LC-MS/MS Measurement of Parathyroid Hormone-Related Peptide.

INTRODUCTION: Parathyroid hormone-related peptide (PTHrP) is involved in activating pathways, allowing tumor cells to form bone metastases. Measurement of PTHrP is used for the diagnosis and clinical management of patients suspected of hypercalcemia of malignancy. We developed an LC-MS/MS method for measuring PTHrP, established sex-specific reference intervals, and assessed the method's performance. METHODS: PTHrP was enriched from plasma samples with rabbit polyclonal anti-PTHrP antibody conjugated to magnetic beads. Enriched PTHrP was digested with trypsin, and PTHrP-specific tryptic peptide was analyzed with 2-dimensional LC-MS/MS in multiple reaction monitoring mode. RESULTS: The lower limit of quantification was 0.6 pmol/L, and the upper limit of linearity was 600 pmol/L. Total imprecision was <10%. Very poor agreement was observed with the RIA (n = 207; Deming regression RIA = 0.059 × LC-MS/MS - 1.8, r = 0.483; Sy|x = 3.9). Evaluation of the clinical performance of the assay using samples from patients with and without hypercalcemia (n = 199) resulted in an area under the ROC curve of 0.874. In sets of consecutively analyzed routine samples of patients assessed for hypercalcemia, the PTHrP positivity rate by RIA (n = 1376) was 1.9%, and 26.6% by LC-MS/MS (n = 1705). Concentrations were below the lower limit of quantification in 95.6% of the samples by RIA and 2.0% by LC-MS/MS. CONCLUSIONS: PTHrP is a normal constituent in circulating blood and its concentrations are substantially underestimated by commercial RIAs, causing false-negative results in samples from patients suspected of hypercalcemia. Our observations suggest a link between increased concentrations of PTHrP in postmenopausal women with low body mass index and increased incidence of osteoporosis.

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase.

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-ß-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.

Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies.

BACKGROUND: Measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC). Difficulty in using Tg as a biomarker of the recurrence of TC in many patients stems from the presence of endogenous anti-Tg autoantibodies (Tg-AAbs), which can interfere with immunoassays (IAs) and cause false-negative results. METHODS: We enriched Tg from serum samples using rabbit polyclonal anti-Tg antiserum and protein precipitation. Unrelated proteins were partially depleted in the process. Enriched proteins were then denatured, reduced, and digested with trypsin after the addition of a winged internal standard peptide. A Tg-specific tryptic peptide was purified by immunoaffinity extraction and analyzed by 2-dimensional LC-MS/MS. Instrument cycle time was 6.5 min per sample. RESULTS: The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL dimer). Total imprecision of triplicate measurements in serum samples over 5 days was <10%. Comparison with a commercial IA using serum samples free of Tg-AAb (n = 73) showed Deming regression, IA = 1.00 * LC-MS/MS - 2.35, r = 0.982, standard error of the estimate (S(y|x)) = 9.52. In a set of Tg-AAb-positive samples that tested negative for Tg using IA (n = 71), concentrations determined by LC-MS/MS were ≥0.5 ng/mL in 23% of samples (median 1.2, range 0.7-11 ng/mL). CONCLUSIONS: The introduced method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between methods was observed in Tg-AAb-positive samples with concentrations <2 ng/mL (determined with LC-MS/MS). The affinity-assisted enrichment strategy used for Tg in this method should be applicable to other biomarkers that have endogenous autoantibodies.

Liquid chromatography-tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric and adult reference intervals.

