LC MS-MS Research Articles

Wisconsin's Screening Algorithm for the Identification of Newborns with Congenital Adrenal Hyperplasia.

Newborn screening for congenital adrenal hyperplasia (CAH) has one of the highest false positive rates of any of the diseases on the Wisconsin panel. This is largely due to the first-tier immune assay cross-reactivity and physiological changes in the concentration of 17-hydroxyprogesterone during the first few days of life. To improve screening for CAH, Wisconsin developed a second-tier assay to quantify four different steroids (17-hydroxyprogesterone, 21-deoxycortisol, androstenedione, and cortisol) by liquid chromatography–tandem mass spectrometry (LC–MSMS) in dried blood spots. From validation studies which included the testing of confirmed CAH patients, Wisconsin established its own reporting algorithm that incorporates steroid concentrations as well as two different ratios—the birth weight and the collection time—to identify babies at risk for CAH. Using the newly developed method and algorithm, the false positive rate for the CAH screening was reduced by 95%. Patients with both classical forms of CAH, salt-wasting and simple virilizing, were identified. This study replicates and expands upon previous work to develop a second-tier LC–MSMS steroid profiling screening assay for CAH. The validation and prospective study results provide evidence for an extensive reporting algorithm that incorporates multiple steroids, birth weight, and collection times.

Development and In-House Validation of an LC–MS and LC–MS/MS Assay for the Determination of Food Fraud for Different Fish Species

Abstract Background: Fish and fish products are one of the most important food sources of high commercial interest. The global food trade and the associated risks are constantly presenting new challenges to consumer protection and public authorities, which, among other things, demand state-of-the-art analytical methods to ensure food authenticity. Objective: The establishment of MS-based strategies plays a decisive role alongside the (further) development of ELISA- or DNA-oriented methods. Methods: In the present work, therefore, the development and in-house validation of an LC–MS and LC–MS/MS-based assay for authenticity testing of certain fish species is described. Results: Based on the execution of a validated bottom-up LC–electrospray–MS and MS/MS assay and multivariate analysis, the commercially available species Lutjanus malabaricus (red snapper) and Sebastes spp. (redfish) are distinguished from each other, whereas an additional 68 samples [nine additional marine species such as pangasius (Pangasianodon hypophthalmus), salmon (Salmo salar), turbot (Scophthalmus maximus), plaice (Pleuronectes platessa), sole (Solea solea), lemon sole (Glyptocephalus cynoglossus), halibut (Reinhardtius hypoglossoides), red salmon (Oncorhynchus nerka), and great scallop (Pecten jacobaeus)] served as blinded negative controls to ensure the specificity of the assay. Conclusions and Highlights: A promising LC–MS and LC–MSMS based assay has been developed that could enable the detection of fish fraud at the protein level in the future.

Bisphenol A and cognitive function in school-age boys: Is BPA predominantly related to behavior?

BackgroundBisphenol A (BPA) has been associated with impairments in children’s behavior, but few studies have investigated its relationship with cognitive function. ObjectiveTo investigate the association of urinary BPA concentrations with cognitive domains and intelligence quotient (IQ) in Spanish boys. MethodsBPA levels were quantified by liquid chromatography-tandem mass spectrometry (LC–MS-MS) in one spot urine sample from 269 boys of the INMA-Granada cohort, in their follow-up at 9–11 years of age. Cognitive function was evaluated by a trained psychologist using a comprehensive neuropsychological test battery (including general intelligence, language skills, working memory, attention, impulsivity, visual-motor coordination, processing speed and executive function, among others). Cross-sectional associations between BPA levels and neuropsychological standardized scores were analyzed by adjusted linear and logistic regression models. ResultsMedian (P25, P75) BPA concentrations were 4.76 (2.77, 9.03) μg/L and 4.75 (2.75, 10.2) μg/g of creatinine (Cr). Boys in the third and fourth quartile of volume-based BPA concentrations showed better processing speed scores than boys in the first quartile (β = 5.47; 95%CI: 1.4, 9.4 and β = 3.57; 95%CI: -0.4, 7.5, respectively); and boys in the third quartile showed better inhibitory control (β = 1.6; 95%CI: -0.3, 3.5) and impulsivity (β= -4.2; 95%CI: -9.0, 0.0). In contrast, boys in the fourth quartile showed poorer working memory scores than those in the first quartile (β= -1.0; 95%CI: -2.1, -0.1). All these associations were attenuated when Cr-standardized BPA concentrations were considered. Cr-based BPA concentrations were also associated with a higher risk of being below the 20th percentile of working memory scores [ORa = 1.51; 95%CI: 1.01, 2.25]. DiscussionOur findings do not support an association between urinary BPA concentrations and cognitive function or IQ among boys, except for working memory. BPA was previously found to be associated with behavior problems in the same study population, suggesting that BPA may predominantly affect the behavior of children rather than their cognitive function, in line with previous epidemiologic studies.

