Development of a New LC-MS/MS Screening Method for Detection of 120 NPS and 43 Drugs in Blood

In the last few years, liquid chromatography coupled with mass spectrometry (LC/MS) has been increasingly used for screening purposes in forensic toxicology. These techniques have the advantages of low time/resource-consuming and high versatility and have been applied in numerous new multi-analytes methods. The new psychoactive substance (NPS) phenomenon provided a great impulse to this wide-range approach, but it is also important to keep the attention on “classical” psychoactive substances, such as benzodiazepines (BDZ). In this paper, a fully validated screening method in blood for the simultaneous detection of 163 substances (120 NPS and 43 other drugs) by a dynamic multiple reaction monitoring analysis through LC-MS/MS is described. The method consists of a deproteinization of 200 µL of blood with acetonitrile. The LC separation is achieved with a 100 mm long C18 column in 35 min. The method was very sensitive, with limits of quantification from 0.02 to 1.5 ng/mL. Matrix effects did not negatively affect the analytical sensitivity. This method proved to be reliable and was successfully applied to our routinary analytical activity in several forensic caseworks, allowing the identification and quantification of many BDZs and 3,4-methylenedioxypyrovalerone (MDPV).

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Screening of 104 New Psychoactive Substances (NPS) and Other Drugs of Abuse in Oral Fluid by LC–MS-MS

Journal of Analytical Toxicology ◽

10.1093/jat/bkaa089 ◽

2020 ◽

Vol 44 (7) ◽

pp. 697-707

Author(s):

Kelly Francisco da Cunha ◽

Karina Diniz Oliveira ◽

Marilyn A Huestis ◽

Jose Luiz Costa

Keyword(s):

Oral Fluid ◽

Drugs Of Abuse ◽

Matrix Effects ◽

Synthetic Cannabinoids ◽

Screening Method ◽

Simultaneous Detection ◽

New Psychoactive Substances ◽

Major Public Health Problem ◽

Psychoactive Substances ◽

Wide Range

Abstract New psychoactive substances (NPS) are a major public health problem, primarily due to the increased number of acute poisoning cases. Detection of these substances is a challenge. The aim of this research was to develop and validate a sensitive screening method for 104 drugs of abuse, including synthetic cannabinoids, synthetic cathinones, fentanyl analogues, phenethylamines and other abused psychoactive compounds (i.e., THC, MDMA, LSD and their metabolites) in oral fluid by liquid chromatography–tandem mass spectrometry (LC–MS-MS). The Quantisal™ oral fluid device was used to collect oral fluid samples. The oral fluid–elution buffer mixture (500-μL sample) was extracted with t-butyl methyl ether, and chromatographic separation was performed on a Raptor™ biphenyl column (100 × 2.1 mm ID, 2.7 μm), with a total run time of 13.5 min. Limits of detection were established at three concentrations (0.05, 0.1 or 1 ng/mL) for most analytes, except for acetyl norfentanyl and mescaline (5 ng/mL). Matrix effects were generally <20% and overall extraction recoveries >60%. The highest matrix effect was observed within the synthetic cannabinoid group (PB22, −55.5%). Lower recoveries were observed for 2C-T (47.2%) and JWH-175 (58.7%). Recoveries from the Quantisal™ device were also evaluated for all analytes (56.7–127%), with lower recoveries noted for 25I-NBOMe, valerylfentanyl and mCPP (56.7, 63.0 and 69.9%, respectively). Drug stability in oral fluid was evaluated at 15, 60 and 90 days and at 25, 4 and −20°C. As expected, greater stability was observed when samples were stored at −20°C, but even when frozen, some NPS (e.g., synthetic cannabinoids) showed more than 20% degradation. The method was successfully applied to the analysis of seven authentic oral fluid samples positive for 17 different analytes. The method achieved good sensitivity and simultaneous detection of a wide range of NPS.

