Liquid chromatography-tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric and adult reference intervals.
Level 4 - case-series / case-control
Analytical assay validation and cross-sectional reference interval study
PubMed 20489135 · doi:10.1373/clinchem.2010.143222
What was done
The authors developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of serum or plasma androstenedione, dehydroepiandrosterone (DHEA), and testosterone. Samples (200 µL) were spiked with isotope-labeled internal standards, extracted using methyl t-butyl ether, derivatized with hydroxylamine, and analyzed using 2-dimensional chromatographic separation with a 3.5-minute run time. Assay performance (imprecision, limit of quantification) was evaluated, and reference intervals were established across pediatric cohorts (6 months to 17 years), men, and women.
What was found
Total imprecision was <11.2% for all three analytes. Limits of quantification were 10 ng/L for androstenedione, 50 ng/L for DHEA, and 10 ng/L for testosterone. Androstenedione and DHEA reached their lowest levels in children aged 2–3 years; adult concentrations were achieved at Tanner stage 3 in girls and Tanner stage 4–5 in boys. Median concentrations in premenopausal vs. postmenopausal women were 810 vs. 360 ng/L for androstenedione, 3000 vs. 1670 ng/L for DHEA, and 270 vs. 180 ng/L for testosterone. In postmenopausal women, testosterone levels remained stable across age groups, whereas androstenedione and DHEA declined with age. In men, median concentrations were 440 ng/L (androstenedione), 2000 ng/L (DHEA), and 3700 ng/L (testosterone); in men older than 40 years, concentrations declined per decade by 5% for androstenedione, 10% for DHEA, and 20% for testosterone.
Why it matters
The method provides a rapid, high-throughput multiplexed assay with the sensitivity required to quantify low androgen concentrations across pediatric and female populations alongside established normative reference intervals.
Limits
The abstract does not state the total number of subjects or detailed demographic characteristics used to establish the reference intervals. Cross-sectional age-group comparisons were used to estimate age-related decline rates rather than longitudinal follow-up, and assay performance in specific clinical disease states was not detailed in the abstract.
Cited by
- supports Testosterone concentrations are approximately 10 times higher in males than in females.