Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone.

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with ß-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur.

First Morning Pregnanetriol and 17-Hydroxyprogesterone Correlated Significantly in 21-Hydroxylase Deficiency.

Background: Biochemically monitoring 21-hydroxylase deficiency (21-OHD) is challenging. Serum/blood 17-hydroxyprogesterone (17OHP) measurements are normally used for this purpose. Urinary pregnanetriol (PT), a urinary metabolite of 17OHP, may also be used. Based on auxological data, we previously reported that the optimal first morning PT value fell in the range of 2.2-3.3 mg/gCr (95% confidence interval of the mean) and 0.59-6.0 mg/gCr (10th - 90th percentile) for monitoring 21-OHD treatment. No report thus far has directly compared the first morning urinary PT value with the 17OHP value at various times during the day. Objective: To explore the correlation between the first morning urinary PT value before glucocorticoid administration and the serum/blood 17OHP value at three time points, namely, before and two and four hours after glucocorticoid administration. Design: This was a prospective study done at two children's hospitals. Methods: In total, 25 patients with 21-OHD aged 3-25 years were recruited. Their urinary PT levels and 17OHP levels were measured for three days within a total period of one week. The first morning PT value was collected on all three days. Dried blood spots and serum were used to measure 17OHP. Results: The range for the first morning PT value for all the samples (n=69) was 0.10-56.1 mg/gCr. A significant, positive correlation was found between the first morning PT and 17OHP values before medication (r=0.87, p<0.01), and weaker correlation was observed between the first morning PT and 17OHP values after medication. Conclusions: The first morning PT correlated more significantly with 17OHP before the morning medication. Measuring the first morning PT value may be more practical and useful for monitoring 21-OHD biochemically.

Isotope ratio mass spectrometry in antidoping analysis: The use of endogenous reference compounds.

RATIONALE: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers. METHODS: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11ß-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution. RESULTS: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway. CONCLUSIONS: For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts.

Modified-Release and Conventional Glucocorticoids and Diurnal Androgen Excretion in Congenital Adrenal Hyperplasia.

Context: The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown. Objective: We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone. Methods: We used urinary steroid metabolome profiling by gas chromatography-mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00-7:00, 7:00-15:00, and 15:00-23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort. Results: Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11ß-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations. Conclusions: Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.

Diagnosis of 21-hydroxylase deficiency by urinary metabolite ratios using gas chromatography-mass spectrometry analysis: Reference values for neonates and infants.

One major issue of newborn screening programs for 21-hydroxylase deficiency (21OHD) is the high rate of false-positive results, especially in preterm neonates. Urinary steroid metabolite analysis using gas chromatography-mass spectrometry (GC-MS) is suitable as a confirmatory diagnostic tool. The objective of this study was to analyze retrospectively diagnostic metabolite ratios in neonates and infants with and without 21OHD using GC-MS with emphasis on glucocorticoid metabolism, and to develop reference values for the steroid metabolite ratios for the diagnosis of 21OHD. We retrospectively analyzed urinary steroid hormone metabolites determined by GC-MS of 95 untreated neonates and infants with 21OHD (1-148 days), and 261 neonates and infants (100 preterms) without 21OHD (0-217 days). Metabolites of 17α-hydroxyprogesterone showed specificities below 98%, whereas the 21-deoxycortisol metabolite pregnanetriolone clearly separated 21OHD from non-21OHD subjects. The best diagnostic ratio for 21OHD was pregnanetriolone to 6α-hydroxy-tetrahydrocortisone. The lowest value of this ratio in the 21OHD group (0.47) was at least eight times higher than the highest values in the non-21OHD group (0.055). We have given appropriate reference values for steroid metabolite ratios in the largest 21OHD cohort so far described. Consideration of glucocorticoid metabolism, especially the use of typical neonatal 6α-hydroxylates metabolites, leads to improvement of diagnostic metabolite ratios.

Pheromone signaling: a pissing contest in tilapia.

