Liquid Chromatography-triple Quadrupole Mass Spectrometry Research Articles

Interindividual Differences in Human Intestinal Microbial Conversion of (-)-Epicatechin to Bioactive Phenolic Compounds.

To quantify interindividual differences in the human intestinal microbial metabolism of (−)-epicatechin (EC), in vitro anaerobic incubations with fecal inocula from 24 healthy donors were conducted. EC-derived colonic microbial metabolites were qualitatively and quantitively analyzed by liquid chromatography triple quadrupole mass spectrometry (LC-TQ-MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS). Quantitative microbiota characterization was achieved by 16S rRNA analysis. The results obtained show 1-(3′,4′-dihydroxyphenyl)-3-(2″,4″,6″-dihydroxyphenyl)-2-propanol (3,4-diHPP-2-ol) and 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone (3,4-diHPV) to be key intermediate microbial metabolites of EC and also revealed the substantial interindividual differences in both the rate of EC conversion and the time-dependent EC metabolite pattern. Furthermore, substantial differences in microbiota composition among different individuals were detected. Correlations between specific microbial phylotypes and formation of certain metabolites were established. It is concluded that interindividual differences in the intestinal microbial metabolism of EC may contribute to interindividual differences in potential health effects of EC-abundant dietary foods or drinks.

Ferrous-activated persulfate oxidation of triclosan in soil and groundwater: The roles of natural mineral and organic matter

Contamination of antimicrobial agents such as Triclosan (TCS) in soil and groundwater possess high risk to human health and ecological systems. Present study systematically studied the degradation of TCS in soil and groundwater by Fe2+ activated persulfate (Fe2+/PS) oxidation process and special attention was paid on revealing the influence of remediation process on soil physicochemical and microbial characteristics. Experimental results demonstrated that TCS was readily degraded in soil upon Fe2+/PS oxidation system. Higher Fe2+/PS concentration and lower pH value may promote the TCS degradation. Besides added Fe2+, the naturally present Fe (III)-O and dissolved Fe from iron containing minerals may also activate PS for TCS degradation. SO4•−, HO•, R• and 1O2 were identified to be involved in the reaction system while addition of Fe2+-chelating agents, e.g., oxalic acid and ethylene diamine tetraacetic acid (EDTA) may slightly promote the degradation. Low concentration of Cl− facilitated TCS degradation and high concentration of Cl− slowed down the degradation. The presence of HCO3− may inhibit the degradation. Fe2+/PS oxidation process may partly reduce the soil organic matter content and diversely affect the composition of various C functional groups on soil. It also induced the breakdown of large soil aggregates and reduced the soil porosity, especially at macroporosity region. Phospholipid Fatty Acid test indicated that soil microbial community structure has been altered and the actinomycetes, fungi and Gram-negative bacteria decreased largely. The feasibility of remediation of TCS using Fe2+/PS oxidation in various natural groundwater samples was evaluated. Finally, five degradation intermediates of TCS by Fe2+/PS oxidation in soil were enriched by solid phase extraction and were identified by liquid chromatography-triple quadrupole mass spectrometry for proposing detailed transformation pathways.

Hypolipidemic effect of Alisma orientale (Sam.) Juzep on gut microecology and liver transcriptome in diabetic rats.

