Validation of an ELISA Synthetic Cannabinoids Urine Assay.

BACKGROUND: Synthetic cannabinoids are touted as legal alternatives to cannabis, at least when first released, and routine urine cannabinoid screening methods do not detect these novel psychoactive substances. Synthetic cannabinoids are widely available, are a major public health and safety problem, and a difficult challenge for drug-testing laboratories. We evaluated performance of the National Medical Services (NMS) JWH-018 direct enzyme-linked immunosorbent assay (ELISA) kit to sensitively, selectively, and rapidly screen urinary synthetic cannabinoids. METHODS: The NMS ELISA kit targeting the JWH-018 N-(5-hydroxypentyl) metabolite was used to screen 2492 urine samples with 5 and 10 mcg/L cutoffs. A fully validated liquid chromatography-tandem mass spectrometry method for 29 synthetic cannabinoids markers confirmed all presumptive positive and negative results. Performance challenges at ±25% and ±50% of cutoffs determined intraplate and interplate imprecision around proposed cutoffs. RESULTS: The immunoassay was linear from 1 to 500 mcg/L with intraplate and interplate imprecision of ≤8.2% and <14.0%, respectively. No interferences were present from 93 common drugs of abuse, metabolites, coadministered drugs, over-the-counter medications, or structurally similar compounds, and 19 of 73 individual synthetic cannabinoids (26%) exhibited moderate to high cross-reactivity to JWH-018 N-(5-hydroxypentyl) metabolite. Sensitivity, specificity, and efficiency results were 83.7%, 99.4%, and 97.6%, as well as 71.6%, 99.7%, and 96.4% with the 5 and 10 mcg/L urine cutoffs, respectively. CONCLUSIONS: This high throughput immunoassay exhibited good diagnostic efficiency and documented that the NMS JWH-018 direct ELISA is a viable method for screening synthetic cannabinoids in urine targeting the JWH-018 N-(5-hydroxypentyl) and related analytes. Optimal performance was achieved with a matrix-matched 5 mcg/L urine cutoff.

Human adaptative behavior to Antarctic conditions: A review of physiological aspects.

The Antarctic environment induces adaptive metabolic and neuroendocrine changes associated with survival, as well as increased risks to physical and mental health. Circadian disruption has been observed in Antarctic expeditioners. The main consequences appear in quality of sleep, which can affect physical and cognitive performance. Physiological adaptation to cold is mediated by the norepinephrine and thyroid hormones (T3 and 3,5-T2 metabolite). The observed changes in the hypothalamic-pituitary-thyroid (HPT) axis of expeditioners varied according to temperature, photoperiod, time spent in the cold environment and stress level. The decrease in T3 levels has frequently been associated with mood swings. Psychological and physical stressors cause disturbances in the hypothalamic-pituitary-adrenal (HPA) axis, with consequent maintenance of high cortisol levels, leading to memory impairment, immunosuppression, and cardiometabolic and reproductive disorders. Preventive measures, such as provision of adequate food, well-established eating times, physical activity and even the use of phototherapy, can all help maintain the circadian rhythm. In addition, the use of high-tech clothing and room temperature control in research stations provide greater protection against the effects of intense cold. However, psychological stress requires a more individualized approach based on the crew's sociocultural characteristics, but it can be mitigated by mental healthcare and training in coping strategies. This article is categorized under: Cardiovascular Diseases > Molecular and Cellular Physiology Cardiovascular Diseases > Environmental Factors Metabolic Diseases > Environmental Factors.

Determination of famprofazone, amphetamine and methamphetamine in liver samples using enzymatic cell dispersion and SPE-LC-ESI-MS.

The use of small amounts of sample presents advantages in chromatographic analyses that have made this a current trend following the development of increasingly sensitive analytical techniques. Biological sample preparation methods, especially for rigid or semi-rigid matrices, are also under constant development, focusing on a more efficient extraction and in obtaining cleaner residues for analysis. In this context, the aim of this study was to present a validated a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of famprofazone and its metabolites, methamphetamine and amphetamine in liver, using enzymatic cell dispersion promoted by collagenase, followed by protein precipitation and solid phase extraction (SPE) for sample extraction, concentration and clean-up. Potentially relevant variables for enzymatic cell dispersion concerning efficiency, such as enzyme concentration, temperature, buffering, agitation, and mechanical effect of stainless-steel spheres were assessed. Recovery evaluations were performed during the optimization of each step to ensure minimal loss of analytes. Linearity, the limit of detection (LOD) and limit of quantification (LOQ), stability, carryover, matrix effect, precision and bias were evaluated using fortified blank samples. An authentic sample was obtained from a controlled daily oral administration of 200 mg famprofazone to pigs for five days. The procedure was optimized for 500 mg of liver tissue, obtaining 99.9 ± 9.3% of digested collagen and 90.2 ± 1.7% of dispersed cells, without the tissue losses that usually ensue during crushing or grinding processes. Precision (CV%) was ≤ 10% and bias was ≤ 13% for all analytes. The LOQ was 5 ng/g for all analytes. The mean famprofazone concentration was 9.3 ± 0.53 ng/g, and mean metabolite concentrations were 16.7 ± 1.67 and 24.3 ± 1.36 ng/g for amphetamine and methamphetamine, respectively.

