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Testosterone, LC/MS/MS

Testosterone, LC/MS/MS

Test Summary

Testosterone, LC/MS/MS


Clinical Use

  • Diagnose and monitor disorders associated with testosterone abnormalities (Table 1).

This table is provided for informational purposes only and is not intended as medical advice. A physician's test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

Table 1. Clinical Use of Testosterone Tests1-5
Test Name Clinical Use
Testosterone, Total, LC/MS/MS


Diagnose disorders of sexual development, including undervirilization in XY infants and infants with ambiguous genitalia


Diagnose hypogonadism
Diagnose early or delayed puberty
Monitor testosterone replacement or deprivation therapy


Test for hyperandrogenism
Diagnose disorders of sexual development
Support or rule out androgen-secreting tumor

Testosterone, Free and Total, LC/MS/MS


Testosterone, Free, Bioavailable, and Total, LC/MS/MS


Limited use


Diagnose androgen deficiency when total testosterone is near lower limit of normal or alteration of SHBG is suspected (eg, aging, obesity, medications)


Diagnose hyperandrogenism in patients with amenorrhea, hirsutism, polycystic ovary syndrome (PCOS), or virilization

Clinical Background

Testosterone is secreted by the testes in men. In women, the adrenal glands secrete 25% of the testosterone and the ovaries secrete another 25%; the remainder is produced by peripheral conversion of androstenedione.6 Most circulating testosterone is bound by sex hormone binding globulin (SHBG) and albumin; approximately 2% of total testosterone is free (not bound to protein).7

SHBG-bound testosterone is so tightly bound that it is not biologically active. Both free and albumin-bound testosterone are biologically active, and together are referred to as the bioavailable fraction. Thus, bioavailable testosterone levels depend on albumin levels to a small extent (low binding affinity) and SHBG levels to a large extent (high binding affinity) in addition to rates of testosterone production and clearance.

In utero, testosterone is necessary for the development of male genitalia in 46, XY fetuses.8 In the absence of testosterone, the fetus tends to develop as a female. Thus, with a disorder of sexual development, a 46, XY newborn may present with external genitalia ranging from nearly normal female to nearly normal male, depending on the severity of the defect. Total testosterone levels can range from absent to increased, depending on the condition. Male infants with hypogonadism or hypopituitarism may display micropenis or cryptorchidism.

Delayed puberty and hypogonadism in boys and men can be associated with primary or secondary testicular failure. Elevated LH and FSH are consistent with primary hypogonadism whereas decreased levels are consistent with secondary or tertiary hypogonadism. As men age, testosterone levels decrease, SHBG levels increase, and these changes result in a decrease in free testosterone levels. Thus, free testosterone measurement offers greater sensitivity than total testosterone for diagnosis of hypogonadism in older men.3

Testosterone therapy may be recommended for adult males with symptomatic hypogonadism.3 In men (‚Č•65 years), clinical studies show that treatment moderately improves in sexual function, mood, and depressive symptoms.9 To assess when levels reach the mid-normal range, guidelines recommend monitoring total testosterone in hypogonadal males during treatment.3 Measurements are recommended 3 to 6 months after initiation of treatment, earlier (at 1 week) for men receiving testosterone enanthate or cypionate injections, and midway between injections thereafter to determine appropriate frequency and dosage.3

Measurement of free or bioavailable testosterone in females offers greater sensitivity for evaluation of mild androgen excess than total testosterone.2,4 In girls and women, excess androgen production is associated with premature adrenarche (ie, appearance of pubic and/or axillary hair before age 8), oligo/amenorrhea, and clinical features of hyperandrogenism (eg, alopecia, severe acne, hirsutism). These features are associated with polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age. Free testosterone levels are elevated in about 70% of PCOS cases.2

Direct immunoassays cannot accurately measure the low serum testosterone levels found in women and children,10 and hypogonadal men.11,12 Thus, the Endocrine Society recommends testosterone methods that use extraction and purification prior to measurement.5 Liquid chromatography tandem mass spectrometry (LC/MS/MS) methods meet these recommendations. In addition, because of increased sensitivity and specificity, LC/MS/MS has emerged as the method of choice in these populations.

Individuals Suitable for Testing

Men, women, and children as discussed in Table 1.


