Liquid Chromatography-triple Quadrupole Mass Spectrometry Research Articles

On the performance of multivariate curve resolution to resolve highly complex liquid chromatography–full scan mass spectrometry data for quantification of selected immunosuppressants in blood and water samples

Liquid chromatography triple-quadrupole mass spectrometry (LC–MS/MS) in multiple reaction monitoring (MRM) mode is a commonly used approach for quantification of target compounds in complex samples. Regarding the multivariate advantages that can be obtained in the light of full spectra recording, the potential and performance of a smart strategy which is a post-processing of LC–MS full scan data sets with multivariate curve resolution-alternating least squares (MCR-ALS) algorithm for determination of cyclosporine-A (CsA) and tacrolimus (TAC) as typical immunosuppressants in human blood and organic micropollutants in surface water samples was investigated in the current study. Before peak resolution and quantification, LC–MS data compression was performed on each individual and augmented data matrices based on searching the regions of interest (ROIs) in the m/z domain. Then, due to presence of matrix effect, MCR-ALS modeling was performed using a matrix-matched calibration strategy. Here, the serious LC–MS issues such as background drift, chromatographic shifts and co-elution of non-calibrated sample constituents were resolved by implementation of MCR-ALS method on ROI based compressed data. It was shown that the strategy is a powerful tool for the quantitation of CsA and TAC in complex matrices and in the presence of uncalibrated interferences with highly overlapped peaks. However, the overall results indicated that performance of the multivariate modeling of LC–MS in full scan mode for drug quantitation strongly depends on the selected drug, as in the current case superiority of the method for analysis of CsA over TAC, with different therapeutic concentration ranges, was observed.

Qualitative and Quantitative NAD+ Metabolomics Lead to Discovery of Multiple Functional Nicotinate N-Glycosyltransferase in Arabidopsis.

The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is a conserved biochemical pathway in land plants. We previously demonstrated that various NA conjugations (mainly methylation and glycosylation) shared the NA detoxification function in all tested plants. It remains unclear whether other NA conjugates with low abundance exist in plants. In this study, we discovered at least two additional NA N-glycosides in Arabidopsis, which was tentatively elucidated as nicotinate N-pentoside (NaNP) and NA N-rhamoside (NaNRha), using liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS) with precursor ion-scanning (PreIS). We further quantitatively profile the NAD+-related metabolites in 24 tissues of Arabidopsis. Biochemical assays of UGT76C family revealed that UGT76C5 (encoded by At5g05890, previously identified as NaNGT) was a multiple functional nicotinate N-glycosyltransferase, with high preference to UDP-xylose and UDP-arabinose. The deficiency of NaNP and NaNRha in ugt76c5 mutant suggested that UGT76C5 is responsible for biosynthesis of NaNP and NaNRha in planta. We also identify one amino acid difference in PSPG (plant secondary product glycosyltransferase) motif is responsible for the divergence of NaNGT (UGT76C4) and UGT76C5. Taken together, our study not only identifies a novel nicotinate N-glycosyltransferase but also paves the way for investigations of the in planta physiological functions of various NA conjugations.

Determination of pendimethalin in water, sediment, and Procambarus clarkii by high performance liquid chromatography-triple quadrupole mass spectrometry.

We established a high-performance liquid chromatography-triple quadrupole mass spectrometry method for the analysis of pendimethalin residues in water, sediments, and Procambarus clarkii (Louisiana crayfish) tissues. Water samples were concentrated on a HLB solid-phase extraction column and eluted with dichloromethane and acetone (1:1). After drying under a stream of nitrogen gas, the sample volume was adjusted to 1 mL with the mobile phase solvent methanol/water/acetic acid (8:20:0.1). Pendimethalin was extracted with ethyl acetate containing 0.1% acetic acid, after rotary evaporation to dryness at 35 °C, the residue was dissolved in mobile phase solvent, purified by a neutral alumina column and graphitized carbon black powder (0.1 g). The mass characterization was conducted in positive ion mode, and the corresponding ions were detected in multi-reaction monitoring mode. The linear equations were y = 1 × 106x + 14275, at pendimethalin levels of 0.05-20 μg L-1 and y = 691029 × - 414368 for 20-200 μg L-1. The detection limits of pendimethalin in water, sediments, and P. clarkii tissues were 1.0 × 10-4μg L-1, 5.0 × 10-3μg kg -1and 5.0 × 10-3 μg kg -1, respectively. The spiked recoveries ranged from 81.6 to 106.3%, and the relative standard deviations ranged from 4.58 to 13.6% (n = 6). The method provided an efficient and low-cost extraction and purification procedure that enabled a sensitive determination of pendimethalin in water as well as complex matrices.

