Using Ultra Performance Liquid Chromatography Research Articles

ULK1 Mediated Autophagy-Promoting Effects of Rutin-Loaded Chitosan Nanoparticles Contribute to the Activation of NF-κB Signaling Besides Inhibiting EMT in Hep3B Hepatoma Cells.

Liver cancer remains to be one of the leading causes of cancer worldwide. The treatment options face several challenges and nanomaterials have proven to improve the bioavailability of several drug candidates and their applications in nanomedicine. Specifically, chitosan nanoparticles (CNPs) are extremely biodegradable, pose enhanced biocompatibility and are considered safe for use in medicine. CNPs were synthesized by ionic gelation, loaded with rutin (rCNPs) and characterized by ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and transmission electron microscopy (TEM). The rCNPs were tested for their cytotoxic effects on human hepatoma Hep3B cells, and experiments were conducted to determine the mechanism of such effects. Further, the biocompatibility of the rCNPs was tested on L929 fibroblasts, and their hemocompatibility was determined. Initially, UV-vis and FTIR analyses indicated the possible loading of rutin on rCNPs. Further, the rutin load was quantitatively measured using Ultra-Performance Liquid Chromatography (UPLC) and the concentration was 88 µg/mL for 0.22 micron filtered rCNPs. The drug loading capacity (LC%) of the rCNPs was observed to be 13.29 ± 0.68%, and encapsulation efficiency (EE%) was 19.55 ± 1.01%. The drug release was pH-responsive as 88.58% of the drug was released after 24 hrs at the lysosomal pH 5.5, whereas 91.44% of the drug was released at physiological pH 7.4 after 102 hrs. The cytotoxic effects were prominent in 0.22 micron filtered samples of 5 mg/mL rutin precursor. The particle size for the rCNPs at this concentration was 144.1 nm and the polydispersity index (PDI) was 0.244, which is deemed to be ideal for tumor targeting. A zeta potential (ζ-potential) value of 16.4 mV indicated rCNPs with good stability. The IC50 value for the cytotoxic effects of rCNPs on human hepatoma Hep3B cells was 9.7 ± 0.19 μg/mL of rutin load. In addition, the increased production of reactive oxygen species (ROS) and changes in mitochondrial membrane potential (MMP) were observed. Gene expression studies indicated that the mechanism for cytotoxic effects of rCNPs on Hep3B cells was due to the activation of Unc-51-like autophagy-activating kinase (ULK1) mediated autophagy and nuclear factor kappa B (NF-κB) signaling besides inhibiting the epithelial-mesenchymal Transition (EMT). In addition, the rCNPs were less toxic on NCTC clone 929 (L929) fibroblasts in comparison to the Hep3B cells and possessed excellent hemocompatibility (less than 2% of hemolysis). The synthesized rCNPs were pH-responsive and possessed the physicochemical properties suitable for tumor targeting. The particles were effectively cytotoxic on Hep3B cells in comparison to normal cells and possessed excellent hemocompatibility. The very low hemolytic profile of rCNPs indicates that the drug could be administered intravenously for cancer therapy.

Greener approaches to improve phenolic acid composition and antioxidant activity of different cultivation methods of Zingiber officinale Roscoe extracts

Zingiber officinale Roscoe, known as ginger (Bentong variety), is an essential spice ingredient used in food and condiments due to its refreshing aroma and intense flavour. The bioactive constituents found in ginger influence the numerous pharmacological effects, including antioxidant, analgesic, anticancer, antipyretic and anti-inflammatory. This work proposed a greener approach than the conventional plant matrices extraction to promote safe, non-toxic, and environment-friendly products. In this study, three different extraction methods: a) water (WE), b) hydrothermal-assisted (HAE) and c) enzymaticassisted (EAE), have been applied to measure the potential of both ginger extracts from different cultivation methods using fertigation technology and conventionally slopegrown. To ascertain the biological content of the extracts, the phenolic acid composition was quantified using ultra-performance liquid chromatography (UPLC). The total phenolic content (TPC) was determined using the Folin-Ciocalteu method, while the antioxidant activities were analysed using ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. The results obtained showed that ferulic acid was significantly highest in EAE extracts of both ginger (fertigation and slope-grown cultivation) at 1.165±0.118 and 0.610±0.062 mg/g. Moreover, EAE of both ginger extracts also found to be significantly highest in TPC (267.481±0.404 mg GAE/g and 247.837±1.675 mg GAE/g), and FRAP value (741.867±2.139 mg AAE/g and 461.733±0.231 mg AAE/g), respectively. However, the highest free radical scavenging activity of fertigated ginger extract through HAE was 134.735±0.021 mg AAE/g. Meanwhile, EAE of the sloped-grown ginger extract was highest at 141.293±0.707 mg AAE/g. This study demonstrates that EAE can be an effective approach in enhancing the TPC and antioxidant activity of ginger extracts. Ginger may therefore be suggested as a more greener and natural antioxidant to lessen oxidation in food products.

