Liquid Chromatography-triple Quadrupole Mass Research Articles

Optimization of ultrasound-assisted extraction of bioactive compounds from coffee pulp using propylene glycol as a solvent and their antioxidant activities

In the cosmetic and pharmaceutical industries, it has been increasingly popular to use alternative solvents in the extraction of bioactive compounds from plants. Coffee pulp, a by-product of coffee production, contains different phenolic compounds with antioxidant properties. The effects of polyols, amplitude, extraction time, solvent concentration, and liquid–solid ratio on total phenolic content (TPC) using ultrasound-assisted extraction (UAE) were examined by single-factor studies. Three main factors that impact TPC were selected to optimize the extraction conditions for total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TTC), and their antioxidant activities using the Box-Behnken design. Different extraction methods were compared, the bioactive compounds were identified and quantified by liquid chromatography triple quadrupole mass spectrometer (LC-QQQ), and the cytotoxicity and cellular antioxidant activities of the extract were studied. According to the response model, the optimal conditions for the extraction of antioxidants from coffee pulp were as follows: extraction time of 7.65 min, liquid–solid ratio of 22.22 mL/g, and solvent concentration of 46.71 %. Under optimized conditions, the values of TPC, TFC, TTC, 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging assay, 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging assay, and Ferric reducing antioxidant power assay (FRAP) were 9.29 ± 0.02 mg GAE/g sample, 58.82 ± 1.38 mg QE/g sample, 8.69 ± 0.25 mg TAE/g sample, 7.56 ± 0.27 mg TEAC/g sample, 13.59 ± 0.25 mg TEAC/g sample, and 10.90 ± 0.24 mg FeSO4/g sample, respectively. Compared with other extraction conditions, UAE with propylene glycol extract (PG-UAE) was significantlyhigher in TPC, TFC, TTC, DPPH, ABTS, and FRAP response values than UAE with ethanol (EtOH-UAE), maceration with propylene glycol (PG-maceration), and maceration with ethanol (EtOH -maceration) (p < 0.05). Major bioactive compounds detected by LC-QQQ included chlorogenic acid, caffeine, and trigonelline. At higher concentrations starting from 5 mg/ml, PG-UAE extract showed higher cell viability than EtOH-UAE in both cytotoxicity and cellular antioxidant assays. The researcher expects that this new extraction technique developed in this work could produce a higher yield of bioactive compounds with higher biological activity. Therefore, they can be used as active ingredients in cosmetics (anti-aging products) and pharmaceutical applications (food supplements, treatment for oxidative stress-related diseases) with minimal use of chemicals and energy.

What once was old is new again: The emergence of traditional stimulants and hallucinogens in Miami-Dade County postmortem casework

