Using Ultra-high Performance Liquid Chromatography Research Articles

Simultaneous Determination of Pesticides and Veterinary Pharmaceuticals in Environmental Water Samples by UHPLC–Quadrupole-Orbitrap HRMS Combined with On-Line Solid-Phase Extraction

Pesticides and veterinary pharmaceuticals are used for effective crop production and prevention of livestock diseases; these chemicals are released into the environment via various pathways. Although the chemicals are typically present in trace amounts post-release, they could disturb aquatic ecosystems and public health through resistance development toward drugs or diseases, e.g., reproductive disorders. Thus, the residues of pesticides and veterinary pharmaceuticals in the environment must be managed and monitored. To that end, we developed a simultaneous analysis method for 41 target chemicals in environmental water samples using ultra-high-performance liquid chromatography (UHPLC)–quadrupole-orbitrap high-resolution mass spectrometry (HRMS) coupled with an on-line solid-phase extraction system. Calibration curves for determining linearity were constructed for 10–750 ng∙L−1, and the coefficient of determination for each chemical exceeded 0.99. The method’s detection and quantitation limits were 0.32–1.72 ng∙L−1 and 1.02–5.47 ng∙L−1, respectively. The on-line solid-phase extraction system exhibited excellent method reproducibility and reduced experimental error. As the proposed method is applicable to the monitoring of pesticides and veterinary pharmaceuticals in surface water and groundwater samples acquired near agricultural areas, it allows for the management of chemicals released into the environment.

The measurement of KRAS G12 mutants using multiplexed selected reaction monitoring and ion mobility mass spectrometry.

RationaleThere is a considerable clinical demand to determine key mutations in genes involved with cancer which necessitates the deployment of highly specific and robust analytical methods. Multiplex liquid chromatography with selected reaction monitoring (LC/SRM) assays offer the ability to achieve quantitation down to levels expected to be present in clinical samples. Ion mobility mass spectrometry (IMS/MS) assays can provide increased peak capacity and hence separation in an extremely short time frame, and in addition provide physicochemical data regarding the collision cross‐section of an analyte which can be used in conjunction with the m/z value of an ion to increase detection specificity.MethodsFor LC/SRM, unlabelled peptides and corresponding stable‐isotope‐labelled standards were spiked into digested human plasma and analysed using ultrahigh‐performance liquid chromatography (UHPLC) coupled to a triple quadrupole mass spectrometer to enable the generation of analyte‐specific calibration lines. Synthetic unlabelled peptides were infused into a Synapt G2 mass spectrometer for travelling wave ion mobility separation and TWCCSN2 values were derived from comparison with previously generated TWCCSN2 calibration values.ResultsLinear calibration lines (0.125 to 25 fmol/μL) were established for each of the KRAS peptides. UHPLC separated the peptides and hence enabled them to be split into different retention time functions/windows. This separation enabled detection of three or four transitions for each light and heavy peptide with at least 10 points per peak for accurate quantitation. All six KRAS G12 peptides were separated using IMS/MS, enabling precise TWCCSN2 values to be determined. Although some of the G12 peptides chromatographically co‐eluted, all the peptides were distinguished by m/z, retention time and/or drift time.ConclusionsThis study advocates that LC/SRM and IMS/MS could both be used to identify single amino acid substitutions in KRAS as an alternative to commonly used methods such as circulating tumour DNA analysis.

Studying method of underivatized determination glyphosate, glufosinate and their metabolites in drinking water

AbstractIn Vietnam, glyphosate, one of the most popular herbicides, has recently been prohibited in agriculture due to indiscriminate use and its negative effect on the environment and human's health. Direct analysis and determination glyphosate, glufosinate and their metabolites in environmental samples such as water has get in numerous difficulties due to their simple structure, high polarity and complex formation with different components in the matrices. Therefore, in this research study, we have initially developed a method using ultra‐high performance liquid chromatography (UPLC) coupled with high resolution mass spectrometry with Q‐Orbitrap mass analyzer to analyze anionic compounds such as glyphosate, glufosinate and their metabolites. The use of this system provides low limit of detection (LOD) and quantification (LOQ) of method were 0.003 mg/kg and 0.015 mg/kg, respectively. With respect of sample treatment, we combined C18 and MAX materials in order to obtain the most effective clean up section and high recovery varying from 70 to 120 %.