Method validation and characterization of stress degradation products of azelastine hydrochloride using LC-UV/PDA and LC-Q/TOF-MS studies.

Azelastine HCl is a second-generation H1 -receptor antagonist approved by the US Food and Drug Administration (US FDA) for treating seasonal allergic rhinitis and non-allergic vasomotor rhinitis. This study encompasses the validation of a liquid chromatography-ultra violet photo diode array (LC-UV/PDA) method for the drug and its extension to liquid chromatography/quadrupole time-of-flightmassspectrometry (LC-Q/TOF-MS) studies for identification and characterization of various stress degradation products of the drug. Stress degradation of azelastine HCl was undertaken under the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) prescribed conditions of hydrolytic, photolytic, oxidative, and thermal stress. The degraded drug solutions were analyzed using Ultra Performance Liquid Chromatography (UPLC) employing a C18 (100 × 4.6mm; 2.6μ, Kinetex) column by isocratic elution. Detection wavelength was 241 nm. The degradation products were identified and characterized using UPLC-MS/TOF studies, and an attempt was made to isolate one of the degradation products by solvent extraction. The drug was found to significantly degrade under acidic/alkaline/neutral photolytic, oxidative, and alkaline hydrolytic conditions. Six degradation products (I-VI) were identified through LC-Q/TOF-MS studies that were adequately resolved from the drug with the developed UPLC method. All degradation products (I-VI) were ionized in the total ion chromatogram (TIC) in the LC-MS studies, and these were identified and characterized, and the degradation pathway of the drug was postulated. One of the oxidation products isolated from the degraded drug solution was characterized through differential scanning calorimetry, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance spectral data. Six degradation products generated from stress degradation studies on azelastine HCl were adequately resolved through LC-UV/PDA studies followed by method validation. These were successfully identified and characterized through LC-Q/TOF-MS studies, and the degradation pathways for the generation of these products from the drug have been postulated.

Comparative Analysis of Glycoform Profiles Between Biosimilar and Originator Monoclonal Antibodies by Liquid Chromatography–Mass Spectrometry

Glycosylation is considered as a critical quality attribute for monoclonal antibodies (mAbs) and needs routine monitoring during production. This study aims to compare the glycoform profiles of biosimilar and four originator mAbs using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionization-quadrupole time of flight-mass spectrometry (ESI/Q-TOF MS). The resultant mass spectrum showed that seven different glycoform pairs, including G0F–GN/G0, G0F–GN/G0F, G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F, and G2F/G2F were identified via intact mass analysis for all tested mAb samples. The correct identification of each glycoform pair was achieved by comparing the observed mass with its theoretical mass using high-resolution mass spectrometry data (with mass accuracies of less than 100 ppm). The most abundant paired glycoforms detected at the intact protein level are G0F/G0F and G0F/G1F, with relative abundance ranges of 38.45 – 43.43% and 19.32 – 22.20%, respectively. The obtained data demonstrated that biosimilar and originators have the same types of glycoform pairs, and the relative abundances of each pair were comparable among biosimilar and four originator mAb samples. Additionally, the reduced mass analysis revealed that five different glycans (G0F–GN, G0, G0F, G1F, and G2F) were attached to the heavy chain of the mAb, and the relative abundance of G0F ranged from 75.21 to 77.90%. The detected mass accuracies for reduced mass analysis were below 25 ppm. The results of the intact and reduced mass analyses showed that the biosimilar is similar to its originator in terms of glycoform percentages and molecular masses.

Abnormal functional connectivity within the prefrontal cortex is associated with multiple plasma lipid species in major depressive disorder