Comparative transcriptome and metabolite profiling of four tissues from Alisma orientale (Sam.) Juzep reveals its inflorescence developmental and medicinal characteristics

Alisma orientale (Sam.) Juzep (A. orientale) is an important medicinal plant in traditional Chinese medicine. In this study, de novo RNA-seq of A. orientale was performed based on the cDNA libraries from four different tissues, roots, leaves, scapes and inflorescences. A total of 41,685 unigenes were assembled, 25,024 unigene functional annotations were obtained by searching against the five public sequence databases, and 3,411 simple sequence repeats in A. orientale were reported for the first time. 15,402 differentially expressed genes were analysed. The morphological characteristics showed that compared to the other tissues, the leaves had more chlorophyll, the scapes had more vascular bundles, and the inflorescences contained more starch granules and protein. In addition, the metabolic profiles of eight kinds of alisols metabolite profiling, which were measured by ultra-Performance liquid chromatography-triple quadrupole-mass spectrometry showed that alisol B 23-acetate and alisol B were the major components of the four tissues at amounts of 0.068~0.350 mg/g and 0.046~0.587 mg/g, respectively. In addition, qRT-PCR validated that farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-CoA reductase should be considered the critical candidate genes involved in alisol biosynthesis. These transcriptome and metabolic profiles of A. orientale may help clarify the molecular mechanisms underlying the medicinal characteristics of A. orientale.

Determination of Flavonoid Glycosides by UPLC-MS to Authenticate Commercial Lemonade.

So far, there is no report on the quality evaluation of lemonade available in the market. In this study, a sample preparation method was developed for the determination of flavonoid glycosides by ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) based on vortex-assisted dispersive liquid-liquid microextraction. First, potential flavonoids in lemonade were scanned and identified by ultra-performance liquid chromatography–time of flight mass spectrometry (UPLC-TOF/MS). Five flavonoid glycosides were identified as eriocitrin, narirutin, hesperidin, rutin, and diosmin according to the molecular formula provided by TOF/MS and subsequent confirmation of the authentic standard. Then, an ultra-performance liquid chromatography–triple quadrupole mass spectrometry (UPLC-QqQ/MS) method was developed to determine these five flavonoid glycosides in lemonade. The results showed that the content of rutin in some lemonade was unreasonably high. We suspected that many illegal manufacturers achieved the goal of low-cost counterfeiting lemonade by adding rutin. This suggested that it was necessary for relevant departments of the state to make stricter regulations on the quality standards of lemonade beverages.

Simultaneous determination of eight bioactive components of Cirsium setosum flavonoids in rat plasma using triple quadrupole LC/MS and its application to a pharmacokinetic study.

Cirsium setosum (Willd.) MB. has been reported to exert significant anti-hemorrhagic, anti-inflammation, antimicrobial, sedative and detoxicating efficacy. It has been widely used to treat gastrointestinal bleeding, uterine bleeding, infectious hepatitis and cardiovascular disease in China. Recent studies have shown that flavonoids are the main active components in C. setosum. Nevertheless, to the best of our knowledge, there is no report concerning the simultaneous determinations and pharmacokinetics of constituents in C. setosum flavonoids in rat plasma. In this study, a rapid, sensitive and selective triple quadrupole liquid chromatography-mass spectrometry method was developed to determine eight analytes from the flavonoids of C. setosum in rat plasma. In addition, the pharmacokinetic study of the eight analytes in rats after oral administration of C. setosum flavonoids was successfully completed through this method. According to the pharmacokinetic parameters of the eight analytes, rutin, naringin, quercetin, acacetin, wogonin were the long-acting components of the C. setosum flavonoids, with long elimination time and high bioavailability. Of note, the method developed in this study fills a blank in pharmacokinetic studies of C. setosum flavonoids. Our findings provide valuable views on the understanding of the absorption mechanism of C. setosum flavonoids and their clinical efficacy.

