Using Ultra Performance Liquid Chromatography Research Articles

Molecular Identification, Nutritional Profile and Heavy Metals Content of Edible Caulerpa from Binuangeun Coast, Banten

Two species of Caulerpa, locally known as “Pedesan” and “Latuh” have been traditionally consumed by coastal communities at Binuangeun, Banten. This study aimed to identify “Pedesan” and “Latuh” using the DNA barcoding method and to evaluate their nutrient and heavy metal contents. Fatty acids were determined by Gas Chromatography Flame Ionization Detector (GC FID), amino acids using Ultra Performance Liquid Chromatography (UPLC), and minerals using Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Based on the tufA gene sequences, the “Pedesan” was identified as Caulerpa racemosa var. macrophysa and “Latuh” as Caulerpa chemnitzia. Thirteen fatty acids were detected in C. racemosa var. macrophysa and twelve fatty acids in C. chemnitzia. Of the total fatty acid content, C. racemosa var. macrophysa contained 41.0% unsaturated fatty acids, dominated by linolenic acid and eicosapentaenoic acid. Meanwhile, C. chemnitzia contained 47.5%, dominated by oleic acid. Both seaweeds contained fatty acids with the w6/w3 ratio lower than 10, which could prevent heart disease risk based on World Health Organization (WHO) recommendation. The primary amino acids content in C. racemosa var. macrophysa were glutamic acid, alanine, serine and aspartic acid, while those in C. chemnitzia were glutamic acid, serine, aspartic acid, and glycine. The high content of glutamic acid in both samples indicated their potential use as food flavor enhancer. The Na/K ratio of C. racemosa var. macrophysa (40.31) and C. chemnitzia (27.48) were higher than those recommended by WHO. Heavy metals were not detected in either “Pedesan” nor “Latuh”, indicating that they are safe for consumption.

Spermidine and spermine exert protective effects within the lung.

Asthma is a heterologous disease that is influenced by complex interactions between multiple environmental exposures, metabolism, and host immunoregulatory processes. Specific metabolites are increasingly recognized to influence respiratory inflammation. However, the role of protein‐derived metabolites in regulating inflammatory responses in the lung are poorly described. The aims of the present study were to quantify polyamine levels in bronchoalveolar lavages (BALs) from healthy volunteers and asthma patients, and to evaluate the impact of each polyamine on inflammatory responses using in vitro models and in a house dust mite (HDM)‐induced respiratory allergy model. Spermidine levels were decreased, while cadaverine levels were increased in BALs from asthma patients compared to healthy controls, using Ultra Performance Liquid Chromatography (UPLC). Both spermine and spermidine inhibit lipopolysaccharide (LPS)‐induced cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) in vitro. In addition, oral gavage with spermine or spermidine modulate HDM‐induced cell infiltration, cytokine secretion, and epithelial cell tight junction expression in murine models. Spermidine also reduces airway hyper‐responsiveness. These results suggest that modulation of polyamine metabolism, in particular spermidine, is associated with respiratory inflammation and these molecules and pathways should be further explored as biomarkers of disease and potential targets for novel therapies.

Validated Simultaneous Gradient Ultra-Performance Liquid Chromatographic Quantification of Some Proton Pump Inhibitor Drug Residues in Saudi Pharmaceutical Industrial Wastewater.

Monitoring and quantification of active pharmaceutical ingredients (APIs) in the environment constitute important and challenging tasks, as they are directly associated with human health. Three commonly used proton pump inhibitors (PPIs), namely, omeprazole sodium (OMP), pantoprazole sodium (PNT), and lansoprazole sodium (LNZ) are well separated and quantified using ultra-performance liquid chromatography (UPLC) in pharmaceutical industrial wastewater. The separation of the studied drugs was performed on a stationary phase with a WatersTM column (100 × 2.1 mm, 1.7 µm). The mobile phase was composed of methanol:0.05 M potassium dihydrogen phosphate buffer (adjusted to pH 7.5 using NaOH) (50:50, v/v). The elution process was done in gradient mode by changing the relative proportions of the mobile phase components with time to get an optimum separation pattern. The flow rate of the developing system was adjusted to 0.8 mL/minute. Detection of the separated drugs was performed at 230 nm. The studied drugs were quantified in the concentration range of 10–200 ng/mL for all drugs. The cited method was fully validated according to the international conference on harmonization (ICH-Q2B) guidelines, then it was applied successfully for quantification of the studied PPIs in real wastewater samples after their solid phase extraction (SPE).

