Liquid Chromatography-evaporative Light-scattering Detection Research Articles

Systematic Chemical Analysis ofCrude Glycan Isolates from the Seven‑herb Decoction Quanzhenyiqitang with Anti-COPD Activity.

The classical Chinese Medicine prescription, Quanzhenyiqitang (QZYQT), containing seven tonic herbs (Shudi, Dangshen, Maidong, Baizhu, Niuxi, Fuzi, and Wuweizi) is clinically used to treat chronic obstructive pulmonary disease (COPD). Although there are studies on the pharmacological effects of QZYQT, little attention has been paid to its active carbohydrate ingredients. We performed a systematic chemical analysis of the crude glycan isolates from the seven-herb decoction (GI-QZYQT) after confirming its anti-COPD activity. GI-QZYQT could enhance lung function, reduce lung damage, and alleviate inflammatory response in mice with COPD. Moreover, two monosaccharides (fructose and glucose) and six oligosaccharides (sucrose, melibiose, 1-kestose, raffinose, mannotriose, and stachyose), accounting for 40.23% of GI-QZYQT, were discovered using hydrophilic interaction liquid chromatography-evaporative light-scattering detection. Inulin-type fructan with an average molecular weight of 2112 Da was identified using high-performance gel-permeation chromatography in combination with monosaccharide mapping analysis, accounting for 20.10% of GI-QZYQT in mass. The comparison study showed that the identified monosaccharides, oligosaccharides, and the inulin-type fructan of GI-QZYQT were mainly derived from herbs of Shudi, Dangshen, Maidong, Baizhu, and Niuxi. These findings provide crucial information on the chemical composition of GI-QZYQT, which is vital for the in-depth understanding of its bioactivity, mechanism, and product development.

Simultaneous determination of six rare sugars in solid foods by high performance liquid chromatography-evaporative light-scattering detection

Excessive sugar consumption is associated with metabolic health problems. Rare sugars are gradually being used as substitutes for sugar, and their consumption is increasing daily, raising food-safety issues such as false advertising, adulteration, and overdosing. The determination of rare-sugar compounds has attracted considerable attention in recent years. However, no standard method for the simultaneous determination of six rare sugars (allulose, tagatose, trehalose, isomaltulose, erythritol, and mannitol) in solid foods is available. Therefore, establishing a suitable analytical method for these sugars is necessary. In this study, high performance liquid chromatography coupled with evaporative light-scattering detection was used to determine rare sugars in solid foods. The optimum chromatographic and detector conditions were determined by evaluating the instrument parameters. Analysis was carried out on a Zorbax Original NH2 column (250 mm×4.6 mm, 5 μm) via flow-rate gradient elution (0-15 min, 1.0 mL/min; 15-18 min, 1.0-2.0 mL/min; 18-25 min, 2.0 mL/min) with acetonitrile-water (80∶20, v/v) as the mobile phase. Sharp and symmetric chromatographic peaks were obtained under these conditions. The resolutions for all the six rare sugars were greater than 1.5. Optimization of the evaporative light-scattering detector was extremely important to the responses of the rare-sugar compounds. The two most significant parameters were the nebulizer carrier gas flow rate and drift tube temperature. The detection system was operated under the following conditions: the drift tube temperature was set to 50 ℃, the nebulizer carrier gas was high-purity nitrogen, the carrier gas flow rate was 1.0 mL/min, the nitrogen pressure was regulated to 275.79 kPa, and the gain factor was set to 3. The sample was extracted with 25 mL of water, shaken and vortexed for 10 min, purified with 200 μL of zinc acetate solution and 200 μL of potassium ferricyanide solution, and centrifuged at 4500 r/min for 10 min. Next, 1 mL of the supernatant was passed through a 0.22 μm aqueous-phase filter membrane, and the filtrate obtained was analyzed using the evaporative light-scattering detector. The six rare sugars were quantitatively analyzed using the external standard method and showed good linearity with coefficients of determination (R2) greater than 0.9985. The limits of detection and quantification were 0.020-0.60 and 0.60-1.8 g/100 g, respectively. In addition, when blank solid food samples were spiked with the analytes at three levels, the average recoveries of the six rare sugars were 92.6%-103.2%, with relative standard deviations (RSDs) of 0.7%-4.4%. An RSD of <5% indicated that the method had good precision. Interference experiments were performed to determine whether the sugars and artificial sweeteners commonly found in solid foods affected the targets. The method established in this study was used to analyze the contents of the six rare sugars in actual solid food samples. The experimental results showed various levels of rare glycoconjugates in different solid foods. Moreover, the actual compositions and labeled of rare glycoconjugates in the solid foods were generally consistent. The proposed method features simple operation, rapid results, high sensitivity, and good reproducibility; thus, it meets the requirements for the detection of the six rare sugars in solid foods. It also provides technical support for the development of methodological standards and detection limits for rare sugars in Chinese foods. The results of this study are of great relevance for the daily monitoring of the levels of the six rare sugars in solid foods.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro

