Reversed-phase Ultra-high Performance Liquid Chromatography Research Articles

An ion mobility-enabled and high-efficiency hybrid scan approach in combination with ultra-high performance liquid chromatography enabling the comprehensive characterization of the multicomponents from Carthamus tinctorius

Liquid chromatography/mass spectrometry (LC/MS) is extensively applied for the untargeted/targeted analyses of the herbal components, utilizing data-dependent acquisition (DDA) or data-independent acquisition (DIA) to record the fragmentation information useful for the structural elucidation. A new trend recently has emerged by integrating DDA and DIA to render the hybrid scan, which, unfortunately, has rarely been reported. Herein, by using the Vion™ ion-mobility quadrupole time-of-flight mass spectrometer, a hybrid scan strategy (HDMSE-HDDDA) was presented and validated by the untargeted characterization of the multicomponents from Carthamus tinctorius (safflower), in combination with reversed-phase ultra-high performance liquid chromatography (RP-UHPLC). Good chromatographic separation was achieved on an HSS T3 column within 26 min, while HDMSE-MS/MS was used to acquire the collision-induced dissociation MS2 data in the negative mode. Automatic workflows (e.g., data correction, precursors/product ions matching, and peak annotation) were well established on UNIFI™ (incorporating an in-house library recording 261 known compounds) to process the obtained MS2 data. Compared with single DDA or DIA, the hybrid approach of HDMSE-HDDDA better balanced between the coverage and reliability, led to high-definition MS spectra, offered useful collision cross section (CCS) information, and showed satisfactory identification performance comparable to MSE. A total of 141 components (involving 41 quinochalcones, 66 flavanols/flavones, 11 flavanones, 6 organic acids, 1 polyacetylene, and 16 others) were characterized from safflower. Moreover, CCS prediction could assist isomers characterization, to some extent. Conclusively, this hybrid scan approach enables a dimension-enhanced MS data acquisition strategy providing the complementary structural information, which more suits the chemical characterization of complex samples.

Stability-Indicating UHPLC Method for the Determination of Desvenlafaxine: Application to Degradation Kinetics

This study was aimed to investigate the degradation behavior and physicochemical stability of desvenlafaxine using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) system. The chromatogram was developed on Eclipse XDB-C8 column (150 x 4.6 mm, 5μm). The eluents were monitored through a photo diode array plus (PDA+) detector at 210 nm using an isocratic method with a flow rate of 1.5 ml/min. Mobile phase composition was 30:70 v/v mixture of 0.1 % trifluoroacetic acid (TFA) in water and methanol. Forced degradation studies were performed on drug substance of desvenlafaxine as per International Conference on Harmonization (ICH) prescribed stressed conditions (Q1A(R2) and Q1B) using hydrolytic (acidic, basic, and neutral), oxidative and photolytic methods. The drug substance was found highly labile to acidic (0.5 N hydrochloric acid, 18.65 % degradation in 2 hours at 70°C), basic (1.0 N sodium hydroxide, 11.01 % degradation in 12 hours at 70°C) and oxidative (3 % hydrogen peroxide, 17.05 % degradation in 2 hours at 50°C) stressed conditions, but a great resistance was observed towards dry heat (maximum degradation 0.27 % in 10 days from ambient to higher temperature, 80°C), moist heat (maximum degradation 0.25 % in 2 hours at 80°C and 75 % relative humidity) as well as in photolytic degradation (maximum degradation 0.23 % in 10 days at UV light of 315 - 400 nm). A pseudo-first order kinetic was followed in acidic, basic and peroxide degradation methods which paved a way to calculate the half-life of the drug substance desvenlafaxine under ICH mentioned stressed conditions. The results were also statistically analyzed and the % RSD values were compared with recommended guidelines. Dhaka Univ. J. Pharm. Sci. 20(2): 167-176, 2021 (December)

Multilayered solid phase extraction and ultra performance liquid chromatographic method for suspect screening of halogenated pharmaceuticals and photo-transformation products in freshwater - comparison between data-dependent and data-independent acquisition mass spectrometry