Comprehensive characterization of Chaenomeles seeds as a potential source of nutritional and biologically active compounds

The nutritional profile of seeds from eighteen Chaenomeles species and cultivars was determined using ultra-performance liquid chromatography (UPLC) and atomic absorption spectrometry (AAS). This is the first comprehensive attempt to determine the profile and content of sugars, organic acids, polyphenols, triterpenes, tocochromanols and minerals in Chaenomeles japonica, speciosa and superba seeds. C. japonica wild #3 seeds were characterized by the highest content of sugars (37.32 mg/g seeds, the dominant sugar was glucose) and organic acids (68.22 mg/g seeds, the highest share from galacturonic acid). The most abundant in phenolic compounds was C. speciosa 'Nivalis' (80.90 mg/g seeds), and the polymeric proanthocyanidins were the dominant group in the total phenolic content. The research revealed that Chaenomeles seeds contain 13 different triterpenes and oleanolic acid accounted for an average of 50 % of their total content. Furthermore, they contained 296−1375 μg of amino acids, 1.65–18.9 μg of tocopherols, and 0.25–2.71 μg of tocotrienols per g of seeds. The seeds are a source of all essential amino acids, and alpha homologs dominate among tocochromanols. Moreover, Chaenomeles seeds are low in sodium, but high in potassium (up to 5434 μg/g seeds) and are a good source of microelements (Fe, Cu, Zn and Mn). Therefore, they can be considered as a source of compounds with nutritional and pharmaceutical uses.

Antioxidant Activities and Profile of Amino Acid of Yoghurt from Beef Milk Fermentation with Dadih Starter

Dadih is naturally fermented buffalo milk in bamboo tubes and known to have antioxidant, antibacterial, and antihypertensive activity. Lactic acid bacteria in dadih can use as a starter to produce yogurt. The study aimed to produce yogurt of cow's milk with dadih as a starter, to determine the antioxidant activity and amino acid composition. Dadih is added with concentration variation of 2.5; 5; 7.5 and 10% (v/v) and it fermented for 48 hours at room temperature. Lactic acid bacteria cell counts of dadih were calculated by the total plate count method. Yogurt was tested by organoleptics with 33 panelists and proximate analysis water, ash, fat and protein contens based on the Association of Official Analytical Chemist (AOAC) in 2005. Antioxidant activities of yogurt were tested by DPPH method (1,1-diphenyl-2-picrylhydrazyl). Amino acid composition of yogurt was analyzed using ultra performance liquid chromatography (UPLC). The research result of amount of LAB on dadih are 1.01 x 1011 CFU/mL, has fulfilled the requirements of SNI 2981: 2009. Yogurt produced with the addition of dadih by 10% (v/v) is most accepted by panelists. The highest antioxidant activity was obtained by yogurt with 2.5% addition of dadih with IC50 value of 78.28 ppm. Yogurt almost contain all essential and non-essensial amino acids including tyrosine and phenylalanine as the antioxidants. Measurement of water, ash, protein and fat content in the sample meets the requirements of SNI 2981: 2009.

Antioxidant Activities and Profile of Amino Acid of Yoghurt from Beef Milk Fermentation with Dadih Starter