BackgroundAbnormalities in functional connectivity (FC) in major depressive disorder (MDD) have been widely reported. Analysis of the relationship between FC and plasma lipid profiles would be meaningful in the exploration of pathophysiological mechanisms and helpful for the identification of biomarkers for MDD. MethodsPatients with MDD (n = 49) and healthy controls (HC, n = 87) were recruited. Resting-state functional magnetic resonance imaging (rs-fMRI) data were collected for FC construction. The plasma lipid profiles were acquired using ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS) analysis and clustered as co-expression modules. The differential FC and lipid modules between HCs and patients with MDD were identified, and then the association between FC and lipid co-expression modules was analyzed using correlation analysis. The modules associated molecular function was explored using metabolite set enrichment analysis (MSEA). ResultsMDD-associated FC and lipid co-expression modules were identified. One module was associated with FC values between the right orbital part of the middle frontal gyrus and the opercular part of the left inferior frontal gyrus, which was enriched in lipid sets of diacylglycerols and fatty alcohols; another module was associated with FC values between the right middle frontal gyrus and the right anterior cingulate and paracingulate gyri, which was enriched in lipid sets of glycerophosphocholines and glycerophosphoethanolamines. ConclusionOur results indicated that abnormal FC in the prefrontal cortex is associated with multiple plasma lipid species, which may provide novel clues for exploring the pathophysiology of MDD.

Effects of Dietary Protein Levels on Sheep Gut Metabolite Profiles during the Lactating Stage.

Diet-associated characteristics such as dietary protein levels can modulate the gut's primary or secondary metabolites, leading to effects on the productive performance and overall health of animals. Whereas fecal metabolite changes are closely associated with gut metabolome, this study aimed to see changes in the rumen metabolite profile of lactating ewes fed different dietary protein levels. For this, eighteen lactating ewes (approximately 2 years old, averaging 38.52 ± 1.57 kg in their initial body weight) were divided into three groups (n = 6 ewes/group) by following the complete randomized design, and each group was assigned to one of three low-protein (D_I), medium-protein (D_m), and high-protein (D_h) diets containing 8.58%, 10.34%, and 13.93% crude protein contents on a dry basis, respectively. The fecal samples were subjected to untargeted metabolomics using ultra-performance liquid chromatography (UPLC). The metabolomes of the sheep fed to the high-protein-diet group were distinguished as per principal-component analysis from the medium- and low-protein diets. Fecal metabolite concentrations as well as their patterns were changed by feeding different dietary protein levels. The discriminating metabolites between groups of nursing sheep fed different protein levels were identified using partial least-squares discriminant analysis. The pathway enrichment revealed that dietary protein levels mainly influenced the metabolism-associated pathways (n = 63 and 39 in positive as well as negative ionic modes, respectively) followed by protein (n = 15 and 8 in positive as well as negative ionic modes, respectively) and amino-acid (n = 14 and 7 in positive as well as negative ionic modes, respectively) synthesis. Multivariate and univariate analyses showed comparative changes in the fecal concentrations of metabolites in both positive and negative ionic modes. Major changes were observed in protein metabolism, organic-acid biosynthesis, and fatty-acid oxidation. Pairwise analysis and PCA reveal a higher degree of aggregation within the D-h group than all other pairs. In both the PCA and PLS-DA plots, the comparative separation among the D_h/D_m, D_h/D_I, and D_m/D_I groups was superior in positive as well as negative ionic modes, which indicated that sheep fed higher protein levels had alterations in the levels of the metabolites. These metabolic findings provide insights into potentiated biomarker changes in the metabolism influenced by dietary protein levels. The target identification may further increase our knowledge of sheep gut metabolome, particularly regarding how dietary protein levels influence the molecular mechanisms of nutritional metabolism, growth performance, and milk synthesis of sheep.

Metabolomic Changes in Plasma of Preterminal Stage of Rhesus Nonhuman Primates Exposed to Lethal Dose of Radiation.

Ionizing radiation exposure is known to induce molecular and cellular injury, inflicting a cascade of potentially catastrophic events leading to tissue and organ damage. Metabolomic analysis allows for the identification and quantification of small molecules downstream of genomic changes induced by radiation exposure. We aimed to characterize metabolomic changes that underscore the prefinal stage of lethally irradiated rhesus nonhuman primates (NHPs). Peripheral blood was drawn at baseline, post-exposure, as well as at the preterminal stage in NHPs (immediately prior to death in moribund NHPs) that did not survive exposure with 7.2 Gy total-body radiation (LD70/60). Herein, we analyzed global metabolomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in plasma samples of NHPs collected at various timepoints in relation to irradiation. The overall goal was to identify metabolic shifts present immediately prior to death. Our findings showed radiation induced significant time-dependent metabolic perturbations when compared to pre-irradiation profiles, particularly in glycerophospholipid metabolism and steroid hormone biosynthesis and metabolism pathways. These findings provide valuable insights for identifying biomarkers for lethality, which may be helpful for triage during a mass casualty scenario.