Since conventional biological wastewater treatments are not admittedly effective to convert pharmaceutical active compounds (PhACs) into nontoxic products, natural abiotic mechanisms such as solar photolysis arises as an important degradation process, especially for halogenated molecules. In the present work, photolysis simulation was carried out in-lab for precursors and their respective photo-transformation products (photo-TPs), which were analyzed through reversed-phase ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (RP-UHPLCHRMS). An in-house library was created in order to provide reference information for target (precursors) and suspect screening (photo-TPs) analysis of freshwater samples from impacted aquatic environments. Strategies in the use of data-dependent acquisition (DDA) and data-independent acquisition (DIA), as well as the data processing software are discussed here for the identification of 6 PhACs and photo-TPs. Because no standards of photo-TPs were available, only the target compounds, i.e. sitagliptin (398 ± 2 ng L−1), iohexol (209 ± 5 ng L−1), lamotrigine (103 ± 10 ng L−1), losartan (43 ± 10 ng L−1), ofloxacin (28 ± 7 ng L−1), and sertraline (25 ± 7 ng L−1) could be quantified through multiple standard additions.

Development and Validation of a Stability-Indicating Reversed-Phase Ultra High Pressure Liquid Chromatography Method for Assay of Delmopinol Hydrochloride and Estimation of Its Related Substances in Commercial Bulk Batches.

Delmopinol hydrochloride is widely used as an active ingredient (AI) in many human oral hygiene products. To the best of our knowledge (via literature search), there is no stability-indicating liquid chromatography method available in the public domain for assay and estimation of related substances of Delmopinol hydrochloride. A fast stability-indicating Reversed-Phase Ultra High-Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for identification, assay and estimation of related substances in commercial bulk batches of this AI. The major peak of the AI and its related compounds are adequately separated in 6min by a gradient elution on a hybrid silica-based C18 column (Waters Acquity UPLC® BEH C18, 50mm × 2.1mm I.D., 1.7μm particle size) maintained at 50°C. Mobile phase-A is composed of aqueous 10mM NH4OH and mobile phase-B is ACN. The AI and its related compounds are detected with UV detection at 220nm and quantitated by an external Delmopinol hydrochloride reference standard. The quantitation limit of the method is 0.1% of the target analytical concentration. This UHPLC method is fast and green and has been demonstrated to be specific, accurate, linear, precise, sensitive and robust as per ICH guidelines.

Highly sensitive quantification of pemetrexed in human plasma using UPLC-MS/MS to support microdosing studies.

Pemetrexed is an antifolate drug approved for the treatment of non‐small‐cell lung cancer and mesothelioma. Assessing pemetrexed pharmacokinetics after administration of a microdose (100 μg) may facilitate drug–drug interaction and dose individualization studies with cytotoxic drugs, without causing harm to patients. Therefore, a highly sensitive bioanalytical assay is required.A reversed‐phase ultra‐high performance liquid chromatography method was developed to determine pemetrexed concentrations in human ethylenediaminetetraacetic acid–plasma after microdosing. [13C5]‐Pemetrexed was used as the internal standard. The sample preparation involved solid‐phase extraction from plasma. Detection was performed using MS/MS in a total run time of 9.5 min.The assay was validated over the concentration range of 0.0250–25.0 μg/L pemetrexed. The average accuracies for the assay in plasma were 96.5 and 96.5%, and the within‐day and between‐day precision in coefficients of variations was <8.8%. Extraction recovery was 59 ± 1 and 55 ± 5% for pemetrexed and its internal standard. Processed plasma samples were stable for 2 days in a cooled autosampler at 10°C.The assay was successfully applied in a pharmacokinetic curve, which was obtained as a part of an ongoing clinical microdosing study.