Alisma orientale (Sam.) Juzep (A. orientale) is a traditional herb that is often used to treat disease including edema and hyperlipidemia. However, the molecular mechanism by which Alisma orientale (Sam.) Juzep exerts its hypolipidemic effects remains unclear. In this study, a diabetic rat model was established by feeding a high-fat and high-sugar diet combined with a low-dose streptozotocin injection (HFS). Then the rats were treated with an A. orientale water extract (AOW), an A. orientale ethanolic extract (AOE) or metform (MET). The gut microflora and liver transcriptome were analyzed by high-throughput next-generation sequencing. Ultra-performance liquid chromatography-triple quadrupole-mass spectrometry was employed to analyze the major compounds in the AOE. The results showed that the serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in rats of the AOE group (2.10 g/kg/day, 14 days) were significantly lower than those in the HFS group (p<0.01). Moreover, AOE treatment altered the gut microecology, particularly modulating the relative abundance of gut microflora involved in lipid metabolism compared with the HFS group. Furthermore, compared with the HFS group, the mRNA expression levels of Fam13a, Mapk7, Mpp7, Chac1, Insig1, Mcpt10, Noct, Greb1l, Fabp12 and Hba-a3 were upregulated after the administration of AOE. In contrast, the mRNA expression levels of Lox, Mybl1, Arrdc3, Cyp4a2, Krt20, Vxn, Ggt1, Nr1d1 and S100a9 were downregulated. Moreover, AOE treatment for two weeks markedly promoted the relative abundance of Lachnospiraceae (p = 0.0013). The triterpenoids contents in AOE were alisol A, alisol A 24-acetate, alisol B, alisol B 23-acetate, alisol C 23-acetate, alisol F, alisol F 24-acetate, and alisol G. Our findings above illustrated that the hypolipidemic effect of the triterpenoids of A. orientale is mediated mainly through alteration of the gut microecology and the regulation of genes involved in cholesterol metabolism, especially Insig1.

Matrix Reference Materials Development for Food Safety Application in Philippine Products

&lt;p&gt;The development of RMs was pioneered in the Philippines by the Industrial Technology Development Institute (ITDI) - DOST as part of its vision to establish metrological traceability and support the RMs needs of chemical testing laboratories in the country. Food safety issues for chemical testing of Philippine products were prioritized in this activity as these could affect the health of consumers and trade issues. Reference methods used for the provision of reference value was developed and validated for contaminants like histamine and benzoic acid in canned tuna and mango juice respectively. The materials were prepared and characterized and found to be homogeneous and stable for at least six (6) months. The reference value assigned to these RMs was done using a higher order method like isotope dilution technique coupled with Liquid Chromatography-Triple Quadrupole Mass Spectrometry (LCMSMS). Results were also cross-checked using gravimetric technique for High Performance Liquid Chromatography (HPLC) analysis. Certified RMs purchased from Metrology Institutes were also used to further support traceability of measurements. Philippine product RMs were developed for histamine and benzoic acid. The measurement traceability was disseminated by ITDI to local testing laboratories through the Proficiency Test schemes organized and conducted for the said analytes. &lt;/p&gt;

Tobacco smoke is a likely source of lead and cadmium in settled house dust

IntroductionEnvironmental exposure to lead (Pb) and cadmium (Cd) are risk factors for adverse health outcomes in children and adults. This study examined whether thirdhand smoke residue contributes to Pb and Cd in settled house dust. MethodsParticipants were 60 multiunit housing residents in San Diego, California. All had indoor smoking bans during the study period, and 55 were nonsmokers. Wipe samples from different surfaces and vacuum floor dust samples were analyzed for nicotine, a marker of thirdhand smoke, and for Pb and Cd using liquid chromatography-triple quadrupole mass spectrometry and inductively coupled plasma-mass spectrometry, respectively. ResultsExamined in each sample type separately, Pb and Cd loadings were significantly correlated (r = 0.73, vacuum floor dust; 0.52, floor wipes; 0.72, window sill/trough wipes; all p < 0.0025). Pb and Cd loadings from different sample types were not correlated (all p > 0.30). Nicotine loading in dust was significantly correlated with Pb and Cd loading in dust (r = 0.49 for Pb; r = 0.39 for Cd, all p < 0.0025). Pb and Cd loadings on floor or window surfaces, showed no association with nicotine loading in dust, on floors, or on furniture (all p < 0.30). ConclusionsTobacco smoke is a likely source of Pb and Cd that accumulates in settled house dust in multiunit housing, suggesting that Pb and Cd are constituents of thirdhand smoke that lingers long after smoking has ended.

