Ultra-performance Liquid Chromatography-electrospray Ionization Research Articles

UPLC-MS/MS method for determination of sofosbuvir in human plasma

A sensitive and rapid method for quantitation of Sofosbuvir in human plasma has been established using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). Sofosbuvir d3 was used as an internal standard. Sofosbuvir and internal standard in plasma sample were extracted using ethyl acetate (liquid liquid extraction). A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of 0.5% formic acid: methanol (30:70, v/v). The reconstituted samples were injected into a Gemini C18 (50×4.6mm, 5μm) column. Using MS/MS in the multiple reaction monitoring mode, Sofosbuvir and Sofosbuvir d3 were detected without severe interferences from human plasma matrix. Sofosbuvir produced a protonated precursor ion ([M+H]+) at m/z 428.35 and a corresponding product ion at m/z 279.26. The internal standard produced a protonated precursor ion ([M+H]+) at m/z 431.38 and a corresponding product ion at m/z 282.37. The calibration curves for the analyte was linear (R2≥0.9956, n=4) over the concentration range of 4.063-8000.010ng/mL. Stability studies revealed that Sofosbuvir was stable in plasma during bench top (7h at room temperature), in injector (20h), at the end of five successive freeze and thaw cycles and long term at -70°C±15°C for 15 days. The developed method was validated as per the guidelines of USFDA and the obtained results were found to be within the limits and could be successfully employed for the determination of Sofosbuvir in human plasma for regular and pharmacokinetic studies.

Quantitative Analysis of Amoxicillin, Amoxicillin Major Metabolites, and Ampicillin in Chicken Tissues via Ultra-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

This manuscript describes a rapid, sensitive, and specific approach that utilizes ultra-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UPLC-ESI/MS/MS) for the selective determination of amoxicillin, amoxicillin metabolites, and ampicillin in chicken tissue samples (muscle, liver, and kidney). This optimized method was validated according to the requirements defined by the European Union and the Food and Drug Administration. The extract obtained after extraction and purification of chicken tissues was injected into a UPLC system coupled to a mass spectrometer operated in the positive electrospray MS/MS mode for analysis. The chicken tissues were spiked with the target compounds at levels of 25.0, 50.0, and 100.0 μg/kg, and the results showed that the extraction recoveries were all higher than 84% and the relative standard deviations did not exceed 20%. The limits of detection and limits of quantification were 0.01–1.36 and 0.05–5.44 μg/kg, respectively. The decision limits were 52.62–57.26 μg/kg, and the detection capabilities were 55.23–64.51 μg/kg. Finally, the new approach was verified through quantitative determination of the analytes in tissues from 30 commercial chickens obtained from local supermarkets.

Effect and mechanism of wedelolactone as antioxidant-coumestan on [formula omitted]-treated mesenchymal stem cells

The antioxidant properties of coumestans have been investigated previously using inappropriate methods, which has resulted in misleading and inaccurate mechanisms being reported to account for their antioxidant behavior. In this study, the phenolic coumestan wedelolactone increased the viability of OH-treated mesenchymal stem cells (MSCs) in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl assay. Mechanistic analysis in vitro suggested that wedelolactone could scavenge various radicals, including OH, O2-, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, as well as reducing Cu2+ and chelating Fe2+. Configuration analysis based on a ball and stick model indicated that the 3′,4′-catechol moiety of wedelolactone was acting as an Fe2+-chelating site. The main product of the reaction between wedelolactone and DPPH was analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry, which gave a mass ion with an m/z value of 709. This peak yielded several fragments with m/z values of 663, 543, 501 and 483. Quantitative evaluation revealed that the antioxidant activity of wedelolactone was 1.62 times higher than that of Trolox. Based on these results, we concluded that (i) wedelolactone can efficiently protect MSCs against OH-induced damage and that this protective effect provides preliminary evidence for the application of wedelolactone in transplantation of MSCs (especially for osteoporosis); (ii) the main antioxidant mechanism of wedelolactone involves direct radical-scavenging via a single electron transfer (SET)→radical adduct formation (RAF) pathway, whereas the Fe2+-chelating activity of wedelolactone represents a minor pathway; and (iii) the direct radical-scavenging and Fe2+-chelating pathways can both be attributed to the catechol moiety rather than the coumestan skeleton.