ObjectiveQili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model. MethodsThe main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia–reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting. ResultsApoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX. ConclusionQLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Phospholipid analysis in whey protein products using hydrophilic interaction high-performance liquid chromatography-evaporative light-scattering detection in an industry setting

The main objective of this work was to develop an analytical method that can be used in a dairy manufacturing facility for the quantitation of phospholipids in dairy products. Total lipids from a dairy matrix were obtained first by Folch extraction. The total lipid extract was then applied to a silica gel-based solid-phase extraction column, and triglycerides and other nonpolar lipids were separated from the phospholipids and sphingolipids. Quantitation was performed by hydrophilic interaction HPLC coupled to evaporative light-scattering detection using a quaternary separation method. The method was validated using a commercial whey protein phospholipid concentrate and was used to analyze phospholipid and sphingolipid composition in buttermilk, whey protein concentrate, whey protein phospholipid concentrate, and several other dairy ingredients. This method was sensitive and reproducible and can be used in the dairy industry as a research tool to develop new value-added dairy phospholipid products, then later as a standard protocol for quality assurance analysis of current and future products.

Detecting kinsenoside from Anoectochilus roxburghii by HPLC-ELSD with dual columns of NH2 and AQ-C18.

Kinsenoside is a characteristic component of Anoectochilus roxburghii and accounts for this herb's medicinal and edible values. No international certified standard method is available for kinsenoside analysis as well as extraction and preservation. To develop a more accurate analytical method of kinsenoside. The effects of extraction and drying methods of A. roxburghii on kinsenoside efficiency were investigated for the first time, as well as to examine the kinsenoside stability. The amino (NH2 ) and AQ-C18 columns for detecting kinsenoside extract was systematically compared by high-performance liquid chromatography evaporative light-scattering detector (HPLC-ELSD) and HPLC-diode-array detector (DAD), respectively. Kinsenoside, its epimer goodyeroside A and the degradation product during preservation were identified through HPLC-electrospray ionization mass spectrometry (ESI-MS). An accurate method of kinsenoside detection by HPLC-ELSD with dual columns of NH2 and AQ-C18 was established. The ratio of Cgoodyeroside A to Ckinsenoside (Y) was determined using the AQ-C18 column method. The concentration detected by the NH2 column was multiplied by 1/(1 + Y) as the corrected result. Using this novel method, the average deviations were reduced by 7.64%. Moreover, the efficiency of kinsenoside extraction with water was almost twice that of extraction with ethanol. Freeze drying also led to a higher extraction efficiency (38.47% increase) than hot-air drying did. Furthermore, the degradation of kinsenoside extract exceeded 70% when stored at 37 °C for 3 months. This study provides a reliable experimental method and theoretical basis for the quality control of kinsenoside from A. roxburghii, as well as other glycosides.

