Rapid indirect separation of glutamine enantiomers by micellar electrokinetic chromatography. Analysis of dietary supplements.

Glutamine is the most abundant free proteinogenic α-amino acid. It is naturally produced in the organism and acts as a precursor for the synthesis of different biologically important molecules (such as proteins or nucleotides). However, under stressful conditions, the organism is unable to produce it in enough amounts to function properly. Thus, glutamine (Gln)-based supplements have become increasingly popular over the last decade. Since legal regulations establish that amino acid-based dietary supplements must contain only the L-enantiomer and not the racemate, adequate chiral methodologies are required to achieve their quality control. In this work, an analytical methodology based on the use of micellar electrokinetic chromatography is proposed for the rapid enantiomeric determination of DL-Gln in dietary supplements. Using (+)-1-(9-fluorenyl)-ethyl chloroformate as a derivatizing agent and ammonium perfluorooctanoate as separation medium, the Gln diastereoisomers formed under optimal conditions were separated in 8 min with a resolution of 2.8. The analytical characteristics of the method were evaluated in terms of linearity, precision, accuracy, and limits of detection/quantitation, and they were found appropriate for the analysis of L-Gln-based dietary supplements.

Screening of CPT1A-Targeting Lipid Metabolism Modulators Using Mitochondrial Membrane Chromatography.

Carnitine palmitoyltransferase 1A (CPT1A), which resides on the mitochondrial outer membrane, serves as the rate-limiting enzyme of fatty acid ß-oxidation. Identifying the compounds targeting CPT1A warrants a promising candidate for modulating lipid metabolism. In this study, we developed a CPT1A-overexpressed mitochondrial membrane chromatography (MMC) to screen the compounds with affinity for CPT1A. Cells overexpressing CPT1A were cultured, and subsequently, their mitochondrial membrane was isolated and immobilized on amino-silica gel cross-linked by glutaraldehyde. After packing the mitochondrial membrane column, retention components of MMC were performed with LC/MS, whose analytic peaks provided structural information on compounds that might interact with mitochondrial membrane proteins. With the newly developed MMC-LC/MS approach, several Chinese traditional medicine extracts, such as Scutellariae Radix and Polygoni Cuspidati Rhizoma et Radix (PCRR), were analyzed. Five noteworthy compounds, baicalin, baicalein, wogonoside, wogonin, and resveratrol, were identified as enhancers of CPT1A enzyme activity, with resveratrol being a new agonist for CPT1A. The study suggests that MMC serves as a reliable screening system for efficiently identifying modulators targeting CPT1A from complex extracts.

Application of SNAP-tag-EGFR cell membrane chromatography model in screening antitumor active components of Silybum marianum (L.) Gaertn.

The SNAP-tag-epidermal growth factor receptor (SNAP-tag-EGFR) cell membrane chromatography (CMC) model is a powerful tool for investigating ligand-receptor interactions and screening active ingredients in traditional Chinese medicine. Most tyrosine kinase inhibitors (TKIs) target epidermal growth factor receptors. However, TKIs associated with significant side effects and drug resistance must be addressed immediately. Therefore, there is an urgent need to develop new TKIs with high efficiency and low toxicity. Because of its low toxicity and side effects, traditional Chinese medicine has been widely employed to treat various diseases, including cancer. Hence, this study aimed to use the SNAP-tag-EGFR/CMC-high-performance liquid chromatography-mass spectrometry (HPLC-MS) two-dimensional system model as the research tool to screen and identify potential EGFR antagonists from the Chinese medicine Silybum marianum (L.) Gaertn. The applicability of the system was verified using the positive control drug osimertinib. Four potential EGFR antagonists were screened from the Chinese medicine Silybum marianum (L.) Gaertn.. They were identified as silydianin, silychristin, silybin, and isosilybin. Additionally, their pharmacological activity was preliminarily verified using a CCK-8 assay. The kinetic parameters of the four active ingredients interacting with EGFR and their binding modes with EGFR were analyzed using nonlinear chromatography (NLC) and molecular docking. This study identified silydianin, silychristin, silybin, and isosilybin from Silybum marianum (L.) Gaertn. and verified their potential antitumor effects on EGFR.