Phenolic Profiles of Leaves, Grapes and Wine of Grapevine Variety Vranac (Vitis vinifera L.) from Montenegro

Vranac, an old autochthonous red grapevine variety of Montenegro, was first mentioned in a historical document published in the 15th century. As currently the study of indigenous varieties is of particular importance, the subject of this work was detailed characterization of phenolic compounds in the autochthonous grapevine variety Vranac, from the Montenegrin Podgorica subregion. Phenolic profiles of leaves, berries (skin, seeds, and pulp were examined separately) and young monovarietal wine were determined using ultra-high performance liquid chromatography (UHPLC) with linear trap quadrupole (LTQ)—Orbitrap XL mass spectrometry (MS). Total phenolic content (TPC) and radical scavenging activity (RSA) were higher for the grape seeds extracts, followed by extracts of grape skins and pulps. As expected, the total anthocyanin content (TAC) was higher in grape skin than in wine. A total of one hundred twenty nine compounds (forty two phenolic acids and their derivatives, twenty three flavan-3-ols, twenty one flavanols, five stilbenes and thirty eight anthocyanins) were identified in the investigated extracts. To our best knowledge, this is the first report of tentative identification of (epi)catechin 3-O-coumarate in grape seed and chalcan-flavan 3-ol dimers in wine and grape seed.

Method development and validation for rapid identification of epigallocatechin gallate using ultra-high performance liquid chromatography.

Although Epigallocatechin gallate (EGCG) is the most available and beneficial catechin found in tea, its auto-oxidation property may lead to toxicity when consumed in large quantities. Thus, there is a need to quantify the EGCG, which enables to study the pharmacological characteristics of the compound. The study aimed to develop and validate a rapid and accurate analytical method for quantitative determination of EGCG. Standard EGCG was used to conduct trials for the optimization of the analytical method using Ultra-High Performance Liquid Chromatography (UHPLC). Tests for validation (specificity, linearity, accuracy, system suitability, method precision, robustness, and ruggedness) were performed. The preliminary trials yielded an analytical method with good peak shape and acceptable system suitability which was further validated. The method was shown to be specific, with a linear correlation coefficient of > 0.9996 and accurate with acceptable recovery rate (99.1% to 100.4%). Acceptable system suitability and method precision were confirmed with a relative standard deviation (less than 2%). Further, robustness and ruggedness experiments also demonstrated the suitability of the present analytical method. The method developed for determination of EGCG was validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and thus can be used in routine compliance tests in the laboratory for further studying/characterizing the properties of EGCG.

Identification and quantification of cytotoxic phenolic acids and flavonoids in Ixora brachiata by UHPLC-DAD and UHPLC-ESI-MS/MS