Postmortem analysis of famprofazone and its metabolites, methamphetamine and amphetamine, in porcine bone marrow.

Forensic toxicologists typically work with body fluids, such as blood and urine, or visceral tissues. The analysis of alternative samples, such as bone marrow, can be requested when the commonly used samples are unavailable due to an extended time lapse between the time of death and collection of the material to be analysed. In this study, a method for the analysis of the lipophilic drug famprofazone (FA) and its metabolites, methamphetamine (MA) and amphetamine (AM), in bone marrow was developed, validated and applied to bone marrow from pigs given controlled doses of famprofazone. This method involves enzymatic bone-cleaning, fragmentation of the bones with the assistance of a micro electric motor, optimization of clean-up and LLE (liquid/liquid extraction) conditions and determination by GC/MS. After evaluation through statistical tests, such as Shapiro Wilk for normality and Cochran for homoscedasticity, a linear model was applied in the range of 100 (LOQ) - 2000 ng g-1. Inter-day precision and bias was always < 4.6%. In real sample analysis, bone marrow FA and MA concentrations ranged from 103 to 232 and from 174 to 267 ng g-1, respectively; AM was not detected. The obtained results are useful for application in forensic toxicological protocols (human autopsy cases) and as a starting point for the development of further analytical tools.

Simultaneous determination of alpha-, beta- and gamma-hydroxybutyric acids in micro-pulverized human hair by GC-MS: Method development, validation and application.

An analytical method was developed and validated for the simultaneous determination of alpha, beta and gamma-hydroxybutyric acids (AHB, BHB and GHB, respectively) in human hair. Hair samples (10 mg) were pulverized and submitted to extraction using methanol (1 mL) for 10 min. The extract was centrifuged, filtered, and evaporated to dryness at 40 °C under a gentle N2 flow, dissolved in ethyl acetate (0.050 mL) and derivatized using 0.050 mL of BSTFA containing 1% TMCS, at °C for 40 min. The derivatives were determined by GC/MS. Aliquots of natural hair ("blank") samples after water extraction (3 steps) were pulverized, spiked with AHB, BHB, GHB or d6-GHB and used for method validation. Figures of merit showed the method's feasibility. The LOQ values were 1 ng mg-1 for BHB and AHB and 0.6 ng mg-1 for GHB, and mean recoveries were 54%, 69% and 75% for AHB, BHB and GHB respectively. Precision and bias were better than 15% and 20% respectively. Calibration curves (LOQ to 20 ng mg-1 for AHB and BHB and LOQ to 16 ng mg-1 for GHB) obtained in 5 days were pooled for statistical analysis, but the data were heteroscedastic, as demonstrated by the White test. Therefore, weighted linear regression and data transformations were applied and the best results were obtained using the Box-Cox transformation [c' = (cλ-1)/λ] for GHB and log transformation for AHB and BHB. Authentic hair samples were collected from the posterior head vertex of 23 volunteers (16 females and 7 males). Four of them declared having type-2 diabetes (T2D). Only female volunteers reported hair treatments such as dyeing, flat ironing, straightening and/or bleaching. Dyeing appeared not to affect GHB levels in hair, but no data were found in the literature about other cosmetic treatments. GHB levels varied from < LOQ to 1.5 ng mg-1. BHB levels varied from < LOQ to 3.4 ng mg-1 and from < LOQ to 1.8 ng mg-1 in non-diabetic volunteers with or without family history of T2D, respectively. However, in diabetics, BHB levels varied from 3.4 to 5.2 ng mg-1. The levels of AHB varied from < LOQ to 1.2 ng mg-1 in non-diabetic individuals, and from 1.1 to 6.2 ng mg-1 in diabetics. Our results showed that AHB levels in hair have potential to as a biomarker for T2D, but this needs to be further studied with larger groups of volunteers. To our knowledge, this is the first published method for the determination of AHB and BHB in human hair.

Particle-size distribution (PSD) of pulverized hair: A quantitative approach of milling efficiency and its correlation with drug extraction efficiency.