Sugar Profile Method by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection in Food, Dietary Supplements, Pet Food, and Animal Feeds: Interlaboratory Validation Study, Final Action 2018.16.

BACKGROUND: A method for sugar profile analysis granted First Action 2018.16 was subjected to a multi-laboratory study. OBJECTIVE: Perform a multi-laboratory study with this method to determine the performance parameters of repeatability and reproducibility against the AOAC Standard Method Performance Requirements (AOAC SMPR 2018.001) for Final Action status. METHODS: Eleven laboratories from three different countries participated in the study. Each laboratory was provided practice materials for successful method setup. Each laboratory then proceeded with analysis of blind duplicates of 10 different products covering the scope of the method. Results were reported to the study directors with any modifications and assessed following the procedures of Appendix D of the AOAC Official Methods of AnalysisSM (guidelines for collaborative study procedures). RESULTS: The majority of results from the study met the SMPR requirements. The data is presented along with any outlying observations or modifications. The method was proven to be flexible across different instrumentation and laboratories, and the method was updated to provide further system suitability and guidelines to maintain the performance of the method across the large scope of matrixes. CONCLUSION: The results from the collaborative study supported the method for Final Action status. The Expert Review Panel reviewed and voted to move the method forward to Final Action and was followed by review from the Official Methods Board and granted approval. HIGHLIGHTS: The method was granted Final Action Official Methods status.

Post-Chromatographic Derivatization Coupled with Mass Spectrometry as a Method of Profiling and Identification of Antioxidants; Ligustrum vulgare Phytocomplex as an Example.

High-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) coupled with radical scavenging assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) can be both used for the detection of the antioxidants in plant extracts. In this study, the ethanolic (70% v/v) extracts from different morphological parts of Ligustrum vulgare collected at different stages of maturity were used as the source of antioxidants. The final identification of antioxidants was performed using high-resolution mass spectroscopy (HRMS). As a result, 19 compounds with antioxidant properties detected with HPLC-ABTS assay and 10 compounds detected with HPTLC-DPPH/ABTS assay were identified, mostly from the group of iridoids, phenylethanoids, and flavonoids. When comparing different L. vulgare samples, it was found that the extracts obtained from leaves contained the greatest number of antioxidants. The results of this study suggest that HPTLC-DPPH/ABTS as well as HPLC-ABTS derivatization coupled with the HRMS can be successfully used for profiling and identification of antioxidants from natural sources. Planar chromatography is more suitable for screening multiple samples because of its simplicity, whereas more challenging liquid chromatography provides more detailed information and is therefore better for a selected set of samples.

Point-of-Care Diagnosis on Selenium Nutrition Based on Time-Resolved Fluorometric Glycoaffinity Chromatography.

Given the lack of timely evaluation of the well-received selenium fortification, a neat lateral-flow chromatographic solution was constructed here by using the recently identified urinary selenosugar (Sel) as a strongly indicative marker. As there are no ready-made receptors for this synthetic standard, phenylboronic acid (PBA) esterification and Dolichos biflorus agglutinin (DBA) affinity joined up to pinch and pin down the analyte into a sandwich-type glycol complex. Pilot lectin screening on homemade glycan microarrays verified such a new pairing between dual recognizers as PBA-Sel-DBA with a firm monosaccharide-binding constant. To quell the sample autofluorescence, europium nanoparticles with efficient long-life afterglow were employed as conjugating probes under 1 µs excitation. After systematic process optimizations, the prepared Sel-dipstick achieved swift and sensitive fluorometry over the physiological level of the target from 0.1 to 10 µM with a detection limit down to 0.06 µM. Further efforts were made to eliminate matrix effects from both temperature and pH via an approximate formula. Upon completion, the test strips managed to quantify the presence of Sel in not just imitated but real human urine, with comparable results to those in the references. As far as we know, this would be the first in-house prototype for user-friendly and facile diagnosis of Se nutrition with fair accuracy as well as selectivity. Future endeavors will be invested to model a more traceable Se-supplementary plan based on the rhythmic feedback of Sel excretion.