Ixora brachiata a plant with ethnomedicinal value is used in the treatment of skin diseases. The present study was carried out to evaluate the cytotoxic effect of methanolic extract of I. brachiata leaf samples against human breast (MDA-MB-231) and pancreatic (PANC-1) cancer cell lines. An attempt was also made to identify and quantify the phenolic acids and flavonoids using ultra high performance liquid chromatography (UHPLC) – diode array detector (DAD) coupled with electrospray ionization mass spectroscopy (UHPLC-ESI-MS/MS). The study revealed dose dependant cytotoxicity with 50% inhibitory (IC50) effect of I. brachiata at a concentration of 115.77 and 80 μg/ml against MDA-MB-231 and PANC-1 cells respectively. The IC50 value of cisplatin was recorded at a concentration of 19.76 and 6.22 μg/ml against MDA-MB-231 and PANC-1 cells respectively. UHPLC-DAD and UHPLC-ESI-MS/MS analysis showed the presence of 13 phenolic acids and 10 flavonoids. Quantitative determination of the compounds revealed the presence of significantly high quantity of protocatechuic acid (20.19 ± 0.17 mg/g) with a total yield of 58.55 ± 0.9 mg/g of phenolics. Similarly, a significantly high quantity of myrcetin (5.03 ± 0.02 mg/g) was detected as a major flavonoid out of the total yield of 5.17 ± 0.02 mg/g. The presence of phenolic acids and flavonoids in the leaf methanol extract of I. brachiata might have contributed towards the cytotoxic effect of this plant. However, further investigations are warranted to confirm the specific phenolic acid/flavonoid responsible for the cytotoxic effect of this plant.

Physicochemical analysis and phenolic profile of polyfloral and honeydew honey from Montenegro.

The research subject of this paper was a detail physicochemical analysis of 28 honey samples from the northern part of Montenegro. The honey from Montenegro has not been previously studied in such detail. Differentiation between samples, such as honeydew honey and polyfloral honey, was based on electrical conductivity, which was higher than 0.8 mS cm−1 for honeydew honey, as was expected. Other investigated physicochemical parameters (water content, free acids, diastase activity, hydroxymethylfurfural (HMF) content and sugar content) have shown great similarity for all honey samples. The main interest of this study was the identification and quantification of phenolic compounds using ultra-high performance liquid chromatography (UHPLC) with mass spectrometry detection. The results show that honey samples are very rich in phenolic compounds, especially quercetin. Among the 31 quantified phenolic compounds, the most dominant were phenolic acids. The highlight was based on p-hydroxybenzoic acid, p-coumaric acid, caffeic acid and ferulic acid. Considering polyphenolic compounds and sugar content, a high nutritional value can be observed in all samples, with an emphasis on polyfloral honeys, as was confirmed with principal component analysis (PCA). In addition, all honey samples were tested for total phenolic content (TPC) and radical scavenging activity (RSA). The results indicate the higher antioxidant ability of honeys from Montenegro in comparison to some honey samples from other countries in the region.

Preparation and Evaluation Pumpkin Seed Oil-based Vitamin E Cream Formulations for Topical Application.

Plant seed oil is often incorporated into the cream emulsions to provide multifunctional effects on the skin. In the current study, pumpkin seed oil (PSO) was used to develop a stable oil-in-water emulsion. The study aimed to optimise PSO cream formulation and determine the synergistic effect of the PSO with vitamin E oil added. The physical properties, antioxidant activities and storage stability of the formulations were analysed. Besides, the synergistic effect of the best formulation was analysed based on α-tocopherol content using ultra-high performance liquid chromatography (UHPLC). The storage stability test was assessed upon storing at 25 ± 2°C and 40 ± 2°C for 12 weeks. The best formulation (20% PSO, vitamin E oil and beeswax) selected showed physically and microbiologically stable. The incorporation of vitamin E oil into the formulation produced with PSO was found to be compatible, as it showed a synergistic effect in the amount of α-tocopherol content (combination index (CI) = 0.98). Thus, PSO had shown its potency to be incorporated into the topical products with a promising potential in delivering additional properties that can nourish the skin.