A Retrospective Analysis of Selected Opioids in Hair of Workplace Drug Testing Subjects.

Opioids, both naturally occurring and semisynthetic, are effective pain management medications, but also possess the potential for abuse. Analyses of over 37,000 head and body hair samples containing codeine, morphine, hydrocodone, hydromorphone, oxycodone or oxymorphone provide a view of use habits of workplace-testing subjects that cannot be obtained from fluid matrices results. Testing was performed using FDA cleared immunoassays using either 2 ng morphine or oxycodone per 10 mg hair as calibrators. Non-negative screening samples were washed with an extended aqueous wash procedure followed by LC-MS-MS confirmation at a cutoff concentration of 2 ng opioid per 10 mg hair. The LC-MS-MS method measured codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone with an administratively established LOQ of 0.50 ng opioid per 10 mg hair. The linear range was 0.50-100 ng morphine per 10 mg hair, and 0.50-150 ng opioid per 10 mg hair for all other measured analytes. For all analytes, within run precision was ≤5.4%, and between-run precision was ≤6.4%. Analysis of samples containing metabolites found that, among codeine positive samples, 97% contained less than 10% morphine metabolite and 88% less than 20% hydrocodone metabolite, among hydrocodone positive samples, 97% contained less than 10% hydromorphone metabolite and 95% of oxycodone positive samples contained less than 10% oxymorphone metabolite. Our analysis of opioid-positive samples may provide guidelines for interpretation of hair opioid levels typically observed in workplace testing.

In Vitro Approach to Addressing Violative Drug Residues in Cattle

Violative residues of the veterinary drug flunixin (FNX) are sometimes found in cattle that were presumably slaughtered in accordance with the withdrawal period prescribed by federal guidelines. A possible cause for violative residues may be slow rates of FNX metabolism associated with sex, age or illness of animals. Experiments were designed to investigate the effects of sex and age on rates of flunixin metabolism using liver microsomal, S9, cytosol, and homogenate fractions from heifers (n=5) and steers (n=6) less than 30 months of age, and cows (n=8) older than 48 months of age. Validation of subcellular fractions was achieved through enzyme analyses: cytochrome P450 (P450), NADPH‐P450 reductase and glucose‐6‐phosphate dehydrogenase (G6PDH) activities under optimized conditions. Similarly, conditions for flunixin metabolism were optimized and fractional activity and metabolite formation was determined. Cows had lower (P < 0.05) concentrations of P450, NADPH‐P450 reductase activity, and FNX metabolism than their younger counterparts (heifers or steers). Using 15 min incubations, 5‐hydroxy FNX was the major metabolite detected by LC MS‐MS. Variables including exact animal age, cattle breed, diet, health and physiological status of animals at slaughter could have influenced results but were not assessed because of limitations associated with tissue collection.Support or Funding InformationAll funding of work was provided by USDA‐ARS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Omega-3 fatty acids and docosahexaenoic acid oxymetabolites modulate the inflammatory response of equine recombinant interleukin1β-stimulated equine synoviocytes