Metabolomics Investigation of Voriconazole-Induced Hepatotoxicity in Mice.

Voriconazole (VCZ) is a widely used triazole drug for the treatment of serious incidence of invasive fungal infections (IFIs), and its most commonly reported clinical side effect is hepatotoxicity. The mechanism of VCZ-induced hepatotoxicity is unclear, and no specific marker can be used for prediction and diagnosis. This study aims to apply the targeted metabolomics approach to identify specific VCZ-induced metabolites related to hepatotoxicity via liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS) in a C57BL/6 mouse model. Mice treated with three repeated doses of 40 mg/kg VCZ by tail vein injection to induce hepatotoxicity (VCZ-induced hepatotoxicity group, n = 8) were compared with mice without treatment (control group, n = 10). Both liver tissue and plasma were collected and analyzed to propose underlying mechanisms associated with VCZ-induced hepatotoxicity. The results indicated that the metabolites associated with oxidative stress were altered, and alterations in the metabolites involved in glutathione biosynthesis were noticed. The ratio of glutamine to glutamate showed a significant reduction in the VCZ-induced hepatotoxicity group compared to the control group, suggesting that glutamine might be transformed into glutamate for glutathione biosynthesis. Accordingly, we proposed that VCZ-induced hepatotoxicity is associated with oxidative stress to cause cell dysfunction, leading to alterations in energy metabolism, the urea cycle, and nucleoside metabolism. To the best of our knowledge, this is the first study to apply metabolomics for investigating the mechanism of VCZ-induced hepatotoxicity.

A rapid UHPLC-MS/MS method for quantitation of phytoestrogens and the distribution of enterolactone in an Alabama estuary

Endocrine disrupting chemicals (EDCs) are natural or synthetic compounds that can interfere with the endocrine systems of humans and wildlife. EDCs can pass through wastewater treatment systems, or run off from urban areas or agricultural operations, into natural water bodies, exposing resident and migratory organisms to complex EDC mixtures. Some phytoestrogenic polyphenolics (PEPP) are known or suspected EDCs; however, their contribution to total EDC burden in natural surface water systems is largely unknown. We describe a rapid, sensitive, and reproducible quantitative method for analysis of 15 PEPP in estuarine sediment and water, using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS). The method provides excellent peak resolution, peak separation, and rapid run times (method separation/total run time: 8/12.5 min). With two exceptions, spiking experiments demonstrated that the percent recoveries for target PEPP in sediment and water samples were within acceptable analytical validation limits. LOD and LOQ values ranged from 0.004 to 0.010 ng/injection and 0.013–0.032 ng/injection, respectively. The validated method was used for PEPP analysis of sediment and water samples collected from 11 locations within the Perdido Bay estuary in coastal Alabama. No PEPP above the LOD were detected in sediment samples. The mammalian-derived lignin enterolactone was observed at low concentrations in water throughout the estuary, and significantly, at elevated concentrations at two locations associated with small-scale septic systems (3.66 ± 0.27 ng L−1 and 4.01 ± 0.33 ng L−1) and a large wastewater treatment system (4.56 ± 0.24 ng L−1 and 5.69 ± 0.43 ng L−1).