Analytical Methods Validation of Retinoic Acid and Hydroquinone Using Ultra High Performance Liquid Chromatography in Medicinal Cream

Hydroquinone and retinoic acid have been widely used as whitening agents in cosmetic creams and have a negative impact if used without a medical prescription. In this study, development of analytical method of retinoic acid (REA) and hydroquinone (HYQ) were carried out using Ultra High Performance Liquid Chromatography (UHPLC) with a PDA detector, Phenomenex Luna® ODS / C18 column (150 mmx4.6 mm), methanol-acetic acid 0.1% (85:15) as mobile phase, flow rate of 1.0 mL/min and detected at 295 nm for HYQ and 341 nm for REA. The result of the validation method showed a retention time of HYQ at 1.55 minutes and REA at 3.73 minutes. Validation parameters performed are selectivity, range, linearity, LOD, LOQ, accuracy, and precision. Linear detector response in the concentration range of 75-250 ppm with a calibration curve equation. The calibration curve equation for HYQ is y = 0.4607x + 1.9014 (r = 0.999), mean while REA is y = 2.3138x + 21.0429 (r = 0.999). LOD and LOQ values of HYQ are 1.615 ppm; 13.98 ppm respectively and REA 1.02 ppm and 3.11 ppm, respectively. The precision (% RSD) ≤ ± 2% and accuracy (% recovery) in the range of 99.0% - 100.6%. This method is valid to be used to analyze HYQ and REA in commercial cosmetic cream samples.

Technology optimization of Gardeniae Fructus processed with ginger juice and composition changes after processing

To optimize the technology of Gardeniae Fructus processed with ginger juice,establish fingerprints and simultaneously determine seven compounds( geniposidic acid,chlorogenic acid,genipin-1-β-D-gentiobioside,geniposide,rutin,crocin Ⅰ,and crocin Ⅱ) by using ultra high performance liquid chromatography( UPLC). Waters ACQUITY UPLC BEH C18( 2. 1 mm×50 mm,1. 7μm) column was used with acetonitrile and 0. 1% formic acid solution as mobile phase for gradient elution at the flow rate of 0. 4 m L·min-1. The data was comprehensively processed and analyzed with similarity evaluation,principal component analysis( PCA) and partial least squares discriminant analysis( PLS-DA) methods. Twenty common peaks were identified in this study,and the similarity of samples was over 0. 97. The results of PCA and PLS-DA showed that there were differences in chemical compositions and contents between the raw Gardeniae Fructus and those processed with ginger juice,with 9 potential differentiated chromatographic peaks. After being processed with ginger juice,the contents of chlorogenic acid,crocin Ⅰ and crocin Ⅱ were less than before and the contents of other four compositions were higher than before. The optimized preparation for Gardeniae Fructus processed with ginger juice was stable and feasible. The methods of UPLC fingerprints and simultaneous determination of seven components can be effectively carried out to distinguish Gardeniae Fructus and Gardeniae Fructus processed with ginger juice.

Effervescent salt and crown ether-assisted matrix solid-phase dispersion extraction of coumarins from Cortex fraxini

A simple, rapid and environmentally friendly method named effervescence-assisted matrix solid-phase dispersion (EA-MSPD) was investigated for the extraction of four coumarins (esculin, esculetin, fraxin and fraxetin) from Crotex fraxini (C. fraxini). In this study, an effervescent tablet containing carbon dioxide sources (100 mg sodium bicarbonate and 200 mg sodium dihydrogenphosphate) and an adsorbent (25 mg benzo-15-crown-5) was prepared. The effervescent tablet was dissolved into an aqueous solution, and as a consequence of the effervescence reaction, carbon dioxide was generated, thus making the sample better a dispersion of the extraction solvent (100 mM sodium dodecyl sulfate). The variables that influenced the extraction efficiency included effervescent salts, extraction solvents and crown ether. These values were represented using ultra- high-performance liquid chromatography (UHPLC). Good linearity was exhibited by coefficients of determination all equal to 1, limits of detection ranging from 1.62 to 3.83 ng·mL−1 and limits of quantification ranging from 5.39 to 12.76 ng·mL−1. The recoveries ranged from 91.37 to 100.29% with relative standard deviations of 0.57–4.58% under optimized conditions. In this work, the proposed EA-MSPD method was successfully applied to extract and identify four coumarins in C. fraxini. Compared with previously reported methods, the method examined herein was faster, greener and more sensitive.

