Electrospray Ionization Tandem Mass Spectrometry Research Articles

Lemongrass (Cymbopogon citratus) oil nanoparticle synthesis, characteristic, and evaluation of antibacterial and antifungal effects and the influence on hardness of acrylic resin

Context: Acrylic resin is used in dentistry as a removable denture base. It can cause various pathologies when not properly cleaned. One of the pathologies is denture stomatitis caused by Candida albicans and Streptococcus mutans accumulation on the acrylic resin surface. Therefore, microbial agents such as denture cleansers are needed. Aims: To evaluate the characteristics of lemongrass (Cymbopogon citratus) nanoparticles as a better antibacterial and antifungal herbal ingredient and their relationship with acrylic hardness. Methods: C. citratus oil nanoparticles (LON) were synthesized and analyzed by transmission electron microscopy (TEM). Electrospray ionization tandem mass spectrometry (ESI-MS) analysis was used to analyze the characteristics of LON bioactive components. Minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) against C. albicans and S. mutans and mechanical hardness test of acrylic were performed. Results: The LON concentration of MIC and MBC against C. albicans and S. mutans was 25 and 100%, respectively. One-way ANOVA showed no significant difference between groups of LON with different concentrations (p=0.687). A paired t-test showed significant differences in acrylic resin hardness before and after treatment of LON with 100% (p=0.022) and 50% (p=0.021) concentration. There was no significant difference in hardness before and after treatment of other concentrations of LON and chlorhexidine as positive control. Conclusions: LON treatment on acrylic resin decreased the growth of C. albicans and S. mutans without altering the mechanical properties (hardness).

Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor-to-production transitions. The developed assay was validated over the range of 1-2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0-t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0-t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.

Insight into the Coextrusion Mechanism between Whey Protein Isolate and Cysteine.

The disulfide cross-linking sites of whey protein isolate (WPI) coextruded with dissolved cysteine (Cys) at concentrations of 0, 20, 40, 60, 80, and 100 mM were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software, and the structure and gel water distribution of WPI during coextrusion (≤50 °C) were also investigated. LC/MS/MS demonstrated that α-La (6) and α-La (120) were the most active sites for intermolecular disulfide cross-linking of α-La. Meanwhile, the molecular weight of protein polymers in coextruded WPI-Cys was the largest at 100 mM Cys, and α-lactalbumin was the main reactant for polymerization from the result of SDS-PAGE and size exclusion chromatography. Additionally, the high concentration of Cys caused the secondary structure of WPI to gradually change from a highly ordered to a disordered structure during coextrusion. In addition, with an increasing concentration of Cys, the free sulfhydryl group of proteins and the binding force to immobilized water gradually increased. Therefore, this work revealed the disulfide cross-linking mechanism between WPI and Cys under low-temperature coextrusion at the molecular level, and the obtained coextruded cross-linked WPI could serve as a novel food ingredient with excellent water-holding capacity for the food industry.

Assessing the stability of daptomycin in icodextrin-based peritoneal dialysis solution.

The stability of antimicrobials in peritonitis during peritoneal dialysis (PD) solutions is a critical factor influencing treatment success. This study investigated the stability of daptomycin (DAP) when combined with icodextrin-based PD solution, by measuring DAP concentrations and observing any structural changes. A dose of DAP (350 mg) was dissolved in 7 mL of saline in a clean bench. The solution was then injected into the large compartment (1,260 mL) of NICOPELIQ® Peritoneal Dialysis Solution and thoroughly mixed. Samples were collected at intervals ranging from 0 to 336 h (7 points in total). The concentration of DAP was quantified using high-performance liquid chromatography (HPLC). The structure of any unidentified peaks was determined using HPLC coupled with electrospray ionization tandem mass spectrometry. DAP maintained 90% of the initial concentration in NICOPELIC® for 72 h at room temperature and 12 h at 37 °C. Unidentified peaks, distinct from DAP, were detected during analysis. Further investigation indicated that these peaks corresponded to anhydrated DAP. The findings from this stability study are expected to enhance the effectiveness of outpatient management and preparation for treating peritonitis using DAP.

Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.

5α-Androstane-3α,17β-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study. To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring. The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels. An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.