Method development for simultaneous analyses of polycyclic aromatic hydrocarbons and their nitro-, oxy-, hydroxy- derivatives in sediments

It is important to establish an available analytical method for polycyclic aromatic hydrocarbons (PAHs), nitrated PAHs (nitro-PAHs), oxygenated forms of PAHs (oxy-PAHs), and hydroxy-PAHs (OH-PAHs) in sediment samples due to the fact that they co-exist in various environmental mediates, particularly in sediment. In this study, an analytical method has been developed and validated for the quantification of PAHs, nitro-PAHs, oxy-PAHs, and OH-PAHs in sediment samples. The sediment samples were extracted by accelerated solvent extraction and cleaned up with SPE alumina-n combining with aminopropyl cartridges. The extracts were further fractionated by using different solvents. The fractionated extracts were analyzed via gas chromatography of single and triple quadrupole mass spectrometry in the electron ionization and negative ion chemical ionization with selective ion monitoring and selective reaction monitoring mode and liquid chromatography-triple quadrupole mass spectrometry. Each group of analytes was determined by different instrument modes such as GC-EI-SIM for PAHs, GC-NICI-SRM for nitro-PAHs, GC-EI-SRM for the oxy-PAHs and LC-ESI-MS/MS for OH-PAHs. A total of 44 analytes (16 PAHs, 14 nitro-PAHs, 9 oxy-PAHs, and 5 OH-PAHs) and 6 deuterated surrogates were performed. Most of recovery percentage varied from 52.8% up to 114.1% for the targeted analytes verified at three concentration levels (100 ng/g, 400 ng/g and 1000 ng/g for PAHs and 10 ng/g, 50 ng/g and 400 ng/g for their derivatives). The repeatability determined by the relative standard deviation percentage of triplicate trials was less than 10% for most analytes. The limit of detection ranged from 0.01 to 0.02 ng/g for PAHs, 0.002–0.067 ng/g for nitro-PAHs, 0.01–0.1 ng/g for oxy-PAHs, and 0.003–0.006 ng/g for OH-PAHs. Most of the compounds were detectable in the sediments collected from a local River, which illustrates the developed method could be a practical and suitable technique for detection of PAHs and their derivatives in real sediment samples.

Determination of ocfentanil and W-18 in a suspicious heroin-like powder in Belgium

Since the early 2010s, synthetic opioids have been on the rise in the illicit drug markets of North America and Europe, often as adulterants or substitutes for heroin. Ocfentanil, an early-onset fentanyl analogue, has been implicated in several fatalities, predominantly in Europe. W-18 is more prevalent in Canada and the United States. We present the findings for an unknown brown powder, advertised and purchased as heroin, which was sent to our laboratory for analysis in the framework of the Belgian Early Warning System on Drugs. The sample was screened using liquid chromatograph coupled to a diode array detector and gas chromatograph coupled to a single quadrupole mass spectrometer. The findings were confirmed by liquid chromatography—triple quadrupole mass spectrometry. No heroin, 6-monoacetylmorphine or morphine was detected. Our analysis detected two new psychoactive substances: the synthetic opioid ocfentanil (1.6% m/m), and W-18 (0.3% m/m). The ocfentanil concentration was similar to those found in previous reports. No reference concentrations have been published for W-18. The presence of these two new psychoactive substances together in the same powder is unprecedented and prompted a warning by the Belgian Early Warning System on Drugs. The unknown powder tested positive for ocfentanil and W-18. To the authors’ best knowledge this is the first case of the combined detection of these two new psychoactive substances in the same powder. We additionally demonstrate the advantage of building an in-house reference library beyond retention time.

1869-P: Nonalcoholic Fatty Liver Disease and Insulin Resistance Are Associated via Xanthine Oxidoreductase Activity