Removal of pharmaceutically active compounds (PhACs) and bacteria inactivation from urban wastewater effluents by UVA-LED photocatalysis with Gd3+ doped BiVO4

In this study, gadolinium doped bismuth vanadate powders were synthesized, characterized, and tested as a potential photocatalyst for the removal of pharmaceutically active compounds (PhACs) and bacterial inactivation in a real wastewater effluent using UVA as irradiation source. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) studies revealed that the bismuth vanadate system was successfully doped with 4% of gadolinium in molar mass leading to the formation of a heterostructured photocatalyst.Up to 98.3 % of diclofenac was removed from pure water after 120 min through photocatalysis. However, the photocatalytic performance of the photocatalyst on wastewater effluent was rather variable due to the complexity of the matrix where 22 different PhACs were detected by means of Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS). High photocatalytic removal efficiency (80–100%) was observed for some of the studied PhACs (e.g. naproxen and furosemide) whereas moderate efficiency (40–70%) was observed for others (e.g. acetaminophen and azithromycin) after 180 min (UVA dose = 139.5 Wh m−2, t30W = 279 min-1). Some of the studied PhACs like clarithromycin and Ibuprofen showed poor removal efficiency (< 30 %). In disinfection tests, Total coliforms, Escherichia coli, Enterococci, and Klebsiella pneumoniae showed inactivation after direct UVA LED photolysis. Nevertheless, higher inactivation was achieved for Enterococci in the presence of the synthesized photocatalyst showing an increase of 41.1 % in kmax.

Comprehensive analysis of synthetic cannabinoids and metabolites in oral fluid by online solid-phase extraction coupled to liquid chromatography-triple quadrupole-mass spectrometry.

The class of novel psychoactive substances known as synthetic cannabinoids (SC) includes illicit compounds that are sprayed on plant material and smoked or sold as liquids to be vaporized in e-cigarettes. In toxicological analysis of SC, fast analytical methods are needed for the detection and confirmation of parent drugs and metabolites at very low levels. While various analytical methods have been developed for SC in blood and urine, few are available for alternative matrices such as oral fluid (OF). There are numerous advantages to using OF as a sample matrix for SC analysis, including non-invasive collection, lesser risk of adulteration, and presence of both parent drug and metabolites. Here we report a validated online solid-phase extraction (online SPE) method coupled to LC-QqQ-MS for rapid confirmation and quantitation of 72 structurally diverse SC parent drugs and metabolites in OF with 2.5min of preconcentration time and a total elution time of < 10min. The use of online SPE for sample pretreatment facilitates rapid and consistent processing and greatly increases sample throughput. The method was fully validated according to relevant guidelines (ANSI/ASB Standard 036). Bias and precision values were within ± 20% for all compounds in human OF matrix. Method detection and quantitation limits ranged from 0.4 to 3.8ng/mL and from 1.1 to 11.6ng/mL, respectively. Recovery, matrix effects, process efficiency, carryover, and stability were also within acceptable limits for the majority of compounds. Successful application of the method was demonstrated using blank human OF fortified with SC in addition to a set of authentic OF specimens previously tested by another laboratory. Graphical abstract.

High-throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction.

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high-throughput assay based on liquid chromatography-triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96-well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5-ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96-well collection plate. With a 10-min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti-Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02-0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

Determination and Pharmacokinetics of Okanin in Rat Plasma by UltraHigh Performance Liquid Chromatography Coupled with Triple-Quadrupole Tandem Mass Spectrometry.

Okanin is a major flavonoid found in Coreopsis tinctoria Nutt., arousing huge interest recently for its considerable biological characteristics including antioxidant, antineurotoxic, and antidiabetic activities. An ultrahigh performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS) was successfully used to determine okanin in rat plasma after oral administration of okanin. Bavachalcone acted as an internal standard (IS). By gradient elution, IS and analyte were separated on a C18 column for 7 min at a flow rate of 0.25 mL/min with acetonitrile-0.1% acetic acid mobile phase. The stability, matrix effect, extraction recovery, accuracy, precision, linearity, and selectivity of the method were firstly demonstrated. The major pharmacokinetic parameters of okanin in rat plasma were then measured using the developed UPLC-MS method. An UPLC-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was finally established to obtain the specific and accurate mass of okanin in rat plasma after oral administration, and its proposed fragmentation was further elaborated.