Dynamic Changes in Flavonoids’ Accumulation Pattern in Tilia miqueliana Flowers at Different Developmental Stages Based on Widely Targeted Metabolomic Analysis

Tilia miqueliana is an endemic species belonging to the genus Tilia L. (Tiliaceae) in China, which is known for its fragrant flowers and nectar, but the dynamic changes in metabolites during its growth and development are still unclear. In this study, the metabolic profiles from T. miqueliana flowers at three developmental stages were detected by performing an ultra-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC-ESI-MS/MS)-based widely targeted metabolomic analysis. A total of 1138 metabolites were detected, with 288 Differentially Accumulated Metabolites (DAMs) determined, flavonoids accounting for the largest proportion. The trend analysis showed that DAMs present seven distinctive patterns, and subclass 5 obtained the largest amount with continuously increased relative content during flower development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analysis of DAMs showed different overlap and variability in metabolic pathways, indicating different directions of flavonoids’ metabolic flux in the three developmental stages. A correlation network analysis further revealed five core metabolites that played essential roles in flavonoid biosynthesis. This research provides comprehensive insights into the exploitation and utilization of T. miqueliana as well as a scientific basis for phylogenetic studies of the genus Tilia.

Polyphenol composition and antioxidant activity of fermentation combined with enzymatic hydrolysis modified Astragalus membranaceus stems

Astragalus membranaceus (AM) roots are a well-known homologous medicine and food in China. AM stems, which are discarded and not used effectively, also contain many active compounds and exhibit beneficial effects. It has the potential to be explored as antibiotic alternative. Fermentation combined with enzymatic hydrolysis (FEH) is an effective strategy for extracting polyphenol and improving the usage of AM stems. Therefore, in this study, the conditions of FEH and extraction for polyphenol in AM stems were screened. The antioxidant activity of extract from AM stems without and with FEH (AMSE and FAMSE) was evaluated. The metabolite profiles of phenolic acids and flavonoids in AMSE and FAMSE were characterized by ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS). The results showed that the highest polyphenol content from AM stems was obtained with cellulase and pectinase (1:1, 2000 U/g), moisture content 43%, fermentation temperature 30 °C, and fermentation time 7 days. Selected extraction conditions of polyphenol were ethanol concentration 50%, ultrasonic power 500 W, extraction temperature 35 °C, and extraction time 40 min. On this condition, compared with AMSE, the polyphenol and flavonoid contents in FAMSE were significantly higher. FAMSE exhibited stronger DPPH, hydroxyl radical scavenging rate and reducing power than AMSE. The relative content of 3-(4-hydroxyphenyl)-propionic acid, dihydroferulic acid, isoferulic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyllactic acid, ferulic acid, vanillic acid, syringic acid, gentisic acid, sinapic acid, apigenin, tricin, acacetin, daidzein, genistein, formononetin, prunetin, pratensein, rhamnocitrin and galangin were significantly upregulated in FAMSE.Graphical

Rapid bromate determination using short-column ion chromatography-mass spectrometry: Application to bromate quantification during ozonation

A streamlined, precise, and dependable method for the concurrent quantification of bromate has been established, employing ion chromatography in conjunction with electrospray ionization tandem mass spectrometry (IC-ESI-MS/MS). In an effort to expedite sample analysis duration, the AG18 short column was utilized for rapid separation of the specified analytes. Utilizing a blend of water and acetonitrile as the mobile phase within an optimized gradient elution setting, it was possible to achieve satisfactory resolution in under 3 minutes. Specifically, the mixing of acetonitrile in water samples improves the sensitivity and immunity of the method. This phenomenon may be attributed to the similar proportions of acetonitrile in the sample and the mobile phase, which effectively reduces the viscosity and surface tension of the droplets during the ESI process. Detection limits and quantification thresholds were 0.14 µg/L and 0.41 µg/L, respectively. Analyte recoveries, when spiked into tap water between 2.5 µg/L to 50 µg/L, ranged from 69.5 % to 116.7 %; precision metrics, measured as intra- and inter-day relative standard deviations, spanned 5.1–7.9 %. The devised approach encountered minimal interference from prevalent water matrix constituents, including chloride (50–200 mg/L), sulfate (25–200 mg/L), bicarbonate/carbonate (1–6 mM), and natural organic matter (1–8 mg/L). In environmental contexts, this methodology effectively tracked the production of bromate in controlled ozonation experiments conducted in the laboratory. Segmented ozone dosing reduced bromate production when the solution contained low concentrations of DOC, which provides an alternative strategy for controlling bromate production.