Iron overload and chelation modulates bisretinoid levels in the retina

AimIron dysregulation in conjunction with other disease processes may exacerbate retinal degeneration. We employed models of iron overload and iron chelation to explore the interactions between iron-catalyzed oxidation and photoreactive bisretinoid lipofuscin.MethodsThe mice were injected intravitreally with ferric ammonium citrate (FAC) or were treated using the iron chelator deferiprone (DFP) from birth to 2 months of age. Short-wavelength fundus autofluorescence (SW-AF) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired. The bisretinoid levels were quantified using ultra performance liquid chromatography (UPLC) and in vivo through quantitative fundus autofluorescence (qAF). In histologic sections, the photoreceptor cell viability was assessed by measuring the thickness of the outer nuclear layer (ONL).ResultsThe levels of bisretinoids, all-trans-retinal dimers, and A2PE were significantly increased in the FAC-injected eyes of C57BL/6J mice. Seven days after FAC injection, hyperautofluorescent foci were visible in fundus autofluorescence (488 nm) images, and in SD-OCT scans, aberrant hyperreflectivity was present in the outer retina and ONL thinning was observed. In FAC-injected Abca4–/– mice with pronounced RPE bisretinoid lipofuscin accumulation, the hyperautofluorescent puncta were more abundant than in the wild-type mice, and the extent of ONL thinning was greater. Conversely, the intravitreal injection of FAC in Mertk–/– mice led to a more modest increase in A2PE after 2 days. In contrast to the effect of iron accumulation, chelation with DFP resulted in significantly increased levels of A2E and A2-GPE and qAF due to the reduced iron-catalyzed oxidation of bisretinoids. In Mertk–/– mice, the A2E level was significantly lower and the ONL area was smaller than in DFP-treated mice. DFP chelation did not impair the visual cycle in BALB/cJ mice.ConclusionIron accumulation was associated with progressive impairment in photoreceptor cells that was associated with the increased formation of a bisretinoid species known to form in photoreceptor outer segments as a precursor to A2E. Additionally, disease features such as the development of hyperautofluorescence puncta in fundus AF images, hyperreflectivity in the outer retina of SD-OCT scans, and ONL thinning were more pronounced when iron was delivered to Abca4–/– mice with a greater propensity for bisretinoid formation. Higher bisretinoid levels and enhanced qAF are indicative of lesser bisretinoid loss due to oxidation.

Molecular formula assignment of dissolved organic matter by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry using two non-targeted data processing approaches: A case study from recirculating aquaculture systems

BackgroundThe accumulation of dissolved organic matter (DOM) poses an issue in the management of the water quality from recirculating aquaculture systems (RAS), but its characterization is often not detailed enough to understand the DOM transformations in RAS. In this study, we investigated the application of two distinct non-targeted data processing approaches using ultra-performance liquid chromatography (UPLC) with quadrupole time-of-flight mass spectrometry (QTOF-MS) and two software with different algorithmic designs: PetroOrg and Progenesis QI to accurately characterize the molecular composition of DOM in RAS by UPLC-QTOF-MS. ResultsThe UPLC-QTOF-MS resolution in combination with PetroOrg and Progenesis QI software successfully assigned 912 and 106 unique elemental compositions, respectively, including compounds containing carbon, hydrogen, and oxygen (CHO) and nitrogen-containing CHO compounds (CHON), in the DOM samples from RAS. The results of these two distinct data processing approaches were consistent with the list of DOM formulas from RAS identified by higher resolution mass spectrometry techniques confirming their reliability. PetroOrg approach revealed only compositional information in the DOM samples from RAS, while Progenesis QI in addition to identifying new elemental compositions, increased their chemical space by giving information about their polarity and their possible key structures. DOM samples from RAS were found to be rich in unsaturated CHO compounds, with tentatively key structures of terpenoids with medium polarity indicating natural origins in their composition. The analysis also revealed probable structures of sucrose fatty acid esters and polyethylene glycol, indicating anthropogenic sources. Significance and NoveltyThe combination of these two non-targeted data processing approaches significantly improves the characterization of the complex mixture of DOM from RAS by UPLC-QTOF-MS reporting for the first time accurate DOM results in terms of its composition, while proposing its key structures. The presented methods can also be used to analyze different DOM samples with other HRMS techniques and software.