Capillary electrokinetic chromatography for studying interactions between ß-blockers and Intralipid emulsion.

Toxicity of ß-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of ß-blockers of different hydrophobicity's (log KD values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed ß-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log KD values of the ß-blockers. The binding and adsorption constants also show that less hydrophobic ß-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of ß-blockers is worth further investigation.

An intelligent extraction approach of feature information from three-dimensional DAD chromatogram for integrated quality control of traditional Chinese medicines: Gardenia jasminoides root as an example.

Traditional Chinese medicine (TCM) fingerprinting, which has the characteristics of holism and ambiguity, is a conventional strategy for the holistic quality control of TCMs. However, the fingerprinting of TCMs at the current stage generally adopts a single wavelength or few wavelengths, lacking the effective utilization of diode-array detector (DAD) chromatogram data. This study proposes an intelligent extraction approach of feature information from a three-dimensional DAD chromatogram to establish a novel bar-form-diagram (BFD) for integrated quality control of TCMs. The BFD was automatically established by the chromatographic and spectral information of a complex hybrid system in a DAD chromatogram. This covered the peak areas of target compositions at the optimal absorption wavelength. Taking 27 batches of Gardenia jasminoides root as samples, the BFD combined with chemometrics was applied for assessing the quality of samples completely, which improved the accuracy of origin classification using hierarchical cluster analysis, principal component analysis, soft independent modeling of class analogy and orthogonal partial least squares discriminant analysis. Single-wavelength fingerprinting and BFD used 23 and 38 common peaks as variables respectively, and the adjusted rand index results of the single wavelength and BFD were 0.559 and 0.819, respectively. Compared with the ergodic methods of each single wavelength, the peak recognition method in this study improved the operation speed from 180 s to 4 s and the computational complexity. The established BFD approach performed more abundant characteristic information of chemical components of TCMs and more accurate origin classification ability, and it had great advantages in the overall quality control of TCMs.

Advances in receptor chromatography for drug discovery and drug-receptor interaction studies.

Receptor chromatography involves high-throughput separation and accurate drug screening based on specific drug-receptor recognition and affinity, which has been widely used to screen active compounds in complex samples. This review summarizes the immobilization methods for receptors from three aspects: random covalent immobilization methods, site-specific covalent immobilization methods and dual-target receptor chromatography. Meanwhile, it focuses on its applications from three angles: screening active compounds in natural products, in natural-product-derived DNA-encoded compound libraries and drug-receptor interactions. This review provides new insights for the design and application of receptor chromatography, high-throughput and accurate drug screening, drug-receptor interactions and more.

Separation and Purification of Hydroxyl-α-Sanshool from Zanthoxylum armatum DC. by Silica Gel Column Chromatography.

Hydroxyl-α-sanshool is the main alkylamide produced by Zanthoxylum armatum DC., and it is responsible for numbness after consuming Z. armatum-flavored dishes or food products. The present study deals with the isolation, enrichment, and purification of hydroxyl-α-sanshool. The results indicated that the powder of Z. armatum was extracted with 70% ethanol and then filtrated; the supernatant was concentrated to get pasty residue. Petroleum ether (60-90 °C) and ethyl acetate at a 3:2 ratio, with an Rf value of 0.23, were chosen as the eluent. Petroleum ether extract (PEE) and ethyl acetate-petroleum ether extract (E-PEE) were used as the suitable enriched method. Afterward, the PEE and E-PEE were loaded onto silica gel for silica gel column chromatography. Preliminary identification was carried out by TLC and UV. The fractions containing mainly hydroxyl-α-sanshool were pooled and dried by rotary evaporation. Lastly, all of the samples were determined by HPLC. The yield and recovery rates of hydroxyl-α-sanshool in the p-E-PEE were 12.42% and 121.65%, respectively, and the purity was 98.34%. Additionally, compared with E-PEE, the purity of hydroxyl-α-sanshool in the purification of E-PEE (p-E-PEE) increased by 88.30%. In summary, this study provides a simple, rapid, economical, and effective approach to the separation of high-purity hydroxyl-α-sanshool.