Simultaneous Determination of Aniline, Benzidine, Microcystins, and Carbaryl in Water Using Ultra-Performance Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

A method for simultaneously determining the levels of aniline, benzidine, microcystin variants (microcystin-LR, RR, and YR) and carbaryl in water was developed based on ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC-MS/MS). The chromatographic conditions were optimized for the trace determination. Without sample enrichment, the method detection limit for all test compounds ranged from 0.040 to 0.155 μg/L; meanwhile, the recoveries for all test compounds were 83.1–114%. Precision, indicated by the relative standard deviation, was <12.9%. The results meet the requirements for the determination of these compounds. Without the need to clean up the samples, the results of the analysis of samples from wastewater and surface water demonstrated that the UPLC-MS/MS method has the capability to analyze complex matrices in the trace-level monitoring of wastewater samples.

Effect and mechanism of Sorbus pohuashanensis (Hante) Hedl. flavonoids protect against arsenic trioxide-induced cardiotoxicity.

The cardiotoxicity of arsenic trioxide (ATO) limits its clinical application in cancer treatment. Evidences suggest that sorbus has antioxidant activity and its consumption has been linked with improved cardioprotection. In this study, we investigated the cardio-protective effect and mechanisms of Sorbus pohuashanensis (Hante) Hedl. flavonoids (SPF) against ATO in BALB/c mice and H9c2 cells. Eleven major flavonoids were confirmed by ultra-performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry (UPLC-ESI-Q-TOF-MS). SPF recovered the ATO-induced disordered electrocardiogram (ECG) and abnormal cardiac structure in the heart of mice. At the same time, SPF significantly reduced levels of creatine kinase (CK), creatine kinase-MB (CK-MB) lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (GOT) against ATO-induced injury and inhibited ATO-induced apoptosis both in vivo and in vitro. In addition, SPF regulated ATO-induced oxidative stress damage by increasing the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in vivo and in vitro, and reducing reactive oxygen species (ROS). Analysis of the oxidative stress pathways showed that SPF prevented the ATO-induced downregulation of phosphorylated protein kinase B (p-Akt) in vivo and in vitro. Pre-treatment of H9c2 cells with SPF inhibited attenuation of nuclear factor (Nrf2) and heme oxygenase (HO-1). Hence, SPF could be used as a preventive and therapeutic plant ingredient against ATO-induced cardiotoxicity.

Evaluation of Methylated Silica Solid-Phase Extraction Sorbent to Retain a Surfactant in the Detection of Pesticides in Water Using Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry

This study examined the matrix effect of a typical surfactant on the detection of pesticides in water using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS). When direct sample injection is employed, surfactants in water samples tend to adsorb onto the chromatographic column, weakening its analyte separation capability and consequently lowering signal detection intensities and raising detection limits. A new sample preparation approach of a tailored solid-phase extraction (SPE) is proposed based on the difference in adsorption affinities between surfactant and pesticide for silylated surfaces. Using sodium dodecyl sulfate (SDS) as a representative surfactant, four types of silylated silica particles with different surface hydrophobicity and functionality were examined for selectively adsorbing and removing SDS from water samples with pesticide analytes to eliminate the matrix effect of the surfactant on liquid chromatographic (LC) separation. It was found that the tailored SPE sample preparation for cleanup of SDS could be effectively achieved based on this concept. Samples prepared with trimethylchlorosilane methylated silica particles in the tailored SPE produced a desirable LC separation and low detection limits for the pesticide analytes. The linear calibration range extended from 0.1 up to 40 μg L−1 (R 2 = 0.9906–0.9998, n = 6). Low limits of quantitation for the 11 pesticides were achieved (0.027–0.090 μg L−1). The precision of the proposed method for the 11 analytes was less than 10%.

Quantification of Piperazine in Chicken Muscle by Ultra-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

An ultra-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UPLC-ESI/MS/MS) method was developed for the detection of piperazine in chicken muscle. Following extraction and purification, the chicken muscle extract was injected into the UPLC system and analyzed. Piperazine detection was performed on a triple-quadrupole mass spectrometer with the ESI interface operating in positive mode. The most sensitive mass transition from the precursor ion to the product ion was 87.1 → 44.1 for piperazine. The coefficient (R2) of piperazine was 0.9995 in the range of 1–200 μg/kg. The recovery and relative standard deviation (RSD) ranged from 102.93 to 111.46% and from 4.57 to 5.28% at 50.0, 100.0 and 200.0 μg/kg adding levels, respectively. Limits of detection (LODs) and limits of quantification (LOQs) were 0.4 and 1.0 μg/kg, respectively. This method was fully validated based on its specificity, sensitivity, linearity, precision, accuracy, matrix effect, and stability results. Compared to other researches, this new method not only omitted the derivative process, which simplify the pre-processing, but also proved a better sensitivity based on its low LOD and LOQ values.

