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Clin Chim Acta. 2014 Sep 25;436:263-7. doi: 10.1016/j.cca.2014.06.009. Epub 2014 Jun 21.

Total testosterone quantitative measurement in serum by LC-MS/MS.

Author information

1
Clinical Chemistry Branch, Division of Laboratory Sciences, Centers For Disease Control and Prevention, 4770 Buford Hwy NE MS F25, Atlanta, GA 30341, USA.
2
Clinical Chemistry Branch, Division of Laboratory Sciences, Centers For Disease Control and Prevention, 4770 Buford Hwy NE MS F25, Atlanta, GA 30341, USA. Electronic address: hvesper@cdc.gov.

Abstract

Reliable measurement of total testosterone is essential for the diagnosis, treatment and prevention of a number of hormone-related diseases affecting adults and children. A mass spectrometric method for testosterone determination in human serum was carefully developed and thoroughly validated. Total testosterone from 100 μL serum is released from proteins with acidic buffer and isolated by two serial liquid-liquid extraction steps. The first extraction step isolates the lipid fractions from an acidic buffer solution using ethyl acetate and hexane. The organic phase is dried down and reconstituted in a basic buffer solution. The second extraction step removes the phospholipids and other components by hexane extraction. Liquid chromatography-isotopic dilution tandem mass spectrometry is used to quantify the total testosterone. The sample preparation is automatically conducted in a liquid-handling system with 96-deepwell plates. The method limit of detection is 9.71 pmol/L (0.280 ng/dL) and the method average percent bias is not significantly different from reference methods. The performance of this method has proven to be consistent with the method precision over a 2-year period ranging from 3.7 to 4.8% for quality control pools at the concentrations 0.527, 7.90 and 30.7 nmol/L (15.2, 228, and 886 ng/dL), respectively. This method provides consistently high accuracy and excellent precision for testosterone determination in human serum across all clinical relevant concentrations.

KEYWORDS:

Hormone; LC-MS/MS; Steroids; Testosterone

PMID:
24960363
PMCID:
PMC4743493
DOI:
10.1016/j.cca.2014.06.009
[Indexed for MEDLINE]
Free PMC Article

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