Determination of the affinity of biomimetic peptides for uranium through the simultaneous coupling of HILIC to ESI-MS and ICP-MS.

Several proteins have been identified in the past decades as targets of uranyl (UO22+) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO22+ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize these sites. In this work, we developed a dedicated analytical method to determine the affinity of biomimetic, synthetic, multi-phosphorylated peptides for UO22+ and evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO22+ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We performed the first separations of several sets of UO22+ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO22+ was determined and made it possible to demonstrate that (i) the increasing number of phosphorylated residues (pSer) promotes the affinity of the peptides for UO22+, (ii) the position of the pSer in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO22+ complexation in comparison with the linear structure. These results are in agreement with those previously obtained by spectroscopic techniques, which allowed to validate the method. Through this approach, we obtained essential information to better understand the mechanisms of toxicity of UO22+ at the molecular level and to further develop selective decorporating agents by chelation.

Purification of total flavonoids from Ginkgo biloba flowers with resin column chromatography and evaluation of antioxidant activities in vitro.

Purification of total flavonoids from Ginkgo biloba flowers (GBF) extracts were studied using six resins. Adsorption-desorption experiments indicated that polyamide resin is the most suitable resin. The optimal purification process of total flavonoids of GBF was as follows: a loading concentration of 5.85 mg/mL, a loading volume of 1 bed volume (BV), a loading flow rate of 2 BV/h, a water volume of 2.67 BV, and a desorption solution of 40% ethanol. Under these conditions, the maximum purity of total flavonoids was 37.1 ± 1.1%. The antioxidant activity of purified flavonoids was further evaluated in vitro. It showed that the 40% ethanol purified fraction (Fr. B) group had the strongest antioxidant activity of the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity concentration for 50% of maximal effect (EC50, 145.4 ± 13.8 µg/mL) and ferric reducing ability (2.5 ± 0.2 mM FeSO4 equivalent mg-1 Fr. B). In addition, at the concentration of 160 µg/mL, the Fr. B strikingly increased the viability rate of hydrogen peroxide stimulated PC-12 cells to normal levels (***p < 0.001). This method provides a basis for the application and development of GBF resources. It indicated that the purified GBF flavonoids can be used as a source of potential antioxidant.

High performance anion exchange chromatographic and colorimetric methods for quality assessment of total and free polysaccharide content in Haemophilus influenzae type b conjugate vaccine containing lactose.

The presence of lactose as a stabilizer in Haemophilus influenzae type b (Hib) conjugate vaccine is a challenge for chromatographic resolution of its total and free poly ribosyl ribitol phosphate (PRP) content. Sample pretreatment using ultrafiltration was performed and had removed ≥95% of lactose in shorter time compared to the conventional dialysis process. Separation of free unconjugated PRP was performed using solid-phase extraction C4 cartridges. Hib conjugate vaccine was then analyzed for determination of total and free PRP, using two validated techniques: high performance anion exchange chromatography with pulsed amperometry (HPAEC-PAD) for ribitol determination and a colorimetric assay for phosphorus determination. Lactose removal had enabled a rapid chromatographic assay via fast depolymerization of PRP using high temperature treatment. Modifying the burning process in the colorimetric assay reduced the analysis time significantly compared to the pharmacopoeial method. Linearity was obtained over the range of 0.10-10.0 µg mL-1 for the HPAEC method and in the range of 1.0-8.0 µg mL-1 for the colorimetric one. Stability of Hib conjugate vaccine was investigated. The HPAEC results revealed about a 35% increase in free PRP content after storage under stressed conditions (moisture and temperature). The proposed methods offered a reliable and economic platform for assessing the immunogenicity, efficacy and stability of Hib conjugate vaccine containing lactose for the biopharmaceutical industry.