Quantitative Analysis of Yeast Phospholipids and Sterols by High-Performance Liquid Chromatography-Evaporative Light-Scattering Detection.

Normal-phase high-performance liquid chromatography (HPLC) is a standard method for separating the major lipid classes in an extract. Owing to the absence of a common property like light absorbance in the various lipid classes, evaporative light-scattering detection (ELSD) is the method of choice for qualitative and quantitative lipid detection. In most cases, neutral lipids and polar lipids are separated by different solvent systems, making it necessary to perform multiple analyses. Compared with other techniques like thin-layer chromatography, normal-phase HPLC-ELSD has better reproducibility and allows a higher degree of automation. Here we describe a method for separating and quantifying yeast neutral lipids and glycerophospholipids in one analytical run.

Application of LC with Evaporative Light Scattering Detector for Biogenic Amines Determination in Fair Trade Cocoa-Based Products

In this study, the separation of eight biogenic amines (cadaverine, serotonin, histamine, spermidine, spermine, tyramine, putrescine and β-phenylethylamine) by a liquid chromatography (LC) method with evaporative light scattering detection (ELSD) was performed. The LC–ELSD method was validated by comparison of the results with those obtained through LC–ultraviolet (UV) determination, based on a pre-column dansyl chloride derivatisation step, and the recorded data showed as both analytical methods can be interchangeably used for biogenic amines determination. LC–ELSD methodology showed good precision and permitted to achieve, for standard solutions, limits of detection (LOD) ranging from 0.01 to 0.02 μg ml−1 and limits of quantitation (LOQ) ranging from 0.03 to 0.05 μg ml−1. The whole methodology, comprehensive of the homogenization–extraction process and LC–ELSD analysis, has been applied in the analysis of several samples of fair trade cocoa derivatives. The most abundant amine found was histamine for a total amount of biogenic amines in the range 5.81–38.82 μg g−1. The highest amounts of biogenic amines (BAs) were found in the most processed products but never representing a possible risk for consumer health, according to the toxicity levels reported in literature and regarded as acceptable.

Colostrum Hexasaccharide, a Novel Staphylococcus aureus Quorum-Sensing Inhibitor

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD), (1)H and (13)C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.

Liquid and gas chromatographic analyses of triacylglycerols for Asian sesame oil traceability

This study aimed to investigate the triacylglycerol (TAG) composition of Asian sesame oils and to differentiate the geographic origins of the oils using the analytical data in combination with canonical discriminant analysis (CDA). The TAG compositions of 51 Korean, 19 Chinese, and 14 Indian sesame oil samples were analyzed by liquid chromatography‐evaporative light scattering detector (LC‐ELSD) and GC‐FID, separately. The TAG content values determined by LC‐ELSD showed strong positive linear correlation to those measured by GC‐FID. The CDA results showed that good discrimination (hit ratio, ∼92%) between the oil samples was achieved by applying the respective canonical variables generated from the LC‐ELSD and GC‐FID data. The CDA using GC‐FID data accurately characterized the origins of all the four additional commercial oil samples (two Korean and two Chinese products), whereas the CDA using LC‐ELSD data correctly characterized the origins of only two samples. This study demonstrated that both LC‐ELSD and GC‐FID analyses of TAG compositions were useful tools for characterizing the geographic origin of the oils and the separation of the Korean sesame oils from the other two groups of oils using the GC‐FID data was more efficient than that using the LC‐ELSD data.Practical applications: The simplicity and universality of GC‐FID and LC‐ELSD make the analysis of TAG composition using these chromatographic systems a practical and feasible procedure for tracing the geographic origin of the sesame oils. The combination of TAG analysis with methods to analyze different constituents in sesame oils would enhance the accuracy of the oil traceability.Score plots of two canonical variables derived from CDA using liquid chromatography‐evaporative light scattering detector (LC‐ELSD) data and GC‐FID data for the Korean, Chinese, and Indian roasted sesame oils.