Abstract 3895: A phase I-II pharmacokinetic drug-drug interaction evaluation of oral palbociclib in combination with vemurafenib in patients suffering metastatic melanoma with BRAF V600 mutated and CDKN2A loss & expression of Rb

Abstract Background: A novel combination of vemurafenib (VM), a selectif inhibitor of BRAF V600 mutated protein + oral palbociclib (Palbo) a highly selective reversible oral inhibitor of cyclin-dependent kinases (CDK) 4 and 6, could provide a synergistic antitumor activity in patients with metastatic melanoma harbouring BRAF V600 mutation and CDKN2A loss. VM has been shown to be an inductor of CYP3A4 which is also mainly involved in Palbo metabolism. In this study we investigated the potential pharmacokinetic (PK) drug-drug interactions (DDI) related to the association of VM and Palbo. Methods: metastatic melanoma harbouring BRAF V600 mutation and CDKN2A loss patients were treated with a 14 days on followed by 7 days off dosing schedule of Palbo + continuous bid dosing of VM. Dose levels [DL, Palbo (mg/day)/VM (mg/bid)] ranged from 25/720 to 200/960. For PK analysis, 7 time point samples were collected on D1C1, D21C1, and D7C2, for Palbo and VM assays. Plasma concentrations of Palbo and VM, were measured using ultra high performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry validated methods. Population PK (PPK) was modeled using a non linear mixed effect model program (Monolix version 2018r) by computing the maximum likelihood estimator of the parameters without any approximation of the model (no linearization). The following parameters were calculated absorption constant (Ka); apparent distribution volume (V/F); apparent clearance (CL/F) Results: A total of 18 patients were treated by the combination of Palbo + VM. VM and palbo plasma concentrations were measured in 236 and 275 blood samples respectively. A one-compartment open model with linear elimination adequately described Palbo concentration-time courses. The inter-individual variabilities (ISV) could be well estimated for all stuctural parameters. The PPK parameters obtained for the structural model were: Ka = 0.791 h-1, CL/F=76.0L/h, V1/F=2830 L. Albuminemia had a significant impact on palbo CL. A one-compartment model adequately fitted the VM plasma concentration-time data. The PPK parameters were CL/F=0.133 L/h, V/F=83.6 L and Ka has been fixed at 0.2 h-1. Body weight (BW) was the best size descriptor when VM CL and V terms were normalized to a mean BW of 70 Kg according to an allometric scaling rule. Conclusions: The PPK modeling satisfactorily described the plasma Plabo and VM time-concentration curves in patients. The main covariate effect was related to BW and albuminemia. There is no significant evidence for drug-drug PK interaction between Palbo and VM. PK-PD modeling will be performed. Citation Format: Samuel Huguet, Thierry Lesimple, Arthur Geraud, Mouna Amini-Adle, Caroline Dutriaux, Laetitia Da Meda, Florence Capelle, Zineb Ghrieb, Samia Mourah, Olivier Madar, Didier Bouton, Mathieu Resche-Rigon, Celest Lebbe, Keyvan Rezai. A phase I-II pharmacokinetic drug-drug interaction evaluation of oral palbociclib in combination with vemurafenib in patients suffering metastatic melanoma with BRAF V600 mutated and CDKN2A loss & expression of Rb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3895.

Study on the correlation between genetic and chemical diversity of Tetrastigma hemsleyanum on the basis of ISSR and UHPLC

Abstract Tetrastigma hemsleyanum is an endangered edible plant with important medicinal value. The intersimple sequence repeat (ISSR) markers was used to analyze genetic differentiation in thirty-two natural accessions of T. hemsleyanum . Eight phenolic contents of the leaves in these accessions were determined using ultrahigh-performance liquid chromatography (UHPLC). T. hemsleyanum accessions exhibited high level of genetic diversity and considerable UHPLC fingerprint variation. The unweighted pair-group method with arithmetic mean cluster analyses of ISSR and UHPLC data showed nearly identical results. Correlation analysis revealed the close relationships between genetic structure and UHPLC fingerprint patterns (R 2 =0.9928 for genetic distance and UHPLC fingerprint). Meanwhile, the antioxidant activity of T. hemsleyanum leaves connected with the phenolic fingerprint type. Results suggested that the analyzed phenolics are likely the antioxidant ingredients, and the chemical components may closely related to genotype.