The phytoequivalence of herbal extracts: A critical evaluation.

The concept of phytoequivalence was developed in Germany in mid-1990 to indicate that a herbal extract is equivalent to another one, more specifically, to a clinically validated one. The pharmacological/physiological activity of an extract is related to its composition, but, due to the presence of many constituents, reliable technique are necessary to compare extracts. While the concept of equivalence among pure chemicals or isolated substances is feasible using the modern chromatographic and spectroscopic technology, specific guideline for herbal extracts or botanicals are still missing. This is mainly due to the multicomponent nature of the herbal extracts and to the natural variability of their constituents. Nevertheless, by using mathematical and/or chemometric approaches, the concept of phytoequivalence can be precisely addressed, as exemplified by a series of examples that are discussed in detail. Helpful practical recommendations based on literature data and personal experience are presented.

In vitro antiplasmodial activity and cytotoxicity of extracts and chromatographic fractions of twigs from Pappea capensis EckI & Zeyh. (Sapindaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: The Vha-Venda people of South Africa use Pappea capensis EckI & Zeyh. (Sapindaceae) twigs to treat malaria and its related symptoms. AIM OF STUDY: The main aim of this study was to evaluate the antiplasmodial and cytotoxic activity of P. capensis extracts and chromatographic fractions. Spectroscopy analysis was conducted using 1H NMR and GC-MS to tentatively identify the major classes of compounds and phytoconstituents that can be attributed to the observed antiplasmodial bioactivity. MATERIALS AND METHODS: Pappea capensis twigs were dried and then ground to fine powder. A solvent mixture of dichloromethane: methanol: water (1:0.5:0.5, v/v) was used to extract. The polar extract was separated from the non-polar. The organic extract was dried to yield a DCM (I = 60 g) extract. The methanol in the aqueous extract was evaporated using a rotary vapour and the remaining water freeze dried to yield a water extract (II = 287 g). Extract I was further partitioned using a solvent mixture of DCM: MeOH (1:1, v/v), separated and concentrated under vacuum to yield dichloromethane (III = 40 g) and methanol (IV = 15 g) extracts. A water-based decoction (V = 10 g) was also prepared to establish the clinical relevance of the preparation administered by Vha-Venda people in South Africa. Extracts II, III and IV were further subjected to silica column chromatography, eluting with a series of different solvents with increasing polarity to yield a total of 25 fractions (A - Y). In vitro antiplasmodial tests on Plasmodium falciparum (NF54) and cytotoxicity screens on mammalian L-6 rat skeletal myoblast cells were performed on all extracts and fractions. Selectivity indices (SI) were also computed for all tested extracts and fractions which were further subjected to 1H NMR spectroscopy and GC-MS analysis for the identification of the major classes of compounds present in the extracts. RESULTS: From the assayed extracts, only extract I (IC50 = 2.93 µg/ml; SI = 14), III (IC50 = 2.59 µg/ml; SI = 21) and IV (IC50 = 3.56 µg/ml; SI = 13) demonstrated the best antiplasmodial activity and selectivity. Of all assayed fractions, only N (0.6 µg/ml; SI = 91), D (0.85 µg/ml; SI = 37) and E (0.91 µg/ml; SI = 30) depicted the best antiplasmodial activity and selectivity. The 1H NMR analysis of the extracts and fractions identified the prominent class of constituents to be aliphatic based which was tentatively identified as terpenoids. When further GC-MS analysis was conducted, the presence of lupin-3-one, lupeol acetate, α-amyrin, and ß-amyrin phytoconstituents were tentatively confirmed. These constituents are triterpenoids with established antiplasmodial activity which can be tentatively attributed to the bioactivity observed in P. capensis twigs. CONCLUSION: The study validates the ethnomedicinal use of P. capensis for malaria treatment. It demonstrated the potential of discovering novel antiplasmodial constituents that could serve as drug hits through dereplication approaches where known compounds with established antimalarial activity can be bypassed to focus on the unknown.