BACKGROUND: Measurement of serum androgens is important in adult, geriatric, pediatric endocrinology, and oncology patients. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of androstenedione, dehydroepiandrosterone (DHEA), and testosterone in these patients. METHODS: We spiked 200 muL of serum or plasma with isotope-labeled internal standards and performed extraction with methyl t-butyl ether. We then derivatized the extracts with hydroxylamine and analyzed them by LC-MS/MS using a 2-dimensional chromatographic separation with a 3.5-min analysis time. RESULTS: Total imprecision for each analyte was <11.2%. Limits of quantification were 10, 50, and 10 ng/L for androstenedione, DHEA, and testosterone, respectively. Reference intervals were established for children (age 6 months to 17 years), men, and women. Androstenedione and DHEA concentrations were lowest in 2- to 3-year-old children. Adult concentrations were achieved in girls at Tanner stage 3 and in boys at Tanner stage 4-5. In premenopausal and (postmenopausal) women the median concentrations of androstenedione, DHEA, and testosterone were 810 (360), 3000 (1670), 270 (180) ng/L, respectively. In postmenopausal women, concentrations of testosterone were age independent, whereas androstenedione and DHEA concentrations decreased with age. In men the median concentrations of androstenedione, DHEA, and testosterone were 440, 2000, and 3700 ng/L, respectively. In men older than 40 years, median concentrations decreased at rates of 5%, 10%, and 20% per decade for androstenedione, DHEA, and testosterone, respectively. CONCLUSIONS: This LC-MS/MS method has the required lower limit of quantification and specificity for analysis of endogenous concentrations of androgens in all groups studied. Reference intervals were established for healthy children and adults.

Association of aromatase (TTTAn) repeat polymorphism length and the relationship between obesity and decreased sperm concentration.

BACKGROUND: Obesity in men is associated with low sperm count, however, this finding is inconsistent. Here, we describe length of the short tandem repeat aromatase (CYP19A1) polymorphism and its relationship to increased weight and sperm count. METHODS: A cohort of 215 men was recruited from the community and BMI, hormone levels and sperm parameters were determined at enrollment. Men (196) were genotyped for length of the tetranucleotide TTTA repeats polymorphism (TTTA(n)), defined as short (S ≤ 7 repeats) or long (L > 7 repeats). Genotypes were categorized using allele combinations as 'low repeats' = S-S, or 'high repeats' = S-L/L-L. Weight and sperm parameters were examined in relation to size of TTTA(n) repeat. RESULTS: Mean (±SD) age was 29.8 ± 8.6 years and mean BMI was 25.6 ± 4.6 kg/m(2). Men with high repeats had higher estradiol (E(2)) levels (98.0 ± 33.36 pmol/l) than men with low repeats (85.9 ± 26.61 pmol/l; P= 0.026). Lower FSH levels tended to be present in men with high repeats versus men with low repeats (P= 0.052). After stratification by genotype, a negative correlation between BMI and sperm count (Pearson's coefficient = 0.406) was seen only among men with high repeats (P= 0.019). Only men with high repeats exhibited increased E(2) with increased weight. A decrease in testosterone: E(2) ratio with increasing BMI was more pronounced in men with high versus low, repeats (R(2) = 0.436 versus 0.281). CONCLUSIONS: Higher TTTA repeat numbers (>7 repeats) in the aromatase gene are associated with a negative relationship between obesity and sperm count. The effect of obesity on E(2) and sperm count appears to be absent in men with low (≤7) repeats.

Competitive surface-enhanced Raman scattering assay for the 1,25-dihydroxy metabolite of vitamin D3.

This paper describes the development and preliminary testing of a competitive surface-enhanced Raman scattering (SERS) immunoassay for calcitriol, the 1,25-dihydroxy metabolite (1,25-(OH)(2)-D(3)) of vitamin D(3). Deficiencies in 1,25-(OH)(2)-D have been linked to renal disease, while elevations are linked to hypercalcemia. Thus, there has been a sharp increase in the clinical demand for measurements of this metabolite. The work herein extends the many attributes of SERS-based sandwich immunoassays that have been exploited extensively in the detection of large biolytes (e.g., DNA, proteins, viruses, and microorganisms) into a competitive immunoassay for the low level determination of a small biolyte, 1,25-(OH)(2)-D(3) (M(w) = 416 g mol(-1)). The assay uses surface modified gold nanoparticles as SERS labels, and has a dynamic range of 10-200 pg mL(-1) and a limit of detection of 8.4 ± 1.8 pg mL(-1). These analytical performance metrics match those of tests for 1,25-(OH)(2)-D(3) that rely on radio- or enzyme-labels, while using a much smaller sample volume and eliminating the disposal of radioactive wastes. Moreover, the SERS-based data from pooled-patient sera show strong agreement with that from radioimmunoassays. The merits and potential utility of this new assay are briefly discussed.