Liver metabolomics reveals potential mechanism of Jieduan-Niwan formula against acute-on-chronic liver failure (ACLF) by improving mitochondrial damage and TCA cycle

BackgroundAcute-on-chronic liver failure (ACLF) is a refractory disease with high mortality, which is characterized by a pathophysiological process of inflammation-related dysfunction of energy metabolism. Jieduan-Niwan formula (JDNWF) is a eutherapeutic Chinese medicine formula for ACLF. However, the intrinsic mechanism of its anti-ACLF effect still need to be studied systematically.PurposeThis study aimed to investigate the mechanism of JDNWF against ACLF based on altered substance metabolic profile in ACLF the expression levels of related molecules.Materials and methodsThe chemical characteristics of JDNWF were characterized using ultra performance liquid chromatography (UPLC) coupled with triple quadrupole mass spectrometry. Wistar rats subjected to a long-term CCL4 stimulation followed by a combination of an acute attack with LPS/D-GalN were used to establish the ACLF model. Liver metabolites were analyzed by LC–MS/MS and multivariate analysis. Liver function, coagulation function, histopathology, mitochondrial metabolic enzyme activity and mitochondrial damage markers were evaluated. The protein expression of mitochondrial quality control (MQC) was investigated by western blot.ResultsLiver function, coagulation function, inflammation, oxidative stress and mitochondrial enzyme activity were significantly improved by JDNWF. 108 metabolites are considered as biomarkers of JDNWF in treating ACLF, which were closely related to TCA cycle. It was further suggested that JDNWF alleviated mitochondrial damage and MQC may be potential mechanism of JDNWF improving mitochondrial function.ConclusionsMetabolomics revealed that TCA cycle was impaired in ACLF rats, and JDNWF had a regulatory effect on it. The potential mechanism may be improving the mitochondrial function through MQC pathway, thus restoring energy metabolism.

Characterization of the varied output from the anthocyanin pathway in Phalaenopsis-type Dendrobium hybrids and its relationship with flower coloration

Floral color is an important Phalaenopsis-type Dendrobium hybrids (Den-Phals) trait in the flower markets. Anthocyanins are key pigments contributing to purple, pink, and violet floral color. However, the impact of anthocyanin composition on flower coloration has not yet been fully elucidated. In this study, the flower color characteristics of 62 Den-Phals were evaluated. Most flowers contained purple, while pure blue, orange, and red floral colors were scarce. In total 35 anthocyanins were identified and two acylated pelargonidin glycosides were tentatively characterized using ultra-performance liquid chromatography (UPLC)-mass spectrum (MS) analysis. Unfortunately, the major anthocyanin components remained unidentified. However, the hydrolysis anthocyanins to their anthocyanidins suggested that the cyanidin was the major component in most hybrids with a relative content over 85 %. Pelargonidin was almost the only pigment in reddish pink flowers. The slightly higher peonidin content was found in purple-violet flowers (up to 6.23 %). The trend indicated that with the increase of total anthocyanidin and cyanidin pigments, the floral color deepened and turned to purple. Flavones or flavonols as co-pigments contributed to brighter and yellower color. From the above results, the varied output from the anthocyanin biosynthetic pathway among Den-Phals was used to identify the rate-limiting steps involved and a strategy for the cultivation of pure red, blue, and violet flowers is proposed. The study provides a basis for screening excellent germplasm and improving floral color in Den-Phals.

The migratory locust (Locusta migratoria) as a potential source of protein and biopolymer compounds in the future

Abstract. Kleden YL, Mukkun L, Ndapamuri MH, Pamatana F. 2023. The migratory locust (Locusta migratoria Meyen) as a potential source of protein and biopolymer compounds in the future. Biodiversitas 24: 5979-5987. The migratory locust Locusta migratoria Linnaeus 1758, is an edible insect posing significant challenges to the agricultural industry in Indonesia, particularly in East Nusa Tenggara (ENT) Province. However, apart from the status of the migratory locust as a highly destructive insect pest, it has great potential as a source of protein, amino acids, and other bioactive compounds. This study aimed to determine the nutritional and other bioactive characteristics of locusts. Proximate analysis was performed using SNI 01-2891-1992 to identify protein, fat, ash, carbohydrates, and energy. The amino acid analysis was conducted using Ultra Performance Liquid Chromatography (UPLC). The chitin from the nymph and imago stages of the locust was obtained through a process including demineralization and deproteinization. Subsequently, the chitin was subjected to deacetylation in order to obtain chitosan. Proximate content analysis shows that locust has promising potential as a source of nutrients in order to address the nutritional problems of the population that are still a problem in Indonesia including in ENT Province. The protein content of locust ranged from 63.72 to 75.26% dry weight. These contents are close to those of egg protein (83%), beef protein (85%), and even most plant proteins, like soybean (81%). The imago stage exhibited the highest total amino acid content, measuring 411.41 mg/100 g, with essential amino acids such as lysine, isoleucine, leucine, valine, phenylalanine, threonine, histidine, and tryptophae. Imago contains chitin from 8.79 to 12.05% dry weight and chitosan from 59.3% to 71.26% by weight of chitin. The study suggests that locusts can be an alternative source of nutrition to dial with stunting in term of their availability of protein and amino acid. Additionally, chitin and chitosan in migratory locusts are promising nutrient sources for agriculture and health applications.