Measurement of atmospheric amines and aminoamides by column adsorption/extraction and hydrophilic liquid chromatography-electrospray-tandem mass spectrometry.

Sampling and chromatography-mass spectrometry methods were investigated to measure atmospheric amines and aminoamides. Amines and their amide derivatives play significant roles in new particle formation (NPF) in the atmosphere, especially diamines and aminoamides have higher NPF potentials compared to monoamines. For amine sampling, silica gel tube collection and formic acid extraction gave good overall recoveries (>93 ± 8%) for mono-, di-, tri-, tetramines, and aminoamides. Two chromatography methods were subjected to analyze the extracted amines. One involved direct analysis using hydrophilic interaction liquid chromatography with carboxyl or diol group functioned separation column (carboxyl-HILIC or diol-HILIC), and the other utilized derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and subsequent reversed-phase chromatography (HPLC). Separated amines were detected by electrospray ionization and tandem mass spectrometry in both cases. DBD-F-HPLC method provided good sensitivity for mono- and all polyamines (limit of detection (LOD) < 4.6nM, relative standard deviation (RSD) for 100nM < 9.2%). However, aminoamides could not be detected by DBD-F-HPLC. Carboxyl-HILIC provided good sensitivities for mono- and diamines and aminoamides (LOD < 1.6nM, RSD < 4.8%). Forest air measurement was performed and data obtained by carboxyl-HILIC and DBD-F-HPLC showed good agreement for 1,3-diaminopropane, 1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine) (R2 = 0.9215-0.9739, n = 7-14). Carboxyl-HILIC method was the best for the amine analysis, and combination with silica gel tube sampling provides atmospheric monitoring available. The developed method can be used not only to study atmospheric chemistry of diamines and aminoamides but also to analyze flavor/odor of foods, flowers and wastes.

A validated LC-MS/MS method for the simultaneous quantification of iothalamate and hippuran in serum and urine for non-radioactive kidney function assessment

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the kidney function markers iothalamate and hippuran in human serum and urine. It is based on protein precipitation with methanol followed by dilution of the supernatant for serum and simple dilution for urine. The polar analytes are chromatographically separated by a 6.5-min gradient on a low-ligand density reversed-phase column; detection is performed by electrospray ionization tandem mass spectrometry in the positive ion mode against stable-isotope labeled internal standards.The results of a thorough method validation show that iothalamate and hippuran can be simultaneously quantified in the concentration ranges 0.500–30.0 ng/mL and 10.0–5000 ng/mL for serum and urine, respectively, with values for CV and absolute bias not exceeding 10 %, and with sufficient stability in all relevant matrices and solvents. The method was successfully applied for the analysis of serum and urine samples of multiple individuals who received both iothalamate and hippuran.

(S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate derivatization combined with UPLC/ESI-MS/MS as a powerful method for dipeptide determination in foods: An application example in fermented cocoa beans

Dipeptides play a crucial role in food flavor, as well as in physiological and nutritional functions. However, as comprehensive methods for determining dipeptides are still under development, the dipeptide composition of foods remains largely unexplored. This study proposed a novel analytical method for dipeptides by applying an amino acid determination technique we previously developed: (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu) derivatization coupled with ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). The DMT-(S)-Pro-OSu derivatization of dipeptides improved both separation in LC and detection sensitivity in MS, allowing for the optimized acquisition of m/z and elution times for 313 available dipeptides, and enabling the absolute quantification of 126 dipeptides in a simultaneous analysis. Subsequent analysis of fermented cocoa beans detected and quantified 42 dipeptides, revealing distinct dipeptide profiles depending on the origin of the beans. Additionally, by conducting in silico analysis of the elution times for 313 dipeptides, a machine learning model was constructed that accurately predicts dipeptide elution times. The DMT-(S)-Pro-OSu-UPLC/MS/MS method and the elution time prediction model developed in this study are expected to contribute to the understanding of dipeptide composition in foods.

