Development and Validation of a High-performance Liquid Chromatography-Tandem Mass Spectrometry Method for Detecting Adrenocortical Hormones and Establishment of Age-stratified Reference Intervals in Reproductive-aged Women from Guangxi, China.

Background Adrenocortical hormones, particularly 11-oxygenated androgens, are pivotal in female reproductive health and fertility. Standardized detection kits and population-specific reference intervals are lacking in China, hindering related clinical applications. Methods A HPLC-tandem mass spectrometry (HPLC-MS/MS) pipeline was developed, rigorously validated, and applied to simultaneously quantify corticosterone, cortisone, cortisol, 18-OH cortisol, androstenedione (A4), 11β-hydroxyandrostenedione (11-OH A4), dehydroepiandrosterone, and dehydroepiandrosterone sulfate in serum samples from 455 reproductive-aged women (18-45 yrs) in Guangxi, China. Age-dependent concentration trends were analyzed, and reference intervals stratified by age (2.5th to 97.5th percentiles) were established. Correlations with body-composition metrics, ethnicity, and the menstrual cycle were investigated. Results The HPLC-MS/MS method demonstrated high precision (intra- and inter-assay CVs <15%), accuracy, and sensitivity. All eight hormones exhibited significant age-related declines (P <0.001 for seven hormones; P =0.001 for 11-OH A4). Notably, 11-OH A4 levels were significantly lower in the 35-45-yr (3.05 nmol/L) and 25-34-yr (3.09 nmol/L) age groups than in the 18-24-yr (3.57 nmol/L) age group, whereas no significant difference was observed between the 35-45-yr and 25-34-yr age groups. Weak negative correlations were observed between the body mass index and corticosterone and cortisone levels, whereas ethnicity and the menstrual cycle showed no significant associations with hormone levels. Conclusions We developed an HPLC-MS/MS-based method for simultaneously quantifying eight adrenocortical hormones, including 11-OH A4, and defined age-specific reference intervals for reproductive-aged Chinese women. These findings advance the clinical utility of adrenocortical hormones in diagnosing and managing reproductive disorders.