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Comparative Study
. 2017 Mar 24:1048:70-76.
doi: 10.1016/j.jchromb.2017.02.006. Epub 2017 Feb 10.

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  1 , Damodara R Mendu  2 , Lakshmi V Ramanathan  1 , Melissa S Pessin  1 , Dean C Carlow  3
Affiliations
Comparative Study

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 et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

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 5pg/mL and 1ng/dL, respectively. The analytical measurement range (AMR) for E2 was 5-600pg/mL and 1-1,170ng/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.

Keywords: CDC HoSt; Estradiol; Liquid chromatography; Mass spectrometry; Method correlation; NIST SRM 971; Testosterone.

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Figures

Figure 1
Figure 1
HPLC-MS/MS ion chromatograms depicting estradiol and testosterone LLOQ’s and specimen collected from male (estradiol) and female (testosterone) patients.
Figure 2
Figure 2
HPLC-MS/MS ion chromatogram of E2, E2-D5, Te, and Te-D3 product ions in a serum sample.
Figure 3
Figure 3
(A) Correlation between the new HPLC-MS/MS assay and the CDC HoSt standardization program; (B) Correlation between the new HPLC-MS/MS assay and the CDC HoSt standardization program at the low end of the AMR from the above data.
Figure 3
Figure 3
(A) Correlation between the new HPLC-MS/MS assay and the CDC HoSt standardization program; (B) Correlation between the new HPLC-MS/MS assay and the CDC HoSt standardization program at the low end of the AMR from the above data.
Figure 4
Figure 4
Correlation between the new HPLC-MS/MS assay and a national reference laboratory.
See this image and copyright information in PMC

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