Since 2011, the Miami-Dade Medical Examiner Department (MDME) has detected over fifty different Novel Psychoactive Substances (NPS) in routine postmortem casework. On average, cases containing NPS account for 15% of the total number of cases analyzed per year at the MDME. While the incidence and variety of NPS has steadily increased in Miami in the past decade, so has the number of cases containing more traditional stimulants and hallucinogens, such as methamphetamine, methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), lysergic acid diethylamide (LSD), and psilocin. Interestingly, this increase in prevalence of traditional stimulants and hallucinogens coincided with the beginning of the pandemic, suggesting a shift in the drug market possibly due to supply chain issues and strict travel restrictions. Screening and quantitative results from the analysis of various postmortem matrices (whole blood, liver, brain, urine, and gastric contents) were collected from 2011 to Q1 2022. All biological specimens were extracted using solid phase extraction (SPE) and screened on a routine basic drug screening method utilizing gas chromatography coupled to a nitrogen phosphorous detector and mass spectrometer (GC-NPD-MS). If needed, cases were also screened/confirmed on a sensitive, targeted method for the detection of stimulants and hallucinogens, utilizing liquid chromatography ion trap mass spectrometry (LC-Ion Trap-MS). Additionally, cases positive for methamphetamine, MDMA, and MDA were quantified utilizing liquid chromatography triple quadrupole mass spectrometry (LC-QQQ-MS). Cases were organized by year, and the data was tabulated for cases where the following analytes were reported as detected: methamphetamine, MDMA, MDA, psilocin, and LSD. Further data analysis was conducted to assess parameters such as quantitative values for the analytes of interest, additional analytes detected, cause of death (COD), manner of death (MOD), race, gender, and age. Since 2020, methamphetamine, MDMA, MDA, LSD, and psilocin have been detected in 366 postmortem cases. All targeted analytes except for LSD had a higher incidence of detection after 2019, with methamphetamine nearly tripling in the rate of detection from 2019 to 2021, and subsequently, more implicated in the cause of death for accidental overdoses. Psilocin had the largest increase in reported cases, with just one case detected in 2018, and eight cases detected in 2020 and 2021. MDMA and MDA both nearly doubled in rate of detection from 2019 to 2020. In addition, in 2020 the MDME reported its highest detected concentration of MDMA in peripheral blood, 62.5 mg/L. LSD detection remained the same, with three cases detected between 2016–2019, and three cases detected in 2020 and 2021. Methamphetamine was the most detected analyte out of the five, with 236 cases detected between 2011–2019, and 246 cases detected between 2020-Q1 2022. Of note, dimethyltryptamine (DMT) was detected for the first time in MDME casework in 2021, and TFMPP was detected in Q1 2022 for the first time since 2011. The introduction of NPS into the drug market has completely altered the landscape for forensic toxicology casework. However, with the implementation of class-wide drug scheduling and strict pandemic restrictions on air and sea travel, drugs that were not often detected in postmortem cases in Miami managed to surge in popularity and contribute to the highest rate of accidental drug overdoses recorded in the county. While monitoring NPS trends and prevalence is still necessary, it is important not to lose sight of analytes that once were considered “old news”, as there is always a possibility for them to make an impact in todays volatile drug supply.

Simultaneous quantification of multiple amino acid adducts from sulfur mustard-modified human serum albumin in plasma at trace exposure levels by ultra-high performance liquid chromatography–triple quadrupole mass spectrometry after propionyl derivatization

Sulfur mustard (HD) is a highly toxic vesicant and is prohibited by the Organisation for the Prohibition of Chemical Weapons (OPCW). HD can modify human serum albumin (HSA) to generate hydroxyethylthioethyl (HETE) adducts, which could be utilized as biomarkers for verifying HD exposure in forensic analysis. Here, five amino acid adducts generated from pronase digestion of HD-exposed human serum albumin (HD–HSA) in plasma were selected as biomarkers to retrospectively detect HD exposure. HD–HSA was precipitated from plasma with acetone, digested by pronase, derivatized with propionic anhydride (PA), and analysed with ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC–TQ MS). The limits of detection (LODs) and limits of quantification (LOQs) of the HD exposure concentrations were evaluated as 1.00 ng/mL at S/N≥3 and 3.00 ng/mL at S/N≥10, respectively, which are approximately 60 times lower than those of the reported method. The approach shows good linearity (R2≥0.997) from 3.00 ng/mL to 10.0 µg/mL of HD-exposed human plasma with satisfactory precision and accuracy. The developed approach was applied to analysing samples from the 6th OPCW Biomedical Proficiency Test (BioPT). The study showed that the developed approach was also suitable for analysing human plasma samples that were exposed to six of HD analogues, which were common impurities in sulfur mustard mixtures. Moreover, the method was successfully applied to plasma from other species, including rabbits, rats and cattle. This study provides a reliable and sensitive tool for the retrospective detection of vesicants exposure based on multiple biomarkers.

A New Muscarine-Containing Inosperma (Inocybaceae, Agaricales) Species Discovered From One Poisoning Incident Occurring in Tropical China.

