Liquid Chromatography-tandem Mass Spectrometry Screening Research Articles

Determination of licit and illicit drugs and metabolites in human sweat by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry screening method in sweat was developed for the simultaneous determination of three licit drugs (nicotine, paracetamol, and caffeine); four illicit drugs (cocaine, ketamine, 25I-NBOMe and methamphetamine) and two metabolites (benzoylecgonine and cotinine). Target drugs were liberated from sweat patches with pH 5 sodium acetate buffer and further purified by solid phase extraction (SPE) utilising Strata-X-Drug B cartridges. Optimal solvent constituents for SPE organic wash and elution were 70% v/v methanol in deionised water and 5% v/v ammonium hydroxide in methanol respectively. Chromatographic separation was achieved using a superficially porous particle C18 column with gradient elution, using (A) 0.1% formic acid in water and (B) acetonitrile as mobile phase constituents. Target drugs were identified using a combination of retention time, and the ion ratios for two precursor-product ion transitions for each analyte monitored in multiple reaction monitoring (MRM) mode. The method was linear for all target drugs from 1.0-150.0 ng mL-1 with corresponding limits of quantitation of 1.0 ng mL-1. Limits of detection were found to range from 0.1-0.6 ng per patch. The method was subsequently applied to the analysis of sweat samples from five male and four female participants aged 20-25 years. Sweat was collected from two areas (right forearm and left thigh) using protected layers of gauze. All eighteen patches tested positive for at least one target analyte. The results of this study not only show a multi-substance screening method was achieved but also that sweat patches can be used to indicate an individual's drug use. Therefore, they can provide an alternative non-invasive technique for forensic applications.

Gastroprotective effects of Caragana ambigua stocks on ethanol-induced gastric ulcer in rats supported by LC-MS/MS characterization of formononetin and biochanin A.

Caragana ambigua has been the part of the dietary routines of the regional people in south-west Pakistan and has traditionally been used for the treatment of diabetes there. There is an increased production of reactive oxygen species in diabetics, leading to gastrointestinal disorders. Natural antioxidants exhibit gastroprotective effects owing to their free-radical scavenging action. C.ambigua possesses appreciable phenolic and flavonoid content; thus, it has the potential to protect against gastrointestinal disorders (e.g. gastric ulcer). This study reports the anti-ulcer potential of C.ambigua. Four different fractions (chloroform, ethyl acetate, butanol, and aqueous) of plant were compared against omeprazole. Ulcer index, ulcer inhibition percentage, gastric pH and volume, total acidity, gastric protein, gastric wall mucus, and histopathology of gastric walls of rats were assessed. All fractions exhibited a reduction in ulcer index and promotion of percentage of ulcer inhibition compared with the ulcer control group. Furthermore, the fractions revealed a significant (P < 0.001) diminution in gastric volume and total acidity with an increase in pH. Among the fractions investigated, the chloroform fraction unveiled the most promising anti-ulcer activity, which is comparable to omeprazole. Liquid chromatography-tandem mass spectrometry screening of fractions revealed the presence of formononetin and biochanin A (isoflavones reported to have anti-ulcer properties) in the chloroform fraction. This study establishes that C.ambigua possesses significant potential in reducing gastric ulcer progression. Formononetin and biochanin A are chiefly responsible for the stated bioactivity due to the fact that these compounds were solely present in the chloroform fraction. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Deletion of SREBF1, a Functional Bone-Muscle Pleiotropic Gene, Alters Bone Density and Lipid Signaling in Zebrafish.