Profiling of ginsenosides in the two medicinal Panax herbs based on ultra-performance liquid chromatography-electrospray ionization-mass spectrometry.

As the king of herb plants, ginseng has been used for nearly 5000 years in medicines in Asia and recently in the West. Ginsenosides, the main active constituents in Panax herbs, have prominent immunoregulatory effects. Although extensively studied in the roots, ginsenosides have not been studied with regard to their profiles and natural variations in the leaf, stem, petiole, lateral root, and main roots during development or among species. In this study, a sensitive ultra-performance liquid chromatography-electrospray ionization–mass spectrometry method with a shorter chromatographic running time was developed and validated for simultaneous quantification of ten ginsenosides. Comparing ginsenoside contents in various parts during different developmental stages revealed part-specific accumulation of most ginsenosides. Further investigation indicated that Rg3 accumulated at significantly higher levels in the petiole of P. ginseng than in that of P. quinquefolius. The relative ratio of ginsenoside Rb2 to Rb1 appears to be a candidate metabolic marker for identifying the ginseng cultivar within a diverse collection of ginseng accessions. In addition, the PCA showed that aboveground parts differed significantly between species and can be considered as species-specific markers rather than roots. This comprehensive survey, providing reliable, affordable and adequate scientific evidence, could be used to differentiate two species and discriminate ginseng cultivar ages.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3427-3) contains supplementary material, which is available to authorized users.

Bioaccessibility of individual phenolic compounds in extra virgin argan oil after simulated gastrointestinal process

The bioaccessibility of individual phenolic compounds in extra virgin argan oil was evaluated using an in vitro digestion method for first time. The analyses were carried out by ultra-performance liquid chromatography-electrospray ionisation tandem mass spectrometry in multiple reaction monitoring (UPLC-ESI-MS/MS). Hydroxyphenylacetic acid (4245 μg/kg), ferulic acid (2478 μg/kg), vanillin (2429 μg/kg) and 3,4-dihydroxybenzoic acid (2247 μg/kg) were the mayor compounds identified in argan oil extracts. After the digestion process, values of bioaccessibility of the different compounds varied from a minimum of 2% (hydroxyphenylacetic acid) to a maximum of 84% (p-coumaric acid), but some such as caffeic acid became detectable, as a result from digestive transformations. The results indicate that the phenolic compounds of the argan oil are strongly affected during the digestive process, and bioaccessibility should be taken into account when assessing polyphenol content of oils.

Effect of high temperature on the lipid composition of Isochrysis galbana Parke in logarithmic phase

Isochrysis galbana is a species of marine microalgae that is principally used to feed the larvae and early juveniles of bivalve mollusks (e.g., ark shells, clams, mussels, oysters and scallops). The artificial hatchery stages of most of these larvae are primarily occur during high-temperature seasons, when the water temperature in the breeding pool frequently exceeds 30 °C and even reaches 35 °C, which may change the lipid composition of I. galbana, thus affecting its nutritional value. In this experiment, we investigated changes in the lipid composition of I. galbana Parke at high temperature using ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. The results showed that the sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), diacylglycerylcarboxyhydroxymethylcholine (DGCC) and digalactosyldiacylglycerol (DGDG) contents increased during growth at 20 and 35 °C. Conversely, the lyso-SQDG, lyso-MGDG, lyso-DGDG, lyso-DGCC and triacylglycerol (TAG) contents clearly decreased. Moreover, higher SQDG, MGDG, DGDG and DGCC contents and lower lyso-lipid and TAG contents were observed in I. galbana Parke grown at 35 versus 20 °C. In addition, the content of docosahexaenoic acid (DHA) improved at 20 and 35 °C during growth, and DHA accumulated to greater levels at 35 °C than at 20 °C at the same time points. These results revealed an increase in DHA content as the temperature rose, which indicated that the nutritional value of I. galbana Parke during logarithmic phase improved in the same cultured time with an increasing temperature.

