Triple Quadrupole Mass Spectrometry Research Articles

Quality uniformity evaluation of Babao Dan capsule based on its 14 main chemical compounds and in vitro anti-inflammatory activity

Babao Dan capsule (BBDC) is a state secrecy traditional Chinese medicine patent prescription (TCMPP) that is widely used for hepatobiliary and urinary diseases. However, its quality uniformity evaluation (QUE) is lacking. It is necessary to develop a novel and multi–dimensional QUE strategy for BBDC based on chemical properties and bioactivities. An ultrahigh performance liquid chromatography–triple quadrupole–mass spectrometry (UHPLC–QQQ–MS/MS) was developed to quantify 14 compounds, and a QUE strategy was to assess intra–batch and inter–batches consistency. Furthermore, the anti-inflammatory activities were measured in vitro by RAW264.7 cell and NF-κB-RE-luc HEK293 cell. The results indicated that 14 compounds from 16 samples (10 batches) were accurately quantified. And the intra–batch consistency values were less than or equal to 20.1%, whereas the largest inter–batches consistency value was 167.5% of glycocholic acid. 10 batches were divided into three categories to distinction inter–batches differences. BBDC had anti-inflammatory activities including inhibition of LPS–induced NO production and TNF α–activated luciferase counts. The NO inhibition difference of 10 batches was 10.87 μM and accounted for 49.8%. Briefly, the anti-inflammatory activities of 10 batches were different, but the dynamic trends within the same batch were similar, which was supported by luciferase counts. And the correlation was confirmed that the main compounds related to anti-inflammatory in BBDC were bile acids and saponins. Hence, a QUE strategy for BBDC was proposed to improve quality and ensure efficacy. This strategy can be utilized as a reference for consistency evaluations of TCMPPs.

Simultaneous determination of three strong polarity herbicides in tea by ion chromatography-triple quadrupole mass spectrometry

Due to lack of chromogenic groups and fluorescence groups, high boiling point, high polarity, low volatility, and small molecular weight of glyphosate, glufosinate and bentazone, the detection of three analyses were limited in all kinds of food. Herein, a method for the simultaneous determination of glyphosate, glufosinate and bentazone in tea by ion chromatography tandem triple quadrupole mass spectrometry (IC-MS) was developed, which is without organic solvent and complex derivatization. The recoveries of three compounds in different teas (black tea, green tea, white tea) ranged from 80.40 % to 107.00 %, and the intraday precision (n = 6) ranged from 0.57 % to 9.90 %, the daytime precision ranged from 1.00 % to 5.30 %, the quantitative limit (LOQ) ranged from 0.36 to 1.30 µg/L, and the detection limit (LOD) ranged from 0.11 to 0.39 µg/L. Furthermore, the detection limit and quantitative limit of glyphosate, glufosinate and bentazone by this method are lower than other methods in real samples. Meanwhile, the established method was successfully applied to determine the terminal residues of the three analytes in twelve tea samples from commercial market. Therefore, this method can provide reliable technical support for the study of residue status in vegetables and fruits.

Comprehensive quality control of Qingjin Yiqi granule based on UHPLC-Q-Orbitrap-MS and UPLC-QQQ-MS.

Qingjin Yiqi granule (QYG) is a prescription medicine of traditional Chinese medicine which is widely used clinically for the recovery of coronavirus patients. However, there is currently limited research on the quality control of QYG. To evaluate the quality of QYG qualitatively and quantitatively by making full use of advanced chromatography-mass spectrometry techniques. Firstly, a multicomponent characterisation of QYG was performed by ultrahigh-performance liquid chromatography coupled with a Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) system using a rapid negative/positive switching mode. Secondly, the co-condition fingerprint analysis of constituted herbal medicines of QYG was performed to unveil active ingredients as the quality markers of QYG. Thirdly, the marker compounds in 10 batches of QYG were quantified by ultrahigh-performance liquid chromatography coupled with a Waters Xevo TQ-S triple quadrupole mass spectrometry (UPLC-QQQ-MS) system. A comprehensive method that combined the inclusion list and data-dependent acquisition (DDA) to achieve a systematic characterisation of QYG was established by UHPLC-Q-Orbitrap-MS. After analysis based on Compound Discoverer software and Global Natural Products Social (GNPS) platform, a total of 332 compounds were detected. Eleven Q-markers were determined for the quality evaluation of QYG by comparison with the fingerprint of nine constituted herbal medicines. An adjusted multiple reaction monitoring (MRM) quantification method was further established to simultaneously determine the 11 Q-markers for holistic quality evaluation of QYG. This is the first study to report comprehensive multicomponent characterisation, identification, and quality assessment of QYG, which could be used for effective guarantee of the quality of QYG.