Endogenous sex hormones and incident fracture risk in older men: the Dubbo Osteoporosis Epidemiology Study.

BACKGROUND: Data on the influence of gonadal hormones on incident fracture risk in elderly men are limited. We prospectively examined the relationship between serum levels of testosterone and estradiol and future fracture risk in community-dwelling men. METHODS: A total of 609 men older than 60 years had been observed between January 1989 and December 2005, with the median duration being 5.8 years (up to 13 years). Clinical risk factors, including bone mineral density and lifestyle factors, were assessed at baseline. Serum testosterone and estradiol levels were measured by tandem mass spectrometry. The incidence of a low-trauma fracture was ascertained during follow-up. RESULTS: During follow-up, 113 men had at least 1 low-trauma fracture. The risk of fracture was significantly increased in men with reduced testosterone levels (hazard ratio [HR], 1.33; 95% confidence interval [CI], 1.09-1.62). After adjustment for sex hormone-binding globulin, serum testosterone (HR, 1.48; 95% CI, 1.22-1.78) and serum estradiol (HR, 1.21; 95% CI, 1.00-1.47) levels were associated with overall fracture risk. After further adjustment for major risk factors of fractures (age, weight or bone mineral density, fracture history, smoking status, calcium intake, and sex hormone-binding globulin), lower testosterone was still associated with increased risk of fracture, particularly with hip (HR, 1.88; 95% CI, 1.24-2.82) and nonvertebral (HR, 1.32; 95% CI, 1.03-1.68) fractures. CONCLUSION: In community-dwelling men older than 60 years, serum testosterone is independently associated with the risk of osteoporotic fracture and its measurement may provide additional clinical information for the assessment of fracture risk in elderly men.

High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol.

High-sensitivity measurement of serum estrogens is important in adult and pediatric endocrinology and oncology. We developed a high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of estrone (E1) and estradiol (E2). Aliquots of 200 muL of serum were spiked with internal standard, extracted, derivatized with dansyl chloride, and analyzed by LC-MS/MS using 2-dimensional chromatographic separation. Total imprecision for the method was less than 11%; the limit of quantitation was 1 pg/mL. Reference intervals were established with samples from more than 900 healthy postmenopausal women, men, girls, and boys. Concentrations of estrogens in children reached adult levels by Tanner stage 3. In men and postmenopausal women, the median concentrations of total estrogens (E1 + E2) were 39 and 22 pg/mL, and the median E2/E1 ratios were 0.98 and 0.55, respectively. The method requires a small sample volume and has adequate sensitivity and specificity for analyzing estrogens in samples from postmenopausal women, men, and children.

Adrenal steroid concentrations in children seven to seventeen years of age.

During puberty, serum steroid concentrations change dramatically. The objective of this study was to determine the adrenal steroid concentrations in children from 7 to 17 years of age. Tanner stage was determined in each child by physical examination. 11-Deoxycortisol, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and testosterone were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Androstenedione and dehydroepiandrosterone sulfate were measured by immunoassay. The median and central 95% of the steroid concentrations were determined for age, gender, and Tanner stage. Except for 11-deoxycortisol, all of the steroids exhibited an increase in concentration after age 7-9 years in both boys and girls. 11-Deoxycortisol, which is made exclusively in the adrenal cortex, declined with age and Tanner stage. This suggests that a rise in gonadal function and decreased efficiency of 11beta-hydroxylase with age may contribute to an increase in the remaining steroids. Testosterone concentrations increased more dramatically in boys, but increases were seen with each Tanner stage in girls.

An exploratory study Evaluating the impact of opioid and non-opioid pain medications on serum/plasma free testosterone and free estradiol concentrations.

