The Simultaneous measurement of serum testosterone and 5α-dihydrotestosterone by gas chromatography-mass spectrometry (GC-MS).

BACKGROUND: Simultaneous measurement of testosterone (T) and 5α-dihydrotestosterone (DHT) is important for diagnosing androgen deficiency states and hyperandrogenism in males and females, respectively. However, immunoassays used for T and DHT determination suffer from inadequate specificity and sensitivity, while tandem mass spectrometry is expensive and demanding in use. METHODS AND RESULTS: We developed a selective gas chromatography-mass spectrometry (GC-MS) method for parallel T and DHT measurement. The assay showed a linear response up to 46.5nmol/L, intra- and interassay imprecision and inaccuracy <15% and recoveries in spiked samples >90% for both analytes. The limit of quantitation was 0.117nmol/L for T and 0.168nmol/L for DHT. Comparison with immunoassays revealed good agreement for T in males, but a bias in favour of immunoassays at low concentrations for T in females and DHT in both sexes. We established reference ranges for T and DHT and suggest interval partitioning for T according to age in men and menstrual cycle in women. Assay validation in a clinical setting suggests that measuring DHT or T/DHT ratio may help identify patients with polycystic ovary syndrome. CONCLUSION: We developed a selective, simple and inexpensive GC-MS method for parallel measurement of T and DHT with potential use in the clinical laboratory.

Determination of serum cholestane-3ß,5α,6ß-triol by gas chromatography-mass spectrometry for identification of Niemann-Pick type C (NPC) disease.

Niemann-Pick type C (NPC) is a neurological disease caused by an intracellular cholesterol accumulation. Cholesterol oxidation product cholestane-3ß,5α,6ß-triol (C-triol) serves as diagnostic biomarker for NPC, but its measurement in the routine laboratory remains difficult. We developed an isotope dilution gas chromatography-mass spectrometry (GC-MS) method permitting screening for NPC in plasma. 1440 plasma samples obtained from clinically suspicious patients were subjected to alkaline saponification. C-triol was extracted with carbon tetrachloride, transformed into the trimethylsilylethers, separated on a fused silica capillary column with a nonpolar silicone stationary phase, and analyzed by GC-MS. NPC diagnosis was confirmed by DNA sequencing. The method was linear over a concentration range of 0.03-200ng/mL with a mean recovery rate of 98.6%. The intra- and inter-day variation coefficients assessed at two concentrations were below 15%. Limits of quantification (LOQ) and detection (LOD) were 0.03ng/mL and 0.01ng/mL, respectively. Receiver operating characteristic (ROC) analysis estimated that the area under curve was 0.997 implying a significant discriminatory power to identify subjects with NPC. Nevertheless, 13 NPC patients and 29 control subjects confirmed by sequencing showed false negative or positive results, respectively. Two patients with cerebrotendinous xanthomatosis showed a 5-10-fold increase in C-triol levels. We developed a quick and sensitive GC-MS method for determination of C-triol, which may serve as a simple and inexpensive diagnostic tool aiding NPC diagnosis in a routine hospital laboratory. As C-triol elevation is not limited to NPC, the NPC diagnosis has to be confirmed by DNA sequencing.