Omega-3 fatty acid (n-3 PUFA) supplementation may have beneficial effects in certain chronic diseases, potentially including osteoarthritis. Favorable effects are attributed, in part, to downstream pro-resolving oxylipid metabolites. We investigated the role of n-3 PUFA and docosahexaenoic acid (DHA)-derived oxylipids (docosanoids) on equine synoviocyte metabolism. We hypothesized that n-3 PUFA and selected docosanoids would modulate inflammatory mediator gene expression by recombinant equine (re)IL-1β-stimulated synovial fibroblasts. Synoviocyte monolayer cultures were prepared from grossly normal equine carpal synovium. Cellular incorporation of eicosapentaenoic acid (EPA) and DHA was determined using LC–MS and docosanoid biosynthesis by LC–MS-MS. The influence of n-3 PUFA and docosanoids on osteoarthritis marker gene expression was determined by quantitative real time polymerase chain reaction (qPCR). Synoviocytes incorporated EPA and DHA in significant amounts and DHA treatment augmented the synthesis of several docosanoids. Synoviocyte cultures pre-treated with EPA or DHA followed by reIL-1β stimulation had significant reductions in expression of ADAMTS4, MMP-1, MMP-13, IL-1β, IL-6 and COX-2. The docosanoids resolvin D1 and D2, maresin 1 and protectin DX, alone and in combination, abrogated ADAMTS4, MMP-1, MMP-13, and IL-6 gene expression in reIL-1β-stimulated synoviocytes. Similarly, both resolvins and maresin 1 stifled COX-2 expression. Our results demonstrate that synoviocytes readily incorporate n-3 PUFA. DHA incorporation was sufficient for biosynthesis of significant concentrations of several docosanoids which modulated the synovial inflammatory response in vitro. These data indicate n-3 PUFA supplementation may prove useful in the prevention or treatment of osteoarthritis.

Ethyl Glucuronide as a Long-term Alcohol Biomarker in Fingernail and Hair. Matrix Comparison and Evaluation of Gender Bias

This work aimed to assess the performance of hair and fingernail ethyl glucuronide (EtG) measurement for use as a biomarker of alcohol consumption in persons with known drinking history across a range of drinking behaviours. EtG concentrations were assessed from the hair and fingernails of 50 study participants. Alcohol consumption of the previous 90 days was assessed by participant interview using the alcohol timeline follow-back method. EtG concentration was determined using LC-MS-MS using a method which was validated and accredited to ISO/IEC 17025 standards. There was significant correlation between alcohol consumption and EtG concentrations found in hair and fingernail samples across the study group (n = 50). From participants testing positive for EtG (male n = 14, female n = 13) no significant difference was found between male and female EtG levels in either hair or fingernails. Across all participants there was no significant difference in hair or fingernail EtG concentration between male (n = 23) and females (n = 27). Our results support the use of EtG to indicate alcohol consumption over the previous 90 days, or ~3 months as is the normal practice in hair analysis. The results confirm that fingernails can be a useful alternative matrix where hair samples are not available.

Development and Validation of Simple LC-MS-MS Assay for the Quantitative Determination of Ticagrelor in Human Plasma: its Application to a Bioequivalence Study.

A sensitive, rapid, reproducible and reliable high-performance liquid chromatography-electrospray ionization tandem mass spectrometric method has been developed and fully validated for the determination of ticagrelor in human plasma using ticagrelor-d7 as an internal standard (IS) after one-step liquid-liquid extraction with ethyl acetate. Ticagrelor and IS were detected in the multiple reaction monitoring mode at m/z transition 523.4 > 127.0 and 530.4 > 127.0, respectively. Chromatographic separation was performed on a Phenomenex Luna® C18 column using a mobile phase consisting of 0.1% formic acid in water-acetonitrile (20:80, v/v) at a flow rate of 0.2 mL/min. The retention times of ticagrelor and IS were 1.03 min and 1.02 min, respectively. The calibration curve was linear [correlation coefficient (r) ≥ 0.9991] over the concentration range of 2-1,500 ng/mL. Intra- and inter-day precisions ranged from 1.0% to 4.9% and from 1.8% to 8.7%, and the accuracy ranged from 97.0% to 105.9% and from 97.5% to 102.9%, respectively. The developed method was fully validated with respect to selectivity, linearity, lower limit of quantitation, recovery, matrix effect and stability. This validated method was successfully applied to the bioequivalence study of ticagrelor in 44 healthy Korean male volunteers.