High-Performance Anion-Exchange chromatography with conductivity detection method for simultaneous determination of nitrogen and phosphorus in polysaccharides.

Capsular polysaccharides of Streptococcus pneumoniae contain a characteristic mix of monosaccharides in their structure resulting in immunologically distinct serotypes. Pneumococcal capsular polysaccharides include sugars such as hexoses, uronic acids, hexosamines, methyl pentoses, other functional groups are attached to the sugars are N and O-acetyl groups, nitrogen and phosphorus. Most of these components can be quantified using different colorimetric methods. However, available methods for quantifying nitrogen and phosphorus are not sensitive enough and laborious. We report a highly sensitive high-performance anion-exchange chromatography-conductivity detector (HPAEC-CD) method for quantifying nitrogen and phosphorus present in pneumococcal capsular polysaccharides. The method is reliable, robust and reproducible with no interference. The LOQ for nitrogen and phosphorus of 3.125 and 62.5 ng/mL, respectively, is highly critical for estimating low levels of total nitrogen and total phosphorus. We have implemented this method to quantify total nitrogen in Typhoid Vi polysaccharide and phosphorus in Haemophilus influenzae type-b polysaccharide. This method has greater application for quantification of nitrogen and phosphorus present in low concentrations in polysaccharide vaccines/biologicals.

Development of immobilized beta1-adrenoceptor chromatography for rapid discovery of ligands specifically binding to the receptor from herbal extract.

The discovery of beta1-adrenoceptor (ß1-AR) ligands is viewed as an enormous demand for fighting ailments mediated by the receptor including cardiovascular diseases. Such pursuit is gravely challenged due to the lack of lead screening methods with high efficiency. This work developed a chromatographic method for pursuing ß1-AR ligand from the herbal extract by fusing epidermal growth factor receptor (EGFR) as a tag at its C-terminus to stably express the fusion receptor in E. coli, immobilizing the expressed EGFR-tagged ß1-AR onto ibrutinib-derivatized amino microspheres, and applying the immobilized receptor in the analysis of ligand-receptor interaction and herbal extract. Comprehensive characterizations like X-ray photoelectron spectroscopy and retention behaviors of canonical drugs demonstrated high specificity and good stability of the immobilized ß1-AR prepared through the covalent reaction between the EGFR and ibrutinib decorated on the microsphere surface. Frontal analysis of atenolol, metoprolol, and esmolol confirmed their bindings to ß1-AR with association constants of 1.07 × 104, 6.54 × 103, and 1.45 × 104 M-1. The thermodynamic analysis provided proof of electrostatic interaction, hydrogen bonds, and van der Waals force driving those interactions. Pulegone was recognized as a bioactive compound that specifically binding to ß1-AR from the extract of Ziziphora clinopodioides Lam by analyzing the retention peak through reverse-phase high performance liquid chromatography coupled with tandem mass spectrometry. These results, taken together, indicated that the current method is possible to provide an alternative for discovering ß1-AR ligands with high efficiency from complex matrices like herbal extract.

Heat-induced aggregation kinetics of potato protein - Investigated by chromatography, calorimetry, and light scattering.