Polyphenol Consumption on Human Bile Acids Metabolism: Preliminary Data of Bile Acid Profiles in Human Biological Samples (P06-131-19)

ObjectivesBile acids (BAs) play a critical role in regulating human health through the activation of BAs receptor farnesoid X receptor (FXR) and membrane G protein coupled bile acid receptor-1 (TGR5). We aimed to develop methods to characterize BAs and their metabolites in human biological samples and characterize changes in BAs profile after chronic polyphenol consumption to help guide investigations on the potential health effects of polyphenols via BAs metabolism. MethodsPlasma, fecal and urine samples from two human studies that included berry intake were used for developing qualitative analysis of BAs using ultra high-performance liquid chromatography (UHPLC) coupled with electrospray ionization quadrupole time of flight (QTOF). The compounds were identified based on the exact mass, fragmentation pattern, available reference standards and database search. To investigate the effect of chronic polyphenol consumption on BAs composition, pooled plasma samples (fasting and postprandial 2 h, n = 6) from a chronic (45 and 90 days) strawberry supplementation study (24 g freeze dried/day) with an older population were analyzed. ResultsAmong 106 BAs and their metabolites which were tentatively identified in the samples used for method development, 70, 55, and 47 BAs species were characterized in plasma, feces and urine samples, respectively. The qualitative analysis of BAs in plasma samples from subjects following the strawberry consumption protocol detected 8 primary and 31 secondary BAs. After 90-days strawberry supplementation, two secondary BAs–glycolithocholic acid and 9(11), (5β)-cholenic acid-3α, 12α-diol were decreased to undetectable levels in the pooled fasting sample and the FRX/TGR5 agonists, including chenodeoxycholic acid, deoxycholic acid, cholic acid, glycodeoxycholic acid and taurocholic acid, showed increasing peak areas at 2 h postprandial compared to fasting. ConclusionsThe changes in BAs profiles in fasting and postprandial plasma samples after chronic strawberry feeding suggest that strawberry polyphenols may alter BAs metabolism and the FXR/TGR5 signaling. Funding SourcesThis work was funded by the California Strawberry Commission, USDA Intramural Funds and various donor funds to the Center for Nutrition Research, IIT.

Identification of Phenolic Metabolites in Human Plasma and Urine After Mango Consumption (P06-021-19)