Identification of somatic embryogenesis (SE) related proteins through label-free shotgun proteomic method and cellular role in Catharanthus roseus (L.) G. Don

Somatic embryogenesis (SE) is an intricate in vitro multi-step biotechnological tool used to develop embryos/plants from a single or a group of somatic cells. It is a model technique for understanding various plant developmental pathways. A lot of research is going on to elucidate the mechanism underlying the process of SE. This study was aimed at the identification of SE related proteins in a medicinally important plant, Catharanthus roseus via label free liquid chromatography–mass spectroscopy (LC–MS). LC–MS is a sensitive and reliable technique than the gel based techniques, using LC–MSMS in tandem for separation and identification of proteins. Here, we are reporting for the first time SE related proteins in C. roseus by using gel free shotgun proteomic approach. The non embryogenic and embryogenic calli of C. roseus were used for comparative proteome analysis. A total of 3573 proteins were identified in both embryogenic and non embryogenic calli of which 1511 proteins were found to be common in both the calli. In non embryogenic callus 982 proteins while in embryogenic callus 1079 proteins were exclusively identified, which were associated with varied cellular functions. The most of these proteins function in different metabolic processes and stress responses. More than 72 stress responsive proteins and isoforms were observed exclusively in embryogenic callus including glutathione S transferase, ascorbate peroxidase, catalase, superoxide dismutase, alkylhydro peroxidase, SOD Fe N domain containing protein, pyridine nucleotide disulphide oxidoreductase, thioredoxin reductase. The role of plant growth regulators (PGRs) in inducing stress cause switching on/off of several genes has been discussed, led biochemical and molecular alterations in acquiring somatic embryogenic competence. Proteomic map of Catharanthus roseus was prepared. A total of 3573 proteins were identified, of which 1079 were embryogenic. These proteins have role in metabolic and stress responses.

Qualitative analysis of the roots of Salvia miltiorrhiza and Salvia yunnanensis based on NIR, UHPLC and LC–MS-MS

Near infrared spectroscopy (NIR) was applied to discriminate the roots of salvia miltiorhiza Bunge (Danshen for short) and Salvia yunnanensis C. H. Wright (Zidanshen for short) by means of principal component analysis (PCA), improved and simplified K nearest neighbors (IS-KNN). Furthermore, an ultra-high performance liquid chromatographic (UHPLC) coupled with photodiode-array detector was developed for building fingerprints of lipophilic components of Danshen and Zidanshen, respectively. Basing on NIR information, both PCA and IS-KNN method classified the two kinds of Chinese medical herbs with 100% accuracy. The chromatographic fingerprints of the lipophilic components of Danshen and Zidanshen have 10 and 12 common peaks, respectively. Liquid chromatography coupled with mass spectroscopy (LC–MS-MS) was applied to identify these peaks. Among these, three small peaks in the fingerprints of Zidanshen are not found in Danshen, one of which was identified as α-lapachone, and the other two compounds were not yet identified; a small peak after tanshinone IIA in the fingerprints of Danshen was not found in Zidanshen, which was identified as miltirone. The two herbs have 10 common lipophilic components. The similarity between the two reference chromatograms of Zidanshen and Danshen is 0.902, but the mean similaritie between Zidanshen (or Danshen) fingerprints and its own reference chromatogram is 0.973 (or 0.976). The contents of main lipophilic components are significantly lower in Zidanshen than in Danshen (P < 0.01 or P < 0.05). The results indicate that the two Chinese medical materials are not only different in NIR spectra, but also different in species and quantities of lipophilic components. NIR spectra analysis can identify Danshen and Zidanshen rapidly and accurately. UHPLC coupled with MS analysis demonstrates the detail differences between the two herbs both in species and contents of their lipophilic components.