Through a genome-wide analysis of bone mineral density (BMD) and muscle mass, identification of a signaling pattern on 17p11.2 recognized the presence of sterol regulatory element-binding factor 1 (SREBF1), a gene responsible for the regulation of lipid homeostasis. In conjunction with lipid-based metabolic functions, SREBF1 also codes for the protein, SREBP-1, a transcription factor known for its role in adipocyte differentiation. We conducted a quantitative correlational study. We established a zebrafish (ZF) SREBF1 knockout (KO) model and used a targeted customized lipidomics approach to analyze the extent of SREBF1 capabilities. For lipidomics profiling, we isolated the dorsal muscles of wild type (WT) and KO fishes, and we performed liquid chromatography-tandem mass spectrometry screening assays of these samples. In our analysis, we profiled 48 lipid mediators (LMs) derived from various essential polyunsaturated fatty acids to determine potential targets regulated by SREBF1, and we found that the levels of 11,12 epoxyeicosatrienoic acid (11,12-EET) were negatively associated with the number of SREBF1 alleles (P = 0.006 for a linear model). We also compared gene expression between KO and WT ZF by genome-wide RNA-sequencing. Significantly enriched pathways included fatty acid elongation, linoleic acid metabolism, arachidonic acid metabolism, adipocytokine signaling, and DNA replication. We discovered trends indicating that BMD in adult fish was significantly lower in the KO than in the WT population (P < 0.03). These studies reinforce the importance of lipidomics investigation by detailing how the KO of SREBF1 affects both BMD and lipid-signaling mediators, thus confirming the importance of SREBF1 for musculoskeletal homeostasis.

Verticillium longisporum Elicits Media-Dependent Secretome Responses With Capacity to Distinguish Between Plant-Related Environments.

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1, NLP2, and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization.

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) - a group of new psychoactive substances (NPS) - is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and finally included to the MS/MS spectra in-house library. Sample preparation was performed using a cheap-and-easy salting-out liquid-liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.

Rapid liquid chromatography tandem mass-spectrometry screening method for urinary metabolites of primary hyperoxaluria.

The primary hyperoxalurias are inherited disorders of glyoxylate metabolism that lead to overproduction of oxalate, urolithiasis and renal failure. Delays in diagnosis can be costly in terms of preserving renal function. Here we present a rapid liquid chromatography tandem mass-spectrometry screening method for the analysis of metabolites (primary hyperoxaluria metabolites) produced in excess by primary hyperoxaluria patients that include glycolate, glycerate and 2,4-dihydroxyglutarate. Assay performance was compared to our existing gas chromatography-mass spectrometry method and clinical utility established by analysis of urine samples from patients with confirmed primary hyperoxalurias (11 PH1, 12 PH2 and 8 PH3) and controls ( n = 12). An additional 67 urine samples from patients with PH3 were used postvalidation to confirm the derived 2,4-dihydroxyglutarate cut-off. Glycolate, glycerate and 2,4-dihydroxyglutarate showed a mean bias of 3.3, -22.8 and 5.7%, respectively, compared to our previously published gas chromatography-mass spectrometry method. The mean total imprecision for glycolate, glycerate and 2,4-dihydroxyglutarate was shown to be 6.4, 10 and 11%, respectively. Clinical assessment confirmed that mean urinary glycolate, glycerate and 2,4-dihydroxyglutarate excretion were significantly elevated in patients with PH1, PH2 and PH3, respectively. The greatest sensitivity and specificity for PH1, PH2 and PH3 was achieved at cut-offs of 193, 100 and 4.9 μmol/mmol for glycolate, glycerate and 2,4-dihydroxyglutarate, respectively. A rapid screening method for the identification and differentiation of patients with suspected PH1, PH2 and PH3 is presented that allows focussing of genetic testing, saving time, money and, with earlier treatment, potential preservation of renal function for these patients.

Screening and Assessment of Low-Molecular-Weight Biomarkers of Milk from Cow and Water Buffalo: An Alternative Approach for the Rapid Identification of Adulterated Water Buffalo Mozzarellas.

Adulteration of Mozzarella di Bufala Campana with cow milk is a common fraud because of the high price and limited seasonal availability of water buffalo milk. To identify such adulteration, this work proposes a novel approach based on the use of species-specific, low-molecular-weight biomarkers (LMWBs). Liquid chromatography-tandem mass spectrometry screening analyses identified β-carotene, lutein, and β-cryptoxanthin as LMWBs of cow milk, while ergocalciferol was found only in water buffalo milk. Adulterated mozzarellas were prepared in the laboratory and analyzed for the four biomarkers. Combined quantification of β-carotene and ergocalciferol enabled the detection of cow milk with a sensitivity threshold of 5% (w/w). The method was further tested by analyzing a certificated water buffalo mozzarella and several commercial products. This approach is alternative to conventional proteomic and genomic methods and is advantageous for routine operations as a result of its simplicity, speed, and low cost.

