Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: total and free testosterone reference ranges

Steroids
Wael A SalamehMichael P Caulfield

Abstract

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 microL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y=1.02x+1.5, r(2)=0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pedi...Continue Reading

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