ObjectivesMango contains considerable amounts of phytochemicals, most notably phenolic acids, mangiferin, carotenoids and gallotannins. However, little is known how these compounds are metabolized in the human body. The objective of this study was to identify the phenolic metabolites in human plasma and urine after mango consumption. MethodsBlood and urine samples were collected at baseline (t = 0 h) and then at 2 h, 8 h, 24 h after mango pulp (500 g) consumption by healthy adults. Pooled plasma and urine samples at different time-points (n = 12; age 30.0 ± 7.6 y and BMI 22.4 ± 1.7 kg/m2) were extracted using solid phase extraction procedure. Phytochemicals in mango pulp were extracted using extraction solution (acetone: water: acetic acid = 70: 29.7: 0.3 v/v). Compounds were identified and quantified using Ultra High Performance Liquid Chromatography (UHPLC) system coupled to iFunnel Quadrupole Time of Flight mass spectrometer (MS) and UHPLC system with Triple Quadrupole MS, respectively. Separation of compounds was achieved by reversed phase chromatography and compounds were identified based on mass accuracy (< 5 ppm), specific fragmentation pattern, previous literature reports and co-chromatography when standards were available. ResultsA total of 31 compounds were tentatively identified and quantified in the mango pulp, including mangiferin (16.48 ± 1.13 μg/100 g fresh weight (FW)), catechin (24.50 ± 8.46 μg/100 g FW), and total gallic acid (2024.2 ± 83.8 mg/100 g FW). A total of 59 and 164 phenolic metabolites were tentatively identified at different time-points in plasma and urine, respectively. The compounds detected in 2 h plasma and urine samples were mostly small phenolic acids such as hippuric acid, cinnamic acid, abscisic acid, gallic acid along with their methyl and glucuronide conjugates. Other compounds such as pyrogalloyl sulfate, methyl and glucuronide conjugates, cinnamic acid, phenylacetic acid and benzoic acid derivatives appeared post 8 and 24 h consumption of mango pulp. ConclusionsOur results suggest that mango polyphenols are absorbed and extensively metabolized resulting in the production of various phenolic metabolites and their conjugates, all together contributing to the bioavailability and potential health benefits associated with mango consumption. Funding SourcesNational Mango Board.

An integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides with application to rheumatoid arthritis

Glycosylation plays an important role in the maintenance of the structure and function of glycoproteins, while aberrant protein glycosylation is correlated with various diseases. Human immunoglobulin G (IgG), which is composed of four subclasses (IgG1, IgG2, IgG3 and IgG4), is one of the most dominant and significant glycoprotein in human serum. The glycosylation on IgG-Fc moiety is known to be alternated with various physiological and pathological states. We herein report an integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides. Firstly, IgG N-glycans were profiled by using mAb-Glyco chip quadrupole time-of-flight mass spectrometry (Q-TOF-MS), resulting characterization of 87 N‑glycans originating from 29 different oligosaccharide compositions. Secondly, subclass-specific glycopeptides were identified on the basis of high-resolution MS and MS/MS data by using ultra high performance liquid chromatography (UHPLC) coupled to Q-TOF-MS. As a result, 83 IgG-Fc glycopeptides from human serum, including 17 sialylated glycopeptides, were identified. In addition, a quantitation method with high sensitivity and repeatability was established by using UHPLC triple quadrupole (QQQ) MS. We applied this approach to carry out quantitative analysis of IgG glycosylation in RA patients. Finally, 36 potential glycopeptide biomarkers, including 13 species from IgG1, 12 species from IgG2/3 and 11 species from IgG4 were identified.

Simultaneous Quantitation of Five Bioactive Ingredients in Raw and Processed Fallopia multiflora by Employing UHPLC-Q-TOF-MS.

Fallopia multiflora is used for treatment of premature graying hair and blood deficiency. In this study, a quantitative method was developed for determination of five bioactive components (emodin, 2,3,5,4'-tetrahydroxy-stilbene- 2-Ο-β-d-glucoside, emodin-8-O-β-d-glucopyranoside, ω-hydroxyemodin and kaempferol) in raw and processed F. multiflora by using ultra-high performance liquid chromatography (UHPLC)-quadrupole time-of-flight mass spectrometry-based method. The sample handling procedure was optimized. Chromatographic separation was carried out on a Thermo Syncronis AQ-C18 UHPLC column with mobile phase consisting of 0.01% aqueous formic acid and acetonitrile. The method was interrogated in terms of linearity, precision, stability and recovery tests. All calibration curves displayed good linearity (R2 > 0.9992). The limit of detection and limit of quantification of these components ranged from 0.01 to 0.03 μg/mL and from 0.03 to 0.07 μg/mL, respectively. The average recoveries of these components were from 98.2 to 102.9% with relative standard deviation values from 0.8 to 2.9% for F. multiflora. The developed method can be applied to quality control of raw and processed F. multiflora.