In many species, males produce elaborate signals used by females in the evaluation of potential mates. Two urinary steroid epimers have now been shown to be components of a courtship display by male Mozambique tilapia that promotes female maturation.

Identity of a tilapia pheromone released by dominant males that primes females for reproduction.

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3-5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20ß-dihydroxypregn-4-en-3-one (17,20ß-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20ß-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20ß-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9-11].

Reduced activity of 11ß-hydroxylase accounts for elevated 17α-hydroxyprogesterone in preterms.

OBJECTIVE: To characterize the urinary steroid metabolome of neonates and infants born either at term or preterm. STUDY DESIGN: We retrospectively analyzed urinary steroid hormone metabolites determined by gas chromatography-mass spectrometry of 78 neonates and infants born at term and 83 neonates and infants born preterm (median 34 weeks of gestational age). The subjects' 11ß-hydroxylase and 21-hydroxylase activities were assessed on the basis of urinary metabolite substrate-to-product ratios. RESULTS: Preterm neonates and infants had elevated urinary concentrations of 17α-hydroxyprogesterone (17OHP) metabolites (P<.001) but lower urinary concentrations of the 21-deoxycortisol metabolite pregnanetriolone (PTO) (P<.01). One reason was lower 11ß-hydroxylase activity in preterms. We could demonstrate a correlation between low 11ß-hydroxylase activity and high urinary concentrations of 17OHP metabolites (r=0.51, P<.001) but low urinary concentrations of the 21-deoxycortisol metabolite PTO (r=-0.24, P=.03) in preterms. CONCLUSIONS: Low 11ß-hydroxylase activity may explain increased 17OHP but decreased 21-deoxycortisol metabolite excretion in preterms. Our analysis clarifies, first, why preterms have higher 17OHP levels and thus higher rates of false-positive screening results for congenital adrenal hyperplasia than do term infants, and, second, why 21-deoxycortisol or its urinary metabolite PTO is more specific than 17OHP for the diagnosis of 21-hydroxylase deficiency.

A 68-year-old phenotypically male patient with 21-hydroxylase deficiency and concomitant adrenocortical neoplasm producing testosterone and cortisol.

The steroidogenic enzyme 21-hydroxylase is necessary for the synthesis of both glucocorticoids and mineralocorticoids. 21-hydroxylase is a cytochrome P-450 enzyme and is encoded by the gene CYP21A2. Here we report a 68-year-old phenotypically 'male' but genetically female patient with 21-hydroxylase deficiency (21OHD) and the concomitant virilizing adrenocortical carcinoma. This patient grew up as a male and has not encountered any episodes of adrenal insufficiency without glucocorticoid replacement in his lifetime. A chromosome test at admission, however, identified the 46, XX karyotype, and serum 17-hydroxyprogesterone and urine pregnanetriolone and 11ß-hydroxyandrostendione were all elevated, consistent with 21OHD. Moreover, serum testosterone was 1.90 ng/ml, much higher than the female standard levels, and serum cortisol was 5.7 µg/ml, slightly lower than standard levels. Genetic analysis identified the patient as a heterozygote of the two pathogenic mutations in the CYP21A2 gene: IVS2-13C(A)>G and R356W. Magnetic resonance imaging (MRI) revealed the presence of left adrenal tumor measuring 6 cm, which was subsequently diagnosed as adrenocortical carcinoma based on the criteria of Weiss. Immunohistochemical analysis of the tumor specimens revealed the expression of various enzymes involved in testosterone production, including 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, and 17ß-hydroxysteroid dehydrogenase. Importantly, the expression of immunoreactive 21-hydroxylase was detected in these tumor cells. The levels of adrenal tumor-derived steroid metabolites were all markedly decreased following the surgery. This is the first report on a virilized 21OHD patient associated with the adrenocortical tumor that produces testosterone. Moreover, the concomitant adrenocortical tumor may ameliorate adrenocortical insufficiency by producing cortisol.

Testicular adrenal rest tumors develop independently of long-term disease control: a longitudinal analysis of 50 adult men with congenital adrenal hyperplasia due to classic 21-hydroxylase deficiency.