Xanthine oxidoreductase (XOR), which is mainly produced by the liver, has been reported to be associated with insulin resistance in addition to disorder of uric acid metabolism, which is common in obese people. Moreover, nonalcoholic fatty liver disease (NAFLD) is known to trigger insulin resistance, but the mechanism has not been sufficiently elucidated. In this study, we used FibroScan equipped with controlled attenuation parameter (CAP) estimation to measure intrahepatic fat accumulation and to evaluate the association of insulin resistance with intrahepatic fat accumulation and XOR activity by adjusting for the effects of visceral fat. Of the 200 individuals who had undergone health checkups at our medical center, 142 individuals (59 men) were included in the study after excluding 58 individuals because of incomplete data, habitual alcohol consumption, or ongoing treatment with hypoglycemic and antihyperuricemic agents. NAFLD was defined as a CAP value ≥236 dB/m. Plasma XOR activity was measured by liquid chromatography-triple quadrupole mass spectrometry using [13C2, 15N2] xanthine as a stable isotope substrate developed by Sanwa Kagaku Kenkyusho Co., Ltd. The association of insulin resistance (HOMA-IR ≥2.5) with NAFLD was analyzed by multivariate logistic analysis while adjusting the effects of other factors. NAFLD, as well as visceral fat area, were found to be an independent explanatory factor for insulin resistance. However, when logarithmic values of XOR activity levels (ln-XOR) were added to the analysis model, ln-XOR, but not NAFLD, was a significant explanatory factor for insulin resistance (odds ratio [95% confidence interval]: 4.36 [2.21-8.60]). This finding suggested that XOR of the liver was involved in the association between NAFLD and insulin resistance. In conclusion, NAFLD and insulin resistance are associated via XOR Activity. Disclosure S. Fukumoto: Research Support; Self; Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Kowa Pharmaceutical Europe Co. Ltd., Mitsubishi Tanabe Pharma Corporation, Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Novo Nordisk Inc., Sanofi K.K. M. Kurajoh: None. T. Nakamura: None. T. Murase: None. Y. Kakutani: None. Y. Yamazaki: None. A. Ochi: None. K. Motoyama: None. T. Morioka: Research Support; Self; Eli Lilly and Company, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Sanofi K.K., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk Inc., Roche Diagnostics K.K., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Other Relationship; Self; Ono Pharmaceutical Co., Ltd. T. Shoji: Research Support; Self; Bayer Yakuhin, Ltd., Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd. Speaker's Bureau; Self; Kissei Pharmaceutical Co., Ltd., Kowa, Kyowa Hakko Kirin Co., Ltd. M. Emoto: Research Support; Self; Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical CO., Ltd., Takeda Pharmaceutical Company Limited. M. Inaba: None. Funding Japan Society for the Promotion of Science

Photocatalytic performance of multi-walled carbon nanotube/BiVO4 synthesized by electro-spinning process and its degradation mechanisms on oxytetracycline

To enhance the photocatalytic performance of bismuth vanadate (BiVO4) and reveal its catalytic mechanism, a multi-walled carbon nanotube photocatalyst (MWCNT/BiVO4) was prepared by sol-gel preparation and an electro-spinning process. Oxytetracycline was used as the target trace persistent pollutant to research its potential degradation pathways catalyzed by MWCNT/BiVO4. X-ray powder diffraction spectroscopy (XRD), Raman spectra, field emission scanning electron microscopy (FE-SEM) and field emission transmission electron microscopy (FE-TEM) were used to observe the crystalline phase, morphological structure of MWCNT/BiVO4. The modification by MWCNTs caused changes of functional groups on the surface and a binding energy shift of Bi4f and V2p, as determined from X-ray photoelectron spectroscopy (XPS). Ultraviolet–visible diffuse reflectometry (UV–Vis DRS) revealed that the band gap became narrower and photoluminescence spectra suggested the recombination of photogenerated electron–hole pairs were inhibited after MWCNT modification. All of the characterizations showed enhancement of catalytic performance of MWCNT/BiVO4. The results from the high-performance liquid chromatography (HPLC) indicated that the degradation rate of oxytetracycline had increased by 42.2% and reached 88.7% within 60 min. Electron paramagnetic resonance (EPR) signals of ·OH and ·O2− were detected and potential photocatalytic mechanisms of the photocatalysts were put forward. Six reaction pathways were proposed based on ultra-performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC–MS/MS) results: demethylation (−14 Da), secondary alcohol oxidation (−2 Da), dehydration (−18 Da), hydroxylation (+16 Da), decarbonylation (−28 Da) and deamination (−15 Da). The present study provides a reference for the application of photocatalytic degradation by MWCNT/BiVO4 on hard degradable substances in aquatic environment.

