Total testosterone: immunoassay versus LC/MS-MS, and the range by age

Total testosterone is the most-ordered hormone test in this population and one of the easiest to over-read. The method matters, the reference range moves with age, and a single draw says less than people think.

What the test measures

Total testosterone is everything in the sample: the fraction bound tightly to sex hormone-binding globulin, the fraction bound loosely to albumin, and the small free fraction. It is one number covering three populations of molecules with quite different availability to tissue, which is why it is a starting point rather than a conclusion.

It is also the number that moves most over a day. Testosterone follows a diurnal rhythm that is pronounced in younger men and flatter with age, which is the reason morning draws are the convention — a result taken at four in the afternoon is not comparable to one taken at eight in the morning.

Immunoassay versus mass spectrometry

Most general panels run an immunoassay. It is fast and cheap and, in the middle of the adult male range, broadly adequate. Where it degrades is at the low end and in samples with unusual binding-protein or steroid profiles, and the direction of the error is not consistent.¹

Established clinical use That the two methods diverge materially at low concentrations is established. The Journal of Clinical Endocrinology and Metabolism requires mass spectrometry for the sex steroid measurements it publishes,² and comparisons of direct immunoassay against LC-MS/MS in men have found disagreement large enough to change how a result reads.¹ The registry note the app carries puts it plainly: do not trend across methods without flagging the switch.

That last point is the practical one. If your first result came from an immunoassay and your second from LC/MS-MS, the difference between them is partly the method, and reading it as a change in you is a mistake. The app keeps the method with the value for exactly this reason.

The range, and why it moves with age

Total testosterone falls gradually with age in most men, and the reference intervals below reflect that — they are the bands the app itself applies, generated by running its own range function rather than transcribed.

Off-label or community practice The optimal band is a different thing from the reference range and is non-diagnostic. Reference intervals are built from a population; an "optimal" band is a convention drawn from clinical literature and community practice about where people tend to feel and function well. Neither is a diagnosis, and a number inside a range does not rule out a problem any more than a number outside one establishes one.

A diagnosis of testosterone deficiency conventionally rests on symptoms plus more than one low morning measurement, not on a single draw — which is worth knowing before acting on one number in either direction.

Reading a result on therapy

On testosterone therapy the number depends heavily on when it was drawn relative to the last injection. A trough drawn the morning of the next dose and a peak drawn two days after one are both "your level", and they can differ by a factor that dwarfs any change you might be trying to detect. Whatever you choose, keep it consistent, and record it — the app stores the draw timing alongside the value.

Off-label or community practice Community practice and most clinics settle on trough draws for monitoring, because a trough is reproducible in a way a peak is not: it is pinned to the injection schedule rather than to how fast a given ester released on a given week. Whether your protocol is right for you is a conversation for the clinician prescribing it, with a consistent draw time and a named assay in front of you.

What the app records for this marker

How the app adjusts this range by sex and age

Generated by running the app's own range function against each band, so this table cannot drift from what the app flags against. The optimal column is non-diagnostic throughout.

Compounds whose monitoring panel includes this marker

Generated by matching this marker's own aliases against the monitoring note on every compound in the app's reference, so the list is the app's, not an author's.

• Testosterone Cypionate Total T, Free T, E2 (sensitive assay), SHBG, Hematocrit, CBC, PSA, Lipids, CMP. Every 6-8 weeks adjusting; every 3-6 months stable.
• Kisspeptin-10 Total T, LH, FSH, E2 every 4 weeks. LH surge 30-60 min post-injection confirms receptor response.
• Anastrozole E2 sensitive assay, Total T, Free T every 6-8 weeks when adjusting.
• Tamoxifen Total T, LH, FSH, E2 at end of PCT. Goal: LH/FSH recovering, T returning toward baseline.
• Clomiphene LH, FSH, Total T, E2 throughout PCT and 4 weeks post-completion.
• HCG E2 (HCG aromatizes significantly), Total T. LH/FSH will be suppressed — this is expected.
• Enclomiphene Total T, Free T, LH, FSH, E2, Semen analysis (if fertility is goal), every 6-8 weeks when adjusting.
• Exemestane E2 sensitive assay, Total T, Free T, Lipid panel, Bone density with long-term use.
• Dutasteride / Finasteride Total T, Free T, DHT levels (if testing available), PSA, sexual function self-assessment monthly.
• DHEA DHEA-S (primary marker), Total T, Free T, E2 (DHEA converts to both), PSA in men, lipid panel.
• Kisspeptin-54 LH, FSH, Total T, E2 every 4 weeks. LH surge 45-60 min post-injection confirms receptor response.
• Gonadorelin LH, FSH (will show some activity unlike HCG protocols), Total T, E2, testicular volume assessment. LH response 30-60 min post-injection confirms pituitary response.
• Testosterone Pellets Total T, Free T, E2, Hematocrit at 4 weeks post-insertion and at trough (just before next insertion). PSA annually. Insertion site monitoring for infection or extrusion.

Questions people actually ask

My level is "normal" but I feel terrible. What does that mean?

It means a population reference interval did not answer your question, which is a limit of the test rather than a contradiction. Reference intervals are wide, they are built from a population that includes people who feel fine and people who do not, and total testosterone does not describe how much is actually available to tissue. Free testosterone and SHBG address that second point directly. What it means for you is a clinical question, not a laboratory one.

Does the draw have to be in the morning?

For a diagnostic workup, conventionally yes — the diurnal rhythm is real and the reference intervals were built on morning samples. On established therapy the more important thing is consistency relative to your injection schedule, so that two results are comparable to each other.

How do I convert nmol/L to ng/dL?

The converter above does it with the app's own factor: nmol/L × 28.84 gives ng/dL. Most non-US reports print nmol/L, and mixing the two units is one of the more common reasons a result looks wildly out of range when it is not.

Sources

Every claim above that is not a definition is either labelled by evidence tier or carries a numbered reference to one of these.

1. Measurement of Serum Testosterone in Nondiabetic Young Obese Men: Comparison of Direct Immunoassay to Liquid Chromatography-Tandem Mass Spectrometry Direct immunoassay compared against LC-MS/MS in men, with the size of the disagreement.
2. Handelsman & Wartofsky, Requirement for Mass Spectrometry Sex Steroid Assays, J Clin Endocrinol Metab 98(10):3971 (2013) The journal requiring mass spectrometry for sex steroid assays in published work.
3. ADLM (formerly AACC), Optimal Testing: laboratory test utilisation guidance Assay-selection guidance for sex steroids at low concentrations.