Guadua angustifolia Kunth leaves as a source for bioactive phenolic compounds: Optimization of ultrasound-assisted extraction using response surface methodology and antioxidant activities

This study evaluated the ultrasound-assisted extraction of phenolic compounds from Guadua angustifolia leaves, along with their optimization using response surface methodology. The effects of two sonication process conditions were determined using a central composite experimental design, with three levels (low, medium, and high) evaluated for time (10, 20, and 30 min) and temperature (20 °C, 35 °C, and 50 °C). A total of 12 experiments with four replicates were conducted at the central point, with the total phenol and flavonoid contents determined using the Folin-Ciocalteu colorimetric method and complexation with AlCl3, respectively. The optimized extract was analyzed using ultra-performance liquid chromatography (UPLC), and the antioxidant capacity of the optimized extract was determined by DPPH• (2,2-Diphenyl-1-Picrylhydrazyl) and ABTS•+ (2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assays. Extraction at 50 °C for 20 min was found to favor the extraction of phenol and total flavonoids. The experimental validation of the total phenol and flavonoid content produced values of 7.39 mg gallic acid equivalents per gram of dry matter and 1.55 mg quercetin equivalents per gram of dry matter, respectively. These values suggest that the extraction process is reproducible, with a relative standard deviation of 22.9 % and 14.1 %, respectively. The chromatographic profile showed that optimization favored the visualization of phenolic compounds compared to the non-optimized extract. The optimized extract had higher antioxidant capacity than the non-optimized extract, with values of 209.23 and 144.76 μmol Trolox per gram extract for the DPPH• and ABTS•+ techniques, respectively. Thus, the conditions evaluated in the ultrasound-assisted extraction were an efficient technique capable of extracting the maximum amount of phenolic compounds with antioxidant activity from the leaves of G. angustifolia, showing its potential application in various industries.

A UPLC-based activity assay method for aminoglycoside phosphotransferase 4-Ia (APH4): A Selectable marker of plant transformation

The aminoglycoside phosphotransferase 4-Ia (APH4) is a hygromycin B phosphotransferase and catalyzes the phosphorylation of the 4-hydroxyl group of the antibiotic hygromycin B, inactivating its antibiotic activity. Therefore, APH4 has utility as a selectable marker for transformation of many plant species. However, suitable methods to measure and quantify plants expressing APH4 enzymes are lacking because of technical challenges, associated with both specificity and sensitivity. Here we describe a highly specific and sensitive APH4 enzymatic activity assay by measuring the production of adenosine 5′-diphosphate (ADP) using ultra performance liquid chromatography (UPLC)-based method. The present assay enabled determination of enzymatic activity of APH4 over a concentration range from 0.05 to 0.8 μg APH4/ml. Method performance validation sample were examined at five concentrations in triplicate in six independent sessions. Average CVs of 5.1 % for intra-assay and 11.7 % for inter-assay over all samples were determined for precision, and 6.0 % was determined for accuracy. Additionally, the method was validated for use to determine APH4 enzymatic activity in both microbially produced and plant-derived samples.

Identification and Evaluation of Diterpenoids from Glandular Trichome Secretions of Air/Sun-Cured Tobacco Germplasm Resources

To explore the multipurpose utilization of tobacco germplasm resources, 80 representative air/sun-cured tobacco germplasms were used as experimental materials to determine the composition and content of the diterpenoids of glandular trichome secretions using ultra-performance liquid chromatography (UPLC). Correlation analysis, cluster analysis, and principal component analysis (PCA) were used to comprehensively evaluate 80 air/sun-cured tobacco germplasms. The results are as follows: (1) 11 chromatographic peaks (Peak1–Peak11) were detected by UPLC, and the coefficient of variation of their contents ranged from 26.3 to 143%. (2) Correlation analysis showed that alpha-cembratriene-diol (α-CBT-diol) and beta-cembratriene-diol (β-CBT-diol) had the highest correlation coefficients (0.97), showing a significantly negative correlation with cis-abienol and a significantly positive correlation with the other diterpenoids. (3) PCA showed that the cumulative contribution rate of the three principal components was 75.70%, and the diterpenoid-rich germplasms were ranked in the following order: X40 (Maiduo) > X48 (Jianpingpiaoba) > X58 (Mianzhu) > X54 (Shifangpipaliu) > X63 (Xuejia5) > X41 (Tangpeng) > X29 (OLOR) > X73 (Criollo) > X44 (Tiebanqing) > X70 (Nicaragua Changxin). The diterpenoid content of X36 was the lowest. These results provide excellent germplasms for plant-based pesticides and fragrance compounds and genetic studies of the diterpenoid metabolism.

