Gas Chromatography Mass Spectrometry (GC-MS) for Identification of Designer Stimulants Including 2C Amines, NBOMe Compounds, and Cathinones in Urine.

Phenethylamine derivatives are being increasingly exploited for recreational use as "designer" stimulants designed to mimic psychostimulant properties of amphetamine or other illicit substances like 3,4-methylenedioxymethamphetamine (MDMA [ecstasy]). Clandestine operations meticulously design phenethylamines so the user can bypass legal action when detected, as many of these are yet to be regulated by government authorities. Substituted phenethylamines or 2C amines, N-methoxybenzyl derivatives of the corresponding 2C amines commonly known as NBOMe compounds, and cathinones are among the most commonly abused phenethylamines. Current FDA-approved assays used in screening for illicit drug use lack the sensitivity needed to detect designer stimulants making it challenging for toxicologists to accurately identify these compounds. Gas chromatography mass spectrometry (GC-MS) is a sensitive method for identifying designer stimulants. This unit describes and compares two qualitative GC-MS methods for identifying 2C amines, NBOMe compounds, and cathinones in urine. © 2017 by John Wiley & Sons, Inc.

LC-MS/MS for Identifying Patients with CYP24A1 Mutations.

BACKGROUND: Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing. METHODS: After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. RESULTS: The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%-15% across the analytical measurement range of 0.1-25 ng/mL (0.2-60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7-35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99-467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS: Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.

Analytical and clinical validation of parathyroid hormone (PTH) measurement in fine-needle aspiration biopsy (FNAB) washings.

BACKGROUND: Parathyroid hormone (PTH) quantitation in fine needle aspirate biopsy (FNAB) saline washings complements current modalities for parathyroid tissue localization. OBJECTIVES: To establish the performance characteristics of the Roche Elecsys intact PTH immunoassay in FNAB needle washings and its diagnostic performance for the identification of parathyroid tissue. DESIGN AND METHODS: Accuracy, precision, reportable range, and analytical specificity and sensitivity for the intact PTH immunoassay in FNAB needle washings were established. For clinical validation, 93 specimens from 79 patients were evaluated. Diagnostic cut-offs were established via receiver operator characteristic (ROC) curve analysis. Performance of PTH in FNAB needle washings was compared to cytology. RESULTS: Measurement of the PTH in FNAB needle washings demonstrated a matrix interference that was overcome by supplementation of the samples with a protein based matrix prior to analysis. ROC area under the curve (AUC) was 0.96 for PTH in FNAB needle washings. A PTH concentration ≥100pg/mL showed 100% specificity and 82% sensitivity for identifying parathyroid tissue. On histology-confirmed parathyroid specimens, 21/38 (55%) were correctly identified by cytology; whereas 31/38 (82%) were identified by PTH. CONCLUSIONS: Measurement of PTH in FNAB washings complements cytology for identification of parathyroid tissue. Analytical validation to exclude interference in the PTH immunoassay and proper localization of the parathyroid tissue by ultrasound is necessary to ensure the robustness of the method.

Iatrogenic vitamin D toxicity in an infant--a case report and review of literature.

Public concern over vitamin D deficiency has led to widespread use of over the counter (OTC) vitamin D (-D3 or -D2) supplements, containing up to 10,000 IU/unit dose (400 IU=10µg). Overzealous use of such supplements can cause hypercalcemia due to vitamin D toxicity. Infants are particularly vulnerable to toxicity associated with vitamin D overdose. OTC supplements are not subject to stringent quality control regulations from FDA and high degree of variability in vitamin D content in OTC pills has been demonstrated. Other etiologies of vitamin D induced hypercalcemia include hyperparathyroidism, granulomatous malignancies like sarcoidosis and mutations in the CYP24A1 gene. The differential diagnosis of hypercalcemia should include iatrogenic and genetic etiologies. C24-hydroxylation and C3-epimerization are two important biochemical pathways via which 25-hydroxyvitamin D3 (25(OH)D3) is converted to its metabolites, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or its C3 epimer, 3-epi-25-OH-D3 respectively. Mutations in the CYP24A1 gene cause reduced serum 24,25(OH)2D3 to 25(OH)D3 ratio (<0.02), elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D3), hypercalcemia, hypercalciuria and nephrolithiasis. Studies in infants have shown that 3-epi-25(OH)D3 can contribute 9-61.1% of the total 25(OH)D3. Therefore, measurements of parathyroid hormone (PTH) and vitamin D metabolites 25(OH)D3, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3 are useful to investigate whether the underlying cause of vitamin D toxicity is iatrogenic versus genetic. Here we report a case of vitamin D3 associated toxicity in a 4-month-old female who was exclusively breast-fed and received an oral liquid vitamin D3 supplement at a dose significantly higher than recommended on the label. The vitamin D3 content of the supplement was threefold higher (6000 IU of D/drop) than listed on the label (2000 IU). Due to overdosing and higher vitamin D3 content, the infant received ∼50,000 IU/day for two months resulting in severe hypercalcemia, hypercalciuria and nephrocalcinosis. We also review the relevant literature on vitamin D3 toxicity in this report.