Dadih is naturally fermented buffalo milk in bamboo tubes and known to have antioxidant, antibacterial, and antihypertensive activity. Lactic acid bacteria in dadih can use as a starter to produce yogurt. The study aimed to produce yogurt of cow's milk with dadih as a starter, to determine the antioxidant activity and amino acid composition. Dadih is added with concentration variation of 2.5; 5; 7.5 and 10% (v/v) and it fermented for 48 hours at room temperature. Lactic acid bacteria cell counts of dadih were calculated by the total plate count method. Yogurt was tested by organoleptics with 33 panelists and proximate analysis water, ash, fat and protein contens based on the Association of Official Analytical Chemist (AOAC) in 2005. Antioxidant activities of yogurt were tested by DPPH method (1,1-diphenyl-2-picrylhydrazyl). Amino acid composition of yogurt was analyzed using ultra performance liquid chromatography (UPLC). The research result of amount of LAB on dadih are 1.01 x 1011 CFU/mL, has fulfilled the requirements of SNI 2981: 2009. Yogurt produced with the addition of dadih by 10% (v/v) is most accepted by panelists. The highest antioxidant activity was obtained by yogurt with 2.5% addition of dadih with IC50 value of 78.28 ppm. Yogurt almost contain all essential and non-essensial amino acids including tyrosine and phenylalanine as the antioxidants. Measurement of water, ash, protein and fat content in the sample meets the requirements of SNI 2981: 2009.

Changes in the Content of Organic Acids and Expression Analysis of Citric Acid Accumulation-Related Genes during Fruit Development of Yellow (Passiflora edulis f. flavicarpa) and Purple (Passiflora edulis f. edulis) Passion Fruits.

Organic acids are key components that determine the taste and flavor of fruits and play a vital role in maintaining fruit quality and nutritive value. In this study, the fruits of two cultivars of passion fruit Yellow (Passiflora edulis f. flavicarpa) and purple (Passiflora edulis f. edulis) were harvested at five different developmental stages (i.e., fruitlet, green, veraison, near-mature and mature stage) from an orchard located in subtropical region of Fujian Province, China. The contents of six organic acids were quantified using ultra-performance liquid chromatography (UPLC), activities of citric acid related enzymes were determined, and expression levels of genes involved in citric acid metabolism were measured by quantitative real-time PCR (qRT-PCR). The results revealed that citric acid was the predominant organic acid in both cultivars during fruit development. The highest citric acid contents were observed in both cultivars at green stage, which were reduced with fruit maturity. Correlation analysis showed that citrate synthase (CS), cytosolic aconitase (Cyt-ACO) and cytosolic isocitrate dehydrogenase (Cyt-IDH) may be involved in regulating citric acid biosynthesis. Meanwhile, the PeCS2, PeACO4, PeACO5 and PeIDH1 genes may play an important role in regulating the accumulation of citric acid. This study provides new insights for future elucidation of key mechanisms regulating organic acid biosynthesis in passion fruit.

Metabolomics study of fibroblasts damaged by UVB and BaP

We have recently shown that both UVB and BaP can induce the production of ROS, apoptosis and even cancer. However, the differences in the metabolic profiles of skin damaged by UVB, BaP or UVB combined with BaP have not been studied. Therefore, we examined the metabolic changes in the human foreskin fibroblast injured by UVB or BaP or the combination of the two, using ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (qTOF-MS). 24 metabolites were altered in the UVB damage group, 25 in the BaP damage group, and 33 in the UVB combined with BaP group. These alterations indicated that the metabolic mechanisms of HFF-1 cells treated with UVB or BaP are related to multiple main metabolites including glycerophosphocholine (PC), lactosylceramide (LacCer), guanidinosuccinic acid (GSA), glutathione(GSH), and lysophosphatidylcholine (LysoPC) and the main mechanisms involved glycerophospholipid and glutathione metabolism. Thus, our report provided useful insight into the underlying mechanisms of UVB and BaP damage to skin cells.

Zanthoxylum bungeanum Seed Oil Elicits Autophagy and Apoptosis in Human Laryngeal Tumor Cells via PI3K/AKT/mTOR Signaling Pathway.