Comparative transcriptomic and plastid development analysis sheds light on the differential carotenoid accumulation in kiwifruit flesh.

Carotenoids are colorful lipophilic isoprenoids synthesized in all photosynthetic organisms which play roles in plant growth and development and provide numerous health benefits in the human diet (precursor of Vitamin A). The commercially popular kiwifruits are golden yellow-fleshed (Actinidia chinensis) and green fleshed (A. deliciosa) cultivars which have a high carotenoid concentration. Understanding the molecular mechanisms controlling the synthesis and sequestration of carotenoids in Actinidia species is key to increasing nutritional value of this crop via breeding. In this study we analyzed fruit with varying flesh color from three Actinidia species; orange-fleshed A. valvata (OF), yellow-fleshed A. polygama (YF) and green-fleshed A. arguta (GF). Microscopic analysis revealed that carotenoids accumulated in a crystalline form in YF and OF chromoplasts, with the size of crystals being bigger in OF compared to YF, which also contained globular substructures in the chromoplast. Metabolic profiles were investigated using ultra-performance liquid chromatography (UPLC), which showed that β-carotene was the predominant carotenoid in the OF and YF species, while lutein was the dominant carotenoid in the GF species. Global changes in gene expression were studied between OF and GF (both tetraploid) species using RNA-sequencing which showed higher expression levels of upstream carotenoid biosynthesis-related genes such as DXS, PSY, GGPPS, PDS, ZISO, and ZDS in OF species compared to GF. However, low expression of downstream pathway genes was observed in both species. Pathway regulatory genes (OR and OR-L), plastid morphology related genes (FIBRILLIN), chlorophyll degradation genes (SGR, SGR-L, RCCR, and NYC1) were upregulated in OF species compared to GF. This suggests chlorophyll degradation (primarily in the initial ripening stages) is accompanied by increased carotenoid production and localization in orange flesh tissue, a contrast from green flesh tissue. These results suggest a coordinated change in the carotenoid pathway, as well as changes in plastid type, are responsible for an orange phenotype in certain kiwifruit species.

Monitoring ascorbic acid oxidation with H2O2 and analyzing the food supplement product using parallel factor analysis model of kinetic UV absorption measurements

Ascorbic acid is a functional compound in maintaining the physiological processes of humans who acquire it from dietary sources, and also functions as an antioxidant. Therefore, the quantitation, and degradation of ascorbic acid in the food matrix and its interaction with coexisting components is a very important issue in terms of food safety and human health. Classical analytical methods may not give the expected results in the analysis of more advanced and complex food products obtained with the developing production technology. In this perspective, the parallel factor analysis (PARAFAC) model using kinetic UV absorption measurements was improved for the co-estimation of the kinetics and quantification of ascorbic acid in a food supplement product, which is a commercial effervescent preparation without requiring a prior separation step. The proposed three-way analysis strategy was validated and applied to the analysis of the relevant samples. For example, in the test samples, percent recovery and relative standard deviation were found to be 99.4 % and 1.36 %, respectively. The proposed chemometric strategy would be an alternative approach with a good selectivity for estimating the kinetics and quantification of ascorbic acid in a commercial supplementary product. The assay results of the PARAFAC model was checked by using ultra-performance liquid chromatography (UPLC).

Analysis of Racecadotril in Pharmaceutical Formulations Using Ultra Performance Liquid Chromatography (UPLC) Method

Analysis of Racecadotril in Pharmaceutical Formulations Using Ultra Performance Liquid Chromatography (UPLC) Method

#5260 PLASMA AND URINE METABOLOMIC PROFILES REVEAL POTENTIAL BIOMARKERS AND PROVIDE VALUABLE INSIGHTS INTO THE MECHANISMS OF MPO-ANCA-GN