Different types of hair were submitted to different milling procedures and their resulting powders were analyzed by scanning electron microscopy (SEM) and laser diffraction (LD). SEM results were qualitative whereas LD results were quantitative and accurately characterized the hair powders through their particle size distribution (PSD). Different types of hair were submitted to an optimized milling conditions and their PSD was quite similar. A good correlation was obtained between PSD results and ketamine concentration in a hair sample analyzed by LC-MS/MS. Hair samples were frozen in liquid nitrogen for 5min and pulverized at 25Hz for 10min, resulting in 61% of particles <104µm and 39% from 104 to 1000µm. Doing so, a 359% increment on ketamine concentration was obtained for an authentic sample extracted after pulverization comparing with the same sample cut in 1mm fragments. When milling time was extended to 25min, >90% of particles were <60µm and an additional increment of 52.4% in ketamine content was obtained. PSD is a key feature on analysis of pulverized hair as it can affect the method recovery and reproducibility. In addition, PSD is an important issue on sample retesting and quality control procedures.

Validation of a SPE-LC-MS/MS method for the determination of ketamine and norketamine in micropulverized hair after a single IV dose.

A SPE-LC-MS/MS method for the determination of ketamine (KET) and norketamine (NKET) was developed and validated. Extensive pulverization (25min at 25Hz) of previously cooled samples (5min in liquid nitrogen) allowed for extraction in a phosphate buffer (pH 6) solution after 10min vortex agitation at room temperature, simplifying the coupling of the extraction to an effective mixed-mode SPE (solid phase extraction) clean-up procedure. The extraction optimization was performed with samples fortified by drug incorporation according to a published procedure involving incubation of blank matrices for 16days. The method was validated for selectivity, matrix effect, linearity, LLQ (lower limit of quantification), precision, accuracy, recovery, carryover and stability after preparation and has proven to be accurate and reliable within a range of 0.02-10ng/mg for KET and 0.04-4ng/mg for NKET, meeting proposed KET cutoffs for discrimination from chronic use. In addition, the method was sensitive enough to detect the drugs after unique small (1mg/kg) intravenous doses received by patients submitted to general anesthesia before surgical procedures. Ketamine levels varied from 0.060 to 0.111ng/mg and norketamine was positive (

Fast and sensitive collagen quantification by alkaline hydrolysis/hydroxyproline assay.

A preparative protein alkaline hydrolysis procedure, as part of a spectrophotometric collagen quantification method, is presented. The procedure is suitable for small amounts of fresh solid or liquid samples. Various aspects of the procedure, such as the NaOH concentration, time needed to hydrolyse different collagen contents, buffer strength of the reagent solution, pH control of the hydrolysate and spectrophotometric conditions, were evaluated. Compared to other procedures that use alkaline hydrolysis, the sensitivity of this procedure was increased by a factor of 5. Compared to the conventionally used Association of Official Analytical Chemists (AOAC) acid hydrolysis method, the reaction time was reduced from 16 h to 40 min and the amount of sample from 4 g to 3-20 mg, producing equivalent results when applied to porcine liver and sausage samples.

Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids.

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time-consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH-018 N-pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two immunoassay cut-off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC-MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5-10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut-off also were investigated to determine performance around the cut-off and intra- and inter-plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r(2) = 0.992) with intra- and inter-plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut-off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut-off 69.3%, 99.8%, and 96.3%, respectively. Cross-reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi-automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH-018 N-pentanoic acid.

Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine.

INTRODUCTION: The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids. METHODS: 2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semi-quantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 µg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated. RESULTS: Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 µg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1-10 µg/L. Performance at 5 µg/L was 92.2%, 98.1% and 97.4%, and for the 20 µg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 µg/L, and documented acceptable linearity from 5-25 µg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 µg/L); 4 showed moderate cross-reactivity (10-50% at 10 µg/L), 30 low cross-reactivity (<10% at 500 µg/L), and 27 <1% cross-reactivity at 500 µg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 µg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 µg/L) but below the manufacturer's recommended cutoff concentration (10 µg/L). CONCLUSION: The Immunalysis HEIA K2 Spice kit required no sample preparation, had a high-throughput, and acceptable sensitivity, specificity and efficiency, offering a viable method for screening synthetic cannabinoids in urine that cross-react with JWH-018 N-pentanoic acid antibodies.

Vigilância toxicológica: comprovação do uso de álcool e drogas através de testes toxicológicos / Toxicological surveillance: evidence of alcohol and drugs by toxicological tests

O objetivo deste livro é ser uma fonte de informação para profissionais que têm que lidar com testes toxicológicos para comprovação do uso de álcool e drogas, no seu dia-a-dia, auxiliando-os na escolha dos mesmos e nos critérios para sua aplicação...