Antifungal and Antiaflatoxigenic Activities of Massoia Essential Oil and C10 Massoia Lactone against Aflatoxin-Producing Aspergillus flavus.

Fungal infection and mycotoxin contamination are major hazards to the safe storage and distribution of foods and feeds consumed by humans and livestock. This study investigated the antifungal and antiaflatoxigenic activities of massoia essential oil (MEO) and its major constituent, C10 massoia lactone (C10), against aflatoxin B (AFB)-producing Aspergillus flavus ATCC 22546. Their antifungal activities were evaluated using a disc diffusion assay, agar dilution method, and a mycelial growth inhibition assay with the AFB analysis using liquid chromatography triple quadrupole mass spectrometry. MEO and C10 exhibited similar antifungal and antiaflatoxigenic activities against A. flavus. C10 was a primary constituent in MEO and represented up to 45.1% of total peak areas analyzed by gas chromatography-mass spectrometry, indicating that C10 is a major compound contributing to the antifungal and antiaflatoxigenic activities of MEO. Interestingly, these two materials increased AFB production in A. flavus by upregulating the expression of most genes related to AFB biosynthesis by 3- to 60-fold. Overall, MEO and C10 could be suitable candidates as natural preservatives to control fungal infection and mycotoxin contamination in foods and feeds as Generally Recognized As Safe (GRAS) in the Flavor and Extract Manufacturers Association of the United States (FEMA), and MEO is a more suitable substance than C10 because of its wider range of uses and higher allowed concentration than C10.

Determination of Oxygen Heterocyclic Compounds in Foods Using Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

The aim of this research was to determine oxygen heterocyclic compounds in twenty-six Citrus- and cinnamon-flavoured foods using supercritical fluid chromatography in combination with triple-quadrupole mass spectrometry (SFC-QqQ-MS). According to the authors' knowledge, this is the first report on the determination of these molecules in foods by means of the SFC-QqQ-MS technique. The analytical technique normally used for their determination in foods is liquid chromatography coupled with a photodiode array detector. However, supercritical fluid chromatography is proving to be a valid alternative approach to investigating coumarins, furocoumarins and polymethoxyflavones. According to the results presented herein, each sample analysed showed the presence of molecules of interest. Coumarin was found in all the cinnamon-flavoured samples analysed in a low concentration. The presence of oxygen heterocyclic compounds in all the Citrus-flavoured samples, according to the label, comfirmed that the foods selected for this research article were prepared with Citrus fruits. Among the samples analysed, mandarin juice was the richest in bioactive compounds, representing a good source of polymethoxyflavones in a diet.

Mutual interference between 3,6-dichlorinated carbazole and p,p'-dichlorodiphenyltrichloroethane in gas chromatography mass spectrometry analysis.

The widespread contamination of the environment by polyhalogenated carbazoles (PHCZs) has been increasingly observed during the past decade. Among numerous PHCZ congeners, 3,6-dichlorocarbazole (36-CCZ) is often among the most frequently detected at higher concentrations. Although the environmental level of the legacy pesticide p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT) has been declining, it continues to be ubiquitously detected. These two compounds were found to interfere with each other during analyses using gas chromatography (GC) coupled with single- or triple-quadrupole low-resolution mass spectrometry (MS or MS/MS). The base peak in the mass spectra was that of m/z 235 for both compounds. In MS/MS with multiple reaction monitoring (MRM), the same transitions (235 → 200 and 235 → 165) were often used. Under the same GC operating conditions, the SH-I-5MS capillary column used in this work did not resolve the two compounds at baseline. Pre-treatment using cleanup column chromatography can fractionate the sample extract, with the two compounds separated in different fractions before instrumental analyses. Reversed-phase HPLC columns also work for resolving 36-CCZ and p,p'-DDT. Possible overlaps in GC retention and similarity in MS spectra might have caused data inaccuracy for 36-CCZ as well as p,p'-DDT in some studies published to date, and steps to avoid the interference should be taken into quality control protocols in future research and environmental monitoring.