Acute Pharmacokinetic Profile of Smoked and Vaporized Cannabis in Human Blood and Oral Fluid.

Currently, an unprecedented number of individuals can legally access cannabis. Vaporization is increasingly popular as a method to self-administer cannabis, partly due to perception of reduced harm compared with smoking. Few controlled laboratory studies of cannabis have used vaporization as a delivery method or evaluated the acute effects of cannabis among infrequent cannabis users. This study compared the concentrations of cannabinoids in whole blood and oral fluid after administration of smoked and vaporized cannabis in healthy adults who were infrequent users of cannabis. Seventeen healthy adults, with no past-month cannabis use, self-administered smoked or vaporized cannabis containing Δ9-tetrahydrocannabinol (THC) doses of 0, 10 and 25 mg in six double-blind outpatient sessions. Whole blood and oral fluid specimens were obtained at baseline and for 8 h after cannabis administration. Cannabinoid concentrations were assessed with enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods. Sensitivity, specificity and agreement between ELISA and LC-MS-MS results were assessed. Subjective, cognitive performance and cardiovascular effects were assessed. The highest concentrations of cannabinoids in both whole blood and oral fluid were typically observed at the first time point (+10 min) after drug administration. In blood, THC, 11-OH-THC, THCCOOH and THCCOOH-glucuronide concentrations were dose-dependent for both methods of administration, but higher following vaporization compared with smoking. THC was detected longer in oral fluid compared to blood and THCCOOH detection in oral fluid was rare and highly erratic. For whole blood, greater detection sensitivity for ELISA testing was observed in vaporized conditions. Conversely, for oral fluid, greater sensitivity was observed in smoked sessions. Blood and/or oral fluid cannabinoid concentrations were weakly to moderately correlated with pharmacodynamic outcomes. Cannabis pharmacokinetics vary by method of inhalation and biological matrix being tested. Vaporization appears to be a more efficient method of delivery compared with smoking.

Cardiovascular Medication Stability in Urine for Non-Adherence Screening by LC-MS-MS.

Biochemical testing in urine is a powerful new tool in the investigation of non-adherence to cardiovascular medications Drug testing using liquid chromatography tandem mass spectrometry (LC-MS-MS) is the mainstay of the laboratory test but may be subject to pre-analytical factors that could impact on test results. The stability of cardiovascular medications in urine is one such factor that has not been fully explored in non-adherence testing and has the potential to result in patients appearing falsely non-adherent to their therapy. The stability of 29 cardiovascular medications in patients' urine samples were assessed at room temperature (RT) and at -80°C using a LC-MS-MS screening method. All drugs and drug metabolites were found to be stable under the storage conditions studied. The findings imply that the medication stability in urine samples does not have any impact on non-adherence results and thus allowing samples to be taken and transported without the need for specialist sample handling procedures. The stability of cardiovascular drugs in urine samples will allow adherence testing to be utilized more widely into routine clinics and research.

In-situ fabrication of needle-shaped MIL-53(Fe) with 1T-MoS2 and study on its enhanced photocatalytic mechanism of ibuprofen