AMINO ACID PROFILING IN EDIBLE SHOOTS OF SOME BAMBOO SPECIES BY USING RP-UHPLC AND THEIR THERAPEUTICAL ASPECTS

Objective: This study was conducted to assess the amino acid profiles of edible shoots of five species of bamboos growing in various geographical regions of India. Methods: Qualitative and quantitative estimation of free amino acids was done by Reverse Phase-Ultra High Performance Liquid Chromatography (RP-UHPLC). Separation of amino acids was carried out using Zorbax eclipse plus C18 Reverse Phase (1.8 μm), 50 mm × 2.1 mm column on quaternary UHPLC system using modified gradient elution. Derivatization was done by using auto-sampler Agilent’s 1290 infinity. Data assortment and processing were accomplished by using Open Lab Chromatography Data System EZchrome edition, version A.04.06 (Agilent technologies, USA). Results: In the study, 19 free amino acids were identified, eight essential, five conditionally essential, and six non-essential amino acids (EAA). The principal amino acids were tyrosine (29.41–74.64 μg/mg d.w.) and asparagine (12.81–111.04 μg/mg d.w.). The amount of EAAs ranged from 0.46 to 21.11 μg/mg d.w. For the 1st time, taurine and ornithine are reported in bamboo shoots. The analysis exhibited significant variation (p&lt;0.05) in free amino acids content among the five studied species. Of the five species, Phyllostachys mannii was best compared to the other species in amino acid content. Conclusion: Results showed superior amino acid composition of bamboo shoots that can be utilized to improve nutritional value of functional foods. Amino acids have various health and therapeutic properties can contribute to the basis for further expansion of these bamboos and imminent potential in applications of food and pharmaceuticals.

Effect of pH on the Antimicrobial Activity and Peptide Population of Pepsin Hydrolysates Derived from Bovine and Porcine Hemoglobins

Slaughterhouse blood is considered a valuable by-product of the meat industry that is mainly derived from the slaughter of cattle and pigs. Its cruor fraction is mainly constituted of hemoglobin, which has been widely described as an important source of several antimicrobial peptides that can be released by hydrolysis with pepsin. Despite the high sequence similarity between bovine (Hb-B) and porcine (Hb-P) hemoglobins, the release of antimicrobial peptides may be affected by varying hydrolysis conditions. This study presents, for the first time, the impact of pH (pH 2, 3, 4, and 5) on the antimicrobial peptide population of hydrolysates derived from the main hemoglobin sources by using pepsin (bovine and porcine). In the same way, their antibacterial, antifungal, and antiyeast activities are compared, as well as their type of mechanism of action (static or cidal effect). These characteristics have not been described previously in the literature. A total of 29 peptides from Hb-B and 35 peptides for Hb-P with potential antimicrobial peptides were identified by reversed-phase ultrahigh-performance liquid chromatography with tandem mass spectrometry (RP-UPLC–MS/MS) and bioinformatics tools. Similarities in the peptide population between Hb-B and Hb-P hydrolysates were observed by PCA, mainly in the enzymatic hydrolysis at pH 2 and pH 3. Both bovine and porcine hydrolysates produced at pH 2 and pH 3 showed antibacterial activity against L. ivanovii (MIC = 0.62–1.25 mg·mL–1) and antiyeast activity against R. mucilaginosa (0.31–1.25 mg·mL–1). Conversely, only antifungal activity against M. racemosus in the porcine hydrolysate at pH 3 was observed (1.25 mg·mL–1). This study confirms the similarities of Hb-B and Hb-P in terms of peptide profiles and antimicrobial activity, which indicates they are suitable sources of antimicrobial peptides with antibacterial, antifungal, and antiyeast activities that could be employed in the preservation of food products.

Effects of water deficit and high N fertilization on wheat storage protein synthesis, gluten secondary structure, and breadmaking quality

The content and composition of wheat storage proteins are the major determinants of dough rheological properties and breadmaking quality and are influenced by cultivation conditions. This study aimed to investigate the effects of water deficit and high N-fertilizer application on wheat storage protein synthesis, gluten secondary structure, and breadmaking quality. Reverse-phase ultrahigh-performance liquid chromatography analysis showed that storage protein and gluten macropolymer accumulation was promoted under both independent applications and a combination of water-deficit and high N-fertilizer treatments. Fourier-transform infrared spectroscopy showed that water deficit and high N-fertilizer treatments generally improved protein secondary structure formation and lipid accumulation, and reduced flour moisture. In particular, high N-fertilizer application increased β-sheet content by 10.4% and the combination of water-deficit and high N-fertilizer treatments increased random coil content by 7.6%. These changes in gluten content and secondary structure led to improved dough rheological properties and breadmaking quality, including superior loaf internal structure, volume, and score. Our results demonstrate that moderately high N-fertilizer application under drought conditions can improve gluten accumulation, gluten secondary structure formation, and baking quality.