Cistus libanotis from Algeria: Phytochemical analysis by GC/MS, HS-SPME-GC/MS, LC–MS/MS and its anticancer activity

In this paper, Cistus libanotis (Cistus clusii Dunal) from Algeria was investigated. This plant is utilized in numerous industries, mostly in perfumery and, more recently, as a raw material for dietary supplements. For the first time, the present study evaluated the anticancer potential of Algerian C. libanotis by performing a comprehensive phytochemical investigation. The volatile fraction of the fresh plant material of C. libanotis was investigated using headspace solid-phase microextraction (HS-SPME) coupled to GC-MS, and the chemical composition of the essential oil (EO) of C. libanotis was examined using GC-MS technique; additionally, the phenolic compounds were identified and quantified using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A healthy cell line (L929) and two cancer cell lines (CAPAN-1 and dld-1) were used to test the ethanolic extract's cytotoxicity. HS-SPME-GC/MS analysis identified sabinene (25.3 %) and α-thujene (23.8 %) as principal components. In EO the major ingredients were γ-terpinene (17.4 %), camphene (16.13 %), α-phellandrene (15.36 %), and 4-carene (13.54 %). Nevertheless, 24 components were identified by LC-ESI-MS/MS analysis and shikimic acid, luteolin and hesperidin were most abundant. Further, the ethanolic extract of C. libanotis showed a significant cytotoxic effect against dld-1 and CAPAN-1 cell lines, while it was less active against the healthy cell line L929.

Effects of Supplementing Different Quantities of Moringa stenopetala Leaves on Plasma Metabolite and Acylcarnitine Profile, Body Condition Score and Milk Yield Performance in Zebu (Bos indicus) Cattle.

The formulation of multi-nutrient blocks based on low-cost and locally available browse feed resources can be a valid feeding strategy in Sub-Saharan Africa, where inadequate feed supply, both in quality and quantity, is a major constraint. We evaluated the four different inclusion percentages (M-0%, M-25%, M-35% and M-45%) of Moringa stenopetala leaf powder to multi-nutrient blocks on their change on blood metabolite of dairy cows under practical, ranging conditions. Multi-nutrient blocks with four inclusion rates of M. stenopetala leaves were applied as complementary feed for free ranging dairy cows. The study was performed on 24 free ranging dairy cows reared around Arba Minch town in the Southern Ethiopian Rift Valley. Blood samples were collected from the jugular vein of dairy cows both before and after supplementation. Plasma glucose, beta hydroxy butyrate (BHB), urea, creatinine, triglycerides and nonesterified fatty acids (NEFA) concentration was quantified spectrophotometrically. Dried serum spots were subject to quantitative electrospray tandem mass spectrometry to estimate changes in nutrient metabolism based on selected carnitines. Based on these measurements, the milk yield and body condition score were increased during the period of multi-nutrient block supplementation. During the supplementation period, the cows got higher plasma glucose, triglyceride and urea concentrations and lower concentrations of BHB, NEFA and creatinine. From the metabolite profiles, a more efficient nutrient use could be concluded. Although no clear dose-response relationship was observed, the highest inclusion of the M. stenopetala leaves in the multi-nutrient blocks gave the best performance. This outcome supports the idea of implementing M. stenopetala based multi-nutrient blocks on tropical smallholder farms that are not easily accessible to conventional extension services.

In-Depth Mass Spectrometry Study of Vanadium(IV) Complexes with Model Peptides.

Investigating the speciation of vanadium complexes in the presence of potential biomolecular targets under physiological conditions remains challenging, and further experimental techniques are needed to better understand the mechanism of action of potential metallodrugs. The interaction of two model peptides (angiotensin I and angiotensin II) with three well-known oxidovanadium(IV) compounds with antidiabetic and/or anticancer activity, [VIVO(pic)2(H2O)], [VIVO(ma)2], and [VIVO(dhp)2] (where pic, ma, and dhp are picolinate, maltolate, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate anions, respectively), was investigated by ESI-MS/MS (electrospray ionization tandem mass spectrometry) and complemented by EPR (electron paramagnetic resonance) spectroscopy measurements and theoretical calculations at the DFT (density functional theory) level. The results demonstrated that vanadium-peptide bonds are preserved after HCD (higher energy collisional dissociation) fragmentation, allowing for the identification of binding sites through a detailed analysis of the fragmentation spectra. Angiotensin I (AT1) and angiotensin II (AT2) exhibited different coordination behaviors. AT1, with two His residues (His6, His9), prefers to form [AT1 + VOL] adducts with both histidine residues coordinated to the metal ion, while AT2, which has only His6, can bind the metal in a monodentate fashion, forming also [AT2 + VOL2] adducts. Insights from this study pave the way to ESI-MS/MS investigations of more complex systems, including target proteins and further development of vanadium-based drugs.