Cerebrospinal fluid in tuberculous meningitis exhibits only the L-enantiomer of lactic acid.

BackgroundThe defining feature of the cerebrospinal fluid (CSF) collected from infants and children with tuberculous meningitis (TBM), derived from an earlier untargeted nuclear magnetic resonance (NMR) metabolomics study, was highly elevated lactic acid. Undetermined was the contribution from host response (L-lactic acid) or of microbial origin (D-lactic acid), which was set out to be determined in this study.MethodsIn this follow-up study, we used targeted ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) to determine the ratio of the L and D enantiomers of lactic acid in these CSF samples.ResultsHere we report for the first time that the lactic acid observed in the CSF of confirmed TBM cases was in the L-form and solely a response from the host to the infection, with no contribution from any bacteria. The significance of elevated lactic acid in TBM appears to be that it is a crucial energy substrate, used preferentially over glucose by microglia, and exhibits neuroprotective capabilities.ConclusionThese results provide experimental evidence to support our conceptual astrocyte–microglia lactate shuttle model formulated from our previous NMR-based metabolomics study — highlighting the fact that lactic acid plays an important role in neuroinflammatory diseases such as TBM. Furthermore, this study reinforces our belief that the determination of enantiomers of metabolites corresponding to infectious diseases is of critical importance in substantiating the clinical significance of disease markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1597-9) contains supplementary material, which is available to authorized users.

Antioxidant Activity andα-Amylase Inhibitory Effect of Polyphenolic-Rich Extract fromOriganum GlandulosumDesf

The polyphenolic-rich extract of Tunisian Origanum glandulosum Desf. was investigated for its phenolic composition, antioxidant activity and potential to contribute to the dietary management of type II diabetes. By mean of an ultra-performance liquid chromatography-electrospray ionization- mass spectrometry (UPLC-PDA-ESI-MS) 16 phenolics were characterized. The major detected compounds are caffeic acid, luteloin glucoside, xanthomicrol and carvacrol. The polyphenolic-rich extract presented a notable antioxidant activity similar to that of the synthetic antioxidant Trolox. It was found to inhibit, in a concentration dependent manner, the α-amylase activity with an IC50 value (8.22 mg/mL) comparable to that of Acarbose (6.12 mg/mL). Overall, our findings suggest that O. glandulosum could be considered as a valuable source of high-value phytochemicals with pharmaceutical and functional food additive applications. Practical Applications Origanum glandulosum Desf., a North African endemic species, is one of the most used plants in folk medicine against whooping cough, fever, bronchitis and diabetes. In the present work, the ability of O. glandulosum to inhibit pancreatic α-amylase in vitro as well as to act as antioxidant food was investigated. The results suggest that O. glandulosum could be considered as a valuable source of phytochemicals with a potent α-amylase inhibition property and antioxidant capacity. Further animal and clinical studies were needed to confirm the potential use of O. glandulosum for the management of type II diabetes.

Distinguishing commercially grown Ganoderma lucidum from Ganoderma lingzhi from Europe and East Asia on the basis of morphology, molecular phylogeny, and triterpenic acid profiles

In China and other countries of East Asia, so-called Ling-zhi or Reishi mushrooms are used in traditional medicine since several centuries. Although the common practice to apply the originally European name ‘Ganoderma lucidum’ to these fungi has been questioned by several taxonomists, this is still generally done in recent publications and with commercially cultivated strains. In the present study, two commercially sold strains of ‘G. lucidum’, M9720 and M9724 from the company Mycelia bvba (Belgium), are compared for their fruiting body (basidiocarp) morphology combined with molecular phylogenetic analyses, and for their secondary metabolite profile employing an ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESIMS) in combination with a high resolution electrospray ionization mass spectrometry (HR-ESI-MS). According to basidiocarp morphology, the strain M9720 was identified as G. lucidum s.str. whereas M9724 was determined as Ganoderma lingzhi. In molecular phylogenetic analyses, the M9720 ITS and beta-tubulin sequences grouped with sequences of G. lucidum s.str. from Europe whereas those from M9724 clustered with sequences of G. lingzhi from East Asia. We show that an ethanol extract of ground basidiocarps from G. lucidum (M9720) contains much less triterpenic acids than found in the extract of G. lingzhi (M9724). The high amount of triterpenic acids accounts for the bitter taste of the basidiocarps of G. lingzhi (M9724) and of its ethanol extract. Apparently, triterpenic acids of G. lucidum s.str. are analyzed here for the first time. These results demonstrate the importance of taxonomy for commercial use of fungi.

Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation.

Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.

Development and characterization of a nanodendritic silver-based solid-phase extraction sorbent for selective enrichment of endocrine-disrupting chemicals in water and milk samples

In this study, 4-[4-phenylazo-phenoxy] butyl-1-thiol (AzSH) functionalized nanodendritic silver (AzS@AgNDs) materials were prepared as a solid-phase extraction (SPE) sorbent for the selective extraction of estrogens. AzS@AgNDs possess an extremely large surface-to-volume ratio and a small average particle size. The performance of the material was evaluated by selective enrichment of hexestrol, diethylstilbestrol, dienestrol and bisphenol A in water and milk samples followed by rapid ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESI–MS) analyses. The results exhibited that AzS@AgNDs had excellent adsorption capability for the targeted estrogens. The limits of detection of the four estrogens ranged from 0.1 to 5.0 pg/mL. The recoveries of the estrogens spiked into tap water were over the range of 83.6–105.3% with relative standard deviations of 2.8–6.0%. The results indicated the capability of this method for the rapid determination of estrogens in milk and other environmental water samples. In addition, this method would be useful for the determination of human exposure and health risk assessments trace level of endocrine-disrupting compounds (EDCs) in drinking water.

Study of pharmacokinetic profiles and characteristics of active components and their metabolites in rat plasma following oral administration of the water extract of Astragali radix using UPLC–MS/MS

Ethnopharmacological relevanceAstragali radix is one of the well-known traditional Chinese herbal medicine, and possesses various biological functions, such as hepatoprotective and anticancer. In present study, to investigate the metabolism and pharmacokinetics of the major constituents of A. radix, a sensitive ultra-performance liquid chromatography–electrospray ionization–mass spectrometry (UPLC–MS/MS) method with shorter chromatographic running time was developed and validated for simultaneous quantification of formononetin, ononin, calycosin, calycosin-7-β-glucoside, astragaloside IV and their glucuronide metabolites in rat plasma after oral administration of water extract of A. radix at two different doses. Materials and methodsThe chromatographic separation was achieved on a C18 column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 0.3mL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring (MRM) via electrospray ionization (ESI) source in positive ionization mode. Samples were pre-treated by a single-step protein precipitation with methanol, and erlotinib was used as internal standard (IS). ResultsThe current UPLC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effects and stability. The lowest limit of quantifications (LLOQ) were 1ng/mL for all analytes. After oral administration, the plasma concentrations of the glucuronides, especially calycosin-3′-glucuronide, were much higher than the parent compounds. The mean half-life (t1/2) was between 1 and 5h, and the metabolites were eliminated faster than the parent constituents. The median (range) time to reach maximum plasma concentration (Tmax) was between 0.5 and 1h. ConclusionsThis is the first study of the pharmacokinetic study of bioactive compounds and their glucuronides in male rat plasma after oral administration of water extract of A. radix. The results demonstrated the biotransformation between the bioactive isoflavonoids and their glucuronides was extensive in rats and provided a significant basis for better understanding the absorption and metabolism mechanism of A. radix. Furthermore, this study could suggest that future studies should focus on the metabolites and biotransformation between the bioactive constituents when conducting a drug efficacy study.

Natural occurrence of four Alternaria mycotoxins in tomato- and citrus-based foods in China.

A total of 70 tomato-based and 86 citrus-based products collected in China were analyzed for alternariol, alternariol monomethyl ether, tentoxin, and tenuazonic acid by ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry. No toxins were found in any fresh tomato or citrus fruit samples. Tenuazonic acid was the predominant toxin detected in all tomato ketchup (10.2–1787 μg/kg) and tomato juice samples (7.4–278 μg/kg). Alternariol was quantitated at higher level than alternariol monomethyl ether with the ratio of alternariol/alternariol monomethyl ether ranging from 0.37 to 104 in 14 alternariol-positive tomato ketchup samples. Tentoxin was detected at much lower levels in all samples analyzed. Some citrus juice samples were positive for tenuazonic acid and alternariol monomethyl ether. It is necessary to conduct a systemic surveillance of Alternaria toxins in raw and processed foods to provide the scientific basis for risk assessment of dietary exposure to these toxins in Chinese populations.