Zanthoxylum bungeanum seed oil (ZBSO) is the main extract of the edible drug Zanthoxylum bungeanum seeds. Recent reports have proved that it has a significant cytotoxic effect on various cancer cells. However, systematic investigation on the role of ZBSO in laryngeal carcinoma (LC) is rare. The aim of the study was to reveal the function of ZBSO on human laryngeal squamous carcinoma cells (Hep-2) and to elucidate its underlying mechanism. In this study, the chemical composition analysis of ZBSO was done using Ultra Performance Liquid Chromatography (UPLC), and the anti-tumor effect of ZBSO on Hep-2 cells was evaluated by cell proliferation, apoptosis and cell cycle experiments. qRT-PCR, immunohistochemistry (IHC) and Western blotting were used for mechanistic investigation at the molecular level. The main compound of ZBSO was identified as polyunsaturated fatty acids. Furthermore, as compared to normal cells, significant inhibitory activities of ZBSO were observed on Hep-2 cells with dose- and timedependency, which induced apoptosis, blocked cell cycle at the S phase, and inhibited cell proliferation. In addition, IHC results showed a difference in the level of protein expression of ZBSO-induced autophagy-related markers. At last, Western blotting results indicated that ZBSO could inhibit the expression and phosphorylation levels of PI3K/AKT/mTOR protein. The anti-LC effect of ZBSO might be intimately associated with the induction of autophagy and the inhibition of the PI3K/AKT/mTOR signaling pathway. ZBSO may be a potential anti-laryngocarcinoma agent.

Hydrolysis Kinetics of a Novel 3,4-Dichloroisothiazole Plant-Activator Candidate LY5-24-2 by UPLC-Q-TOF

Hydrolysis characteristics of a novel 3,4-dichloroisothiazole based fungicide with activating plant defense responses as a candidate plant-activator LY5-24-2 were investigated under different conditions (pH and temperature) using ultra-performance liquid chromatography (UPLC) and quadrupole Time-of-Flight (Q-TOF). The hydrolysis case complied with the first-order kinetic model, with half-lives ranging from 4.8h to 3.2days at pH 4, 7, 9 and temperature at 25 and 50℃. One of the hydrolysis metabolite 3,4-dichloroisothiazole-5-carboxylic acid (metabolite 1, M1) was determined and quantified using authentic standard. The other hydrolysate 3-chloro-5-(trifluoromethyl) pyridin-2-amine (metabolite 2, M2) was determined and identified according to accurate mass information, fragmentation patterns and principle component analysis (PCA). By utilizing high-resolution mass spectrometry and multivariate statistical analysis, hydrolysis dynamic of the metabolites was characterized and figured out. This research provided a non-target screening method to analyze hydrolysis metabolites of a new plant-activator and to find its degradation products in aqueous solution.

Automated PET-RAFT Polymerization Towards Pharmaceutical Amorphous Solid Dispersion Development.

In pharmaceutical oral drug delivery development, about 90% of drugs in the pipeline have poor aqueous solubility leading to severe challenges with oral bioavailability and translation to effective and safe drug products. Amorphous solid dispersions (ASDs) have been utilized to enhance the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). However, a limited selection of regulatory-approved polymer excipients exists for the development and further understanding of tailor-made ASDs. Thus, a significant need exists to better understand how polymers can be designed to interact with specific API moieties. Here, we demonstrate how an automated combinatorial library approach can be applied to the synthesis and screening of polymer excipients for the model drug probucol. We synthesized a library of 25 random heteropolymers containing one hydrophilic monomer (2-hydroxypropyl acrylate (HPA)) and four hydrophobic monomers at varied incorporation. The performance of ASDs made by a rapid film casting method was evaluated by dissolution using ultra-performance liquid chromatography (UPLC) sampling at various time points. This combinatorial library and rapid screening strategy enabled us to identify a relationship between polymer hydrophobicity, monomer hydrophobic side group geometry, and API dissolution performance. Remarkably, the most effective synthesized polymers displayed slower drug release kinetics compared to industry standard polymer excipients, showing the ability to modulate the drug release profile. Future coupling of high throughput polymer synthesis, high throughput screening (HTS), and quantitative modeling would enable specification of designer polymer excipients for specific API functionalities.

A Rapid UPLC Method for the Simultaneous Quantitation of Caffeic Acid Derivatives in Dried Extracts of Echinacea Purpurea.

