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

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 (E) 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 C column and mass spectrometry was performed in both positive and negative ion modes.

RESULTS

The lower limits of quantitation (LLOQs) of E and Te were 5pg/mL and 1ng/dL, respectively. The analytical measurement range (AMR) for E 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 E and Te levels in serum by HPLC-MS/MS without chemical derivatization and using commercially available calibrators.