Chronic use of opioid medications has been reported to cause altered sexual function. It is not known if non-opioid pain medications have similar effects. Assessment of this effect through the measurement of concentrations of free hormones is limited. Positivity of opioid medications (hydrocodone, oxycodone, morphine, methadone, tramadol and fentanyl) and non-opioid pain medications (gabapentin or pregabalin) in human serum and plasma samples from adult men and women were evaluated for association with concentrations of free testosterone (fTe) and free estradiol (fE2) measured using equilibrium dialysis-liquid chromatography-tandem mass spectrometry methods. Lower concentrations of fTe (p = 0.0253) were observed in samples positive for the hydrocodone, oxycodone and morphine group compared to age matched controls. The presence of methadone, tramadol, fentanyl and pregabalin had no effect on fTe. When compared with age-matched controls, women between 48-55 years of age showed reduced fE2 concentrations in samples positive for tramadol, fentanyl and gabapentin (p = 0.0243, 0.0045 and 0.0050, respectively). Particular opioid medications such as methadone, tramadol or fentanyl and non-opioid medications such as pregabalin or gabapentin may offer advantages over opioid medications for treating pain with fewer endocrinologic side effects. Measurement of free hormones in pain medication users could be important in determining their association with sexual function. Copyright © 2017 John Wiley & Sons, Ltd.

Direct Measurement of Free Estradiol in Human Serum and Plasma by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry.

We describe a direct method of measurement of free estradiol using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum aliquots and internal standards are extracted by liquid-liquid extraction using methyl-tert-butyl ether (MTBE) followed by derivatization with dansyl chloride. An API 5500 mass spectrometer operated in positive electrospray mode is used for detection.

High Sensitivity Measurement of Pancreatic Polypeptide and Its Variant in Serum and Plasma by LC-MS/MS.

Aliquots of serum or plasma samples are combined with stable isotope labeled internal standard. Pancreatic polypeptide (PP) and its truncated variant PP3-36 are enriched by incubation with anti-PP antibody conjugated to magnetic beads. Peptides are eluted from beads in acidic buffer and the samples analyzed using liquid chromatography coupled with tandem mass spectrometry. Instrumental analysis of PP and PP3-36 is performed using electrospray ionization ESI in positive ion mode and multiple reaction monitoring (MRM) acquisition.

Exploratory study of the association of steroid profiles in stimulated ovarian follicular fluid with outcomes of IVF treatment.

Steroid concentrations in stimulated follicular fluid (sFF) samples have been linked to the quality of oocytes used in IVF treatments. Most of the published studies focused on evaluating the association of the IVF outcomes with only a few of the steroids, measured by immunoassays (IA). We performed a treatment outcome, prospective cohort study using stimulated FF sampled from 14 infertile women undergoing IVF treatment; single oocyte was used per IVF cycle. Fourteen endogenous steroids were analyzed in 22 ovarian follicle aspirations, which corresponded to the embryos used in the IVF. Ten oocytes were associated with live birth (LB) and 12 with no pregnancy (NP). Steroids were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Differences in distribution of concentrations in association with the pregnancy outcome (LB or NP), and receiver operating characteristic (ROC) curves analysis were performed for the entire cohort and for within-women data. The predominant androgen and estrogen in stimulated sFF were androstenedione (A4) and estradiol (E2), respectively. Lower concentrations of pregnenolone (Pr), lower ratios of A4/ dehydroepiandrosterone (DHEA), testosterone (Te)/DHEA, and greater ratios of E2/Te, and estrone/A4 were observed in sFF samples associated with LB. Among the oocytes associated with NP, in four out of 12 samples total concentration of androgens was above the distribution of the concentrations in the oocytes corresponding to the LB group. Observations of the study indicated increased consumption of precursors and increased biosynthesis of estrogens in the follicles associated with LB. Our data suggest that potentially steroid profiles in sFF obtained during oocyte retrieval may serve as biomarkers for selection of the best embryo to transfer after IVF.

Mid-pregnancy, perinatal, and neonatal reproductive endocrinology: a prospective cohort study in twins and singleton control subjects.