CONTEXT: Testicular adrenal rest tumors (TARTs) and hypogonadotropic hypogonadism are the two most common causes for male infertility in classic 21-hydroxylase deficiency. Current hypotheses suggest the quality of disease control to be one of the main pathogenic factors for TART development. OBJECTIVE: The aim was to study long-term predictors for TART development in a retrospective longitudinal study. DESIGN: Fifty men with classic 21-hydroxylase deficiency (31 salt wasting, 19 simple virilizing) were investigated. Testicular ultrasound at a median age at investigation of 27 years detected TARTs in 28 of 50 subjects (19 salt wasting, 9 simple virilizing). TART presence was correlated with long-term parameters of disease control during childhood and adolescence obtained from patients' charts: 24-hour urine pregnanetriol, serum 17-hydroxyprogesterone, onset and stage of pubic hair development, testicular growth, and bone age in relation to chronological age. RESULTS: There was no difference in pregnanetriol excretion over lifetime between patients with and without TARTs. Similarly, neither development of pubic hair and testicular volume (Tanner) nor bone age in relation to chronological age differed between the two groups. Furthermore, the two groups had the same body mass index and the same impairment of final height in relation to midparental target height. CONCLUSION: Our longitudinal analysis demonstrates no association between TART presence and parameters of disease control. These data, therefore, argue for other mechanisms more relevant for TART induction including those occurring during fetal development.

LC/MS/MS of steroids having vicinal diol as electrospray-active boronates.

A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4ß-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50°C within 1 h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.

Two new compounds from Scolopendra multidens Newport.

Two new compounds, 5ß-pregnane-2α,6α,20(S)-triol (1) and 8-hydroxyl-3-methoxyl-2(1H)-quinolone (2), were isolated from Scolopendra multidens Newport. Their structures were elucidated on the basis of spectroscopic methods including 1D and 2D NMR and HR-TOF-MS.

Reference ranges for the urinary steroid profile in a Latin-American population.

The urinary steroid profile has been used in clinical endocrinology for the early detection of enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age, exercise, diet, and ethnicity, among others; laboratories own reference ranges might compensate for ethnic differences among population and inter-laboratory biases. This paper shows the reference ranges obtained in the Antidoping Laboratory of Havana for the following steroid profile parameters: ten androgens (testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstan-3α,17ß-diol, 5ß-androstan-3α,17ß-diol, dehydroepiandrosterone, epiandrosterone, 11ß-hydroxyandrosterone and 11ß-hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and tetrahydrocortisol). The urine samples (male: n = 2454 and female: n = 1181) and data obtained are representative of population from Latin-American countries like Cuba, Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were prepared by solid-phase extraction followed by enzymatic hydrolysis and liquid-liquid extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were established for each sex, allowing the determination of abnormal profiles as a first diagnostic tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the Caucasian population confirms that the urinary steroid profile is influenced by ethnicity.

Two-step biochemical differential diagnosis of classic 21-hydroxylase deficiency and cytochrome P450 oxidoreductase deficiency in Japanese infants by GC-MS measurement of urinary pregnanetriolone/ tetrahydroxycortisone ratio and 11ß-hydroxyandrosterone.

BACKGROUND: The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD. METHODS: We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5ß-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11ß-hydroxyandrostenedione (11OHAD4) by GC-MS. RESULTS: At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11ß-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine. CONCLUSIONS: A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

Role of a disordered steroid metabolome in the elucidation of sterol and steroid biosynthesis.

