Taylor, Robert L.

Abstract Background: Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines. Methods: We added 250 ng of d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 °C water bath, neutralized, and prepared by solid-phase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column. Results: Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of 10–5000 μg/L. Interassay CVs were 5.7–8.6% at mean concentrations of 90–4854 μg/L for MN and NMN. The detection limit was 10 μg/L. Recovery of MN and NMN (144–2300 μg/L) added to urine was 91–114%. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y = 0.81x − 0.006 (r = 0.822; n = 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y = 1.09x + 0.05 (r = 0.998; n = 40). Conclusions: The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography–mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

Nonadherence with anti-inflammatory treatment is a frequent cause of continued symptoms in asthmatic patients. Clinical assessments including patient-reported medication administration may provide the asthma specialist incomplete information regarding actual adherence to anti-inflammatory medications. The objective of this report was to describe the first case where adherence to inhaled asthma therapy was assessed by direct analysis of glucocorticoids in induced sputum. The patient's blood, urine, and sputum were tested for synthetic corticosteroids using mass spectrometry. To evaluate a clinical suspicion of poor adherence, sputum, urine, and blood were used to assess for current compliance to medication use. We report a case where asthma specialists attributed poorly controlled asthma to nonadherence to medical therapy. After modification of the medical regimen, adherence with oral and inhaled steroids was assessed–via examination of the urine, blood, and sputum. Direct analysis of glucocorticoids in sputum is feasible and in theory could provide a novel tool to document current medication adherence. Concomitant assessment of glucocorticoids and eosinophils in the same induced sputum specimen could provide insight into possible steroid resistance in select referral patients with difficult asthma.

Abstract Context: Prenatal androgen excess can cause a phenocopy of polycystic ovary syndrome (PCOS) in mammals. Retrospective studies have suggested that girls at risk for PCOS have low birth weight, and prospective studies have suggested an increased prevalence of small-for-gestational-age offspring in women with PCOS. Objective: The objective of the study was to determine whether infants of women with PCOS have reduced birth weight or increased intrauterine androgen levels. Design: This was a prospective case-control study. Participants: Thirty-nine PCOS and 31 control women and their infants participated in the study. Main Outcome Measures: Birth weight and mixed cord blood testosterone, androstenedione (A), dehydroepiandrosterone, 17-hydroxyprogesterone, estradiol (E2), and dihydrotestosterone levels were measured. Results: Mean birth weight did not differ, but there was a significant increase in the prevalence of large-for-gestational-age infants in the PCOS group. Cord blood E2 and A levels were lower (P < 0.05), but testosterone to E2 ratios did not differ in female PCOS compared with control offspring. There was no difference in E2 and A levels in the male PCOS and control offspring. There was no difference in 17-hydroxyprogesterone or other androgen levels in either male or female PCOS offspring compared with their respective control group. Conclusion: Infants of women with PCOS were more likely to be large for gestational age. Female offspring of affected women have lower cord blood A levels; other cord blood androgen levels do not differ compared with female control offspring. Cord blood E2 levels are also significantly decreased in PCOS, without any difference in the testosterone to E2 ratio, suggesting decreased fetal or placental production of steroids.

Abstract Context: We have recently introduced liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 25-hydroxyvitamin D2 (25OHD2) and 25OHD3 testing. During subsequent clinical use, we identified significantly elevated results in some infants. We hypothesized this might represent assay interference caused by C-3 epimers of 25OHD2 or 25OHD3. Objective: Our aims were to 1) determine the prevalence of C-3 epimers of 25OHD2 or 25OHD3 in human serum, and 2) identify the patient populations that might be affected. Study Design: We modified our LC-MS/MS method to allow detection of C-3 epimers. We retested specimens from four patient groups with the new method and an extracted RIA: 1) children less than 1 yr old, 2) children 1–18 yr old, 3) adults aged 20–87 yr with liver disease, and 4) adults aged 19–91 yr without liver disease. Results: In 172 children from group 1 with detectable 25OHD2 or 25OHD3, we identified C-3 epimers in 39 (22.7%). The epimers contributed 8.7–61.1% of the total 25-OHD. There was an inverse relationship between patient age and epimer percentage (r = 0.48; P < 0.002). The RIA gave accurate 25-OHD results that correlated with the modified LC-MS/MS method. No C-3 epimers were detected in any of the other groups. Conclusions: Significant concentrations of C-3 epimers of 25OHD2 or 25OHD3 are commonly found in infants. This can lead to overestimation of 25-OHD levels. Measurements in children less than 1 yr should therefore be performed with an assay that allows accurate detection of 25-OHD in the presence of its C-3 epimers.

Abstract Background: Measurements of serum or urine concentrations of synthetic glucocorticoids are useful for assessing suspected iatrogenic hypothalamic-pituitary-adrenal axis suppression and Cushing syndrome. We have developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantitative analysis of beclomethasone dipropionate, betamethasone, budesonide, dexamethasone, fludrocortisone, flunisolide, fluorometholone, fluticasone propionate, megestrol acetate, methylprednisolone, prednisolone, prednisone, triamcinolone, and triamcinolone acetonide. Methods: Stable isotopes of cortisol-9,11,12,12-d4 and triamcinolone-d1 acetonide-d6 were added as internal standards to calibrators, controls, and unknown samples. After acetonitrile precipitation, these samples were extracted with methylene chloride, and the extracts were washed and dried. Reconstituted extract (15 μL) was injected on a reversed-phase column and analyzed by LC-MS/MS in positive-ion mode. Assay precision, accuracy, linearity, and sample stability were determined by use of enriched samples. Clinical validation included analysis of 8 serum and 20 urine samples from patients with undetectable cortisol concentrations and analysis of different types of tablets. Results: Functional assay sensitivity was as low as 0.6–1.6 nmol/L for all compounds except for triamcinolone (7.6 nmol/L). Interassay CVs were 3.0–20% for concentrations of 0.6–364 nmol/L for all analytes. Recoveries of all analytes (except triamcinolone in serum) were 82–138% at 19.2–693 nmol/L. All but one of the serum and urine samples from patients who were tested because of suppressed cortisol concentrations contained at least one synthetic steroid. Tablet analysis recovered 75% of the synthetic steroids in suspected drugs. Conclusions: LC-MS/MS allows simultaneous quantitative detection of various synthetic steroids in serum, plasma, urine, and tablets. This provides a valuable tool for evaluating the clinical effects of topical and systemic synthetic corticosteroids.