Photocatalytic degradation of pharmaceutically active compounds (PhACs) in urban wastewater treatment plants effluents under controlled and natural solar irradiation using immobilized TiO2

The degradation of pharmaceutically active compounds (PhACs) under controlled and natural solar irradiation using immobilized TiO2 was studied in model solution and real urban wastewater effluents. The TiO2 coatings were prepared via the sol–gel method with two different configurations, using simple (thin films) and the “sandwich” approach. Prepared coatings were characterized employing X-ray diffraction (XRD), scanning electron microscopy (SEM), ellipsometry and UV–Vis diffuse reflectance spectroscopy. Decomposition of PhACs detected in urban wastewater effluents (diclofenac, carbamazepine, atenolol, propranolol, albuterol, ofloxacin, ciprofloxacin, azithromycin, erythromycin, hydrochlorothiazide, and furosemide) was monitored using Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS). Among detected PhACs three are included in the Watch List (EU Decision 2018/840). The “sandwich” coatings (deposited on frosted support) with the following structure: anatase layer, TiO2 P25 nanoparticles and anatase layer (sealing film) demonstrated the highest photocatalytic activity among all tested TiO2 coatings and were used for elimination of PhACs from real urban wastewater effluents. Photocatalytic treatment of urban wastewater effluents under controlled and natural solar irradiation led to moderate and high removal (>40%) of all detected PhACs except carbamazepine and atenolol, for which poor removal efficiency was observed (~20%). Some of the PhACs compounds present in wastewater effluents, such as diclofenac, ofloxacin, ciprofloxacin, hydrochlorothiazide and furosemide were sensitive to solar photolysis. The efficiency of solar photocatalytic inactivation of Escherichia coli present in urban wastewater effluent was similar to that of solar disinfection (SODIS).

Analytical method development for α-amanitin and β-amanitin in plasma at ultra-trace level by online solid phase extraction-high performance liquid chromatography-triple quadrupole mass spectrometry and its application in poisoning events

α-Amanitin and β-amanitin are the main fatal mushroom toxins. The toxins metabolize rapidly in blood and are reported hard to be detected 24 h after poisoning. The main challenge is that of developing a highly sensitive method at sub-pg mL−1 level in blood to diagnose intoxication cause and to study the poisoning mechanism and blood toxicity kinetics. An analytical method for α-amanitin and β-amanitin at ultra-trace level was developed in this study by online solid phase extraction-high performance liquid chromatography-triple quadrupole mass spectrometry (online SPE-LC–MS/MS). Simple protein precipitation and liquid-liquid extraction were introduced to resolve the sample preparation problem of the online SPE-LC–MS/MS system with large-volume injection. A quick valve-switching technique with a quantitative loop as interface was used in the online system. This design can ensure the independence of flow path and pressure between the SPE and LC–MS/MS modules and can obtain the precise cleanup of the toxins. The limits of detection for α-amanitin and β-amanitin in plasma were both 0.02 ng mL-1. The linear ranges were 0.05–20 ng mL-1 with a correlation coefficient r >0.99. The average recoveries at three spiking levels were 82.9 %–92.2 % with the relative standard deviations (RSD) of 5.4 %–8.0 % for α-amanitin and 84.5 %–93.9 % with RSDs of 4.5 %–7.8 % for β-amanitin. The composition and concentration of the toxins in plasma from 18 patients in 5 mushroom poisoning events caused by aminitins were studied. The developed method has high positive confirmation ability and can identify toxins in plasma 40 h after poisoning.