Endocrine disrupting chemicals in wastewater treatment plants in Kenya, East Africa: Concentrations, removal efficiency, mass loading rates and ecological impacts

This study investigated the levels, mass loadings, removal efficiency, and associated ecotoxicological risks of selected endocrine disrupting chemicals (EDCs), namely, dibutylphthalate (DBP), diethylhexylphthalate (DEHP), dimethylphthalate (DMP), linuron (LNR) and progesterone (PGT) in wastewater, sludge, and untreated dry biosolid (UDBS) samples from twelve wastewater treatment plants (WWTPs) in nine major towns in Kenya. Analysis was done using high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS). All the wastewater influents had quantifiable levels of EDCs with DBP being the most abundant (37.49%) with a range of 4.33 ± 0.63 to 19.68 ± 1.24 μg L−1. DEHP was the most abundant in sludge and accounted for 48.2% ranging between 278.67 and 9243.49 ng g−1 dry weight (dw). In the UDBS samples, DEHP was also the most abundant (40%) of the total EDCs detected with levels ranging from 78.77 to 3938.54 ng g−1 dw. The average removal efficiency per pollutant was as follows: DMP (98.7%) > DEHP (91.7%) > PGT (83.4%) > DBP (77.9%) > LNR (72.2%) which can be attributed to sorption onto the biosolid, biological degradation, photolysis, and phytoremediation. The pH was negatively correlated to the EDC concentrations while total dissolved solids (TDS), chemical oxygen demand (COD), biochemical oxygen demand (BOD5), and electrical conductivity (EC) were positively correlated. The mass loadings were as high as 373.33 g day−1 of DBP in the treatment plants located in densely populated cities. DEHP and PGT had their Risk Quotients (RQs) > 1, posing a high risk to biota. DMP, DBP, and LNR posed medium risks as their RQ values were between 0.1 and 1. EDCs are therefore loaded to environmental compartments through either the effluent that loads these pollutants into the receiving aquatic ecosystem or through the UDBS, which are used as fertilizers in agricultural farmlands causing potential toxicological risks to aquatic and terrestrial life.

Fermentation of Betaphycus gelatinum Using Lactobacillus brevis: Growth of Probiotics, Total Polyphenol Content, Polyphenol Profile, and Antioxidant Capacity.

Little information is available regarding polyphenol variations in the food processing of edible and medicinal red seaweed, Betaphycus gelatinum. This study investigated the effects of Lactobacillus brevis fermentation on total polyphenol content (TPC), polyphenol profile, and antioxidant activity in Betaphycus gelatinum pretreated by ultrasound-assisted mild acid hydrolysis for the first time. During 60 h of fermentation, the viable colony number significantly increased, pH significantly decreased, and reducing sugar content significantly decreased initially, then significantly increased. Free TPC significantly increased to 865.42 ± 29.29 μg GAE/g DW (163.09% increase) with increasing antioxidant activity, while bound TPC significantly decreased to 1004.90 ± 87.32 μg GAE/g DW (27.69% decrease) with decreasing antioxidant activity. Furthermore, 27 polyphenol compounds were identified by ultra-high-performance liquid chromatography with Xevo triple quadrupole mass spectrometry. In total, 19 and 23 free polyphenols and 24 and 20 bound polyphenols were identified before and after fermentation, respectively. Before fermentation, bound trans-cinnamic acid (56.75%), bound rosmarinic acid (26.62%), and free trans-cinnamic acid (3.85%) were the main components. After fermentation, free rosmarinic acid (43.57%), bound trans-cinnamic acid (15.19%), bound rosmarinic acid (13.33%), and free trans-cinnamic acid (5.99%) were the main components. These results provide information for the food processing of Betaphycus gelatinum.

Metabolic regularity of bioactive compounds in Bufei Jianpi granule in rats using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry analysis technology.