Olopatadine hydrochloride loaded Kollidon® SR nanoparticles for ocular delivery: Nanosuspension formulation and in vitro–in vivo evaluation

IntroductionAllergic eye diseases includes a spectrum of diseases, with each state being characterized by a complicated immunopathology. In the present study, polymeric Kollidon® SR nanoparticles loaded with olopatadine hydrochloride (OLO) were formulated as a suspension for ocular drug delivery. The formulations were developed to treat ocular allergic disease and characterized by their improved duration of corneal retention. MethodsOLO-loaded Kollidon® SR nanoparticles were prepared using the spray-drying method. The active agent was quantified using ultra-high performance liquid chromatography (U-HPLC). The nanoparticles were characterized according to entrapment efficiency, particle size, zeta potential, morphology, solid-state characterizations, and drug release. The in vitro release studies on OLO-loaded nanoparticles were examined in simulated tear fluid (pH 7.4). In vivo studies were conducted on healthy female/male Anatolian Merino sheep to investigate the retention time of the active agent. Results and DiscussionCharacterization studies showed that OLO was successfully loaded into the nanoparticle system and that the suspension formulation is suitable for ocular administration. Using DDSolver, the in vitro release of the OLO from nanoparticle formulations followed the Korsmeyer-Peppas model. ConclusionsThe proposed spray-drying method can be successfully applied for preparing OLO-loaded nanoparticles without adding crosslinkers. OLO-loaded nanoparticles appear to be a promising extended-release drug delivery system that can be used to treat ocular diseases with a more prolonged drug-residence time on the ocular surface.

Validated comprehensive metabolomics and lipidomics analysis of colon tissue and cell lines

Current untargeted approaches for metabolic fingerprinting of colon tissue and cell lines lack validation of reproducibility and/or focus on a selection of metabolites as opposed to the entire metabolome. Yet, both are critical to ensure reliable results and pursue a fully holistic analysis. Therefore, we have optimized and validated a platform for analyzing the polar metabolome and lipidome of colon-derived cell and tissue samples based on a consecutive extraction of polar and apolar components. Peak areas of selected targeted analytes and the number of untargeted components were assessed. Analysis was performed using ultra-high performance liquid-chromatography (UHPLC) coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (HRMS). This resulted in an optimized extraction protocol using 50% methanol/ultrapure water to obtain the polar fraction followed by a dichloromethane-based lipid extraction. Using this comprehensive approach, we have detected more than 15,000 components with CV < 30% in internal quality control (IQC) samples and were able to discriminate the non-transformed (NT) and transformed (T) state in human colon tissue and cell lines based on validated OPLS-DA models (R2Y > 0.719 and Q2 > 0.674). To conclude, our validated polar metabolomics and lipidomics fingerprinting approach could be of great value to reveal gastrointestinal disease-associated biomarkers and mechanisms.

Isolation, Characterization and Structural Elucidation of Polybutylene Terephthalate Cyclic Oligomers and Purity Assessment Using a 1H qNMR Method

The use of polybutylene terephthalate (PBT) as a food contact material is increasing over the last years. Typical contaminations in the final PBT product include its cyclic oligomers, which are allowed as additives in food contact plastics according to Regulation (EU) No. 10/2011. Their investigation is currently limited by the lack of analytical standards and physical-chemical information. Therefore, four PBT cyclic oligomers have been isolated and purified from a PBT raw material with an automated preparative HPLC-DAD system. Comprehensive characterization of the compounds was performed using Ultra-High Performance Liquid Chromatography (UHPLC) with high resolution time-of-flight mass spectrometry, Fourier-Transform Infrared spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and Nuclear Magnetic Resonance (NMR) spectroscopy. The purity of each oligomer was assessed using a 1H qNMR method and ranged from 96.1% to 97.0% for PBT tetramer and trimer respectively. The availability of pure and well characterized PBT cyclic oligomer standards will facilitate future studies of release from plastic food packaging materials.