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Efficient Matrix Cleanup of Soft-Gel-Type Dietary Supplements for Rapid Screening of 92 Illegal Adulterants Using EMR-Lipid dSPE and UHPLC-Q/TOF-MS

Pharmaceuticals ◽

10.3390/ph14060570 ◽

2021 ◽

Vol 14 (6) ◽

pp. 570

Author(s):

Beomhee Kim ◽

Wonwoong Lee ◽

Youlee Kim ◽

Jihyun Lee ◽

Jongki Hong

Keyword(s):

Dietary Supplements ◽

Matrix Effects ◽

Limit Of Detection ◽

Solid Phase ◽

Multiple Reaction Monitoring ◽

Rapid Screening ◽

Wide Range ◽

Matrix Removal ◽

Soft Gels ◽

Tof Ms

An efficient matrix cleanup method was developed for the rapid screening of 92 illegal adulterants (25 erectile dysfunction drugs, 15 steroids, seven anabolic steroids, 12 antihistamines, 12 nonsteroidal anti-inflammatory drugs (NSAIDs), four diuretics, and 17 weight-loss drugs) in soft-gel-type supplements by ultra-high performance liquid chromatography-quadrupole/time of flight-mass spectrometry (UHPLC-Q/TOF-MS). As representative green chemistry methods, three sample preparation methods (dispersive liquid-liquid microextraction (DLLME), “quick, easy, cheap, effective, rugged, and safe” dispersive solid-phase extraction (QuEChERS-dSPE), and enhanced matrix removal-lipid (EMR-Lipid) dSPE) were evaluated for matrix removal efficiency, recovery rate, and matrix effect. In this study, EMR-Lipid dSPE was shown to effectively remove complicated matrix contents in soft-gels, compared to DLLME and QuEChERS-dSPE. For the rapid screening of a wide range of adulterants, extracted common ion chromatogram (ECIC) and neutral loss scan (NLS) based on specific common MS/MS fragments were applied to randomly collected soft-gel-type dietary supplement samples using UHPLC-Q/TOF-MS. Both ECICs and NLSs enabled rapid and simple screening of multi-class adulterants and could be an alternative to the multiple reaction monitoring (MRM) method. The developed method was validated in terms of limit of detection (LOD), precision, accuracy, recovery, and matrix effects. The range of LODs was 0.1–16 ng/g. The overall precision values were within 0.09–14.65%. The accuracy ranged from 81.6% to 116.6%. The recoveries and matrix effects of 92 illegal adulterants ranged within 16.9–119.4% and 69.8–114.8%, respectively. The established method was successfully applied to screen and identify 92 illegal adulterants in soft-gels. This method can be a promising tool for the high-throughput screening of various adulterants in dietary supplements and could be used as a more environmentally friendly routine analytical method for screening dietary supplements illegally adulterated with multi-class drug substances.

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Research activities for the monitoring of genetically modified organisms in Japan

Pure and Applied Chemistry ◽

10.1351/pac-con-09-02-02 ◽

2010 ◽

Vol 82 (1) ◽

pp. 139-147

Author(s):

Kazumi Kitta

Keyword(s):

Quality Control ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Screening Method ◽

Simultaneous Detection ◽

Cauliflower Mosaic Virus ◽

Detection Methods ◽

Internal Quality ◽

Research Activities ◽

Labeling System

The Japanese government introduced a labeling system for genetically modified (GM) foods. To ensure the authenticity of the labeling system, we have developed and validated detection methods for newly approved GM events. One was the development of quantitative analytical methods utilizing plasmid DNAs as calibrators, which enabled us to obtain an unlimited supply of calibrators of consistent quality and also to obtain a stable standard curve to quantify GM organisms (GMOs) in samples. The significance of quality control has been recognized among relevant stakeholders, and in response we launched a project to distribute certified reference materials (CRMs) to the users of our methods for the purpose of internal quality control. In addition to these activities, we have developed time- and cost-effective detection methods, such as a new screening method to simultaneously detect the sequence of Cauliflower mosaic virus 35S promoter (p35S) and the construct-specific sequence of GA21 event utilizing multiplex real-time polymerase chain reaction (PCR). We also developed a qualitative nonaplex PCR detection method, which allows the simultaneous detection of eight events of GM maize lines. Because the influx of any unapproved and unknown GMOs into the Japanese market is not permitted, we continue to explore this issue.