Currently, mushroom poisoning still poses a huge problem to humans' health and life globally. Poisoning incidents caused by Inosperma spp. were reported continuously in tropical China in recent years. In this study, a new poisonous Inosperma species, discovered from a poisoning incident, was described in tropical China based on morphological, molecular, and toxin detection evidence; detailed descriptions, photographs, and comparisons to closely related species were provided. For qualitative analysis, through targeted screening using ultra-high liquid chromatography triple quadrupole mass spectrometry (UPLC-MS/MS), the new species contains muscarine and no other toxins (two isoxazole derivatives, two tryptamine alkaloids, three amatoxins, and three phallotoxins). For quantitative analysis, muscarine contents in the pileus and the stipe were 2.08 ± 0.05 and 6.53 ± 1.88 g/kg, respectively.

Intra- and Inter-individual Differences in the Human Intestinal Microbial Conversion of (-)-Epicatechin and Bioactivity of Its Major Colonic Metabolite 5-(3',4'-Dihydroxy-Phenyl)-γ-Valerolactone in Regulating Nrf2-Mediated Gene Expression.

(-)-Epicatechin (EC) is one of the most popular polyphenols present in various food products in daily life. Upon intake, it is intensively metabolized by microbiota in the large intestine. In the present study, intra- and inter-individual variations in this gut microbial conversion of EC and the concomitant formation of its major metabolites, including 5-(3′,4′-dihydroxy phenyl)-γ-valerolactone (3,4-diHPV), were identified and quantified via liquid chromatography triple quadrupole mass spectrometry (LC-TQ-MS) in anaerobic fecal incubations. In addition, the bioactivity of EC and 3,4-diHPV in activating Nrf2-mediated gene expression was tested quantifying their effects in the U2OS Nrf2 CALUX assay (a reporter gene assay that is used to test the potency of chemicals in activation of Nrf2 signaling), and on the expression levels of Nrf2-related proteins in Hepa1c1c7 and Caco-2 cells via nanoLC-MSMS. A quantitative real-time polymerase chain reaction (RT-qPCR) was carried out to confirm selected Nrf2-regulated gene expressions at the mRNA level. Results obtained show that both intra- and inter-individual differences exist in human gut microbial EC degradation and 3,4-diHPV formation, with inter-individual differences being more distinct than intra-individual differences. The metabolite, 3,4-diHPV, showed higher potency in the U2OS Nrf2 CALUX assay than EC itself. Among the obviously altered Nrf2-related proteins, 14 and 10 Nrf2-associated proteins were upregulated to a higher extent upon 3,4-diHPV treatment than in the EC treated group for Hepa1c1c7 and Caco-2 cells, respectively. While only three and four of these Nrf2-associated proteins were induced at a higher level upon EC than upon 3,4-diHPV treatment for Hepa1c1c7 and Caco-2 cells, respectively. RT-qPCR results showed that indeed Nrf2-mediated genes (e.g., Nqo1 and Ugt1a) were only induced significantly in 3,4-diHPV treated and not in EC treated Hepa1c1c7 cells. Taken together, the results suggest that the major colonic EC metabolite, 3,4-diHPV, was more capable of inducing Nrf2-mediated gene expression than its parent compound EC. This implies that the evident inter- and intra-individual differences in the microbial conversion of EC to this major metabolite 3,4-diHPV may affect the overall health-promoting effects of EC consumption related to the Nrf2 pathway activation.

Microwave-based preparation of γ-Fe2O3/SrTiO3 photocatalyst for efficient degradation of organic pollutants in water