Application of high performance liquid chromatography-evaporative light scattering detection in determination of water-soluble sugars and sorbitol in tobacco flavourings and casings

A simultaneous determination method of water-soluble sugars (rhamnose, xylose, fructose, glucose, sucrose, maltose) and sorbitol in tobacco flavourings and casings based on high performance liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) was established. The analytes were extracted with ultrasonic assisted extraction into water and cleaned-up by Isolute ENV + solid phase extraction (SPE) cartridge, and then separated on Prevail Carbohydrate ES column (250 mm x 4.6 mm, 5 microm) with acetonitrile and water (75: 25, v/v) as mobile phase. The temperature of the drift tube in ELSD was 79 degrees C and the flow rate of carrier gas of nitrogen was 2.0 L/min. The calibration curves were plotted on the double logarithmic scales with the mass concentrations of analytes in the range of about 0.06 - 1.2 g/L. The limits of detection (LODs) were in the range of 12 - 26 mg/L. And the spiked recoveries in real samples were 88% - 109%. Based on the double logarithmic calibration of analytes, experiments were carried out to indicate that the physical and chemical characteristics of analytes, the retention behaviors on column and the elution ability of mobile phase can all influence the response of ELSD.

Calibration of an Evaporative Light-Scattering Detector for the Universal Quantitative Analyses in Liquid Chromatography—Application to the Determination of Triacylglycerols in Cucurbitaceous Oils

The goal of this work is to validate a quantitative method to analyze 15 triacylglycerols (TAG) which are present in cucurbitaceous oils using a new liquid chromatography-evaporative light-scattering detector (LC-ELSD) methodology. For the majority of the TAG, the absence of chromophoric groups absorbing in a spectral zone different from those of the high performance liquid chromatography mobile phases limits the use of UV detectors. The most convenient detectors for this type of analyses are the “universal” aerosol-based detectors, and the most frequently used one among these detectors is the ELSD. TAG responses using ELSD can vary according to their structure and the analyst may not possess all the 15 different TAG standards. Therefore, the determination of these analytes in unknown samples cannot be accurate and depends on the nature of the available standards. We propose here a new quantification methodology by LC-ELSD using the standard response model A = a × mb, where a and b are numerical coefficients which are specific for each solute and both correlated. The method uses a sample dilution procedure which provides for each compound to quantify an accurate b value, and by calculation, the corresponding a value. The quantification of both coefficients a and b easily gives the direct mass determination of each TAG. Such LC-ELSD results are validated by the comparison with the fatty acid methyl ester (FAME) analysis using the conventional capillary gas chromatography-flame ionization detector that transforms non-volatile TAG in FAME after a transesterification step. To perform this validation, we compared the experimental FAME composition to the theoretical one calculated from the TAG results obtained by LC-ELSD. For each TAG, theoretical and experimental values were statistically similar, which proves and validates the relevance of the new quantification methodology proposed in this work. The concordance between the two different methods also highlights the great advantage of the direct LC-ELSD method which additionally provides more information on TAG structures.

Sipeimine-Producing Endophytic Fungus Isolated from

Ten strains of endophytic fungi were isolated from the bulbs of the traditional Chinese medicinal plant Fritillaria ussuriensis. The extract from one of them, Fu7, showed a positive reaction with Dragendorff's reagent and the same Rf value in thin-layer chromatography (TLC) analysis as authentic sipeimine. A further TLC scan and high-performance liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) showed that one ingredient of the extract of strain Fu7 had a similar absorption curve in the range 200-700 nm and the same retention time as authentic sipeimine. Thus, the fungus produces the bioactive ingredient sipeimine, as does its host plant, and could be used for the production of sipeimine by fermentation.