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Rice (Oryza sativa L.) germplasm with better seedling emergence under direct sowing in flooded paddy field

Plant Genetic Resources ◽

10.1017/s1479262118000011 ◽

2018 ◽

Vol 16 (4) ◽

pp. 352-358 ◽

Cited By ~ 1

Author(s):

Junichi Kashiwagi ◽

Koji Hamada ◽

Yutaka Jitsuyama

Keyword(s):

Paddy Field ◽

Oryza Sativa L ◽

Seedling Emergence ◽

Screening Method ◽

Paddy Fields ◽

Rice Germplasm ◽

Geographical Regions ◽

Wide Range ◽

Cultivation Practice ◽

Direct Sowing

AbstractDirect sowing of rice in a flooded paddy field is a beneficial cultivation practice for water use and labour efficiency, compared to the transplanted cultivation. However, a drastic reduction in seedling emergence under flooded paddy fields is a serious constraint especially when the seeds fell at deeper soil layers. Suitable rice germplasm for the direct sowing in flooded paddy fields could ensure the success of this cultivation practice. Instead of laborious field-based screening systems, a pot-based screening method was adopted for simplicity and efficient evaluation of seedling emergence of a subset of world rice germplasm (n = 75) at different sowing depths. As a result, two rice genotypes, ‘Vary Futsi’ (landrace from Madagascar, non-glutinous, subspecies Indica) and ‘Dahonggu’ (landrace from China, non-glutinous, subspecies Indica), with consistently better seedling emergence were identified from a wide range of rice germplasm. These genotypes could serve as excellent parents for the breeding program in developing new rice cultivars with the improved seedling emergence in flooded paddy fields. There were no significant differences in the seedling emergence rate in flooded paddy conditions among the groups from various agro-geographical regions.

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Tyrosol and Hydroxytyrosol Determination in Extra Virgin Olive Oil with Direct Liquid Electron Ionization–Tandem Mass Spectrometry

Separations ◽

10.3390/separations8100173 ◽

2021 ◽

Vol 8 (10) ◽

pp. 173

Author(s):

Veronica Termopoli ◽

Maurizio Piergiovanni ◽

Achille Cappiello ◽

Pierangela Palma ◽

Giorgio Famiglini

Keyword(s):

Olive Oil ◽

Matrix Effects ◽

Multiple Reaction Monitoring ◽

Virgin Olive Oil ◽

Standard Addition ◽

Extra Virgin Olive Oil ◽

Electron Ionization ◽

Free Form ◽

Multiple Reaction Monitoring Mode ◽

Health Promoting

Extra virgin olive oil (EVOO) is one of the main ingredients of the Mediterranean diet. It is claimed as a functional food for its unique content of health-promoting compounds. Tyrosol (Tyr), Hydroxytyrosol (Htyr), and their phenolic derivatives present in EVOO show beneficial properties, and their identification and quantification, both in their free form and after the hydrolysis of more complex precursors, are important to certify its quality. An alternative method for quantifying free and total Tyr and Htyr in EVOO is presented using an LC–MS interface based on electron ionization (EI), called liquid electron ionization (LEI). This method requires neither sample preparation nor chromatography; the sample is diluted and injected. The selectivity and sensitivity were assessed in multiple reaction monitoring mode (MRM), obtaining confirmation and quantification in actual samples ranging from 5 to 11 mg/Kg for the free forms and from 32 to 80 mg/Kg for their total amount after hydrolysis. Two MS/MS transitions were acquired for both compounds using the Q/q ratios as confirmatory parameters. Standard addition calibration curves demonstrated optimal linearity and negligible matrix effects, allowing a correct quantification even without expensive and difficult to find labeled internal standards. After several weeks of operation, the system’s repeatability was excellent, with an intraday RSD (%) spanning from five to nine and an interday RSD (%) spanning from 9 to 11.

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HCR-FlowFISH: A flexible CRISPR screening method to identify cis-regulatory elements and their target genes

10.1101/2020.05.11.078675 ◽

2020 ◽

Author(s):