PSIV-13 Cell Wall Bound Ferulic and Coumaric Acids in Diets Based on Corn and Soybean Meal or Corn, Soybean Meal, and Distiller Dried Grains with Solubles Are Poorly Fermented in the Intestinal Tract of Pigs

Abstract Phenolic acids are bound to cell wall polymers in corn and distillers dried grains with solubles (DDGS) and contribute to the low digestibility of fiber in corn-based ingredients. Phenolic acids link arabinoxylans to lignin and may substitute sidechains of arabinoxylans, which precludes fermentation. To develop enzymes to aid in separating phenolic acids from arabinoxylans, the natural fermentation of phenolic acid in the intestinal tract of pigs needs to be known. Therefore, an experiment was conducted to test the hypothesis that phenolic acids are poorly fermented by pigs. A corn-soybean meal (SBM) diet and a corn-SBM-DDGS diet were fed to 24 pigs (initial body weight: 61.71 ± 5.39 kg) in a randomized complete block design with 4 blocks and a total of 12 pigs per diet. Diets were fed for 22 d; feces were collected during the last 5d. Concentrations of phenolic acids were analyzed in diets and feces using reversed phase ultrahigh-performance liquid chromatography coupled with mass spectrometry. Data were analyzed using the PROC MIXED of SAS with pig as the experimental unit. Results indicated that concentrations of bound phenolic acids were greater (P &amp;lt; 0.05) in feces from pigs fed the corn-SBM-DDGS diet than from pigs fed the corn-SBM diet (Table 1). Disappearance (%) of free coumaric acid and bound ferulic acid in the intestinal tract of pigs was not different between the 2 diets. In contrast, disappearance of bound coumaric acid was greater (P &amp;lt; 0.05) from the corn-SBM diet than from the corn-SBM-DDGS diet, but disappearance of bound ferulic acid and bound coumaric acid was less than 50% confirming that phenolic acids may hinder fermentation. In conclusion, ferulic acid and coumaric acid appear to be barriers for fermentation of arabinoxylans in pigs and enzymes that release phenolic acids may be needed to increase fermentation of corn fiber.

Direct enantioselective gradient reversed-phase ultra-high performance liquid chromatography tandem mass spectrometry method for 3-hydroxy alkanoic acids in lipopeptides on an immobilized 1.6μm amylose-based chiral stationary phase.

3-Hydroxy fatty acids are important chiral building blocks of lipopeptides and metabolic intermediates of fatty acid oxidation, respectively. The analysis of the stereochemistry of such biomolecules has significant practical impact to elucidate and assign the enzymatic specificity of the biosynthesis machinery. In this work, a new mass spectrometry compatible direct chiral ultra high performance liquid chromatography separation method for 3-hydroxy fatty acids without derivatization is presented. The application of amylose tris(3,5-dimethylphenyl carbamate) based polysaccharide chiral stationary phase immobilized on 1.6μm silica particles (CHIRALPAK IA-U) allows the enantioseparation of 3-hydroxy fatty acids under generic electrospray ionization mass spectrometry friendly reversed phase gradient elution conditions. Adequate separation factors were achieved with both acetonitrile and methanol as organic modifiers, covering hydrocarbon chain lengths between C6 and C14 . Elution orders were derived from rhamnolipid (R-95) of which enantiomerically pure or enriched (R)-3-hydroxy fatty acids were recovered after ester hydrolysis. The S-configured acids consistently eluted before the respective R-enantiomers. The method was successfully applied for the elucidation of the absolute configuration of 3-hydroxy fatty acids originating from a novel lipopeptide with unknown structure. The work furthermore demonstrates that gradient elution is a viable option also in enantioselective (ultra)high performance liquid chromatography, even for analytes with modest separation factors, although less commonly exploited.