High photocatalytic hole-electron pairs separation efficiency and the wide use of inexpensive and earth-abundant materials as cocatalysts in most semiconductor-based photocatalytic systems are desired for improving the photocatalytic activity and practical application. Herein, we report a facile one-pot solvothermal approach of integrating stable metallic nonmetal materials 1T-MoS2 nanosheets with MIL-53(Fe) to form needle-shaped 1T-MoS2@MIL-53(Fe) (TSMF) composites. Interestingly, the introduction of 1T-MoS2 turns nonspecial-structured MIL-53(Fe) into needle-like structure and the BET analysis reveals that the optimal TSMF composites possess abundant coexistence of micropores and mesopores with a large surface area of 337 m2 g−1, which is about 16 folds higher than that of the pure MOFs. Meanwhile, it is remarkable that the photocatalytic rate of ibuprofen (IBP) by optimal TSMF nanocomposites has improved 7.5 and 9.4 times compared to the pristine MIL-53(Fe) and 1T-MoS2, respectively. The photocatalytic efficiency of TSMF composites enhances due to the emerging micropores, which can provide more adsorption and reaction sites. In addition, the formed compact and uniform interface contact between 1T-MoS2 sheets and MOF may dramatically accelerate the separation of the photo-induced charges, thus enhance the photocatalytic activity. We also study the photocatalytic mechanism combined the corresponding electrochemical testing and the photo-degradation intermediates identified by ion chromatography (IC) and LC–MS-MS, indicating that superoxide radicals (O2–), hydroxyl radical (OH) and electrons (e−) are the main active radicals in IBP photocatalysis and decarboxylation and hydroxylation are the main degradation pathways of IBP.

Development and Validation of a Novel All-Inclusive LC-MS-MS Designer Drug Method.

Designer drugs including synthetic cannabinoids and synthetic cathinones are an increasing problem due to the ease of access to these compounds. They present analytical challenges inasmuch as the compound structures are numerous and growing within each class. Typically each class of designer compounds is analyzed separately due to differences in chemistry, desired cut-offs or other reasons. Physicians treating "high-risk" patients typically order tests for all "illicit" substances which can span several test classes. Despite that multiple classes of designer drugs are ordered together, there has not been a comprehensive confirmatory test developed to date. Presented here is a novel comprehensive designer drug LC-MS-MS method that combines synthetic cannabinoids and synthetic cathinones, etizolam, a designer benzodiazepine and mitragynine (kratom), a natural product analgesic. This method improves laboratory throughput with a cycle time of ~4.5 min which affords resolution of crucial isomers, such as ethylone and butylone. Development of this method also provided an opportunity to update the list of compounds within the method. Analytes with fewer than five positive specimens in a year of testing with previous separate methods were removed as old and not current. New analytes were added based on reports from NMS Laboratories and the US Drug Enforcement Administration testing and drug seizures, which included etizolam, its major metabolite α-hydroxyetizolam as well as newer synthetic cannabinoids (5-fluoro ADB metabolite 7, AB-FUBINACA metabolite 3, AB-FUBINACA metabolite 4 and MDMB-FUBINACA metabolite M1) and synthetic cathinones (N-ethyl pentylone). Finally, the impact of the new analytes and cut-off changes are discussed in context with patient results from the first 4 months of testing after implementation of the method in the lab.

Tobacco Use Classification by Inexpensive Urinary Cotinine Immunoassay Test Strips.

Urinary cotinine is one of the most commonly measured biomarkers reflecting recent exposure to nicotine. In some cases a simple qualitative dichotomization of smokers and non-smokers is all that is required. NicAlert® test strips have been evaluated for this purpose, but other recently introduced, inexpensive single-line test strips have not. In this study we evaluated two such strips with nominal cutoffs of 200 and 10 ng/mL. A total of 800 urine samples with known cotinine concentrations determined by an LC-MS-MS method were examined, including 400 urine samples ranging from 0.23 to more than 24,000 ng/mL by the 200 ng/mL strip, and 400 samples with concentrations <200 ng/mL by the 10 ng/mL cutoff strip. Both test strips performed well in these evaluations. Classification relative to LC-MS-MS by the 200 ng/mL strips had a sensitivity of 99.5% and specificity of 92%, with 95.8% accuracy. The 10 ng/mL strips had a sensitivity of 98.7% and specificity of 90.1%, with 93.3% accuracy. The positive predictive value for the 200 ng/mL strips was 92.6% and the negative predictive value was 99.5%. For the 10 ng/mL strips, the corresponding values were 85.4 and 99.2%, respectively. The prevalence of positive samples was 50% in the 200 ng/mL group, and 37% in the 10 ng/mL set. Each strip was read by two readers with an overall agreement of >98%. Our results suggest that these simple and inexpensive lateral flow immunoassay test strips can provide useful qualitative estimates of nicotine exposures for appropriate applications within the inherent limitations of sensitivity and precision of the immunoassay test strip format.