Estradiol assays--The path ahead.

Estradiol quantitation is useful in the clinical assessment of diseases like hypogonadism, hirsutism, polycystic ovary syndrome (PCOS), amenorrhea, ovarian tumors and for monitoring response in women receiving aromatase inhibitor therapy. Physiologically relevant serum estradiol concentration in women can span across four orders of magnitude. For example, in women undergoing ovulation induction serum estradiol concentration can range between 250-2000 pg/mL whereas aromatase inhibitor therapy can decrease serum estradiol concentration to <5 pg/mL. While high-through-put automated un-extracted (direct) immunoassays accommodate the growing clinical need for estradiol quantitation, are amenable to implementation by most hospital clinical laboratories, they display a significant loss of specificity and accuracy at low concentrations. Most clinical scenarios (example: estradiol monitoring in fertility treatments) place a modest demand on accuracy and precision of the assay in use but accurate quantitation of estradiol in certain clinical scenarios (pediatric and male patients and for monitoring aromatase inhibitor therapy) can be challenging using currently available immunoassays since the direct immunoassays are prone to issues with sub-optimal accuracy and specificity due to cross reactivity with estradiol conjugates and metabolites. In this review we discuss the bases for the evolution of estradiol assays from extracted (indirect) radio-immunoassays to direct immunoassays to liquid-chromatography tandem mass spectrometry (LC-MS/MS) based assays, discuss technical factors relevant for development and optimization of a LC-MS/MS assay for estradiol and present the details and performance characteristics of an ultra-sensitive LC-MS/MS estradiol assay with a limit of quantitation of 0.2 pg/mL.

Clinical applications of LC-MS sex steroid assays: evolution of methodologies in the 21st century.

PURPOSE OF REVIEW: The purpose of this review is to summarize why and how liquid chromatography tandem mass spectrometry (LC-MS/MS) is increasingly replacing other methodologies for the measurement of sex steroids. RECENT FINDINGS: Measurement of sex steroids, particularly testosterone and estradiol, is important for diagnosis or management of a host of conditions (e.g. disorders of puberty, hypogonadism, polycystic ovary syndrome, amenorrhea, and tumors of ovary, testes, breast and prostate). Historically, metabolites of testosterone and estradiol were measured as ketosteroids in urine using colorimetric assays that lacked sensitivity and specificity due to endogenous and exogenous interferences. Extracted competitive manual radio-immunoassays provided improved, but still imperfect, specificity, and offered increased sensitivity. As testing demand increased, they were displaced by automated immunoassays. These offered better throughput and precision, but suffered worse specificity problems. Moreover, agreement between different immunoassays has often been poor and they are all compromised by a limited dynamic measurement range. To overcome these problems, LC-MS/MS methods have been developed and validated for quantitation of sex steroids. These methods reduce interferences, provide better specificity, improve dynamic range, and reduce between-method bias. SUMMARY: Endocrine Society and Urology Society guidelines have highlighted the limitations of the immunoassays for sex steroids and have provided convincing evidence that mass spectrometric methods are preferable for measurement of sex steroid hormones. In this review, we describe LC-MS/MS methods for measurement of testosterone and estradiol.

The sclerostin-bone protein interactome.

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.