Total Testosterone, LC/MS/MS

Testosterone is an androgen steroid hormone and the principle male sex hormone. Testosterone plays a key role in development of the male reproductive system and promotes secondary sexual characteristics. It is primarily secreted from the testes. In females, low levels of testosterone are secreted by the ovaries. Small amounts are also secreted by the adrenal gland in both genders.

Testosterone deficiency affects approximately 30% of men aged 40 to 79 years. There is a growing body of evidence that strongly associates deficiency with aging and common medical conditions including obesity, diabetes, and hypertension. Testosterone replacement therapy in hypogonadal men can lead to improvement of metabolic syndrome indicators and cardiovascular risk factors. Maintaining testosterone concentrations in the normal range also contributes to bone health, more lean muscle mass, and better physical and sexual function.

An abnormal testosterone concentration can cause premature or delayed puberty in children and hirsutism, as well as virilization in women.

A total testosterone measurement is made up of free and bound testosterone. Circulating testosterone is primarily bound to carrier proteins like albumin and sex hormone binding globulin. Only a small percentage of total testosterone (~2%) remains unbound or “free”. Sex hormone binding globulin binds testosterone with high affinity and acts as a reservoir for storage and transport. Albumin, on the other hand, binds testosterone with weak affinity. Free and albumin-bound fractions are available for immediate binding to an available receptor; therefore, the sum of free and albumin-bound testosterone is termed bioavailable testosterone.

Testosterone reference intervals vary according to age and gender. Different labs may have slightly different reference intervals based on their own studies. The CDC’s hormone standardization project is leading the nation’s testosterone standardization effort, which may be able to harmonize testosterone results across different labs.

Using LC/MS/MS technology, we are able to achieve higher sensitivity and specificity in complex matrices such as human serum and plasma. While immunoassay has been the dominant testing platform, LC/MS/MS technology is now widely accepted as the gold standard for small molecule determinations. LC/MS/MS is applicable to steroid measurements, testing for inborn errors of metabolism, and toxicology testing, particularly for therapeutic drug monitoring and pain management.

For women, children, and hypogonadal males, in whom we expect very low testosterone concentrations, the advantages of LC/MS/MS technology are very important. LC/MS/MS can separate out interfering and structurely similar compounds. Direct immunoassays can suffer significant cross-reactivity from testosterone-like compounds, causing erroneous results. Therefore, they are not recommended for low level testosterone quantitation. In 2007, the Endocrine Society recommended use of extraction and chromatography followed by either immunoassay or mass spectrometry for measuring low testosterone concentrations. Direct immunoassays, however, are still suitable for measuring testosterone levels in adult males, in whom the levels are expected to be higher. In addition, immunoassays have typically a faster turnaround time.

Most healthy males have a testosterone to epitestosterone ratio of about 1:1. Since exogenous administration of testosterone does not affect epitestosterone levels in the body, the ratio of testosterone to epitestosterone has been used to identify athletes who might be using illegal anabolic steroids. Epitestosterone may potentially interfere with LC/MS/MS determinations. Therefore, we designed our assay to separate epitestosterone from testosterone. We do NOT measure or report epitestosterone in our current LC/MS/MS testosterone method. If an epitestosterone measurement is required, we suggest you approach a testing site that uses a World Anti-doping Agency (WADA)-approved assay.