Liquid Chromatography-Tandem Mass Spectrometry Screening Method for the Detection of Radical-Scavenging Natural Antioxidants from the Whole Scutellariae (Radix, Stem and Leaf).

A novel free radical reaction combined with high-performance LC-photodiode array-ESI-MS/MS screening method was developed for the detection and identification of natural antioxidants from whole Scutellariae. Six compounds and whole Scutellariae extracts were found to possess a potential antioxidant capacity, and their free radical-scavenging activities were investigated in detail. The six compounds were identified as baicalin, baicalein, scutellarin, scutellarein, wogonoside and chrystin-7-glucoronide. The present study reveals that the radical-scavenging capacities of the whole Scutellariae extracts are as follows: hydroxyl radical > superoxide radical > peroxy radical. Wogonoside showed the strongest capability for scavenging hydroxyl radical. Baicalein not only showed the strongest capability for scavenging superoxide radical but also showed capability for lipid radical; Scutellarein (Peak 5) exhibited the highest reactivity in the lipid peroxidation processes. Based on these studies, the current paper accomplishes the evaluation of activities of some important anti-radical substances extracted from the radix, stem and leaf of the whole Scutellariae.

Loading of free radicals on the functional graphene combined with liquid chromatography–tandem mass spectrometry screening method for the detection of radical-scavenging natural antioxidants

A novel free radical reaction combined with liquid chromatography electrospray ionization tandem mass spectrometry (FRR-LC–PDA-ESI/APCI-MS/MS) screening method was developed for the detection and identification of radical-scavenging natural antioxidants. Functionalized graphene was prepared by chemical method for loading free radicals (superoxide radical, peroxyl radical and PAHs free radical). Separation was performed with and without a preliminary exposure of the sample to specific free radicals on the functionalized graphene, which can facilitate reaction kinetics (charge transfers) between free radicals and potential antioxidants. The difference in chromatographic peak areas is used to identify potential antioxidants. The structure of the antioxidants in one sample (Swertia chirayita) is identified using MS/MS and comparison with standards. Thirteen compounds were found to possess potential antioxidant activity, and their free radical-scavenging capacities were investigated. The thirteen compounds were identified as 1,3,5-trihydroxyxanthone-8-O-β-d-glucopyranoside (PD1), norswertianin (PD2), 1,3,5,8-tetrahydroxyxanthone (PD3), 3, 3′, 4′, 5, 8-penta hydroxyflavone-6-β-d-glucopyranosiduronic acid-6′-pentopyranose-7-O-glucopyranoside (PD4), 1,5,8-trihydroxy-3-methoxyxanthone (PD5), swertiamarin (PS1), 2-C-β-d-glucopyranosyl-1,3,7-trihydroxylxanthone (PS2), 1,3,7-trihydroxylxanthone-8-O-β-d-glucopyranoside (PL1), 1,3,8-trihydroxyl xanthone-5-O-β-d-glucopyranoside (PL2), 1,3,7-trihydroxy-8-methoxyxanthone (PL3), 1,2,3-trihydroxy-7,8-dimethoxyxanthone (PL4), 1,8-dihydroxy-2,6-dimethoxy xanthone (PL5) and 1,3,5,8-tetramethoxydecussatin (PL6). The reactivity and SC50 values of those compounds were investigated, respectively. PD4 showed the strongest capability for scavenging PAHs free radical; PL4 showed prominent scavenging capacities in the lipid peroxidation processes; it was found that all components in S. chirayita exhibited weak reactivity in the superoxide radical scavenging capacity. The use of the free radical reaction screening method based on LC–PDA-ESI/APCI-MS/MS would provide a new approach for rapid detection and identification of radical-scavenging natural antioxidants from complex matrices.

Development and validation of a multi-residue and multiclass ultra-high-pressure liquid chromatography-tandem mass spectrometry screening of antibiotics in milk

A multi-residue screening method for 33 antibiotics from five different families was employed to simultaneously determine sulphonamide, tetracycline, macrolide, quinolone and chloramphenicol antibiotics using ultra high pressure liquid chromatography tandem mass spectrometry. A simple sample preparation method was developed using protein precipitation, centrifugation and solid phase extraction and was optimised to achieve the best recovery for all compounds. The methodology was validated for quantitative screening methods, by evaluating the detection capability (CCβ), specificity, selectivity, precision, applicability and ruggedness. Precision, in terms of relative standard deviation, was under 21% for all compounds. Because CCβ was determined for screening purposes and, according to maximum residue limit, the limit of detection of the method was calculated and ranged from 0.010 μg kg−1 to 3.7 μg kg−1. This validation provided evidence that the method is suitable to be applied in routine analysis for the detection of antibiotics in bovine, caprine and ovine milk.