Identification of potential peptide markers for the shelf-life of Pacific oysters (Crassostrea gigas) during anhydrous preservation via mass spectrometry-based peptidomics

The shelf-life of live oysters is a quality-concerned issue along with the supply chain of the products. In present study, mass spectrometry-based peptidomics has been employed to investigate changes of endogenous peptides of Pacific oysters (Crassostrea gigas) occurred during anhydrous preservation with the purpose of discovering shelf-life indicators of live oysters. Samples of Crassostrea gigas harvested freshly were acclimated to laboratory environment first and then put under defined anhydrous preservation conditions (being packaged in sterile plastic bags and stored at 4 ± 1 °C in low-temperature incubators). After regular intervals, samples were randomly collected for peptidomics analysis using ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry and potential peptide markers were mined by chemometric tools and verified by a multi-reaction monitoring method with ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry. Approximately 8526 peptides, in total, were characterized and 4 endogenous peptides, SSSTGEVGTYSGTTN, TARNEANVNI, VGIIKGSSSEEA, and TARNEANVNIY, were finally proposed as potential markers for shelf-life evaluating. The proposed markers were applied to real-life samples and expected trends of the markers during anhydrous preservation were observed, implying applicability of the endogenous peptides for shelf-life evaluating. The findings of this study provide favorable evidences of mass spectrometry-based peptidomics in quality assessing for aquaculture products.

Stepwise mass spectrometry-based approach for confirming the presence of benzoxazinoids in herbs and vegetables.

Benzoxazinoids (BXs) are plant phytochemicals that have both defensive properties in plants and therapeutic effects in humans. The presence of BXs has been largely studied in the Poaceae family (monocots). To study the presence or absence of BXs in dicotyledons and monocotyledons outside the Poaceae family, parts of 24 plant species at several growth stages were selected for analysis, some of which were already known to contain BXs. To devise a stepwise mass spectrometry-based approach for confirming the presence of BXs in plant samples, and to use the method to explore the status of BXs in selected plant species. Plant samples were extracted using accelerated solvent extraction and analysed using triple-quadrupole liquid chromatography-mass spectrometry. The use of different columns, double mass transitions, and ion ratios proved to be a robust tool for confirming the presence of BXs in different plant species. By this method, the presence of BXs was confirmed in three of the 24 species. Double-hexose forms of BXs, which have not been reported before in dicotyledons, were confirmed to be present in the dicotyledon plants Acanthus mollis and Lamium galeobdolon, and the presence of BXs in the seeds of Consolida orientalis is reported for the first time here. High concentrations of BXs were found in the aerial parts of Acanthus mollis and Lamium galeobdolon, at 20 and 32 μmol/g plant dry weight, respectively. The stepwise approach described in this work confirmed the presence of BXs in new samples.

Rapid Detection of Anabolic and Narcotic Doping Agents in Saliva and Urine By Means of Nanostructured Silicon SALDI Mass Spectrometry.

Novel doping agents and doping strategies are continually entering the market, placing a burden on analytical methods to detect, adapt, and respond to subtle changes in the composition of biological samples. Therefore, there is a growing interest in rapid, adaptable, and ideally confirmatory analytical methods for the fight against doping. Nanostructured silicon (nano-Si)-based surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) can effectively address this need, allowing fast and sensitive detection of prohibited compounds used in sport doping. Here, we demonstrate the detection of growth hormone peptides, anabolic-androgenic steroids, and narcotics at low concentrations directly from biological matrices. Molecular confirmation was performed using the fragmentation data of the structures, obtained with the tandem mass spectrometry capabilities of the SALDI instrument. The obtained data were in excellent agreement with those obtained using leading triple quadrupole liquid chromatography-mass spectrometry instruments. Furthermore, nano-Si SALDI-MS has the capacity for high-throughput analysis of hundreds of biological samples, providing opportunities for real-time MS analysis at sporting events.

Quantification of 54 Benzodiazepines and Z-Drugs, Including 20 Designer Ones, in Plasma.