Echinacea purpurea is a traditional medicinal plant widely used as adjuvant for the treatment of respiratory and urinary infections. Caffeic acid derivatives are considered the main active markers, such as chicoric acid, caftaric acid and chlorogenic acid. An analytical method using ultra performance liquid chromatography (UPLC) and diode array detector was developed and validated, to quantify caffeic acid derivatives in commercial dried extracts of EP. UPLC method was developed using a C18 column (50× 2.1mm, 1.8μm), at 30°C. Mobile phase was composed of acetonitrile and 0.05% (v/v) formic acid aqueous solution (10:90), flow rate 0.2mL/min. Injection volume was 10μL and detection was performed at 300 and 330nm. The developed method complied with all required validation parameters, and showed to be linear, precise, accurate, selective and robust for all caffeic acid derivatives. Using the validated method, the levels of caftaric acid (0.110-0.507%w/w), chicoric acid (0.040-0.179%w/w) and chlorogenic acid (0.013-0.084%w/w) were determined in five commercial dried extracts of E. purpurea, with significant variation in the contents between different samples, indicating the need of standardization and control of individual caffeic acid derivatives in commercial extracts.

Effects of Shengmai San on key enzymes involved in hepatic and intestinal drug metabolism in rats

Ethnopharmacological relevanceShengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions. Aim of the studyThe studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model. Materials and methodsThe SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined. ResultsSchisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity. ConclusionsThese findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A.

Saengmaeksan, a traditional herbal formulation consisting of Panax ginseng, ameliorates hyperuricemia by inhibiting xanthine oxidase activity and enhancing urate excretion in rats

BackgroundSaengmaeksan (SMS) is a traditional Korean medicine composed of three herbs, Panax ginseng, Schisandra chinensis, and Liriope platyphylla. SMS is used to treat respiratory and cardiovascular disorders. However, whether SMS exerts antihyperuricemic effects is unknown. MethodsEffects of the SMS extract in water (SMS-W) and 30% ethanol (SMS-E) were studied in a rat model of potassium oxonate-induced hyperuricemia. Uric acid concentrations and xanthine oxidase (XO) activities were evaluated in the serum, urine, and hepatic tissue. Using renal histopathology to assess kidney function and uric acid excretion, we investigated serum creatinine and blood urea nitrogen concentrations, as well as protein levels of renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), and organic anion transporter 1 (OAT1). The effects of SMS on in vitro XO activity and uric acid uptake were also evaluated. The components of SMS were identified using Ultra Performance Liquid Chromatography (UPLC). ResultsSMS-E reduced serum uric acid and creatinine concentrations, and elevated urine uric acid excretion. SMS-E lowered XO activities in both the serum and liver, and downregulated the expression of renal URAT1 and GLUT9 proteins. SMS-E reduced renal inflammation and IL-1β levels in both the serum and kidneys. SMS-E inhibited both in vitro XO activity and urate uptake in URAT1-expressing oocytes. Using UPLC, 25 ginsenosides were identified, all of which were present in higher levels in SMS-E than in SMS-W. ConclusionSMS-E exhibited antihyperuricemic effects by regulating XO activity and renal urate transporters, providing the first evidence of its applicability in the treatment of hyperuricemia and gout.

Purple Sweet Potatoes from East Java of Indonesia Revealed the Macronutrient, Anthocyanin Compound and Antidepressant Activity Candidate.

Introduction:The development of new antidepressant is crucial to overcome the remission rate limitation. Anthocyanin on purple sweet potatoes (PSP) from East Java cultivar previously demonstrated a behavioural effect. However, the certain mechanism and the nutritional compound need further exploration.Aim:This study aimed to characterize macronutrient content, amino acids, anthocyanin, and revealed the potential of PSP from East Java-Indonesia as antidepressant agent through D2-dopamine receptor (D2DR).Methods:This study was characterized the macronutrient content using proximate analysis. The amino acids were analysed using Ultra-Performance Liquid Chromatography (UPLC) and High-Performance Liquid Chromatography (HPLC). Anthocyanin was identified using Ultra High-Performance Liquid Chromatography (UHPLC). Molecular docking was conducted to predict the interaction between anthocyanins and D2 dopamine receptor.Results:We were found the predominance of water on proximate analysis. Alanine was demonstrated as the highest content of amino acid. Cyanidin, cyanidin-3-O-glucoside and peonidin-3-O-glucoside were identified as major anthocyanin content. Molecular docking was showed that cyanidin bound to similar binding site with dopamine on D2DR with stronger interaction than cyanidin-3-glucoside.Conclusion:Current study was indicated that cyanidin as major anthocyanin from purple sweet potatoes has potential health beneficial as antidepressant potential candidate.