Bufei Jianpi granule (BJG) is clinically effective for treating chronic obstructive pulmonary disease (COPD). At present, there is no report regarding the drug metabolism of BJG in vivo. This work developed an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method with high accuracy and sensitivity to determine drug metabolism of this compound in vivo. After continuous administration of BJG, the concentrations of 10 components in rat plasma, namely betaine, peimine, peiminine, astragaloside A, sinensetin, nobiletin, naringin, calycosin, formononetin, and magnolol, were determined at different time points. Meanwhile, the pharmacokinetic parameters and metabolic rules of these 10 components were evaluated: Cmax , 8.624-574.645 ng/mL; Tmax , 0.250-8.667 h; AUC0-t , 17.640-8947.393 ng h/mL; T1/2 , 3.405-66.014 h; mean residence time (MRT), 6.893-11.223 h. All these components possessed anti-inflammatory, antioxidant, and other biological activities to varying degrees, contributing to improving lung function, mitigating pneumonia and pulmonary fibrosis, and preventing and treating chronic obstructive pulmonary disease. Exploring the pharmacokinetic parameters and the laws of chemical components in BJG forms the scientific basis for applying the compound clinically and identifying quality markers for the control of the compound.

Rapid identification of chemical composition and quality markers of Gynostemma pentaphylla by ultra-performance liquid chromatography combined with triple quadrupole mass spectrometry connected with high-performance liquid chromatography

Gynostemma pentaphylla, one of the traditional Chinese medicinal materials, which has gradually become a research hotspot due to its rich pharmacological activities such as hypolipidemic properties, anti-inflammatory, anti-cancer and etc. The main active ingredients of Gynostemma pentaphyllum are saponins (gypenoside), flavonoids and polysaccharides. The pharmacologically active dose depends on the content of the active ingredient, so it is necessary to establish a reliable quality evaluation standard for Gynostemma pentaphyllum. However, the existing Gynostemma pentaphyllum quality control methods are not perfect, Ginsenoside Rb1 was regarded as a signature ingredient of Gynostemma pentaphylla in local standards. Surprisingly, the high-performance liquid chromatogram results displayed that the content of Ginsenoside Rb1 was not suitable as a quality marker. Therefore, a triple quadrupole time-of-flight tandem mass spectrometer method was developed to explore other potential quality markers in Gynostemma pentaphylla. As a result, a total of 24 compounds was identified tentatively. While another stable and reliable component Gypenoside XLVI was found and the content was carried out. It was expected to replace Ginsenoside Rb1 to establish a more reliable and precise quality standard of Gynostemma pentaphylla.

Reactivity to allergenic food contaminants: A study on products on the market.

The frequency and severity of reactions in food-allergic consumers exposed to unintentional food allergen contamination during production is unknown. To warn allergic consumers, it has been suggested for pre-packaged foods to be precautionary labelled when the food allergen contamination may exceed the amount to which 1%-5% of the population could react (ED01-ED05). ED01 for hazelnut and milk have been estimated at 0.1 and 0.2mg, respectively, by the Voluntary Incidental Trace Allergen Labelling (VITAL) initiative. The respective reference doses recommended by the FAO/WHO Codex consultation are 3 and 2mg. We evaluated the reactivity to potential traces of milk and hazelnut allergens in allergen-free pre-packaged products by children affected by severe allergies to milk and hazelnuts. Oral Food Challenges with commercially available hazelnut-free wafer biscuits and milk-free chocolate pralines were administered to patients with severe food allergies to hazelnut and cow's milk, respectively. Contamination levels of milk or hazelnut allergens were measured using chromatographic separation interfaced with triple quadrupole mass spectrometry. No hazelnut allergic patient showed allergic reactions to exposure to biscuits, nor any milk allergic patient displayed allergic reactions to the dark chocolate praline. While no hazelnut trace was detected in biscuits, the praline was found to be contaminated by milk at concentrations ranging between 8 and 35mg total protein/kg food. In our dose model, these amounts exceeded 1.5-10 times the VITAL ED01 and reached the threshold suggested by the FAO/WHO Codex consultation. Upon the consumption of food products available on the market, many patients with severe food allergies tolerate significantly higher doses of allergen than reference doses indicated in the VITAL system used for precautionary allergen labelling. These doses support the safety of the FAO/WHO recommended reference doses.