Benzodiazepines are widely used in the treatment of sleep and anxiety disorders, as well as epileptic seizures and alcohol withdrawal because of their broad therapeutic index and low cost. Due to their central nervous system depressant effects they are also often implicated in traffic accidents and drug-related intoxications. With an increasing number of designer benzodiazepines used in a recreational setting, there is a need for analytical methods to be able to quantify both the prescribed and designer benzodiazepines. A liquid chromatography-triple quadrupole mass spectrometry method was developed for the quantification of 34 prescribed and 20 designer benzodiazepines in plasma. Different sample preparation strategies, including protein precipitation, liquid-liquid extraction, solid-phase extraction and mini-QuEChERS, were tested. The best recoveries for all compounds of interest were obtained with a liquid-liquid extraction using methyl-tertiary-butyl-ether and 500μL plasma. The method was fully validated according to the European Medicines Agency guidelines for all compounds, except pivoxazepam, which is included for qualitative purposes only. In-sample stability issues were observed for cloxazolam, both at ambient temperature and during long-term storage at -20°C. Due to the large number of compounds included, the simple and time-efficient sample preparation and the relatively inexpensive instrumentation used, the presented method can be readily implemented in both therapeutic drug monitoring and forensic analyses.

A high‐throughput and broad‐spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography–high‐resolution mass spectrometry

A high-throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide-conjugated drug targets. This is followed by extraction using solid-supported liquid extraction (SLE) and analysis using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The entire set-up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC-HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography-triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post-race or out-of-competition urine samples collected from racehorses. This method was developed for pre-race urine testing in Hong Kong; however, it is also suitable for testing post-race or out-of-competition urine samples, especially when a quick total analysis time is desired.

Simultaneous determination of cucurbitacin B, E and I in plasma, urine, and melon and fruit vegetables by ultra performance liquid chromatography-triple quadrupole mass spectrometry using atmospheric pressure chemical ionization

A method for the determination of cucurbitacin B (CuB), cucurbitacin I (CuI) and cucurbitacin E (CuE) in plasma, urine and melon and fruit vegetables was developed by ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS). The target analytes in plasma and urine were extracted and cleaned-up by solid supported liquid-liquid extraction, while those in melon and fruit vegetables were extracted with acetonitrile and then diluted with water. CuB, CuI and CuE were separated on an XBridge BEH C18 column (100 mm×3.0 mm, 2.5 μm) with gradient elution using mobile phases of methanol and 0.025% (v/v) ammonia aqueous solution. An atmospheric pressure chemical ionization interface was used as the ion source and the analysis was performed in negative ionization multiple reaction monitoring (MRM) mode. The cucurbitacins in plasma and urine were quantified by the matrix working standard curve internal standard method, while those in melon and fruit vegetables were quantified by the solvent standard curve external standard method. Oleandrin was used as the internal standard. The average recoveries were 89.0%-113% for the three cucurbitacins in plasma and urine, with RSDs of 1.7%-12.2% (n=6). The average recoveries were 87.6%-114% for the three cucurbitacins in melon and fruit vegetables, with RSDs of 4.1%-11.1% (n=6). The limit of detection (S/N=3) of the three cucurbitacins was 0.03 μg/L in plasma and urine, and 5-10 μg/kg in melon and fruit vegetables. The method is simple, sensitive and accurate. It has been used for the determination of cucurbitacins in bitter bottle gourd and in the plasma and urine of patients poisoned by bitter bottle gourd, CuB was successfully detected.

Determination of ibotenic acid and muscimol in plasma by liquid chromatography-triple quadrupole mass spectrometry with bimolecular dansylation