In 1937 Butler and Marrian found large amounts of the steroid pregnanetriol in urine from a patient with the adrenogenital syndrome, a virilizing condition known to be caused by compromised adrenal secretion even in this pre-cortisol era. This introduced the concept of the study of altered excretion of metabolites as an in vivo tool for understanding sterol and steroid biosynthesis. This approach is still viable and has experienced renewed significance as the field of metabolomics. From the first cyclized sterol lanosterol to the most downstream product estradiol, there are probably greater than 30 steps. Based on a distinctive metabolome clinical disorders have now been attributed to about seven post-squalene cholesterol (C) biosynthetic steps and around 15 en-route to steroid hormones or needed for further metabolism of such hormones. Forty years ago it was widely perceived that the principal steroid biosynthetic defects were known but interest rekindled as novel metabolomes were documented. In his career this investigator has been involved in the study of many steroid disorders, the two most recent being P450 oxidoreductase deficiency and apparent cortisone reductase deficiency. These are of interest as they are due not to mutations in the primary catalytic enzymes of steroidogenesis but in ancillary enzymes needed for co-factor oxido-reduction A third focus of this researcher is Smith-Lemli-Opitz syndrome (SLOS), a cholesterol synthesis disorder caused by 7-dehydrocholesterol reductase mutations. The late George Schroepfer, in whose honor this article has been written, contributed greatly to defining the sterol metabolome of this condition. Defining the cause of clinically severe disorders can lead to improved treatment options. We are now involved in murine gene therapy studies for SLOS which, if successful could in the future offer an alternative therapy for this severe condition.

Monitoring of therapy in congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations. CONTENT: We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and 17-ketosteroids as well as serum measurements of 17-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays. SUMMARY: Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and 17-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.

Adiponectin levels are high in children with classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency.

OBJECTIVE: It has been shown that adiponectin serves as an insulin-sensitizing adipokine. Serum concentrations of adiponectin are low in children with obesity, and increase with fat mass loss, indicating that adiponectin can serve as a biomarker. Since the prevalence of overweight and obesity is increased in children with congenital adrenal hyperplasia (CAH), our study aimed to evaluate serum levels of adiponectin in a cohort of CAH children and adolescents, and their associations with clinical parameters such as chronological age (CA), body mass index (BMI), Tanner stage (TS), medication and metabolic control. PATIENTS AND METHODS: We studied 51 patients, aged between 5.6 and 19.6 years (median 11.8; 30 females, 21 males), cross-sectionally. All patients had genetically confirmed CAH and received standard steroid substitution therapy. Adiponectin was measured by an enzyme linked immunoassay. Since BMI SDS of the CAH cohort were significantly higher compared to the reference population, we built matched pairs with healthy Caucasian subjects from a normal representative cohort for sex, Tanner stage, chronologic age and BMI. RESULTS: Adiponectin concentrations were significantly higher in CAH patients (median 11 microg/L) compared to the matched controls (6.7 microg/L, p < 0.0001). Correlation analyses in CAH patients revealed a significant inverse relationship between adiponectin and CA, TS, BMI, serum DHEAS and serum testosterone, but no correlation with hydrocortisone and fludrocortisone dosage. CONCLUSION: Currently, the importance of the elevated adiponectin concentrations in CAH children for risk assessment is not clear. However, our data imply that besides adequate metabolic control of glucocorticoid substitution, a long-term follow-up of other metabolic markers of insulin resistance should be conducted in CAH patients.

Steroid measurement with LC-MS/MS in pediatric endocrinology.

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an increasingly common tool in the clinical laboratory. Established applications include routine assays for detecting inborn errors of metabolism and for monitoring therapeutic drugs and steroids. Steroid profiling is a very effective method for distinguishing almost all steroid related disorders. It allows accurate diagnosis and is very useful in many clinical situations. Most methods for the determination of steroid hormones are based on immunoassays, which are rapid and easy to perform. However, the reliability of steroid immunoassays has been shown to be doubtful because of the lack of specificity and of matrix effects. Immunological methods, especially direct assays, often overestimate true steroid values. This is of particular importance in the newborn period and in early infancy. Problems with steroid immunoassays have further been reported for female patients or when analysing different media, e.g. saliva. Patient follow-up over time or between laboratories, as well as longitudinal studies are extremely difficult. In contrast to immunoassays, which allow the measurement of only a single steroid at a time, LC-MS/MS has the advantage that a wide spectrum of steroid hormones can be measured simultaneously. The applicability for clinical samples and problems in pediatric endocrinology will be discussed.