Fungal microbiome related to mycotoxin contamination in medicinal and edible seed Semen Persicae

Medicinal and edible seed Semen Persicae is susceptible to mycotoxin and fungal contamination. However, the occurrence of mycotoxin contamination and fungal infection is still unclear. In this paper, ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry and high-throughput sequencing were conducted to determine the mycotoxin contents and fungal abundances of Semen Persicae. 42.86% of samples were positive for aflatoxin B1 (26.48–48.37 μg/kg) and 28.57% of samples were positive for aflatoxin B2 (1.47–4.82 μg/kg). Ochratoxin A and fumonisin B1 were only detected in one sample (91.02 and 34.61 μg/kg, respectively). Chao 1 and Shannon indices were significantly higher in the Dalian of Liaoning, Baotou of Innermongolia and Langfang of Hebei regions than in other groups. Ascomycota, Basidiomycota, Wallemia, Candica, Saccharomyces and Aspergillus were the predominant fungi and they were significantly region-specific. Simultaneously, the diversity, composition and co-occurrence network complexity in the mycotoxin-free group were significantly higher than those in the mycotoxin-contaminated group. Spearman correlation analysis showed aflatoxins, ochratoxin A and fumonisins contents were positively and significantly correlated with the abundances of Aspergillus, Rhodotorula, Wallemia and Candida. In conclusion, this study reported the prevalence of mycotoxin contamination and the great diversity of fungi associated with Semen Persicae for the first time, providing an early warning for subsequent potential mycotoxin biosynthesis.

Developing an Improved Strategy for the Analysis of Polychlorinated Dibenzo-p-Dioxins/Furans and Dioxin-like Polychlorinated Biphenyls in Contaminated Soils Using a Combination of a One-Step Cleanup Method and Gas Chromatography with Triple Quadrupole Mass Spectrometry.

Soils contaminated with polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), and dioxin-like (dl) polychlorinated biphenyls (PCBs), known as persistent organic pollutants (POPs), have garnered global attention because of their toxicity and persistence in the environment. The standard method for target analytes has been used; however, it is an obstacle in large-scale sample analysis due to the comprehensive sample preparation and high-cost instrumental analysis. Thus, analytical development of inexpensive methods with lower barriers to determine PCDDs/Fs and dl-PCBs in soil is needed. In this study, a one-step cleanup method was developed and validated by combining a multilayer silica gel column and Florisil micro-column followed by gas chromatography with triple quadrupole mass spectrometry (GC-QqQ-MS/MS). To optimize the separation and quantification of 17 PCDDs/Fs and 12 dl-PCBs in soils, the sample cleanup and instrumental conditions were investigated. For quantification method validation, spiking experiments were conducted to determine the linearity of the calibration, recovery, and method detection limit of PCDDs/Fs and dl-PCBs using isotopic dilution GC-QqQ-MS/MS. The applicability of the simultaneous determination of PCDDs/Fs and dl-PCBs was confirmed by the recovery of native target congeners and labeled surrogate congeners spiked into the quality-control and actual soil samples. The results were in good agreement with the requirements imposed by standard methods. The findings in this work demonstrated the high accessibility of the sample cleanup and analysis methods for the efficient determination of PCDDs/Fs and dl-PCBs in contaminated soils.

Optimization of bioactive compounds by ultrasound extraction and gas chromatography - mass spectrometry in fast-growing leaves

Fast-growing crops are of great economic importance in the production of paper pulp and energy. The commercialization of the bioactive components from underutilized parts could provide additional value to these crops. In the present study, chromatography gas coupled to mass spectrometry triple quadrupole (GC–MS/MS) and ultrasound extraction technique was used in the bioactive compounds determination in fast-growing crops leaves. Twenty-one terpenes and eight polyphenolic bioactive compounds were studied. Prior to GC–MS/MS, polydimethylsiloxane (PDMS) - stir bars preconcentration and trimethylsilane (TMS) derivatization steps were used for terpenes and polyphenolic compounds, respectively. Several parameters (pH, temperature, ethanol–water mixtures, ultrasound power, and ultrasound time) of the extraction step were optimized by central composite experimental design. Ethanol content, ultrasound power, and ultrasound time were the parameters that most influence the extraction efficiency of terpenes in biomass residues, while for polyphenolic compounds it was temperature and ultrasound time.The optimal ultrasound extraction conditions for terpenes were 60% ethanol, pH 4, 80 W, 40 °C, and 15 min, whereas for polyphenolic compounds they were 60% ethanol, pH 4, 120 W, 50 °C, and 15 min. The detection limits (LOD) were in the range of 0.200–3.02 µg kg−1 and 7.9–540 µg kg−1 for terpenes and polyphenolic compounds, respectively.The developed analytical method was applied to twelve fast-growing leaves (Leucaene (diversifolia and leucocephala), Eucalyptus (globulus and urograndis), Populus (I214 and AF2), Prosopis (alba and juliflora), Robinia, Tagasaste, Ulmus pumila, and Paulownia). Eucalyptol was the mayor terpene present in all the plants studied, while chlorogenic and quinic acid were the mayor phenolic compounds.