Presence of Parent Cocaine in the Absence of Benzoylecgonine in Urine.

Cocaine (COC) is widely abused and associated with significant adverse effects. Forensic and clinical laboratories often test for COC intake through detection of the primary metabolite, benzoylecgonine (BZE) in urine. Testing for BZE alone may result in false-negative determinations in situations where COC is recently administered or metabolism is impaired. To our knowledge, no data have been provided demonstrating the utility of adding parent COC to urine confirmation testing in routine analyses. For this study, random urine specimens from patients undergoing treatment for pain management and/or addiction were collected over six months from 800 clinics across 39 states. A total of 7,587 urine specimens tested positive for a COC marker (COC and/or BZE). A positive result was determined using a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with a limit of quantitation of 50 ng/mL. Of the positive specimens, 26% and 97% were positive for COC and BZE, respectively. Positive BZE-only specimens represented 74% of total positive specimens. However, 231 of the 7,587 urine specimens (3% of positive specimens) were positive for COC in the absence of BZE. The 231 COC-only positive specimens were collected from 206 patients, and two of these patients provided four COC-only positive specimens. Of a select group of COC-only specimens tested by both LC-MS-MS and immunoassay (IA) (N = 32), 81% were negative by IA, demonstrating the limitation of screening with BZE-specific IAs. A false-negative COC result can have profound impacts such as a delay in patient referral to addiction treatment, unintentional prescribing of a controlled substance to a patient actively abusing an illicit substance, or undetected cocaine use in the workplace. This study highlights the importance of testing for COC in addition to BZE in forensic and healthcare settings.

Screening of Novel Psychoactive Substances in Postmortem Matrices by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS).

Novel psychoactive substances (NPS) are newly emerging compounds, natural and synthetic, that are often sold as "legal" alternatives to controlled substances. These substances can include phenethylamines, tryptamines, synthetic cathinones, piperazines and others. Most novel psychoactive substances and similar designer drugs are often based on the chemical backbone of classic drugs of abuse but are not regularly encountered in postmortem casework. These compounds may go undetected in forensic toxicology labs where suitable identification methods for NPS may not exist. In order to keep up with this ever-expanding list of pharmacologically active and toxicologically hazardous substances, there is an increased need for a screening panel suitable for postmortem human samples such as whole blood and urine. In order to address this increased prevalence of NPS, a method was developed and validated to identify 50 NPS in postmortem samples using liquid chromatography triple quadrupole mass spectrometry. The data presented here represents a validated liquid chromatography triple quadrupole mass spectrometry method to qualitatively identify NPS in postmortem samples. The limit of detection for all compounds was set at 2.5 ng/mL. To evaluate the prevalence of NPS in the region, blood and urine specimens from 110 postmortem cases were submitted for analysis based on initial screening results and/or case history. Of those cases, 28% were positive for several NPS, though only a few were included in the cause of death. The robustness of this method proves that it is suitable for the continual addition of newer substances, ensuring up-to-date simultaneous drug detection.

Drug screening using the sweat of a fingerprint: lateral flow detection of Δ9-tetrahydrocannabinol, cocaine, opiates and amphetamine.

