion modes. Results The lower limits of quantitation (LLOQs) of E2 and Te were 5 pg/mL and 1 ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5–600 pg/mL and 1–1,170 ng/dL for Te. Assay accuracy was determined both by comparison with a LC–MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N = 10) and between-day (N = 20) CVs at concentrations spanning the AMR were less than 7% for both analytes. Conclusion We have developed an accurate and highly sensitive assay to measure E2 and Te levels in serum by HPLC–MS/MS without chemical derivatization and using commercially available calibrators.

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