Residues of sulfoxaflor and its metabolites in floral and extrafloral nectar from Hibiscus rosa-sinensis L. (Malvaceae) with or without co-application of tebuconazole

Systemic pesticide exposure through nectar is a growing global concern linked to loss of insect diversity, especially pollinators. The insecticide sulfoxaflor and the fungicide tebuconazole are currently widely used systemic pesticides which are toxic to certain pollinators. However, their metabolisms in floral or extrafloral nectar under different application methods have not yet been well studied. Hibiscus rosa-sinensis was exposed to sulfoxaflor and tebuconazole via soil drenching and foliar spraying. Sulfoxaflor, tebuconazole, and their main metabolites in floral and extrafloral nectar, soil, and leaves were identified and quantified using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ MS). The chemical compositions of unexposed and contaminated H. rosa-sinensis floral nectar or extrafloral nectar were compared using regular biochemical methods. The activities of two pesticide detoxifying enzymes, glutathione-s-transferase and nitrile hydratase, in H. rosa-sinensis nectar were examined using LC-MS and spectrophotometry. The floral nectar proteome of H. rosa-sinensis was analysed using high-resolution orbitrap-based MS/MS analysis to screen for sulfoxaflor and tebuconazole detoxifying enzymes. H. rosa-sinensis can absorb sulfoxaflor and tebuconazole through its roots or leaf surfaces and secrete them into floral nectar and extrafloral nectar. Both sulfoxaflor and tebuconazole and their major metabolites were present at higher concentrations in extrafloral nectar than in floral nectar. X11719474 was the dominant metabolite of sulfoxaflor in the nectars we studied. Compared with soil application, more sulfoxaflor and tebuconazole remained in their original forms in floral nectar and extrafloral nectar after foliar application. Sulfoxaflor and tebuconazole exposure did not modify the chemical composition of floral or extrafloral nectar. No active components, including proteins in the nectar, were detected to be able to detoxify sulfoxaflor.

Comparing the sensitivity of a low- and a high-resolution mass spectrometry approach for xenobiotic trace analysis: An exposome-type case study

The chemical exposome consists of environmental exposures experienced throughout a lifetime but to date analytical approaches to investigate the plethora of low-abundance chemicals remain very limited. Liquid chromatography high-resolution mass spectrometry (HRMS) is commonly applied in untargeted exposome-wide analyses of xenobiotics in biological samples; however, human biomonitoring approaches usually utilize targeted low-resolution triple quadrupole (QQQ) mass spectrometry tailored to a small number of chemicals. HRMS can cover a broader chemical space but the detection of molecules from low-level exposure amidst a background of highly-abundant endogenous molecules has proven to be difficult. In this study, a triple quadrupole (QQQ) and a high-resolution mass spectrometer (HRMS) with identical chromatography were utilized to determine the limits of quantitation (LOQ) of >100 xenobiotics and estrogenic hormones in pure solvent and human urine. Both instrumental platforms are currently applied in exposure assessment studies and were operated in their most frequently used acquisition mode (full scan for HRMS and multiple reaction monitoring for QQQ) to mimic typical applications. For HRMS analyses, the median LOQ was 0.9 and 1.2 ng/mL in solvent and urine, respectively, while for low-resolution QQQ measurements, the median LOQ was 0.1 and 0.2 ng/mL in solvent and urine, respectively. To evaluate the calculated LOQs in complex biological samples, spot urine samples from 24 Nigerian female volunteers were investigated. The higher LOQ values for HRMS resulted in less quantified low-abundance analytes and decreased the number of compounds detected below the LOQ. Even at chronic low-dose exposure, such compounds might be relevant for human health because of high individual toxicity or potential mixture effects. Nevertheless, HRMS enabled the additional screening for exposure to unexpected/unknown analytes, including emerging compounds and biotransformation products. Therefore, a synergy between high- and low-resolution mass spectrometry may currently be the best option to elucidate and quantify xenobiotics in comprehensive exposome-wide association studies (ExWAS).