Quantitative determination and characterization of polyphenols from Cissus quadrangularis L. and dietary supplements using UHPLC-PDA-MS, LC-QToF and HPTLC

Stem and leaf of Cissus quadrangularis L. (Vitaceae), indigenous to Asia and Africa, were used for medicinal and dietary purposes with limited information about the plant's phytochemistry. Stem and leaf samples were assessed for the simultaneous determination of polyphenolic compounds (catechin, epicatechin, quercetin-3-O-β-glucopyranoside, kaempferol-3-O-β-glucoside, quercetin-3-O-β-rhamnoside, leachianol F, amurensin A, pallidol, resveratrol, and quadrangularin A), using UHPLC-PDA-MS. The validation data showed that the method is precise, specific, accurate, and linear over the range of 0.5−100 μg/mL. Reversed-phase ultra-high performance liquid chromatography (UHPLC) fingerprints of the crude methanolic stem and leaf extracts of C. quadrangularis were obtained at different wavelengths based on their λmax. Polyphenolics were characterized using both UHPLC-PDA-MS and LC-QToF analysis. From liquid chromatography quadrupole time of flight-electrospray ionization mass spectrometry (LC-QToF) spectra, over 40 components were structurally correlated, and confirmation was based on the fragmentation characteristics and also from the information available in the literature. In addition to the LC-QToF method, a simple, fast HPTLC method was developed as a visual aid for the rapid qualitative analytical tool to help establish the quality assessment of botanical raw materials and dietary supplements claiming to contain Cissus.

The analysis of chromatographic behavior of homoandrostane derivatives in reversed-phase ultra-high performance liquid chromatography

Homoandrostane derivatives, as compounds with significant bioactivity, were studied in terms of their chromatographic behavior in reversed-phase ultra-high performance liquid chromatography (RP-UHPLC). In the present study, five androstane derivatives from the series of homoandrostanes were analyzed, including: 3?-hydroxy-17-oxa-17a-homoandrost-5-en-16-one, 3?,5?-dihydroxy-17- oxa-17a-homoandrostane-6,16-dione, 17-oxa-5?,6?-epoxy-17a-homoandrostane-3,16-dione, 5?-hydroxy- 17-oxa-17a-homoandrostane-6,16-dione-3?-yl acetate and 3?-hydroxy-17-oxa-5?,6?-epoxy-Dhomoandrostan- 16-one. The compounds were analyzed by applying methanol-water mobile phases with different volume fractions of methanol, as a polar protic solvent, and logk0 parameters of each compound were determined. The outstanding correlations between in silico logP descriptors and logk0 parameters were obtained, as well as between in silico logD descriptors and logk0 parameters. The logk0 parameters are very well correlated with polar surface area (PSA) descriptor as well. The studied compounds and lipophilicity descriptors (including the chromatographic lipophilicity parameters - logk0) were clustered applying hierarchical cluster analysis (HCA) in the form of clustered heat map known as double dendrogram. Furthermore, the sum of ranking differences (SRD) method was used for the ranking of the lipophilicity measures of the analyzed homoandrostane derivatives so the most suitable lipophilicity measures of this series of compounds can be selected.

Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV.

Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.

Holistic quality evaluation of Saposhnikoviae Radix (Saposhnikovia divaricata) by reversed-phase ultra-high performance liquid chromatography and hydrophilic interaction chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry-based untargeted metabolomics

Untargeted metabolomics more suits the quality evaluation of TCM because of its holistic property. To assess the holistic quality difference of Saposhnikoviae Radix (the roots of Saposhnikovia divaricata), we integrate ultra-high-performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay. A BEH C18 column in the reversed-phase mode and a BEH Amide column in Hydrophilic Interaction Chromatography (HILIC) mode were utilized for metabolites profiling, which enabled high coverage of the non-polar to polar components in Saposhnikoviae Radix. Moreover, the application of major components knockout strategy enlarged the exposure of those minor components. Integrated use of high-definition MSE (HDMSE) and data-dependent acquisition (DDA) could enhance the metabolites characterization by providing reliable fragmentation information and collision cross section values. Computational in-house library-driven automated peak annotation of the HDMSE and DDA data assisted to characterize 104 components from Saposhnikoviae Radix. Chemometric analyses of the commercial Saposhnikoviae Radix samples (64 batches collected from 11 cultivars aging from 1 to 4 years), based on the positive MSE data, in general could indicate large discrimination between Guan-Fang-Feng (from Heilongjiang) and the others, but negligible difference among Saposhnikoviae Radix from the other ten provinces of China and with different ages. Quantitative assays of prim-O-glucosylcimifugin and 4′-O-β-D-glucosyl-5-O-methylvisamminol, by a rapid and fully validated UHPLC-UV method, could primarily deduce that Guan-Fang-Feng aging 2 and 3 years exhibited better quality. The methods established can holistically assess the quality of TCM with wide spans of plant metabolites covered.