Abstract Background and Aims Currently, the precise mechanism of myeloperoxidase- anti-neutrophil cytoplasmic antibody associated glomerulonephritis (MPO-ANCA-GN) remains elusive. And there is a lack of dependable biomarkers for evaluating disease activity, therapeutic resistance, and risk of relapse. We try to seek valuable biomarkers and explore the mechanism of MPO-ANCA-GN by metabolomics. Method Urine and plasma samples from 63 MPO-ANCA-GN, 31 lupus nephritis (LN), 15 IgA nephropathy (IgAN), and 15 minimal change disease (MCD) patients and 30 HC were collected for metabolomics analysis. The samples were analyzed using Ultra Performance Liquid Chromatography (UPLC) and Tandem mass spectrometry (MS/MS). The different groups of samples were compared using Partial Least Squares-Discriminant Analysis. A Receiver Operator Characteristic (ROC) curve was constructed to assess the performance of the metabolite model in identifying active renal vasculitis. KEGG annotation and enrichment analysis were performed for key pathways. Results All metabolites in urine and plasma samples including hydrophilic compounds and hydrophobic compounds were identified. Compared with the disease control (DC) and HC, we observed 79 significantly upregulated metabolites (VIP>1, FDR<0.05, FC>1.5) and 32 downregulated metabolites (VIP>1, FDR <0.05, FC<0.67) in the plasma samples of MPO-ANCA-GN patients. KEGG enrichment analysis suggested that these upregulated metabolites were mainly associated with phenylalanine metabolism and galactose metabolism and downregulated metabolites were mainly associated with bile secretion, steroid biosynthesis, and ovarian steroidogenesis. Compared with the DC and HC, we identified a total of 122 differential metabolites in the urine samples of MPO-ANCA-GN patients, 40 of which were significantly upregulated (VIP>1, FDR<0.05, FC>1.5) and 68 of which significantly were downregulated (VIP>1, FDR <0.05, FC<0.67). Subsequently, KEGG analysis revealed that these upregulated metabolites were mainly enriched in glycerolipid metabolism, lipid and atherosclerosis, cholesterol metabolism, insulin resistance, regulation of lipolysis in adipocytes, vitamin digestion and absorption, fat digestion and absorption (Corrected P < 0.05). Downregulated metabolites were mainly enriched in steroid hormone biosynthesis (Corrected P < 0.05). Nine MPO-ANCA-GN patients with paired plasma and six MPO-ANCA-GN patients with paired urine during the active and remission phase were included in the study. A total of 504 differential metabolites in plasma samples were identified between the active and remission phase based on VIP value >1, P < 0.05, and FC >1.5 or FC < 0.6. Of these, 425 metabolites were downregulated, while 79 lipid metabolites were upregulated in the active group. MPO-ANCA-GN patients were divided into two groups according to Mayo Clinic/Renal Pathology Society Chronicity Score: Minimal-mild group and Moderate-severe group. In plasma samples, our data showed that 3-Methoxysalicylic Acid, Carnitine C20:5, and isoxanthopterin presented good ability in distinguishing between the minimal-mild group and the moderate-severe group, with an AUC > 0.7. In urine samples, cyclohexylamine and arachidonic acid (AUC > 0.8) can distinguish the two groups well. Conclusion We identified many novel potential metabolic biomarkers in urine and plasma samples for evaluating disease activity, therapeutic resistance, and risk of relapse in patients with MPO-ANCA-GN.

Development of UPLC-UV-ELSD Method for Fatty Acid Profiling in Polysorbate 80 and Confirmation of the Presence of Conjugated Fatty Acids by Mass Spectrometry, UV Absorbance and Proton Nuclear Magnetic Resonance Spectroscopy

Polysorbate 80 (PS80), a chemical substance composed of sorbitol, ethylene glycol, and fatty acids, is commonly used in pharmaceutical drug products to stabilize formulations. However, recent studies have demonstrated that PS80 may hydrolyze over time and the released free fatty acids (FFAs) may lead to particle formation. Naming conventions of fatty acids in current pharmacopeia and in products’ certificates of analysis (CoA) of PS80 do not typically distinguish between isomeric species of fatty acids in PS80. Thus, methods to fully characterize the fatty acid species present in PS80 raw materials are needed to enhance quality control strategies of pharmaceuticals using PS80. Here, extended effort is taken to characterize fatty acids in hydrolyzed PS80 raw materials and elucidate the identities of isomeric fatty acid species. In this work, a method was developed and optimized for separation and detection of fatty acids in alkaline hydrolyzed PS80 raw materials using ultra performance liquid chromatography (UPLC) with ultra-violet (UV) detection and evaporative light scattering detection (ELSD). Fatty acids not specified in the current pharmacopeias were detected in PS80 raw material by the developed LC-UV-ELSD method including conjugated forms of linoleic and linolenic fatty acid species. Their identities were orthogonally confirmed by retention time agreement with analytical standards, accurate mass by high resolution mass spectrometry, UV absorbance, and proton nuclear magnetic resonance spectroscopy. The detected conjugated fatty acids are theoretically more hydrophobic and less soluble than their unconjugated counterparts and may increase the propensity of PS80 to form particles upon hydrolysis. This work highlights the need for better quality control of PS80 raw material, as it may eventually play a critical role in product quality of therapeutic proteins.