OBJECTIVE: To answer the questions: Are perinatal reproductive hormone profiles different in case of a twin compared with a singleton pregnancy? Are reproductive endocrine profiles of twin girls influenced by their male co-twin and vice versa? DESIGN: Prospective cohort study from January 2004 to October 2009. SETTING: Not applicable. PATIENT(S): A total of 204 mothers of twins and 248 singleton control subjects, aged >18 years, pregnant with a twin or singleton and no endocrine disease or malignancy. INTERVENTION(S): Blood samples were collected at mid-gestation from the mother and at delivery from the mothers and the umbilical cords. Estrogens, androgens, sex hormone-binding globulin, progesterone, and gonadotropins were measured. MAIN OUTCOME MEASURE(S): Hormonal profiles were compared between singletons and twins, different types of twins, and opposite-sex and same-sex twins. RESULT(S): Estrogen and progesterone concentrations were higher in mothers of twins compared with singletons, but twin babies had lower estrogen and progesterone concentrations at birth. Opposite-sex twin girls did not have higher androgens in cord blood compared with same-sex twin girls. Boys of an opposite-sex twin had lower luteinizing hormone concentrations compared with dizygotic twin boys with a brother as a co-twin. CONCLUSION(S): Children from a twin are not overexposed to sex steroids at the time of birth, despite higher concentrations in their mothers, and girls from opposite sex twins do not show androgenic influences from their male co-twin. The female co-twin may influence the hypothalamic-pituitary-testicular axis of her brother via central inhibition.

Pseudo-Cushing syndrome caused by fenofibrate interference with urinary cortisol assayed by high-performance liquid chromatography.

Urinary free cortisol (UFC) excretion over 24 h reflects the production rate of cortisol and is used commonly in the diagnosis of Cushing syndrome. We report on two patients evaluated for Cushing syndrome who had elevated UFC when analyzed by HPLC but normal values for the analysis performed by RIA and HPLC-mass spectrometry/mass spectrometry (HPLC-MS/MS). Other laboratory testing was inconsistent with the diagnosis of Cushing syndrome and raised doubts about the diagnosis. We identified a probable cause of analytical interference as coming from fenofibrate (Tricor), medication taken by the patients. Fenofibrate peak overlapped with the HPLC peak of cortisol and produced an MS/MS transition overlapping the major transition of cortisol. A second MS/MS transition was free from interference. In summary, fenofibrate administration may cause false elevation of UFC values determined by HPLC or HPLC-MS/MS in patients evaluated for Cushing syndrome. An HPLC-MS/MS method using multiple mass transitions, rather than a single transition, allows accurate quantitation of urinary cortisol in patients taking fenofibrate.

The interrelationships between thyroid dysfunction and hypogonadism in men and boys.

Thyroid hormone deficiency affects all tissues of the body, including multiple endocrine changes that alter growth hormone, corticotrophin, glucocorticoids, and gonadal function. Primary hypothyroidism is associated with hypogonadotropic hypogonadism, which is reversible with thyroid hormone replacement therapy. In male children follicle-stimulating hormone (FSH) is elevated and associated with testicular enlargement without virilization. Men with primary hypothyroidism have subnormal responses of luteinizing hormone (LH) to gonadotropin-releasing hormone (GnRH) administration and normal response to human chorionic gonadotropin (hCG). Free testosterone concentrations are reduced in men with primary hypothyroidism and thyroid hormone replacement normalizes free testosterone concentrations. In men with primary hypothyroidism, prolactin is not consistently elevated (except in men and children with longstanding severe primary hypothyroidism), but prolactin declines following thyroid hormone replacement therapy. Thyroid hormone is known to affect sex hormone-binding hormonal globulin (SHBG) concentrations. Men with hyperthyroidism have elevated concentrations of testosterone and SHBG. Thyroid hormone therapy in normal men may also duplicate this elevation. In addition estradiol elevations are observed in men with hyperthyroidism, and gynecomastia is common in them as well. In contrast to patients with primary hypothyroidism, men with hyperthyroidism exhibit hyperresponsiveness of LH to GnRH administration and subnormal responses to hCG. Radioactive iodine therapy (RAI) of men treated for thyroid cancer produces a dose-dependent impairment of spermatogenesis and elevation of FSH up to approximately 2 years. Permanent testicular germ cell damage may occur in men treated with high doses of RAI. RAI commonly increases serum concentrations of FSH and LH while reducing inhibin B levels without affecting serum concentrations of testosterone. Thus, radioiodine therapy transiently impairs both germinal and Leydig cell function that usually recover by 18 months posttherapy.