Characterization of bioactive thiophenes from the dichloromethane extract of Echinops grijisii as Michael addition acceptors

Michael addition acceptors are considered as biologically active molecules, which regulate many signal pathways in cells. In the present study, it was demonstrated that the dichloromethane extract of Echinops grijisii had phase II detoxifying enzyme-inducing and nuclear factor-kappaB (NF-kappaB)-inhibiting activities, which might be attributed to the modification of key cysteine residues in Keap1 and NF-kappaB by Michael addition acceptors in it. To screen these Michael addition acceptors, glutathione (GSH) was employed, and a simple liquid chromatography-tandem mass spectrometry screening method was established to investigate the formation of GSH conjugates. Three thiophenes, 5-(penta-1,3-diynyl)-2-(3,4-diacetoxybut-1-ynyl)-thiophene (6), 2-(penta-1,3-diynyl)-5-(4-hydroxybut-1-ynyl)-thiophene (7), and 2-(penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene (8) were demonstrated to react with GSH. Then NAD(P)H/quinone oxidoreductase1(NQO1) induction assay and an ultrafiltration mass spectrometric screening method were performed to investigate whether the above three compounds had NQO1-inducing and NF-kappaB (p65) alkylating activities. The result indicated that compounds 6-8, which had a common structural moiety, a penta-1,3-diynyl group, had strong NQO1-inducing activities, and compounds 7 and 8 could effectively alkylate the cysteine residues in NF-kappaB (p65).

Validation of a liquid chromatography-tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach

A multi-residue method was developed for monitoring antibiotic residues in milk using liquid chromatography coupled to a tandem quadrupole mass spectrometer (LC/MS-MS). Two very short extractions followed by two LC/MS-MS acquisitions allowed the screening of 58 antibiotics belonging to eight different families (penicillins, cephalosporins, sulfonamides, macrolides, lincosamides, aminoglycosides, tetracyclines, and quinolones). This method is currently implemented in the laboratory in a qualitative way, i.e. monitoring the presence or absence of residue in a sample and identification of the analyte before the confirmation step. In order to assess the performance of this method, a validation strategy described in an internal guideline for the validation of screening methods was applied. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest (maximum residue limit, MRL) at least. According to European Commission Decision 2002/657/EC, the suitable sensitivity of a screening method can be demonstrated when the CCβ is below or equal to the MRL and so the false-compliant rate below or equal to 5% at the MRL level. The validation scheme was established in order to take into account various variability factors: the apparatus response, the interday repeatability, the matrix effect, etc. The results of the validation clearly demonstrate the suitability of this method for the detection and identification of more than 50 antibiotics and they are in agreement with the results obtained in routine analysis.

Detection of anabolic steroids in dietary supplements: The added value of an androgen yeast bioassay in parallel with a liquid chromatography–tandem mass spectrometry screening method

Recently we constructed a recombinant yeast cell that expresses the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. When exposed to testosterone, the concentration where half-maximal activation is reached (EC 50) was 50 nM. Eighteen different dietary supplements, already analysed by a liquid chromatography–tandem mass spectrometry method (LC–MS/MS) for the presence of anabolic steroids, were screened for androgenic activity. Eleven samples containing at least one anabolic steroid, with a concentration that was around or above 0.01 mg unit −1 according to LC–MS/MS, were also positive in the bioassay. Seven samples did not contain any of the 49 compounds screened for in LC–MS/MS. In contrast two of them were positive in the bioassay. Bioassay-directed identification, using the bioassay as an off-line LC-detector and LC–time of flight-MS with accurate mass measurement was carried out in these two samples and revealed the presence of 4-androstene-3β,17β-diol and 5α-androstane-3β,17β-diol in the first and 1-testosterone in the second supplement, showing the added value of the bioassay in comparison with a LC–MS/MS screening method alone.