LC-ESI/MS determination of xanthone and secoiridoid glycosides from in vitro regenerated and in vivo Swertia chirayita

A rapid analytical method has been developed to determine xanthone and secoiridoid glycoside in in vitro and in vivo Swertia chirayita extracts. Ultra performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) was applied and validated for the analysis of xanthone and secoiridoid glycoside a potential active component isolated from methanolic extracts of in vitro and in vivo Swertia chirayita plantlets. Chromatographic separation was achieved on a RP-C18 column using gradient elution. Mangiferin (Xanthone), Amarogentin and Swertiamarin (Secoiridoid glycosides) were identified in both the extracts. In the LC/ESI-MS spectra, major [M + H] (+) and [M + Na] (+) ions were observed in positive ion mode and provided molecular mass information. An ultra-performance liquid-chromatography in combination with electrospray ionization tandem mass spectrometry involving metal cationisation was successfully utilized for the rapid identification of xanthone and secoiridoid glycosides. This method is suitable for the routine analysis, as well as for the separation and identification of known and novel secoiridoid glycoside and xanthone.

Ultra-high performance liquid chromatography tandem mass spectrometry for the detection of durum wheat contamination or adulteration.

In this work, an ultra-performance liquid chromatography electrospray ionization (UPLC-ESI)-MS/MS methodology based on multiple reaction monitoring (MRM) for the selective and sensitive detection and quantification of durum wheat adulteration has been developed and fully validated. The targeted analysis was performed by monitoring specific transitions at m/z 543.7 > 657.4 and m/z 543.7 > 299.2 of a species-specific marker derived from a tryptic peptide of puroindoline a (Pin-a), a cysteine-rich protein selectively present only in common wheat. In addition, two transitions at m/z 500.4 > 725.4 and m/z 500.4 > 561.9 of a reference peptide belonging to purothionin A-1, present in both species, were also monitored. The calibration curves obtained on binary mixtures with known percentages of common/durum wheat flours showed linearity (coefficient of regression, r ≥ 0.99) over concentrations that ranged between 80 and 1%. The limit of detection (LOD) and limit of quantification (LOQ) for the Pin-a marker in wheat flours were 0.01 and 0.03%, respectively. The identified Pin-a marker was also found to be highly diagnostic for the quantification of common wheat in raw materials (kernels) and processed products (pasta), thus offering new opportunities to assess food authenticity.

Quantitative evaluation of collagen crosslinks and corresponding tensile mechanical properties in mouse cervical tissue during normal pregnancy.

The changes in the mechanical integrity of the cervix during pregnancy have implications for a successful delivery. Cervical collagens are known to remodel extensively in mice with progressing gestation leading to a soft cervix at term. During this process, mature crosslinked collagens are hypothesized to be replaced with immature less crosslinked collagens to facilitate cervical softening and ripening. To determine the mechanical role of collagen crosslinks during normal mouse cervical remodeling, tensile load-to-break tests were conducted for the following time points: nonpregnant (NP), gestation day (d) 6, 12, 15, 18 and 24 hr postpartum (PP) of the 19-day gestation period. Immature crosslinks (HLNL and DHLNL) and mature crosslinks (DPD and PYD) were measured using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). There were no significant changes in the total immature crosslink density (HLNL+DHLNL mol per collagen mol) throughout normal mouse gestation (range: 0.31–0.49). Total mature crosslink density (PYD+DPD mol per collagen mol) decreased significantly in early softening from d6 to d15 (d6: 0.17, d12: 0.097, d15: 0.026) and did not decrease with further gestation. The maturity ratio (total mature to total immature crosslinks) significantly decreased in early softening from d6 to d15 (d6: 0.2, d15: 0.074). All of the measured crosslinks correlated significantly with a measure of tissue stiffness and strength, with the exception of the immature crosslink HLNL. This data provides quantitative evidence to support the hypothesis that as mature crosslinked collagens decline, they are replaced by immature collagens to facilitate increased tissue compliance in the early softening period from d6 to d15.