A case report of a suspicious, heroin-like powder in Belgium, revealing the presence of ocfentanil and W-18

An unknown brown powder, advertised and purchased as heroin, was sent to the Toxicological Centre at the University of Antwerp for analysis in the framework of the Belgian Early Warning System on Drugs. Since the early 2010s, synthetic opioids have been on the rise in the illicit drug markets of North America and Europe, often as adulterants or substitutes for heroin. [1] Ocfentanil, an early-onset fentanyl analogue, has been implemented in several fatalities, predominantly in Europe. W-18 is more prevalent in Canada and the United States. The sample was screened using liquid chromatography coupled to a diode array detector and gas chromatography coupled to a single quadrupole mass spectrometer. The findings were confirmed by liquid chromatography–triple quadrupole mass spectrometry. No heroin, 6-monoacetylmorphine or morphine was detected. Our analysis detected two new psychoactive substances: the synthetic opioid ocfentanil (1.6% m/m), and W-18 (0.3% m/m). The common cutting agents paracetamol (56% m/m), caffeine (24% m/m), benzoic acid (9.5% m/m) and quinine (trace amounts) were also detected. The ocfentanil concentration was consistent with those found in similar brown powders in Belgium (2.5% m/m) and Switzerland (0.9% m/m). [2] , [3] There have been no other reported sightings of W-18 in Western Europe. A combination of analytical techniques was necessary to ensure the highest confidence in the findings, particularly given a spectral mismatch for W-18 between our in-house library and a publically available one. The unknown powder tested positive for ocfentanil and W-18. To the authors’ best knowledge this is the first case of the combined detection of these two new psychoactive substances in the same powder. These findings prompted an alert warning by the Belgian Early Warning System on Drugs.

Determination of seven urinary metabolites of benzene, toluene and xylene by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry

Objective: To develop a method using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to determine the urinary metabolites of benzene, toluene and xylene. The selected metabolites are S-phenylmercapturic acid (S-PMA) , trans, trans-muconic acid (t, t-MA) , 8-hydroxy-2 deoxyguanosine (8-OHdG) , hippuric acid (HA) , 2-methylhippuric acid (2-MHA) , 3-methylhippuric acid (3-MHA) and 4-methylhippuric acid (4-MHA) . Methods: The urine sample was pretreated using methanol to precipitate the proteins. HSS T3 chromatographic column was used to separate the metabolites. The mass spectrometric acquisition was carried out using multiple reaction monitoring (MRM) after ionization with ESI source. External standard method was used for quantification. Results: All the standard curves showed good linear relation, and r of the seven metabolites was all above 0.999. The detection limits and quantitative limits of the seven metabolites were 0.01-500 ng/ml and 0.02-1 000 ng/ml (based on the actual dilution ratio) , respectively. The average spiked recoveries of four loadings ranged from 85.8% to 109.9%. The intra-day and inter-day precisions were 0.2%-4.5% and 0.6%-9.5%, respectively. The samples can be kept for at least 14 days at both 4 ℃ and -20 ℃. Conclusion: This method is simple, rapid and highly sensitive with low cost, and its accuracy, precision and stability can meet the daily test requirements. It can be applied for the determination of urinary S-PMA, t, t-MA, 8-OHdG, HA, 2-MHA, 3-MHA and 4-MHA for the occupational population exposed to benzene, toluene and xylene.

Comparative quality of the forms of decoction pieces evaluated by multidimensional chemical analysis and chemometrics: Poria cocos, a pilot study