Comprehensive off-line CCC × LC-DAD-MS separation of Cyclopia pubescens Eckl. & Zeyh. phenolic compounds and structural elucidation of isolated compounds.

The minor phenolic constituents of Cyclopia pubescens Eckl. & Zeyh. are unknown and one dimensional (1D) liquid chromatography (LC) is unable to provide sufficient separation. A two-dimensional (2D) LC method incorporating normal-phasehigh performance countercurrent chromatography (NP-HPCCC) in the first dimension (1 D) and reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) as the second dimension (2 D) was developed. The analytical HPCCC method was subsequently scaled up to semi-preparative mode and fractions pooled based on phenolic sub-groups. The phenolic compounds in selected fractions were subsequently isolated using RP-HPLC on a C18 column. Isolated compounds were identified by nuclear magnetic resonance (NMR) spectroscopy. The absolute configurations of compounds were determined by optical rotation and electronic circular dichroism spectra. Sugars were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The comprehensive off-line 2D CCC × LC method gave a good spread of the phenolic compounds. Orthogonality calculated using both the convex hull and conditional entropy methods were 81%. High-resolution mass spectrometric fragmentation spectra obtained from a quadrupole-time-of-flight instrument and ultraviolet-visible (UV-vis) spectral data were used to (tentatively) identify 32 phenolic compounds from the analytical CCC fractions. Of the seven isolated compounds, (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl]eriodictyol (3) and (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl]-5,7,3',4'-tetrahydroxyflavan (4) were newly identified in all plants. The other isolated compounds were identified as (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl]naringenin (1), R-neo-eriocitrin (2), 3-O-α-l-arabinopyranosyl-3,4-dihydroxybenzoic acid (5), 4-O-β-d-glucopyranosyl-Z-4-hydroxycinnamic acid (6) and 4-(4'-O-β-d-glucopyranosyl-4'-hydroxy-3'-methoxyphenyl)-2-butanone (7). Among the 32 compounds (tentatively) identified, only six were previously identified in Cyclopia pubescens using 1D LC. Most of the isolated compounds were also identified for the first time in Cyclopia spp., improving the knowledge of the minor phenolic compounds of this genus.

Development of a rapid and accurate UHPLC-PDA-FL method for the quantification of phenolic compounds in grapes

Grapes are a great source of phenolic compounds, which have excellent antioxidant properties. Efficient analytical methods are necessary to selectively and precisely determine these compounds content in grapes. In this study, a reverse-phase ultra-high performance liquid chromatography (UHPLC) method with fluorescence and photodiode array detection has been developed to determine and quantify 27 of the main phenolic compounds present in grapes. An ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm i.d., 1.7 mm particle size) column was employed. A gradient method was developed and column temperature (25–55 °C), as well as flow rate (0.6–0.75 mL min−1), were optimized. The optimum conditions allowed the separation of all the compounds in less than 9 min. The method was validated and demonstrated excellent detection and quantification limits, precision, and selectivity. Finally, several grape varieties were studied in order to demonstrate the applicability of the method to the analysis of real matrix samples.

Development of a rapid reversed-phase liquid chromatographic method for total free thiol quantitation in protein therapeutics

Free thiols, or unpaired cysteines, are important product quality attributes in the therapeutic proteins due to their potential impact on the protein structure, bioactivity and stability. While many free thiol quantitation methods were developed for specific therapeutic formats, an unmet need still exists for a multiproduct, high-throughput method for free thiol quantitation. In this study, a workflow was established that combines N-cyclohexylmaleimide (NcHM) derivatization and high-throughput reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) separation with superficially porous particle (SPP) column for quantitating total free thiols in monoclonal antibodies (mAbs), fragment antigen-binding (Fab), and bispecific antibodies (BsAbs). The NcHM derivatization increases the hydrophobicity of the free thiol variants and allows the further separation and quantitation with RP-UHPLC. A thorough evaluation of sample preparation, column selection, chromatographic condition and LC–MS peak identification was performed to optimize and characterize the method outputs. Method optimization resulted in a 42-minute total analysis time. Method qualification demonstrated suitable accuracy, precision, linearity, specificity and robustness. This high-throughput method is not only used for quantitation of total free thiols for both in-process testing and drug substance/drug product batch testing, but also for provide the positional distribution of the free thiols in the protein.

