Blood Spot LCMS Hormones 5 with SHBG

Testosterone is the principal androgen in males, synthesised by Leydig cells under LH control, and in smaller amounts by the adrenal cortex and, in females, by the ovaries and peripheral conversion of precursors. It circulates mostly protein-bound (to SHBG and albumin), with 1–3 per cent free; dried blood spot reflects the total circulating pool equilibrated into capillary whole blood at the time of collection. Liquid chromatography tandem mass spectrometry delivers the specificity required to quantify testosterone reliably across the physiological range, including the low female and child concentrations where immunoassays often fail.

Testosterone measurement underpins the evaluation of hypogonadism in males, hyperandrogenism in females, delayed or precocious puberty, and adrenal or gonadal tumours. LC-MS analysis of dried blood spot offers a convenient collection route without venepuncture, particularly useful in paediatric, anxious, or remote patients, though reference intervals are method- and matrix-specific and not interchangeable with serum.

Estradiol is the most potent naturally occurring oestrogen, produced by ovarian granulosa cells in premenopausal women, by peripheral aromatisation of testosterone in males and postmenopausal women, and by the placenta during pregnancy. It regulates the menstrual cycle, maintains bone density, modulates cardiovascular and cognitive function, and in males contributes to skeletal maturation and fertility. Dried blood spot LC-MS captures the unbound and protein-bound circulating pool, with sensitivity adequate for low postmenopausal and male concentrations.

Estradiol is measured to confirm ovarian function or failure, assess menstrual irregularities, investigate infertility, monitor ovulation-induction or hormone-replacement therapy, and evaluate gynaecomastia or suspected aromatase-excess states in males. The dried blood spot matrix permits at-home collection but requires platform-specific reference data.

Progesterone is secreted by the corpus luteum after ovulation, preparing the endometrium for implantation and maintaining early pregnancy until placental production begins. In the absence of ovulation progesterone remains low. Small amounts are produced by the adrenal cortex in both sexes. Dried blood spot progesterone equilibrates from the circulating pool and is quantified by LC-MS to avoid the cross-reactivity that plagues immunoassays of related steroids.

Progesterone measurement confirms ovulation, assesses luteal-phase adequacy, investigates infertility and recurrent pregnancy loss, and monitors progesterone-replacement therapy. A mid-luteal value above 5 ng/mL generally indicates ovulation has occurred; lower values suggest anovulation or luteal insufficiency.

DHEA-S is the sulphated, water-soluble reservoir form of dehydroepiandrosterone, synthesised almost exclusively in the adrenal zona reticularis under ACTH control. Its long half-life (7–10 hours for DHEA-S versus minutes for DHEA) and high circulating concentration make it a stable marker of adrenal androgen output, with minimal diurnal variation. It serves as a precursor for peripheral conversion to more potent androgens and oestrogens.

DHEA-S is used to distinguish adrenal from ovarian or testicular androgen excess in hirsutism and virilisation, to screen for adrenal tumours or late-onset congenital adrenal hyperplasia, and to assess adrenal reserve. Functional practitioners also track age-related decline, though the clinical significance of supplementing physiological decline remains debated.

Cortisol is the principal human glucocorticoid, secreted by the adrenal zona fasciculata in a pulsatile and circadian pattern with peak levels shortly after waking and a nadir near midnight. It circulates largely bound to cortisol-binding globulin, with 5–10 per cent free; dried blood spot reflects total circulating cortisol at the moment of collection. LC-MS avoids synthetic-steroid cross-reactivity that affects immunoassays.

Cortisol measurement underpins the diagnosis of Cushing syndrome, adrenal insufficiency, and evaluation of the hypothalamic–pituitary–adrenal axis. Because cortisol is highly time-dependent, a single random dried blood spot is less diagnostically robust than timed saliva or 24-hour urinary free cortisol; it serves better as a snapshot within a longitudinal or multi-point functional assessment.

SHBG is a hepatic glycoprotein that binds testosterone and oestradiol with high affinity, regulating the bioavailable fraction of sex steroids. Its synthesis is increased by oestrogens and thyroid hormone and decreased by androgens, insulin, and obesity. SHBG concentration influences the free and bioavailable hormone pools, so measurement alongside total testosterone or oestradiol refines interpretation of androgen and oestrogen status.

SHBG is measured to calculate free or bioavailable testosterone, to investigate hyperandrogenism (low SHBG amplifies androgen action) and apparent hypogonadism (high SHBG may reduce bioavailable hormone despite normal total levels), and to assess metabolic syndrome, where low SHBG is a common feature and independent cardiovascular risk marker.

Free testosterone represents the unbound, biologically active fraction, approximately 1–3 per cent of total testosterone. Because direct free-testosterone immunoassays are unreliable, free testosterone is calculated from total testosterone and SHBG using empirical equations (Vermeulen or similar) that estimate the partition between bound and free hormone.

Calculated free testosterone refines the assessment of androgen status when SHBG is abnormal — high SHBG can mask bioavailable androgen deficiency, low SHBG can mask hyperandrogenism. The calculation improves diagnostic sensitivity for hypogonadism and polycystic ovary syndrome, but its accuracy depends on the total testosterone and SHBG assays and the equation used.