Many Chinese medicinal materials (CMMs) are parts of plants or fungi that have been processed into different physical forms, termed decoction pieces, that are typically boiled in water for consumption. One CMM may have several decoction pieces forms, e.g., slices, small cubes (dice), or grains. The specifications that have different morphological parameters (shape, size and thickness) for these various decoction pieces have been developed over, in some cases, centuries of practice. Nevertheless, whether and how the form of decoction pieces affects the extraction (decoction) dynamics, and quality stability during storage has not been studied. Here, we investigated Poria cocos (PC) as a pilot study; we explore how the form of PC decoction pieces affects its chemistry using multidimensional chemical evaluation such as ultra-performance liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry (UHPLC-PDA-QTOF-MS/MS), ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) and high performance gel permeation chromatography coupled with charged aerosol detector (HPGPC-CAD), combined with analysis of variance (ANOVA), principal component analysis (PCA), factor analysis (FA) and hierarchical cluster analysis (HCA). The results indicated that different specifications had significant differences, and these specifications could be divided into four groups. The comprehensive results of the chemical analyses undertaken here indicate that the highest potentially available quality of PC decoction pieces was in the forms of curl, ultra-small grains and small grains, followed by thin slices. This information not only is conducive to promoting the standardization of the specification/form of PC decoction pieces and maximizing the benefits from its utilization, but also provide a promising strategy for assessing other CMM decoction pieces in different forms.

Targeted UPLC-MS/MS high-throughput metabolomics approach to assess the purine and pyrimidine metabolism

Purines and pyrimidines, the important components of DNA and RNA, are closely related to metabolic syndrome and disorder, such as renal disease, gout and diabetic nephropathy etc. Given the importance of the biological significance of purines and pyrimidines, it is necessary to further develop a rapid and sensitive method for practical detection of a large-scale analyses. In this study, based on 96-well solid phase extraction plates-ultra performance liquid chromatography-triple quadrupole mass spectrometry (SPE-UPLC-QqQ-MS/MS), a novel approach for simultaneous determination of 23 purines and pyrimidines in biological samples was developed. First, plasma samples were pretreated by SPE using 96-well plates, which lead to an automated, simplified and rapid sample preparation process. In the methodology development, a large-scale test was performed to evaluate the stability and reliability of the approach. Finally, the levels of purines and pyrimidines in the biological samples were analyzed by this strategy. Experimental results showed that lowest limit of quantification (LLOQ) range from 6.678 × 10−2 μg/mL to 4.275 × 10−6 μg/mL; intra- and inter-day precision are <15% for all analytes. The stability and maximal capability of a single analytical batch could be extended to at least 431 injections (about 70 h). Analysis time of a single run was controlled in 10 min. Under the optimized conditions, wide linear ranges and good correlation coefficients (R2 > 0.99) were acquired. The successful development of this method provides a feasible protocol for a large-scale metabolomics study and it also lays the foundation of quantitative analysis in endogenous analytes.

Stable isotope dilution assay for the accurate determination of tricaine in fish samples by HPLC-MS-MS.

Tricaine methanesulfonate is one of most commonly used anesthetics in fish during blood sampling, artificial propagation and long-distance transportation. In this study, an accurate method for the quantitative determination of tricaine in fish samples by a stable isotope dilution assay coupled with high-performance liquid chromatography-triple quadrupole mass spectrometry was developed. Tricaine-D5 was synthesized and used as an isotopically labeled internal standard for the determination of tricaine. The analytical performance of the method was validated for tricaine determination in marine fish and freshwater fish. The determination of tricaine was linear in the range of 2.0-200.0 μg L-1 . The limit of detection and limit of quantitation for fish muscle tissues were 1.0 and 4.0 μg kg-1 , respectively. Good recoveries were obtained in the range of 92.08-97.50%. The inter- and intra-assay relative standard deviations (RSD values) were investigated, and the values were 0.39-3.01 and 0.85-2.77%, respectively. The values of CCα and CCβ were 10.21-10.43 and 10.42-10.87 μg kg-1 , respectively. The clearance of MS-222 from grass carp was further studied using our method. The results demonstrate that MS-222 could be well absorbed and rapidly eliminated after bath administration.