Enhancement of specificity of aldosterone measurement in human serum and plasma using 2D-LC-MS/MS and comparison with commercial immunoassays.

BACKGROUND: Accurate measurement of aldosterone is important for the standardized testing of primary hyperaldosteronism. Commercial immunoassays show substantial between-method variations resulting in significant clinical consequences. We developed a specific two dimensional (2D)-LC-MS/MS method for measuring aldosterone in human serum and plasma and compared it with three commercial immunoassays and an LC-MS/MS method. METHODS: 250 µL samples, controls and calibrators spiked with d4-aldosterone were subjected to liquid-liquid extraction. The samples were analyzed using negative mode electrospray and 2D-LC followed by MS detection using an ABSciex 5500 mass spectrometer and compared with immunoassays of Siemens (Coat-A-Count), DiaSorin (CLIA-LIAISON), and IBL (ELISA). Data was acquired using multiple reaction-monitoring mode. RESULTS: LOQ and LOD of the method were 0.04 and 0.02 nmol/L respectively. The assay was linear up to 166 nmol/L. Inter and intra-assay imprecision at 0.13, 1.38 and 8.30 nmol/L were <10%. Interferences were absent and no differences were observed between serum and plasma matrices. Method recovery ranged from 95% to 113%. Ion suppression was not observed. Evaluated immunoassays showed positive biases ranging between 22% and 37% when compared with the developed method. CONCLUSIONS: We developed and validated an accurate method for measurement of aldosterone in human serum and plasma using 2D-LC-MS/MS which is suitable for clinical purposes. The method is faster than previously published LC-MS/MS methods, uses less sample, has adequate sensitivity while being able to preserve high specificity in a cost effective manner. Linearity of the assay makes it promising for urine and adrenal venous samples. Comparison with three commercial immunoassays demonstrates the advantages of the developed method.

Direct measurement of free estradiol in human serum by equilibrium dialysis-liquid chromatography-tandem mass spectrometry and reference intervals of free estradiol in women.

BACKGROUND: Measurement of free estradiol offers a better representation of the bioactive fraction of the hormone. We describe a direct equilibrium dialysis-liquid chromatography-tandem mass spectrometry (ED-LC-MS/MS) method for serum free estradiol. METHODS: Two hundred fifty microliter aliquots of serum were dialyzed for 22h followed by liquid-liquid extraction and derivatization with dansyl chloride. Free estradiol was measured using LC-MS/MS with an AB SCIEX 5500 mass spectrometer in positive ion and multiple reaction monitoring (MRM) mode. RESULTS: The limits of detection and quantification for free estradiol were 0.25 and 0.5pg/ml (0.9 and 1.8pmol/l) respectively. Total imprecision was less than 10%. Results of method comparison showed 3 times overestimation using indirect methods of measurement. Reference intervals in pre-menopausal women in follicular, mid-cycle, and luteal phases of cycle were <2.4, <3.1 and <2.6pg/ml (8.8, 11.4, 9.5pmol/l) respectively; in post menopausal women the concentrations were ≤0.5pg/ml (1.8pmol/l). CONCLUSIONS: ED-LC-MS/MS is a direct method for accurately measuring free estradiol, independent of total estradiol or sex hormone binding globulin concentrations. Imprecision and sensitivity of the method are adequate for clinical diagnostic applications. The degree of variation observed in the method comparison reinforces the relevance of method specific reference ranges.