Monoaminergic and Kynurenergic Characterization of Frontotemporal Dementia and Amyotrophic Lateral Sclerosis in Cerebrospinal Fluid and Serum

Exploring the neurochemical continuum between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) with respect to monoamines and kynurenines in cerebrospinal fluid (CSF) and serum, may be useful to identify possible new research/therapeutic targets. Hence, we analysed monoamines and kynurenines in CSF and serum derived from patients with FTD (n = 39), ALS (n = 23), FTD-ALS (n = 4) and age-matched control subjects (n = 26), using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) with electrochemical detection (ECD) and liquid chromatography tandem mass spectrometry, respectively. We noted a shared dopaminergic disturbance in FTD and ALS when compared to CONTR, with significantly increased serum DA levels and decreased DOPAC concentrations, as well as decreased DOPAC/DA ratios in both disease groups. In CSF, significantly reduced DOPAC concentrations in FTD and ALS were observed as well. Here, a significant increase in DA levels and decrease in DOPAC/DA ratios was only found in FTD relative to CONTR. With respect to the kynurenine pathway (KP), we only found decreased HK/XA ratios, indicative for vitamin B6 status, in serum of ALS subjects compared to FTD. The dopaminergic commonalities observed in FTD and ALS might relate to a disturbance of dopaminergic nerve terminals in projection areas of the substantia nigra and/or ventral tegmental area, although these findings should first be confirmed in brain tissue. Lastly, based on the results of this work, the KP does not hold promise as a research/therapeutic target in FTD and ALS.

Modulation of Glucosinolate Composition in Brassicaceae Seeds by Germination and Fungal Elicitation.

Glucosinolates (GSLs) are of interest for potential antimicrobial activity of their degradation products and exclusive presence in Brassicaceae. Compositional changes of aliphatic, benzenic, and indolic GSLs of Sinapis alba, Brassica napus, and B. juncea seeds by germination and fungal elicitation were studied. Rhizopus oryzae (nonpathogenic), Fusarium graminearum (nonpathogenic), and F. oxysporum (pathogenic) were employed. Thirty-one GSLs were detected by reversed-phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP-UHPLC-PDA-ESI-MSn). Aromatic-acylated derivatives of 3-butenyl GSL, p-hydroxybenzyl GSL, and indol-3-ylmethyl GSL were for the first time tentatively annotated and confirmed to be not artifacts. For S. alba, germination, Rhizopus elicitation, and F. graminearum elicitation increased total GSL content, mainly consisting of p-hydroxybenzyl GSL, by 2–3 fold. For B. napus and B. juncea, total GSL content was unaffected by germination or elicitation. In all treatments, aliphatic GSL content was decreased (≥50%) in B. napus and remained unchanged in B. juncea. Indolic GSLs were induced in all species by germination and nonpathogenic elicitation.

Data-Dependent Acquisition and Database-Driven Efficient Peak Annotation for the Comprehensive Profiling and Characterization of the Multicomponents from Compound Xueshuantong Capsule by UHPLC/IM-QTOF-MS.

The state of the art ion mobility quadrupole time of flight (IM-QTOF) mass spectrometer coupled with ultra-high performance liquid chromatography (UHPLC) can offer four-dimensional information supporting the comprehensive multicomponent characterization of traditional Chinese medicine (TCM). Compound Xueshuantong Capsule (CXC) is a four-component Chinese patent medicine prescribed to treat ophthalmic disease and angina. However, research systematically elucidating its chemical composition is not available. An approach was established by integrating reversed-phase UHPLC separation, IM-QTOF-MS operating in both the negative and positive electrospray ionization modes, and a “Component Knockout” strategy. An in-house ginsenoside library and the incorporated TCM library of UNIFITM drove automated peak annotation. With the aid of 85 reference compounds, we could separate and characterize 230 components from CXC, including 155 ginsenosides, six astragalosides, 16 phenolic acids, 16 tanshinones, 13 flavonoids, six iridoids, ten phenylpropanoid, and eight others. Major components of CXC were from the monarch drug, Notoginseng Radix et Rhizoma. This study first clarifies the chemical complexity of CXC and the results obtained can assist to unveil the bioactive components and improve its quality control.