Determination of Unsulfonated Aromatic Amines in FD&C Yellow No. 5 and FD&C Yellow No. 6 by Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

Background: This paper describes a simple and sensitive ultra-HPLC-triple quadrupole MS (LC-MS/MS) method for the determination of six unsulfonated aromatic amines in the color additives FD&C Yellow No. 5 (Y5) and FD&C Yellow No. 6 (Y6). The six amines determined by this method are aniline (ANL), benzidine (BNZ), 4-aminobiphenyl (4ABP), 4-aminoazobenzene (4AAB), 2-aminobiphenyl (2ABP), and 4-aminobenzonitrile (4ABN). Objective: This method is intended for use in batch certification of the color additives by the U.S. Food and Drug Administration (FDA) to ensure that each lot meets published specifications for coloring foods, drugs, and cosmetics. Methods: A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure is used for extraction of the amines. Quantitative determination was performed in electrospray positive ionization and multiple-reaction monitoring modes. Results: Validation of the method demonstrated overall recovery of 101-115% and precision of 1.74-9.78% for all analytes. Excellent regression coefficients were obtained, with values >0.999. Conclusions: The validated method was successfully used for the analyses of 30 Y5 and Y6 samples and provided results that are consistent with results from the current method used by FDA, with greater sensitivity and low matrix effects. Highlights: The validation results demonstrate that the new LC-MS/MS method is applicable for use in routine batch certification.

Enhancement of liquid chromatography-ion trap mass spectrometry analysis of 4(5)-methylimidazole in biscuits through derivatization with dansyl chloride

The presence of potential carcinogen 4(5)-Methylimidazole (4-MeI) in foods causes much concern, stressing the need of a sensitive determination means. Here, we proposed a high-sensitivity method to determine 4-MeI in biscuits using dansyl chloride derivatization and disperse liquid-liquid micro-extraction (DLLME) followed by a liquid chromatography-ion trap mass spectrometry (LC-IT-MS) analysis. This developed method was subsequently compared to the solid phase extraction combining with liquid chromatography-triple quadrupole mass spectrometry (SPE-LC-QqQ-MS) method. The optimized derivatization conditions were 30 °C and 10 min. Results suggested that the column retention time (RT) was significantly extended, and the MS signal response of 4-MeI-dansyl derivative was also amplified. The developed DLLME-LC-IT-MS method in 4-MeI determination provided satisfactory linearity in a range of 0.5–300 ng/mL (R2 > 0.9991), inter-day accuracy (87-102%) and precision (%RSD ≤ 13.6%) with the quantification limit of 0.2 ng/mL, which obtained similar results comparing to the conventional SPE-LC-QqQ-MS method.

Simultaneous determination of seven effective components of Tripterygium glycosides in human biological matrices by ultra performance liquid chromatography–triple quadrupole mass spectrometry

This paper developed a novel, sensitive, and selective ultra-performance liquid chromatography–triple quad mass spectrometry method to simultaneously determine seven effective constituents (triptolide, triptophenolide, celastrol, wilforgine, wilforine, wilfordine and wilfortrine) of Tripterygium glycosides (GTW) in human serum and urine. The chromatographic separation was performed on the C18 column using an ammonium acetate buffer solution–acetonitrile (both containing 0.1% formic acid) in a gradient program with a flow rate of 0.3 mL/min. Monitoring reaction mode was applied to target compounds quantitative analysis in the positive electrospray ionization (ESI) mode. The analysis process took 11 min in total. This method was fully validated with a linear range of 1–200 ng/mL for triptolide, 0.4–80 ng/mL for celastrol, 0.1–20 ng/mL for triptophenolide, wilforgine, wilforine, wilfordine, and wilfortrine. The intra-day and inter-day accuracy and precision of the target compounds all met the 15% criterion in both serum and urine. Extraction recovery, matrix effect, and dilution integrity were also validated. The short-term and long-term stability results indicated that all the constituents were stable in human serum and urine under the investigated storage conditions. 10 patients' specimens were collected and analyzed. Most of the compounds exhibited the tendency of higher concentration in urine than that in serum. The concentration that was detected in the serum and in the urine of alkaloids showed a positive-correlation property. This is the first time that triptophenolide was quantified in human bio-matrices. The method is feasible for multi-components therapeutic monitoring or pharmacokinetics study in clinical pharmaceutical research of Tripterygium glycosides.