Ibotenic acid (IBA) is an amino acid and muscimol (MUS) is the decarboxyl derivative of IBA. They are mushroom neurotoxins with high polarity and low molecular weight. Only one transition (159–>113 for IBA and 115->98 for MUS) can be found when directly measured by high performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Therefore, the identification and quantification of trace amount of the toxins in biomaterial are difficult. A highly sensitive and accurate analytical method for IBA and MUS in plasma was developed by LC-MS/MS with the application of bimolecular dansylation and internal standard calibration. Acetonitrile was used for protein precipitation and for toxin extraction from plasma. The toxins and internal standards (L-tyrosine-13C9,15N for IBA and tyramine-d4 for MUS) were derivatized with dansyl chloride (DNSCl). The reaction conditions of the bimolecular dansylation were optimized and the fragmentation pathways of the derivatives in MS/MS were studied. Method validation was carried out according to the Bioanalytical Method Validation Guidance for Industry (FDA, USA, 2018). The limits of detection for IBA and MUS in plasma were 0.3 ng mL−1 and 0.1 ng mL−1, respectively. The linear ranges in plasma were 1–500 ng mL−1 and 1–200 ng mL−1 with the correlation coefficients of 0.998 and 0.999 for IBA and MUS, respectively. The recoveries at three spiked levels were 90.7–111.4% with relative standard deviations (RSDs) of 6.4–10.3% for IBA and the results were 85.1–94.2% with RSDs of 5.0–8.9% for MUS. The toxin levels in patients’ plasma samples under different poisoning degree were presented.

Derivatization assisted LC-p-MRM-MS with high CID voltage for rapid analysis of brassinosteroids

In this study, a derivatization-assisted pseudo-multiple reaction monitoring with high CID voltage (HV-p-MRM) strategy was proposed for the analysis of brassinosteroids (BRs) by liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ MS). The concept of the HV-p-MRM strategy was proposed on the basis of an assumption that the precursor ion of analyte is stable in collision cell and less prone to fragmentation at high CID voltage, while co-existing ions (impurity) of easy fragmentation can break down into smaller fragment ions. In such case, by increasing the CID voltage, the co-existing ions that are introduced due to the low resolution of the quadrupole 1 (Q1) can be filtered out by quadrupole 3 (Q3), while the stable precursor ion of analyte will pass through Q3, thereby that the signal-to-noise ratio (S/N) of the analysis can be improved. As a proof-of-concept study, BRs were derivatized with rhodamine B-boronic acid (RhB-BA) and then the derivatives were used to investigate their variations in MS signal, background noise, and S/N upon the CID voltage and MS scanning resolution. The results showed that S/N of these derivatives can be improved in HV-p-MRM mode. To further demonstrate the feasibility of HV-p-MRM strategy, a RhB-BA derivatization assisted LC-HV-p-MRM-MS method was developed for the analysis of BRs. Using this method, rapid and sensitive determination of BRs in different organs of rape flower was achieved. It can be expected that HV-p-MRM may be suitable for the analytes that are stable or can be converted into compounds of high stability in collision cell at high CID voltage.

Evaluation of the pharmacokinetics of 2-carba-cyclic phosphatidic acid by liquid chromatography-triple quadrupole mass spectrometry

Cyclic phosphatidic acid (cPA) is a lysophospholipid mediator that suppresses cancer metastasis and osteoarthritis. It also has neuroprotective roles in diseases such as multiple sclerosis and delayed neuronal death following transient ischemia. In order to take advantage of the properties of cPA for the development of new therapeutic strategies, we have synthesized several cPA derivatives and discovered 2-carba-cPA (2ccPA) as a promising candidate. To develop 2ccPA as a therapeutic agent, we investigated the pharmacokinetic profile of 2ccPA by liquid chromatography-triple quadrupole mass spectrometry in this study. When 2ccPA was administered intraperitoneally to mice at a dose of 1.6 mg/kg, the half-life of 2ccPA in plasma was 16 min. The 2ccPA, dosed intraperitoneally to mice at 16 mg/kg, distributed to each organ including brain at 20 min after dosing. It was found that 2ccPA was stable in neutral or alkaline conditions (e.g., intestine) but unstable in acidic conditions (e.g., stomach). When 2ccPA was orally administrated to rats as a gastro-resistant form using an enterosoluble capsule, plasma 2ccPA levels peaked at 2 h, slowly declined thereafter and persistently detected even at 10 h after administration. Here, we present the findings on the effect of the continuous release of 2ccPA from the capsule to reduce the lysophospholipase D activity and also decrease plasma levels of lysophosphatidic acid in rat. These findings will be useful in further studies for evaluating the application of 2ccPA in several disorders.