Photocatalysis is considered one of the most promising wastewater treatment technologies. In this work, a magnetic photocatalytic γ-Fe2O3/SrTiO3 composite with high performance and stability were synthesized by a one-step microwave method, and the amount of γ-Fe2O3 on methylene blue (MB) and tetracycline (TC) degradation performance of composite photocatalyst was investigated under simulated sunlightand. The materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), photoluminescence (PL), X-ray photoelectron spectroscopy (XPS), ultraviolet–visible diffuse reflectance spectra (UV–vis DRS) and electron spin resonance (ESR). The degradation of MB and TC over 0.3% γ-Fe2O3/SrTiO3 was found to be very effective, and the degradation reached 84.91% (150 min) and 92.03% (60 min), respectively. After five consecutive cycles of reusability test under visible light irradiation, the photocatalytic degradation efficiency remained unchanged. According to the results of free radicals capture experiments and ESR, superoxide radical and hole were found to be the main active species for MB degradation. The degradation products were identified by liquid chromatography triple quadrupole mass spectrometry (LC-MS), and a possible degradation pathway of TC was proposed. Thus, magnetic composites provide a new strategy for the design of photocatalysts with excellent catalytic performance, which is conducive to recovery.

Multiresidue analysis and probabilistic dietary risk assessment of 241 pesticides in wheatgrass (Triticum sp.) using LC-MS/MS in combination with QuEChERS extraction.

Wheatgrass is consumed as an important nutritious herbal food supplement across the globe; however, limited studies have been reported analyzing multiclass pesticides in this complex, nutrient-rich natural product. An analytical method was developed for the estimation of 241 pesticides in random wheatgrass samples collected from Delhi Northern Capital Region (Delhi-NCR). Extraction was performed by QuEChERS, cleaning was performed by dispersive solid-phase extraction and the extracts were analyzed using triple quadrupole liquid chromatography mass spectrometry. The limit of quantification was 0.5μg/kg, which is well below the European Union Maximum Residue Level. The coefficient of determination was >0.991 across a calibration range of 0.5-100 μg/kg. The relative standard deviation values for 231 pesticides based on 10 replicates of samples spiked at 10μg/kg were <5%. Among random samples, 54% confirmed the presence of at least one pesticide. The results indicated the presence of eight different pesticides among 38% of the total population with metribuzin at 299.7μg/kg and carfentrazone-ethyl at 19.47 μg/kg exceeding the permissible limits among 6% of the total estimated population. The chronic and acute risk quotients as calculated were <1, indicating nonsignificant dietary risk to consumers. However, the presence of pesticides above the permissible limit is likely to result in adverse health effects to the consumers of herbal supplements from an urban population and incorporating measures would be useful to ensure the quality and safety of wheatgrass consumption.

Potential Misidentification of Natural Isomers and Mass-Analogs of Modified Nucleosides by Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many naturally modified nucleosides identified to date (>160), the discrimination of isomers and mass-analogs can be problematic and is often overlooked. This study analyzes 17 nucleoside standards by LC-TQ-MS with separation on three different analytical columns and discusses, with examples, three major causes of analyte misidentification: structural isomers, mass-analogs, and isotopic crosstalk. It is hoped that this overview and practical examples will help to strengthen the accuracy of the identification of modified nucleosides by LC-TQ-MS.

Distinct Urinary Metabolic Biomarkers of Human Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with high mortality rate due to its poor diagnosis in the early stage. Here, we report a urinary metabolomic study on a cohort of CRC patients (n =67) and healthy controls (n =21) using ultraperformance liquid chromatography triple quadrupole mass spectrometry. Pathway analysis showed that a series of pathways that belong to amino acid metabolism, carbohydrate metabolism, and lipid metabolism were dysregulated, for instance the glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, glycolysis, and TCA cycle. A total of 48 differential metabolites were identified in CRC compared to controls. A panel of 12 biomarkers composed of chenodeoxycholic acid, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid, and homocitrulline were identified by Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis classification model and validated by Gradient Boosting (GB), Logistic Regression (LR), and Random Forest diagnostic model, which were able to discriminate CRC subjects from healthy controls. These urinary metabolic biomarkers provided a novel and promising molecular approach for the early diagnosis of CRC.

Serum Amino Acid Profiles Predict the Development of Hepatocellular Carcinoma in Patients with Chronic HBV Infection.