SK Reilly ◽

SJ Gosai ◽

A Gutierrez ◽

JC Ulirsch ◽

M Kanai ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Screening Method ◽

Cell Types ◽

Regulatory Elements ◽

Hybridization Chain Reaction ◽

Genome Wide ◽

Wide Range ◽

Causal Variants ◽

Endogenous Loci

AbstractCRISPR screens for cis-regulatory elements (CREs) have shown unprecedented power to endogenously characterize the non-coding genome. To characterize CREs we developed HCR-FlowFISH (Hybridization Chain Reaction Fluorescent In-Situ Hybridization coupled with Flow Cytometry), which directly quantifies native transcripts within their endogenous loci following CRISPR perturbations of regulatory elements, eliminating the need for restrictive phenotypic assays such as growth or transcript-tagging. HCR-FlowFISH accurately quantifies gene expression across a wide range of transcript levels and cell types. We also developed CASA (CRISPR Activity Screen Analysis), a hierarchical Bayesian model to identify and quantify CRE activity. Using >270,000 perturbations, we identified CREs for GATA1, HDAC6, ERP29, LMO2, MEF2C, CD164, NMU, FEN1 and the FADS gene cluster. Our methods detect subtle gene expression changes and identify CREs regulating multiple genes, sometimes at different magnitudes and directions. We demonstrate the power of HCR-FlowFISH to parse genome-wide association signals by nominating causal variants and target genes.

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Handheld Portable Energy-Dispersive X-Ray Fluorescence Spectrometry (pXRF)

The Oxford Handbook of Archaeological Ceramic Analysis ◽

10.1093/oxfordhb/9780199681532.013.41 ◽

2016 ◽

pp. 362-381 ◽

Cited By ~ 1

Author(s):

Elisabeth Holmqvist

Keyword(s):

Production Systems ◽

Matrix Effects ◽

Chemical Characterization ◽

High Sensitivity ◽

Limited Range ◽

Energy Dispersive ◽

Analytical Sensitivity ◽

X Ray ◽

Non Destructive

Handheld portable energy-dispersive X-ray fluorescence (pXRF) spectrometry is used for non-destructive chemical characterization of archaeological ceramics. Portable XRF can provide adequate analytical sensitivity to discriminate geochemically distinct ceramic pastes, and to identify compositional clusters that correlate with data patterns acquired by NAA or other high sensitivity techniques. However, successful non-destructive analysis of unprepared inhomogeneous ceramic samples requires matrix-defined scientific protocols to control matrix effects which reduce the sensitivity and precision of the instrumentation. Quantification of the measured fluorescence intensities into absolute concentration values and detection of light elements is encumbered by the lack of matrix matched calibration and proper vacuum facilities. Nevertheless, semi-quantitative values for a limited range of high Z elements can be generated. Unstandardized results are difficult to validate by others, and decreased analytical resolution of non-destructive surface analysis may disadvantage site-specific sourcing, jeopardize correct group assignments, and lead to under-interpretation of ceramic craft and production systems.

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METHOD DEVELOPMENT AND VALIDATION OF LERCANIDIPINE IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2018v10i4.26544 ◽

2018 ◽

Vol 10 (4) ◽

pp. 87

Author(s):

Yahdiana Harahap ◽

Norma Andriyani ◽

Harmita .

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Method Development ◽

Matrix Effects ◽

Multiple Reaction Monitoring ◽

Tandem Mass ◽

Mass Detection ◽

Column Temperature ◽

Z Value

Objective: To obtain an optimum and validated method for analyzing lercanidipine in plasma using Ultra Performance Liquid Chromatography of Tandem Mass Spectrometry (UPLC-MS/MS).Methods: The separation was carried out using 1.7μm (2.1 x 100 mm) Waters AcquityTM UPLC C18 column, a mobile phase of the 0.1% formic acid-methanol mixture (20:80 v/v) with isocratic elution, 30 °C column temperature, 0.2 ml/min flow rate and amlodipine as an internal standard. Mass detection was performed with a positive XBL TQD type Electrospray Ionization (ESI) in Multiple Reaction Monitoring modes. Lercanidipine was detected at m/z value of 612.11>280.27 and amlodipine was detected at m/z value 409.1>238.15. The optimum sample preparation method was a liquid-liquid extraction using 5 ml of n-hexane-ethyl acetate (50:50 v/v), vortex mixed for 3 min, centrifuged at 4000 rpm for 20 min, evaporated with nitrogen at 50 °C for 30 min, and the residue was reconstituted with 100 μl of mobile phase.Results: The method was linear in the range of 0.025-10 ng/ml with r ≥ 0.9986. Accuracy and precision within-run and between-run met the requirements with %diff and %CV, not exceeding ± 15% and not more than ± 20% for Lower Limit of Quantification (LLOQ) concentration.Conclusion: It was concluded that the developed method met the requirements of selectivity, carry over, stability, the integrity of dilution, and matrix effects under the Guideline on Bioanalytical Method Validation by the European Medicines Agency in 2011.