Utilizing ESI-MS/MS and ion mobility spectrometry for structural characterization and isomer differentiation of 1, 2-unsaturated (1-4) linked disaccharide derivatives and their 2-C-functionalized analogues.

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

SIL-IS LC-ESI-MS/MS method for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma: Development, validation and its application to a pharmacokinetics study.

A liquid chromatography electrospray ionization tandem mass spectrometry method with amoxicillin-d4 as the stable isotope-labeled internal standard for simultaneous quick detection of amoxicillin and clavulanic acid in human plasma was developed and validated. Chromatographic separations were performed on a Hedera ODS-2 column (2.1 × 150 mm, 5μm). The mobile phases for gradient elution were aqueous solution containing 0.2% acetic acid (AA) (mobile phase A) together with organic phase solution (acetonitrile and methanol mixed solution, mobile phase B). Mass spectrometry was performed using negative electrospray ionization in multiple reaction monitoring mode. The target fragment ion pairs of amoxicillin, clavulanic acid and amoxicillin-d4 were m/z 364.1 → 223.1, 198.1 → 135.9 and 368.1 → 227.1, respectively. The linear ranges of this method were 40-5,000 ng/ml for amoxicillin and 30-2,500 ng/ml for clavulanic acid, with coefficient of determination > 0.9900. This method validation included selectivity, standard curve, lower limit of quantitation, accuracy, precision, recovery, matrix effect (hemolytic matrix and hyperlipidemic matrix), carryover, stability, dilution reliability and incurred sample reanalysis study. A successful application of this method was realized in a pharmacokinetic study after administration of amoxicillin-clavulanic acid potassium granules.

Determination of azodicarbonamide in flour samples using high-performance liquid chromatography–tandem mass spectrometry with xanthydrol pre-column derivatisation

Azodicarbonamide (ADA) is approved as a food additive in flour products due to its oxidising and bleaching properties. However, it is prohibited in Australia and Europe on account of its toxicity and the risk of causing asthma in humans. A method was developed to determine ADA in actual flour samples. This work presents an optimised methodology based on derivatisation and clean-up procedures followed by ultra-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry (UPLC–ESI-MS/MS). The analytical method was successfully validated. An excellent result was obtained for the linearity of matrix-matched calibration curves (R 2 > 0.99) in the concentration range of 0.10–80 mg/kg. The recovery rate varied from 81.7% to 102.3%. The relative standard deviations (RSDs) of repeatability (n = 6) were 1.3–4.1%, and inter-day RSDs (n = 6) were 2.2–4.8%. The limit of detection and the limit of quantification were 0.014 and 0.042 mg/kg, which were significantly lower than the requirement of 45 mg/kg stipulated in the Chinese National Food Safety Standard (GB 2760-2014). The detection rate of ADA in 26 flour samples was 23.1%, with the concentration ranging from 0.023 to 23.2 mg/kg.

A Flavonoid Concentrate from Moringa Oleifera Lam. Leaves Extends Exhaustive Swimming Time by Improving Energy Metabolism and Antioxidant Capacity in Mice.