Background: The study aimed to find out the alterations in serum amino acid (AA) profiles and to detect their relationship with carcinoma formation. Methods: Targeted metabolomics based on ultraperformance liquid chromatography triple quadrupole mass spectrometry to quantitatively analyze serum AA levels in 136 hepatitis B (CHB) patients and 93 hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients. Results: It was shown that decreased serum levels of leucine, lysine, threonine, tryptophan, valine, serotonin, and taurine were observed in more HCC patients than CHB patients, but the serum phenylalanine level was increased. Serum valine and serotonin were lower in Class C than Class A and Class B in HCC patients. Accompanied with the higher score of Model for End-Stage Liver Disease, serum phenylalanine was increased not only in CHB patients but also in HCC patients. The serum level of phenylalanine increased in the decompensated stage more than in the compensated stage, while serum leucine and serotonin significantly decreased. Serum serotonin still had significant differences between CHB and HCC both in the HBV desoxyribonucleic acid (HBV-DNA) negative group and in the HBV-DNA positive group. Furthermore, it was shown that the tryptophan ratio, branched-chain amino acids (BCAA)/aromatic amino acids ratio, BCAAs/tyrosine ratio, Fischer’s ratio, and serotonin-to-tryptophan ratio significantly decreased, while the tyrosine ratio and the kynurenine-to-tryptophan ratio increased in HCC patients more than those in CHB. Conclusions: A distinct metabolite signature of some specific serum amino acids was found between CHB and HCC patients, which may help predict the development of HCC at an early stage.

11 Determination of Milk Concentrations and Pharmacokinetics of Salicylic Acid Following Acetylsalicylic Acid (Aspirin) Administration in Postpartum Dairy Cows

Abstract Administration of non-steroidal anti-inflammatory drugs, such as the widely available drug aspirin, or acetylsalicylic acid (ASA), may improve health and milk production in postpartum dairy cows. However, plasma pharmacokinetic (PK) data for salicylic acid (SA), the active metabolite of ASA, are unrepresentative of lactating dairy cows and current treatment regimens, and there are currently no published milk residue and minimal pharmacodynamic data available for ASA or SA in dairy cows. The objectives of this descriptive study were to (1) describe the pharmacokinetics of SA in the milk and plasma of postpartum dairy cows following oral ASA administration; (2) to estimate a milk withdrawal period for dairy cows treated with ASA; and (3) to determine the impact of ASA on plasma prostaglandin E2 metabolite (PGEM) concentrations. Primiparous (n = 3) and multiparous (n = 7) postpartum Holstein dairy cows received 2 oral treatments with ASA at 200 mg/kg, 24 h apart. Plasma and milk SA concentrations from 0 h through 120 h after ASA administration were analyzed using ultra-performance liquid chromatography triple quadrupole mass spectrometry. Plasma PK analysis was performed using a non-compartmental approach using the free software, PKSolver, in Excel. An estimated milk withdrawal interval of 168 h was determined using the FDA Milk Discard App in R (RStudio, Boston, MA) using a 99th percentile upper tolerance limit with 95% confidence. Plasma PGEM concentrations from 0 h to 24 h after ASA administration were determined in duplicate using ELISA and were reduced compared with baseline for up to 12 h after ASA administration. A mean reduction in PGEM of -49.3% was observed at 2 h. Results from this study suggest that the current milk withhold recommendation of 24 h for dairy cows administered ASA should be revised and that ASA administration may mitigate postpartum inflammation through reduction in prostaglandin production.

Production of Hydroxy Fatty Acids, Precursors of γ-Hexalactone, Contributes to the Characteristic Sweet Aroma of Beef.