Sustainable chromatographic quantitation of multi-antihypertensive medications: application on diverse combinations containing hydrochlorothiazide along with LC–MS/MS profiling of potential impurities: greenness and whiteness evaluation

Cardiovascular disorders are among the leading causes of death worldwide, especially hypertension, a silent killer syndrome requiring multiple drug therapy for appropriate management. Hydrochlorothiazide is an extensively utilized thiazide diuretic that combines with several antihypertensive drugs for effective treatment of hypertension. In this study, sustainable, innovative and accurate high performance liquid chromatographic methods with diode array and tandem mass detectors (HPLC–DAD and LC–MS/MS) were developed, optimized and validated for the concurrent determination of Hydrochlorothiazide (HCT) along with five antihypertensive drugs, namely; Valsartan (VAL), Amlodipine besylate (AML), Atenolol (ATN), Amiloride hydrochloride (AMI), and Candesartan cilextil (CAN) in their diverse pharmaceutical dosage forms and in the presence of Chlorothiazide (CT) and Salamide (DSA) as HCT officially identified impurities. The HPLC–DAD separation was achieved utilizing Inertsil ODS-3 C18 column (250 × 4.6 mm, 5 μm) attached with photodiode array detection at 225.0 nm. Gradient elution was performed utilizing a mixture of solvent A (20.0 mM potassium dihydrogen phosphate, pH 3.0 ± 0.2, adjusted with phosphoric acid) and solvent B (acetonitrile) at ambient temperature. Linearity ranges were 0.1–100.0 µg/mL for HCT, VAL, AML and CAN, 0.05 –100.0 µg/mL for both ATN and AMI and 0.05–8.0 µg/mL for both CT and DSA. Additionally, this work describes the use of liquid chromatography–electrospray–tandem mass spectrometry for the accurate detection and quantification of the impurities; CT and DSA in the negative mode utilizing triple quadrupole mass spectrometry. The linearity ranges for those impurities were 1.0–200.0 ng/mL and 5.0–200.0 ng/mL for CT and DSA, respectively. Developed methods’ validation was achieved in accordance with International Conference on Harmonization (ICH) guidelines. Upon applying liquid chromatographic techniques for the drug analysis, a green and sustainable assessment have to be handled due to the consumption of energy and many solvents. Through the use of the HEXAGON, Analytical Greenness (AGREE) and White Analytical Chemistry (WAC) tools, greenness and sustainability have been statistically assessed. The optimized HPLC–DAD and LC–MS/MS methods were fast, accurate, precise, and sensitive, and consequently could be applied for conventional analysis and quality control of the proposed drugs in their miscellaneous dosage forms for the purpose of reducing laboratory wastes, time of the analysis time, effort, and cost.Graphical

Elucidating the chemical interaction effects of herb pair Danshen-Chuanxiong and its anti-ischemic stroke activities evaluation