Here, we describe the use of a fluorescence based lateral flow competition assay for the screening of four classes of drugs, viz, Δ9-tetrahydrocannabinol (THC), cocaine (through the detection of benzoylecgonine, BZE), opiates (through the detection of morphine, MOR) and amphetamine (AMP) present in the sweat of a fingerprint. The Drug Screening Cartridge was specifically developed for fingerprint sample collection and analysis. For this study, the cut-offs were set at: 190, 90, 68 and 80 pg/fingerprint for THC, BZE, MOR and AMP, respectively. Working with three UK coroners, the Drug Screening Cartridge, together with its fluorescence reader, was applied to the detection of drugs in the sweat of a fingerprint from deceased individuals. The study shows that there was sufficient sweat present on the fingertips to enable analysis and that the Drug Screening Cartridge could detect the presence, or absence, of each drug. The presence of the drugs was confirmed using LC–MS-MS analysis of a second fingerprint sample collected simultaneously. Excellent correlation was achieved between the results obtained from the Drug Screening Cartridge and the LC–MS-MS analysis of the fingerprint samples obtained from 75 individuals. The accuracy of the results was: 99% for THC; 95% for BZE; 96% for MOR and 93% for AMP. The results obtained using the Drug Screening Cartridge were also compared to toxicological analysis of blood and urine samples with good correlation. The accuracy of the results between the Drug Screening Cartridge and blood was: 96%, 92%, 88% and 97% for THC, BZE, MOR and AMP, respectively. The comparison with urine showed an accuracy ranging between 86% and 92%. This fingerprint sample method has a collection time of just 5 s and a total analysis time of <10 mins. These results show that the lateral flow Drug Screening Cartridge is an excellent screening test to provide information on drug use from the sweat in a single fingerprint sample.

Risk assessment of pharmaceutically active compounds (PhACs) in the Klang River estuary, Malaysia.

The concentration profile, distribution and risk assessment of pharmaceutically active compounds (PhACs) in the coastal surface water from the Klang River estuary were measured. Surface coastal water samples were extracted using offline solid phase, applying polymeric C18 cartridges as extraction sorbent and measuring with liquid chromatography mass spectrometry-mass spectrometry (LC MS-MS) technique. Extraction method was optimized for its recovery, sensitivity and linearity. Excellent recoveries were obtained from the optimized method with percentage of recoveries ranging from 73 to 126%. The optimized analytical method achieved good sensitivity with limit of detection ranging from 0.05 to 0.15ngL-1, while linearity of targeted compounds in the LC MS-MS system was more than 0.990. The results showed that amoxicillin has the highest concentration (102.31ngL-1) followed by diclofenac (10.80ngL-1) and primidone (7.74ngL-1). The percentage of contribution (% of total concentration) for the targeted PhACs is in the following order; amoxicillin (92.90%) > diclofenac (3.95%) > primidone (1.23%) > dexamethasone (0.75%) > testosterone (0.70%) > sulfamethoxazole (0.33%) > progesterone (0.14%). Environmental risk assessment calculated based on deterministic approach (the RQ method), showed no present risk from the presence of PhACs in the coastal water of Klang River estuary. Nonetheless, this baseline assessment can be used for better understanding on PhACs pollution profile and distribution in the tropical coastal and estuarine ecosystem as well as for future comparative studies.

Adduct Formation-Supported Two-Way Electrospray Ionization Strategy for the Determination of Urinary Creatinine Concentration with LC-MS-MS in Abstinence Control.

In this work, an alternative LC-MS-MS strategy for the analysis of urinary creatinine in abstinence control was presented and discussed. The two-way electrospray ionization consisted of two different precursor ions in which fragmentation was used in multiple reaction monitoring experiments. A creatinine adduct ion with sodium and sodium acetate together with the conventional analyte protonation was investigated. Adduct formation and fragmentation was explored by appropriate infusion experiments performed with analyte solutions prepared in different concentrations. The analytical signal was compensated by the application of appropriate isotopically labeled internal standard. The advantages of information carried by precursor ions separated in the mass spectra were pointed out. Sample preparation based solely on sample dilution performed in the final HPLC vial directly. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column together with a mobile phase consisted of H2O/methanol = 3%/97% (v/v) with 10 mmol/L ammonium acetate and 0.1% acetic acid (flow = 0.4 mL/min) were used for the separation performed during a run of 5 min. The linearity was examined in the range of 100-3,000 mg/L. The limit of detection (13 mg/L), limit of quantification (43 mg/L) together with method precision/accuracy, selectivity, stability and matrix effect were tested to be appropriate for forensic applications. The applicability of water as surrogate matrix for method calibration was also examined successfully. The presented strategy was used in the analysis of real samples. No interferences with the creatinine peak eluted at about 1.0 min could be recorded.