Aroma is an essential factor for meat quality. The meat of Japanese Black cattle exhibits fine marbling and a rich and sweet aroma with a characteristic lactone composition. The mechanism of lactone formation associated with beef aroma has not been elucidated. In this study, we examined the precursors of γ-hexalactone, an indicator of the sweet aroma of beef and identified the mechanism underlying γ-hexalactone production. A low-temperature vacuum system was used to prepare beef tallow from Japanese Black cattle and Holstein cattle. The odor components were identified using headspace–gas chromatography. The analysis revealed that γ-hexalactone, γ-dodecalactone, δ-tetradecalactone, and δ-hexadecalactone were present as sweet aroma components of beef tallow prepared from marbling and muscle. Since we previously reported that γ-hexalactone formation correlates with linoleic acid content in beef, we analyzed ten oxidized fatty acids derived from linoleic acid by liquid chromatography–triple quadrupole mass spectrometry and detected two hydroxy-octadecadienoic acids (9S-HODE and 13S-HODE) in beef tallow. Significant differences in arachidonic acid 15-lipoxygenase and cyclooxygenase protein expression levels among subcutaneous fat, intramuscular fat, and muscle tissue were observed. Our results suggest that the combination of linoleic acid and the expression of lipid oxidase derived from beef muscle and intramuscular fat produce hydroxy fatty acids that result in a sweet aroma.

Is there any biological insight (or respite) for insects exposed to plastics? Measuring the impact on an insects central carbon metabolism when exposed to a plastic feed substrate

Insects used to treat organic waste streams and produce valuable protein products are increasingly exposed to plastic contaminated source material assimilating plastic carbon into organic biomass, which is pervasive and hazardous to organisms. Our understanding of this increased insect-plastic interaction remains limited and needs urgent scientific attention if plastic biodegradation and production rates of quality protein are to be improved. Herein, we investigated the biochemical impact of various plastics using three insect models. Black Soldier Fly (BSF), Mealworm (MW), and Wax Moth (WM) larva were each exposed to a plastic substrate (PET, PE, PS, Expanded PE, PP, and PLA) as the primary carbon source for five days to explore any positive metabolic benefits in terms of insect performance and plastic degradation potential. Central carbon metabolism (CCM) metabolites were analyzed via a targeted tMRM liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) method. Unique expressed pathways were observed for each insect model. When reared on PET, BSF larvae were found to have an elevated pyrimidine metabolism, while the purine metabolism pathway was strongly expressed on other plastics. BSF also exhibited a downregulated Vitamin B6 metabolism across all plastics, indicating a likely gut-symbiont breakdown. The MW and WM model insects were metabolically more active on PLA and expanded foam plastics. Further, WM exhibited an elevation in Vitamin B6 metabolism. This data suggests a positive insect-specific interaction towards certain plastic types that warrants further investigation. It is anticipated that through deeper insight into the metabolic impact and benefits afforded from certain plastics, an insect biotransformation pipeline can be established that links fit-for-purpose insect models to individual plastic types that address our growing plastic waste issue.

Analysis of the Formation of Sauce-Flavored Daqu Using Non-targeted Metabolomics.

Sauce-flavored Daqu exhibits different colors after being stacked and fermented at high temperatures. Heiqu (black Daqu, BQ) with outstanding functions is difficult to obtain because its formation mechanism is unclear. In this study, we compared the metabolites in different types of Daqu using ultra-high-performance liquid chromatography triple quadrupole mass spectrometry to explore the formation process of BQ. We found that 251 differential metabolites were upregulated in BQ. Metabolic pathway analysis showed that “tyrosine metabolism” was enriched, and most metabolites in this pathway were differential metabolites upregulated in BQ. The tyrosine metabolic pathway is related to enzymatic browning and melanin production. In addition, the high-temperature and high-humidity fermentation environment of sauce-flavored Daqu promoted an increase in the melanoidin content via a typical Maillard reaction; thus, the melanoidin content in BQ was much higher than that in Huangqu and Baiqu. By strengthening the Maillard reaction precursor substances, amino acids, and reducing sugars, the content of Daqu melanoidin increased significantly after simulated fermentation. Therefore, the enzymatic browning product melanin and Maillard reaction product melanoidin are responsible for BQ formation. This study revealed the difference between BQ and other types of Daqu and provides theoretical guidance for controlling the formation of BQ and improving the quality of liquor.

Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review

Harmful algal blooms (HABs) and their toxins are a significant and continuing threat to aquatic life in freshwater, estuarine, and coastal water ecosystems. Scientific understanding of the impacts of HABs on aquatic ecosystems has been hampered, in part, by limitations in the methodologies to measure cyanotoxins in complex matrices. This literature review discusses the methodologies currently used to measure the most commonly found freshwater cyanotoxins and prymnesins in various matrices and to assess their advantages and limitations. Identifying and quantifying cyanotoxins in surface waters, fish tissue, organs, and other matrices are crucial for risk assessment and for ensuring quality of food and water for consumption and recreational uses. This paper also summarizes currently available tissue extraction, preparation, and detection methods mentioned in previous studies that have quantified toxins in complex matrices. The structural diversity and complexity of many cyanobacterial and algal metabolites further impede accurate quantitation and structural confirmation for various cyanotoxins. Liquid chromatography–triple quadrupole mass spectrometer (LC–MS/MS) to enhance the sensitivity and selectivity of toxin analysis has become an essential tool for cyanotoxin detection and can potentially be used for the concurrent analysis of multiple toxins.

The Screening Approaches for 34 Common Drugs and Metabolites in Biological Samples by Liquid Chromatography Orbital Trap Mass Spectrometry

To establish a high-performance liquid chromatography orbital trap mass spectrometry (HPLC-Obitrap MS) method for screening 34 common drugs and metabolites in biological samples. The target analytes in urine and blood samples were extracted with ethyl acetate, concentrated by nitrogen blowing and redissolved. The hair samples were washed with water and acetone, dried and cut into bits of about 1 mm, and then crushed in a freezing grinder. The analytes were extracted with methanol, and after filtration, the filtrate was used for instrumental analysis. Hypersil Gold PFP (2.1 mm×100 mm, 3 µm) column was used for chromatographic separation. Methanol and 5 mmol/L ammonium acetate solution were used as mobile phase with gradient elution at a flow rate of 400 μL/min. Mass spectrometry was done by electrospray positive and negative ion alternation mode. The data were collected using Full MS and Full MS/dd-MS2 mode. Xcalibur 4.0 software was used to control instruments and to collect data, and TraceFinder 3.3 was used for screening and identification. The method's detection limits for 34 drugs and their metabolites in blood, urine and hair samples were 3.30-10700 ng/L, 4.43-5440 ng/L, 0.0350-4.21 μg/kg, respectively. The intra-day and inter-day precisions of the spiked samples at the levels of 5.0, 10, and 20 μg/L were 3.50%-6.00% and 4.18%-9.90%, respectively. A total of 1125 biological samples of urine, blood and hair were collected and screened. The results showed that 96.7% of the drug users were taking a single drug, while 3.3% were mixed drug users. The main types of drug of abuse were methamphetamine (75.8%), heroin (18.5%), ketamine (2.4%) and other drugs (3.3%), and 87.9% of the positive samples were from male users. Compared with the results of high-performance liquid chromatography triple quadrupole mass spectrometry, this method can be used to identify more types of drugs in one run and to conduct retrospective analysis. The method established in the study is simple and sensitive and is well suited for the screening of common drugs and metabolites in biological samples.

Changes in the free amino acid profile of pickling cucumber during lactic acid fermentation.