In this study, the heat-induced aggregation behavior of patatin rich potato protein isolate (PPI) was investigated by reversed-phase high-pressure liquid chromatography (RP-HPLC), differential scanning calorimetry (DSC), and dynamic light scattering. It could be shown that aggregation already occurs at low temperatures, despite low degrees of unfolding. The unfolding temperature, determined by DSC, coincided with a change in the reaction kinetics, which is determined by the unfolding step below a critical temperature up to the point, where the proteins are completely unfolded. The reaction rate k as a function of the absolute temperature T is then determined by diffusion of unfolded proteins forming aggregates. This change can be visualized in the Arrhenius diagram by a change of the slope of the relationship k âˆ¼ 1/T. A change in pH from 7 to 6 shifted the critical temperature towards higher values and resulted in larger aggregate sizes, due to reduced electrostatic repulsion.

Characterization of chemical composition variations in raw and processed Codonopsis Radix by integrating metabolomics and glycomics based on multiple chromatography-mass spectrometry technology.

Codonopsis Radix, a popular food homology medicine, is widely used in clinical traditional Chinese medicine and food supplement, raw products and three types of processed products are the main forms of decoction pieces in China. However, there is no scientific basis for comprehensive chemical characterization of raw and three types of processed products. Herein, we investigated qualitatively and quantificationally secondary and primary metabolites in raw Codonopsis Radix and three types of processed products by metabolomics and glycomics employing multiple chromatography-mass spectrometry technology combined with chemometric analysis further to look for differential compounds and propose the processing-induced chemical mechanisms. The results indicated that Codonopsis Radix became dark-colored and the smell of burnt incense odor was observed after processing. The principal component analysis demonstrated that secondary metabolome and glycome were significantly altered between raw and processed products, and 36 differential secondary metabolites and 11 differential primary metabolites were finally screened through orthogonal partial least-squares-discriminant analysis. The main types of compounds are alkaloids, terpenoids, glycosides, amino acids, monosaccharides, oligosaccharides, and furfural derivatives. Meanwhile, Chemical mechanisms could be involved, including oxidation, glycosidic hydrolysis, esterification, dehydration, and Maillard reaction. This work supplies a chemical basis for the application of various types of Codonopsis Radix decoction pieces.

Sweeping-micellar electrokinetic chromatography with tandem mass spectrometry as an alternative methodology to determine neonicotinoid and boscalid residues in pollen and honeybee samples.

In this work, it is proposed for the first time an electrophoretic approach based on micellar electrokinetic chromatography coupled with tandem mass spectrometry (MEKC-MS/MS) for the simultaneous determination of nine neonicotinoids (NNIs) together with the fungicide boscalid in pollen and honeybee samples. The separation was performed using ammonium perfluorooctanoate (50 mM, pH 9) as both volatile surfactant and electrophoretic buffer compatible with MS detection. A stacking strategy for accomplishing the on-line pre-concentration of the target compounds, known as sweeping, was carried out in order to improve separation efficiency and sensitivity. Furthermore, a scaled-down QuEChERS was developed as sample treatment, involving a lower organic solvent consumption and using Z-Sep+ as dispersive sorbent in the clean-up step. Regarding the detection mode, a triple quadrupole mass spectrometer was operating in positive ion electrospray mode (ESI+) under multiple reaction monitoring (MRM). The main parameters affecting MS/MS detection as well as the composition of the sheath-liquid (ethanol/ultrapure water/formic acid, 50:49.5:0.5 v/v/v) and other electrospray variables were optimized in order to achieve satisfactory sensitivity and repeatability. Procedural calibration curves were established in pollen and honeybee samples with LOQs below 11.6 µg kg-1 and 12.5 µg kg-1, respectively. Precision, expressed as RSD, lower than 15.2% and recoveries higher than 70% were obtained in both samples. Two positive samples of pollen were found, containing imidacloprid and thiamethoxam. Imidacloprid was also found in a sample of honeybees. The obtained results highlight the applicability of the proposed method, being an environmentally friendly, efficient, sensitive and useful alternative for the determination of NNIs and boscalid in pollen and honeybee samples.