Ethnopharmacological relevanceSalvia miltiorrhiza Bunge (Danshen) and Ligusticum chuanxiong Hort. (Chuanxiong) is the core herb pair in traditional Chinese medicines (TCMs) formulae for treating ischemic stroke. However, the synergistic effect of Danshen-Chuanxiong against anti-ischemic stroke and its compatibility mechanism remains unclear. Aim of the studyThis study aimed to uncover the compatibility mechanism of Danshen-Chuanxiong against ischemic stroke through chemical profiling, pharmacodynamics evaluation, network pharmacology and experimental validation. Materials and methodsUltra-high performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS) and UHPLC connected with tandem triple quadrupole mass spectrometry (QQQ-MS) were utilized to conduct the chemical interaction analysis. Then the synergistic effects of Danshen-Chuanxiong against ischemic stroke were comprehensively evaluated by the middle cerebral artery occlusion reperfusion (MCAO/R) mice model, zebrafish ischemic stroke model and glutamic acid-induced PC12 cells injury model. Afterwards, network pharmacology and molecular docking were applied to dissect the significant active compounds and potential mechanisms. Finally, the key target proteins were experimentally validated by Western blot. Results83 compounds were characterized in Danshen-Chuanxiong by UHPLC-QTOF-MS analysis, and 4 compounds were tentatively identified for the first time. The quantification results (24 accurately identified compounds) in 13 proportions of Danshen-Chuanxiong revealed that Danshen significantly increased the dissolution of most phthalides (from Chuanxiong), while Chuanxiong facilitated the dissolution of most phenolic acids (from Danshen) in solution. The anti-ischemic stroke effects of Danshen-Chuanxiong were significantly better than Danshen or Chuanxiong in attenuating infarct size, reducing brain edema and neurological scores in MCAO/R mice. Also, compared with single herbs, this herb pair exerted better effects of suppressing the incidence of cerebral thrombosis in zebrafish, and increasing the cell viability of glutamic acid-induced PC12 cells. In network pharmacology, 7 effective compounds (rosmarinic acid, chlorogenic acid, salvianolic acid B, (Z)-ligustilide, ferulic acid, caffeic acid, tanshinone IIA) and 5 hub targets (AKT, TNF−α, IL-1β, CASP3 and BCL2) as well as 4 key pathways were predicted. Western blot results showed that Danshen-Chuanxiong exert therapeutic effects mainly through decreasing the protein expressions of TNF-α, IL-1β and Cleaved-CASP3, elevating the levels of p-AKT and BCL2. ConclusionsThis work provided an integration strategy for uncovering the synergistic effects and compatibility mechanism of Danshen-Chuanxiong herb pair for treating ischemic stroke, and laid foundation for the further development and utilization of this herb pair.

Targeted Workflow Investigating Variations in the Tear Proteome by Liquid Chromatography Tandem Mass Spectrometry.

Proteins in tears have an important role in eye health and have been shown as a promising source of disease biomarkers. The goal of this study was to develop a robust, sensitive, and targeted method for profiling tear proteins to examine the variability within a group of healthy volunteers over three days. Inter-individual and inter-day variabilities were examined to contribute to understanding the normal variations in the tear proteome, as well as to establish which proteins may be better candidates as eventual biomarkers of specific diseases. Tear samples collected on Schirmer strips were subjected to bottom-up proteomics, and resulting peptides were analyzed using an optimized targeted method measuring 226 proteins by liquid chromatography-scheduled multiple reaction monitoring. This method was developed using an in-house database of identified proteins from tears compiled from high-resolution data-dependent liquid chromatography tandem mass spectrometry data. The measurement of unique peptide signals can help better understand the dynamics of each of these proteins in tears. Some interesting trends were seen in specific pathways or protein classes, including higher variabilities for those involved in glycolysis, glutathione metabolism, and cytoskeleton proteins and lower variation for those involving the degradation of the extracellular matrix. The overall aim of this study was to contribute to the field of tear proteomics with the development of a novel and targeted method that is highly amenable to the clinical laboratory using high flow LC and commonly used triple quadrupole mass spectrometry while ensuring that protein quantitation was reported based on unique peptides for each protein and robust peak areas with data normalization. These results report on variabilities on over 200 proteins that are robustly detected in tear samples from healthy volunteers with a simple sample preparation procedure.

Simultaneous LC-MS determination of glucose regulatory peptides secreted by stem cell-derived islet organoids.

For studying stem cell-derived islet organoids (SC-islets) in an organ-on-chip (OoC) platform, we have developed a reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method allowing for simultaneous determination of insulin, somatostatin-14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl-C18 separations using 2.1mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2µg/L for human insulin, 0.1µg/L for somatostatin-14, and 0.05µg/L for glucagon. The here-developed RPLC-MS/MS method showed that the SC-islets have an insulin response dependent on glucose concentration, and the SC-islets produce and release somatostatin-14 and glucagon. The RPLC-MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC-islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC-islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well-plate. Taken together, RPLC-MS/MS method is well suited for multi-hormone measurements of SC-islets on an OoC platform.