Free amino acid (FAA) profiles of fresh, acidified, naturally fermented, and starter culture fermented cucumbers were analyzed by liquid chromatography triple quadrupole mass spectrometry. Fermented cucumbers contained more total FAA than acidified cucumbers (1,302 ± 102mg/kg and 635 ± 35mg/kg, respectively). Total FAA content of fermented cucumber was similar regardless of brine salt levels (2-6% NaCl) and starter culture addition. Glutamine (1491.4± 69.3mg/kg), γ-aminobutyric acid (GABA, 269.6± 21.4mg/kg), asparagine (113.0± 6.4mg/kg), and citrulline (110.3±8.5mg/kg) were the most abundant FAA in fresh pickling cucumber, whereas GABA (181.3±21.5mg/kg), isoleucine (165.2± 11.2mg/kg), leucine (129.8± 10.9mg/kg), and lysine (110.9± 5.0mg/kg) were the most abundant in fermented cucumber. GABA and ornithine were produced during fermentation, indicating glutamate decarboxylase and arginine deiminase activities. Notably, ornithine was significantly higher in natural (63.3±31.5mg/kg) versus starter culture fermented cucumbers (3.0±0.7mg/kg). This new information on FAA composition of fresh and fermented pickling cucumbers shows the impact of fermentation conditions on cucumber amino acid profiles while providing insight for manipulating fermentations for health promotion and consumer acceptance. PRACTICAL APPLICATION: This study reports changes in the free amino acid profiles of raw, fermented and acidified cucumbers, which may be valuable for understanding the impact of these foods on human health and nutrition. This information is useful for food microbiologists studying the metabolism of lactic acid bacteria during fermentation and/or designing starter cultures and could contribute to the development of novel fermented cucumber pickle products with enhanced nutritional value.

Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography–tandem mass spectrometry

PurposeZolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography–triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOFMS).Methods7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS.ResultsThe presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems.ConclusionsThe presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

A critical comparison between an ultra-high-performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QqQ-MS) method and an enzyme assay for anti-cholinesterase pesticide residue detection in cereal matrices.

Analytical method development for the control of pesticide residues occurring in significant dietary foodstuffs is of utmost importance considering their potential impact on consumer health and food market sustainability. Depending on the purpose, either instrumental analysis, mainly chromatographic methods, or screening assays, mostly using biorecognition affinity, are commonly used, featuring different advantages and drawbacks. To practically compare these two different types of analytical strategies, we applied them for the detection of (i) 97 organophosphate (OP) and carbamate (CM) pesticide residues in wheat flour and (ii) carbofuran (a carbamate insecticide) in wheat, rye and maize flour samples. Regarding high-end analysis, an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) method was developed and validated achieving low limits of quantification (LOQs, from 0.002 to 0.040 mg kg-1) and a short chromatographic run (12 min). In terms of bioanalytical methods, a fast (17 min) and cost-efficient (∼0.01€ per sample) acetylcholinesterase (AChE) microplate assay for carbofuran screening was utilized. Importantly, carbofuran was the strongest of the 11 OP and CM tested pesticides achieving a half maximal inhibitory concentration (IC50) of 0.021 μM whilst the assay detectability was at the parts per billion level in all three cereal matrices. Based on the attained results, a critical discussion is presented providing the analytical merits and bottlenecks for each case and a wider outlook related to the application of analytical methods in the food safety control analytical scheme.

Groundwater CSI: Unravelling Pollution Sources in Complex Environments with Liquid Chromatography Triple Quadrupole Mass Spectrometry

Wastewater treatment plants (WWTP) are used worldwide to purify domestic and industrial wastewater before it is returned to the environment. Even after treatment, wastewater may still contain a variety of contaminants, including nutrients (usually nitrogen and phosphorus) and organic pollutants, such as endocrine disruptors and pesticides. These compounds can leak into groundwater via old and/or damaged infrastructure, leaching from biosolids storage areas and/or release of effluents. However, similar contaminant impacts can come from other sources, such as agriculture. It is often very difficult to distinguish the true source of such contamination, especially where wastewater treatment plants are located in agricultural areas. Robust and sensitive techniques are needed to characterize impacts where there are multiple potential sources. This study analyzed a variety of synthetic chemicals using liquid chromatography triple quadrupole mass spectrometry (LC–QQQ–MS) to generate unique fingerprints of pollution. These were used to distinguish the impact on the local groundwater of a WWTP in southeast Victoria (Australia) from that of local agriculture. The use of such novel tracers could become a valuable tool in environmental monitoring, management, and remediation in the future.