Rhaponticin contained Rheum officinale root extract improved Postmenopause symptom of Ovariectomized Rat.

ABSTRACTPostmenopausal women have decreased levels of the hormone estrogen. Reduced estrogen levels will often involve many symptoms that reduced quality of life. This research aims to analyze the effects of Rheum officinale root extract on postmenopausal model rats. To this end, thirty rats underwent ovariectomy (OVX) surgery and six rats were operated without having their ovaries removed. The OVX was confirmed by body weight–uterus weight ratio and a vaginal swab. Six groups of the rats were performed: SHAM group and negative control groups are given vehicle; the positive control was assigned tamoxifen; and the extract has been given three doses 7, 35, and 175 mg/200 g BW, respectively, for 30 days. The calcium content of bone ash was measured using atomic absorption spectrophotometer. Blood pressure was evaluated using CODA®, and the metabolites in the blood were assessed using gas chromatography–mass spectrometry (MS) and high-performance liquid chromatography. As a result, using ultra-performance liquid chromatography (UPLC)-MS, we found that the extract's major component was rhaponticin and its metabolites. The bone calcium levels increased with increasing doses of the extract. In the OVX group, the bone calcium content was decreased significantly 51.56% ± 8.9% g compared with the SHAM group 62.97% ±5.6% g, and the administration of Rheum extract could restore the calcium content of the bone to become 69.27% ± 3.8% g. From the above data, we concluded that Rheum root extracts contain astrigin, rhaponticin, rhapontigenin, and desoxyrhaponticin. Rheum root extract could improve calcium content and lipid profiles of OVX rats by stimulation osteoblastogenesis. Rheum root extracts could control the blood pressure of OVX rats by reducing lipid profiles.

A comparison between univariate and multivariate statistical techniques to determine source of pen inks using ultra-performance liquid chromatography (UPLC) chromatograms

Forensic ink analyst is frequently asked to determine the source of questioned ink entries. Ultra-performance liquid chromatography (UPLC) technique is useful for profiling organic compounds of pen inks. However, interpreting the UPLC chromatograms requires the use of statistical approaches in order to ensure the objectivity and reliability of the results. This work aims to compare the performances of univariate and multivariate statistical techniques in determining the source of ballpoint pen inks based on UPLC chromatograms. Two sets of UPLC chromatograms comprising of four blue and black ballpoint pen inks were, respectively, treated like a mini ink database for predicting unknown blue and black pen inks. The pitfalls and merits of descriptive and inferential statistics, principal component analysis (PCA) and hierarchical clustering analysis (HCA) in interpreting the source of pen inks based on the mini database were critically discussed. The results strongly indicated the superiority of HCA over the other statistical techniques. The descriptive statistics presented the worst prediction results whereas the one-way ANOVA-LSD test rarely produced definite identification. In conclusion, HCA appears to be the most robust statistical-based approach in the inferring source of pen ink based on UPLC chromatograms.

Extracellular and intracellular zilpaterol and clenbuterol quantification in Hep G2 liver cells by UPLC-PDA and UPLC–MS/MS