Bifunctional Building Blocks for Glyco-Architectures by TiCl4-Promoted Ring Opening of Cyclodextrin Derivatives

During our studies on the preparation of blocklike substituted 1,4-glucans by cationic ring-opening polymerization,1,2 we found that TiCl4 behaves differently from common initiators like Et3O+X- (X = PF6, SbCl6), BF3.Et2O, or methyl triflate, causing only ring opening under formation of alpha-maltooligosyl chlorides bearing one free hydroxyl group (4-OH) at the nonreducing end. These compounds are valuable building blocks for the preparation of new glyco-architectures since they are easily accessible starting materials for direct glycosylations or the preparation of a variety of oligomeric glycosyl donors like alkyl glycosides, thioglycosides, or azides. We successfully carried out and optimized the TiCl4-promoted ring opening with per-O-methylated, per-O-ethylated, and temporarily protected per-O-allylated cyclodextrins of various ring size. 1H NMR spectroscopy and high-pressure liquid chromatography-evaporative light-scattering detection (HPLC-ELSD) were used to characterize the products.

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb ®, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30–70 °C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC–ELSD and LC-APCI–MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.

Comparison of liquid chromatography and capillary electrophoresis methods for quantification of sodium residuals

Liquid chromatography (LC) and capillary electrophoresis (CE) methods were developed to perform the determination of residual sodium in mother liquors and successive washes of an active pharmaceutical ingredient (API). The addition of sodium chloride to the product solution results in rapid and complete crystallization of the API. The LC method was coupled to evaporative light scattering detection (ELSD) while the CE approach was based on indirect UV detection. Both methods were fully validated. Selectivity, response function, trueness, precision, accuracy, linearity and limits of detection (LOD) and quantification (LOQ) were the criteria investigated. The LC–ELSD method was found to be more sensitive than the CE/indirect UV approach. The methods were found to be valid over concentration ranges of 62–500 and 235–1500 ppm for the LC and the CE methods, respectively. Both methods were compared and used for the determination of actual samples coming from different batches of the same API chemical synthesis.

Direct detection and quantification of Alternaria alternata lycopersici toxins using high-performance liquid chromatography-evaporative light-scattering detection

Alternaria alternata lycopersici toxins are produced by the tomato pathogenic fungus A. alternata f. sp. lycopersici. They are primary determinants for stem canker disease of tomato and induce apoptosis in animals and plants. Due to the lack of a UV chromophore, their detection has relied on derivatizations or immunoassays, which require tedious pre-assay processes. Here, we describe a method for direct and quantitative detection of the toxins. The method employs an evaporative light-scattering detector (ELSD) coupled to HPLC. It gives a linear response in the range of 400–4000 ng toxin, with a detection limit of about 6 ng/μl. The detection of all major toxin congeners by HPLC-ESLD was confirmed by liquid chromatography-mass spectrometry. Thus, the method provides a simple and sensitive tool for the studies of this group of host-specific mycotoxins.

Post-column addition as a method of controlling triacylglycerol response coefficient of an evaporative light scattering detector in liquid chromatography–evaporative light-scattering detection

The non-linear response is generally the main limitation to the general quantitative use of evaporative light-scattering detection (ELSD). In the particular case of triacylglycerol (TG) analysis, we present a preliminary paper dealing with the use of post-column additives as a means of monitoring the response of such a detector. As TG can form molecular association complexes (ligand–ligate associations) with either cholesterol, urea or silver nitrate, we report the influence of the concentration of each of these chemical compounds in the liquid phase directed towards the ELSD system. The results show that the response coefficient b of the calibration curve either decreases from 1.25–1.30 to 0.51 or increases from 1.25–1.30 to 1.78 according to the nature and concentration of post-column additive. The use of cholesterol as additive, at a discrete concentration, may lead to a linear response curve ( b=1), i.e. to the direct proportionality of ELSD response versus the TG concentration, making quantitative analysis of such solutes easier. On the other hand, to improve sensitivity, the addition of silver nitrate may be chosen for an increase in b value.