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

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 analysed 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.

Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry

“…The LOD of the method is 11.0 pM (2.99 pg/mL) and 0.03 nM (0.75 ng/dL), for E2 and TT respectively. These LODs are similar to those reported for methods using derivatization procedures (40, 41), and lower than some reported for methods without derivatization using higher sample volume (42, 43). The method was found to be linear within the ranges of 11.0–5138 pM (2.99–1400 pg/mL) for E2 ( r 2 > 0.999) and 0.03–48.5 nM (0.75–1400 ng/dL) for TT ( r 2 > 0.999).…”

Data to support a simultaneous testosterone and estradiol assay in serum by LC-MS/MS

“…The LC-MS/MS assay used to obtain the data set is given in [1]. The immunoassay used for testosterone is the Abbott Architect 2nd Generation Assay (Chicago, IL, USA).…”

“…The LC-MS/MS parameters used in this study are given [1]. In order to rule out interference from structurally related and/or isobaric compounds, Chromsystems level III quality control from their MassCheck ® Steroid Panel 1 [4] and Panel 2 [5] were combined.…”

“…The organic extract was dried, reconstituted and injected into the LC/MS-MS system for analysis [1]. For immunoassay analysis human serum was placed onto the analyzer according to the manufacturer׳s guidelines [2], [3].Experimental featuresSpecimens analyzed by LC-MS/MS for estradiol and testosterone were compared with a chemiluminescencent immunoassays.…”

Current Trends of Liquid Chromatography Tandem Mass Spectroscopy Use in Clinical Gynecology

“…As a poster of the method's high yield and ease of use, the assay is reportedly routinely done several times a week with 80 patient sample plates, standards and controls, automated sample extraction being completed in around 90 minutes and 5-minute LC-MS/MS sample to sample injection time. There are numerous published studies on LC-MS/MS methods for testosterone quantitation that highlight the multiplex capabilities of the technique and mainstream becoming [20][21][22][23].…”