Moringa oleifera Lam. leaves contain various nutrients and bioactive compounds. The present study aimed to assess the anti-fatigue capacity of a flavonoids concentrate purified from M. oleifera Lam. leaves. The total flavonoids in the purified extract were analyzed by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS). The mice were supplemented with purified M. oleifera Lam. leaf flavonoid-rich extract (MLFE) for 14 days. The weight-loaded forced swimming test was used for evaluating exercise endurance. The 90-min non-weight-bearing swimming test was carried out to assess biochemical biomarkers correlated to fatigue and energy metabolism. UPLC-MS/MS analysis identified 83 flavonoids from MLFE. MLFE significantly increased the swimming time by 60%. Serum lactate (9.9 ± 0.9 vs. 8.9 ± 0.7), blood urea nitrogen (BUN) (8.8 ± 0.8 vs. 7.2 ± 0.5), and nonesterified fatty acid (NEFA) (2.4 ± 0.2 vs. 1.7 ± 0.3) were significantly elevated; phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GCK), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression were significantly downregulated; and heme oxygenase 1 mRNA expression was significantly upregulated in muscle after swimming. MLFE supplement significantly decreased serum lactate (8.0 ± 1.0 vs. 9.9 ± 0.9), BUN (8.6 ± 0.4 vs. 8.9 ± 0.8), and NEFA (2.3 ± 0.4 vs. 2.4 ± 0.2) and increased the protein and mRNA expression of GCK, PEPCK, and Nrf2. The enhancement of glucose metabolism and antioxidant function by MLFE contributes partly to its anti-fatigue action.

Abstract P209: The novel angiotensin Alamandine-(1-5) is increased in plasma of pediatric patients with primary nephrotic syndrome

Background and Aim: The Renin Angiotensin System (RAS) has an important role in the pathophysiology of primary nephrotic syndrome (NS). It was previously found that urinary levels of angiotensin converting enzyme 2 (ACE2) are reduced in patients with NS in comparison to health controls. The aim of this study was to measure the novel angiotensin peptide, Alamandine-(1-5), in plasma samples of patients with primary NS and to compare with sex and age-matched controls. Methods: Blood samples of pediatric patients with primary NS (n=11) and of sex and age-matched health controls (n=11) were collected in sterile tubes with 7.5% (w/v) EDTA. After plasma separation and purification by solid phase extraction, the circulating levels of Alamandine and of Alamandine-(1-5) were measured by Mass Spectrometry using multiple reaction monitoring on an ACQUITY I-Class UPLC system coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS/MS Xevo TQ-S). Data were compared between patients and controls and associated with laboratory findings. Continuous variables were presented as mean ± standard error of the mean. Results: A total of 11 patients (7 males) with NS with mean age of 10.65±3.66 were compared to 11 controls (6 males) and with 10.86±4.35 years. In terms of disease response, 5 patients were in partial remission, 5 patients in total remission and only 1 patient was in disease relapse. All patients have normal renal function. Plasma levels of Alamandine-(1-5) were significantly higher in patients when compared to controls (28.03±7.54 vs. 10.68±3.02 pg.mL-1 in controls). Despite a mild decrease in patients, plasma levels of Alamandine did not differ between groups (19.54±6.33 vs 26.09±6.12 pg.mL-1 in controls). No correlations were found between RAS molecules and proteinuria and with the medications in use. However, Alamandine-(1-5) negatively correlated with plasma levels of albumin (r= -0.695; p=0.038). In addition, Alamandine negatively correlated with total cholesterol (r=-0.700; p = 0.036), LDL (r=-0.800; p=0.004), and HDL (r = -0.762; p = 0.038). Conclusion: The novel RAS peptide Alamandine-(1-5) is significantly increased in primary NS and inversely correlated with plasma albumin levels. These findings suggest a potential role of Alamandine-(1-5) in NS

Quantitative analysis of phenolics in Trifolium pratense L. flowers and evaluation of antioxidant activity by sensory

Trifolium pratense L. flowers (TPF) were collected and dried in shade in this study. After extraction in methanol, a diluted solution was applied to the liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) device to determine the bioactive compounds quantitatively. Isoquercitrin (38.64 mg/g extract), coumarin (13.66 mg/g extract), and catechin (12.52 mg/g extract) were verified as major products. Antioxidant activity of TPF was performed using a potentiometric PVC membrane sensor to evaluate 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content of TPF. TPF inhibited the DPPH radical as 31.4 ± 0.02% at the 500-ppm concentration. However, the DPPH activity of gallic acid was determined as 81.43 ± 0.07% and 92.44 ± 0.1% at the TPF concentrations of 12.5 ppm and 25 ppm, respectively. In addition, the total phenolic content was calculated to be 82.4 ± 0.15 mg gallic acid eq/g extract. It was observed that TPF has the potential to be an antioxidant and a valuable source of isoquercitrin, coumarin, and catechin.