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Commercial Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Molecular Assays: Superior Analytical Sensitivity of cobas SARS-CoV-2 Relative to NxTAG CoV Extended Panel and ID NOW COVID-19 Test

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2020-0283-sa ◽

2020 ◽

Vol 144 (11) ◽

pp. 1303-1310 ◽

Cited By ~ 1

Author(s):

Run Jin ◽

Matthew A. Pettengill ◽

Nicole L. Hartnett ◽

Herbert E. Auerbach ◽

Stephen C. Peiper ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

E Gene ◽

Molecular Assays ◽

Performance Variation ◽

Wide Range ◽

Reliable Detection ◽

Testing Algorithms ◽

Low Levels

Context.— We implemented multiple nucleic acid amplification test platforms because of the limited availability of test kits for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the early stages of the pandemic. Interpretation of results generated by different platforms and prioritization for testing algorithms required cross-comparison. Objective.— To compare the analytical sensitivity of 3 commercial SARS-CoV-2 molecular assays, selected samples were studied in parallel with Cobas SARS-CoV-2 test, NxTAG CoV Extended Panel, and ID NOW COVID-19 assays. Design.— A total of 8043 SARS-CoV-2 tests performed from March 22 to April 19, 2020, were included in this study. For all 1794 positive specimens detected by the cobas SARS-CoV-2 assay, the cycle threshold (Ct) values were manually tracked and plotted to demonstrate the distribution of sample viral levels. Additionally, 50 and 63 low-positive specimens (Ct values >32) as well as 50 and 61 consecutive positive specimens by the cobas assay were tested with NxTAG and ID NOW, respectively, to estimate their relative sensitivities. Results.— The Ct values of cobas SARS-CoV-2–positive samples were evenly distributed throughout ranges of 13.32 to 39.50 (mean, 25.06) and 13.60 to 42.49 (mean, 26.45) for ORF1 and E gene targets, respectively. NxTAG reliably detected only specimens with E gene Ct values lower than 33, and is estimated to detect 89.4% of positive specimens detected by cobas assay. ID NOW had performance variation independent of Ct value and is estimated to detect 83.5% of cobas positives. Conclusions.— Clinical specimens exhibit a wide range of viral burden, with a significant portion at low levels. Analytical sensitivity of testing platforms is critical for reliable detection of SARS-CoV-2 and uniform care to patients.

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LC-MS/MS Quantification of Nevirapine and Its Metabolites in Hair for Assessing Long-Term Adherence

Molecules ◽

10.3390/molecules25235692 ◽

2020 ◽

Vol 25 (23) ◽

pp. 5692

Author(s):

Haoran Yang ◽

Liuxi Chu ◽

Yan Wu ◽

Wei Wang ◽

Jin Yang ◽

...

Keyword(s):

Population Analysis ◽

Multiple Reaction Monitoring ◽

Simultaneous Detection ◽

Oral Drug ◽

Limit Of Quantification ◽

Selective Method ◽

Coefficients Of Variation ◽

Adherence Assessment ◽

High Consistency

The adherence assessment based on the combination of nevirapine (NVP) and its two metabolites (2-hydroxynevirapine and 3-hydroxynevirapine) would more comprehensively and accurately reflect long-term adherence than that of a single prototype. This study aimed to develop a specific, sensitive and selective method for simultaneous detection of the three compounds in hair and explore whether there was consistency among the three compounds in assessing long-term adherence. Furthermore, 75 HIV-positive patients who were taking the NVP drug were randomly recruited and divided into two groups (high-and low-adherence group). All participants self-reported their days of oral drug administration per month and provided their hair strands closest to the scalp at the region of posterior vertex. The concentrations of three compounds in the hair were determined using a developed LC-MS/MS method in multiple reaction monitoring. This method showed good performances in limit of quantification and accuracy with the recoveries from 85 to 115% and in precision with the intra-day and inter-day coefficients of variation within 15% for the three compounds. The population analysis revealed that patients with high-adherence showed significantly higher concentrations than those with low-adherence for all three compounds. There were significantly moderate correlations of nevirapine with 2-hydroxynevirapine and 3-hydroxynevirapin and high correlation between 2-hydroxynevirapine and 3-hydroxynevirapin. The two NVP’s metabolites showed high consistency with NVP in evaluating long-term adherence.

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