Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice.
BACKGROUND
Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory.


METHODS
We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h.


RESULTS
Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak.


CONCLUSIONS
The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.
Cawood
Marion L
ML
SAS Centre for Steroid Hormones (Leeds), Department of Clinical Biochemistry and Immunology, Leeds General Infirmary, Leeds, United Kingdom. marion.cawood@leedsth.nhs.uk
Field
Helen P
HP
Ford
Clive G
CG
Gillingwater
Scott
S
Kicman
Andrew
A
Cowan
David
D
Barth
Julian H
JH
D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D011863:Radioimmunoassay; D011865:Radioisotope Dilution Technique; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000097:blood*
D013739:Testosterone; D003903:Deuterium