Zilpaterol and Clenbuterol are β-adrenergic agonists that have been widely used to feed cattle. Although the use of Zilpaterol has been approved, Clenbuterol is still used illegally at unknown doses. However, the research of both substances has been based mainly on the evaluation of residues. To our knowledge, this is the first time that a cellular model using Hep G2 cells treated with Zilpaterol and Clenbuterol is presented as an alternative approach to quantify both drugs at the cellular level. Thus, a complete analytical methodology has been developed for the accurate quantitation of these β-adrenergic agonists in both cellular compartments. We propose the use of ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) for extracellular determinations while UPLC coupled to a tandem mass spectrometer (UPLC-MS/MS) for intracellular analysis. The methods were fully validated in terms of selectivity, linearity, accuracy, and precision, limits of detection and quantitation (LOD and LOQ, respectively), stability, carryover, and matrix effect. The method for intracellular content was linear ranging from 0.25 to 8 ng/mL while for extracellular content, the concentration of Zilpaterol and Clenbuterol ranged from 0.125 to 4 μg/mL, with correlation coefficients of R > 0.98 and >0.99, respectively. The combination of the two methodologies in the cellular model showed intracellular concentrations of 0.344 ± 0.06 μg/mL and 2.483 ± 0.36 μg/mL for Zilpaterol and Clenbuterol, respectively. Extracellular concentration was 0.728 ± 0.14 μg/mL and 0.822 ± 0.11 μg/mL for Zilpaterol and Clenbuterol, respectively. This work shows the potential applications of cellular modelling in the study of toxicity for the mentioned drugs.

Herbal Combination of Phyllostachys pubescens and Scutellaria baicalensis Inhibits Adipogenesis and Promotes Browning via AMPK Activation in 3T3-L1 Adipocytes.

To investigate the anti-obesity effects and underlying mechanism of BS21, a combination of Phyllostachys pubescens leaves and Scutellaria baicalensis roots was used to investigate the effects of BS21 on adipogenesis, lipogenesis, and browning in 3T3-L1 adipocytes. The expression of adipocyte-specific genes was observed via Western blot, and the BS21 chemical profile was analyzed using ultra-performance liquid chromatography (UPLC). BS21 treatment inhibited adipocyte differentiation and reduced the expression of the adipogenic proteins peroxisome proliferator-activated receptor γ (PPAR-γ), CCAAT/enhancer-binding protein (C/EBP-α), and adipocyte protein 2 (aP2), as well as the lipogenic proteins sterol regulatory element-binding protein 1c (SREBP-1c) and fatty-acid synthase (FAS). BS21 enhanced protein levels of the beta-oxidation genes carnitine palmitoyltransferase (CPT1) and phospho-acetyl-coA carboxylase (p-ACC). BS21 also induced protein expressions of the browning marker genes PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α), and uncoupling protein (UCP) 1, and it induced the expression of the thermogenic gene UCP2. Furthermore, BS21 increased adenosine monophosphate-activated protein kinase (AMPK) activation. UPLC analysis showed that BS21 contains active constituents such as chlorogenic acid, orientin, isoorientin, baicalin, wogonoside, baicalein, tricin, wogonin, and chrysin. Our findings demonstrate that BS21 plays a modulatory role in adipocytes by reducing adipogenesis and lipogenesis, increasing fat oxidation, and inducing browning.

Region-Specific Characterization of N-Glycans in the Striatum and Substantia Nigra of an Adult Rodent Brain.

N-glycan alterations in the nervous system can result in different neuropathological symptoms such as mental retardation, seizures, and epilepsy. Studies have reported the characterization of N-glycans in rodent brains, but there is a lack of spatial resolution as either the tissue samples were homogenized or specific proteins were selected for analysis of glycosylation. We hypothesize that region-specific resolution of N-glycans isolated from the striatum and substantia nigra (SN) can give an insight into the establishment and pathophysiological degeneration of neural circuitry in Parkinson's disease. Specific objectives of the study include isolation of N-glycans from the rat striatum and SN; reproducibility, resolution, and relative quantitation of N-glycome using ultra-performance liquid chromatography (UPLC), weak anion exchange-UPLC, and lectin histochemistry. The total N-glycomes from the striatum and SN were characterized using database mining (GlycoStore), exoglycosidase digestions, and liquid chromatography-mass spectrometry. It revealed significant differences in complex and oligomannose type N-glycans, sialylation (mono-, di-, and tetra-), fucosylation (tri-, core, and outer arm), and galactosylation (di-, tri-, and tetra-) between striatum and SN N-glycans with the detection of phosphorylated N-glycans in SN which were not detected in the striatum. This study presents the most comprehensive comparative analysis of relative abundances of N-glycans in the striatum and SN of rodent brains, serving as a foundation for identifying "brain-type" glycans as biomarkers or therapeutic targets and their modulation in neurodegenerative disorders.