  • Total Testosterone

   Turbulent flow liquid chromatography tandem mass spectrometry (LC/MS/MS)

   Analytical sensitivity: 1.0 ng/dL

   Analytical specificity: no cross-reactivity with 30 testosterone-related steroid compounds

   Analytical Measurement Range: 1.0 ng/dL to 2,000 ng/dL

  • Free and Total Testosterone

   Total: LC/MS/MS

   Percent free: equilibrium dialysis

   Free: calculated based on total and percent free testosterone

  • Free, Bioavailable, and Total Testosterone

   Total: LC/MS/MS

   Free: calculated based on constants for the binding of testosterone to SHBG and albumin

   Bioavailable: calculated based on constants for the binding of testosterone to SHBG and albumin

   SHBG: immunochemiluminometric assay (ICMA)

   Albumin: spectrophotometry

Interpretive Information

In newborns, the results of testosterone measurement and chromosome analysis can help diagnose the cause of external genitalia abnormalities (Table 2).

Table 2. Testosterone Levels and Disorders of Sexual Development1,3,8
Condition Genotype

External Genitalia


Complete androgen insensitivity syndrome XY


Normal or elevated male range

Partial androgen insensitivity syndrome XY


Normal male range

Complete gonadal dysgenesis XY



Partial gonadal dysgenesis XY



5α-reductase deficiency XY


Normal or elevated male range

Complete testosterone biosynthetic defect XY



Partial testosterone biosynthetic defect XY



Micropenis XY



Congenital adrenal hyperplasia XX



Klinefelter syndrome XXY

Small penis

Decreased or normal, depending on age

Turner syndrome XO



45XO,46XY mosaicism  



In adolescent males and females, elevated testosterone may be diagnostic of precocious puberty; in boys, a decreased concentration may be indicative of hypogonadism.

In men, decreased testosterone levels may be due to primary testicular failure, secondary or tertiary hypogonadism, or treatment of prostate cancer with gonadotropin releasing hormone analogs or antiandrogens. Elevated testosterone levels may result from androgen-secreting tumors of the adrenal gland, late-onset congenital adrenal hyperplasia, or Cushing syndrome.

For symptomatic hypogonadal males receiving testosterone therapy, recommended total testosterone levels are within the mid-normal range observed in healthy younger men3 (280 to 873 ng/mL for men 19 to 40 years9). For men receiving testosterone injections, recommended levels are 400 to 700 ng/dL.3

In women, elevated serum testosterone levels can be due to androgen-secreting tumors of the adrenal gland or ovary (>150 ng/dL),2 PCOS, late-onset congenital adrenal hyperplasia, or Cushing syndrome.

Testosterone results should be interpreted in conjunction with other laboratory and clinical findings. Medical conditions altering serum concentrations of SHBG or albumin may affect the bioavailable testosterone level. Albumin levels decrease with liver disease, kidney disease, and nutritional deficiency, whereas SHBG levels decrease with obesity, diabetes mellitus, chronic illness, hypothyroidism, and use of glucocorticoids, progestins, and androgenic steroids.3 SHBG levels increase with aging in men, liver disease, hyperthyroidism, and use of anticonvulsants and estrogens.3


  1. Ahmed SF, Achermann JC, Arlt W, et al. Society for Endocrinology UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development (Revised 2015). Clin Endocrinol (Oxf). 2016;84:771-788.

  2. Azziz R, Carmina E, Dewailly D, et al. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril. 2009;91:456-488.

  3. Bhasin S, Cunningham GR, Hayes FJ, et al. Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010;95:2536-2559.

  4. Martin KA, Chang RJ, Ehrmann DA, et al. Evaluation and treatment of hirsutism in premenopausal women: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2008;93:1105-1120.

  5. Rosner W, Auchus RJ, Azziz R, et al. Position statement: utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab. 2007;92:405-413.

  6. Burger HG. Androgen production in women. Fertil Steril. 2002;77 Suppl 4:S3-5.

  7. de Ronde W, van der Schouw YT, Pols HA, et al. Calculation of bioavailable and free testosterone in men: a comparison of 5 published algorithms. Clin Chem. 2006;52:1777-1784.

  8. Ogilvy-Stuart AL, Brain CE. Early assessment of ambiguous genitalia. Arch Dis Child. 2004;89:401-407.

  9. Snyder PJ, Bhasin S, Cunningham GR, et al. Effects of testosterone treatment in older Men. N Engl J Med. 2016;374:611-624.

  10. Taieb J, Mathian B, Millot F, et al. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem. 2003;49:1381-1395.

  11. Wang C, Nieschlag E, Swerdloff R, et al. Investigation, treatment and monitoring of late-onset hypogonadism in males: ISA, ISSAM, EAU, EAA and ASA recommendations. Eur J Endocrinol. 2008;159:507-514.

  12. Wang C, Catlin DH, Demers LM, et al. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab. 2004;89:534-543.

Content reviewed 04/2017

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