Preparative Liquid Chromatography Research Articles

Matrix solid-phase dispersion combined with micro-fractionation bioactivity evaluation screening polymethoxylated flavones from Citrus peels.

Polymethoxyflavones were a unique class of natural and safe flavonoids containing two or more methoxy groups, which were also the most abundant edible part in Citrus peel. The optimum condition in the process of selective extraction of polymethoxylated flavones from Citrus peel by matrix solid-phase dispersion (MSPD) was as follows: SBA-15 as adsorbent, ethyl acetate as eluent, the mass ratio of adsorbent to sample 1:1, and the mixture of sample and adsorbent was ground for 3min. Twelve antioxidants were successfully screened by micro-fractionation bioactivity evaluation assay, in which four of them were flavonoid glycosides, seven of them were polymethoxylated flavones, and one was phenylpropanoid. 1-sinapoly-β-D-glucopyranoside (1) was reported for the first time in Citrus peel. And antioxidant capacity of 1-sinapoly-β-D-glucopyranoside, 5, 7, 8, 3', 4', 5'-hexamethoxyflavone (6), hexamethoxyflavone (11), and 5, 6, 7, 4'-tetramethoxyflavone (7) were reported for the first time. Nobiletin (compound 8), 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone (9) and tangeretin (10) were isolated and purified by countercurrent chromatography combined with preparative liquid chromatography. Antioxidant activity evaluation indicated that the three isolated polymethoxylated flavones owned similar antioxidant activity. This study indicated that MSPD combined with micro-fractionation bioactive evaluation was efficient in screening bioactive compounds for rapid extraction and effective pinpointing bioactive substances in natural products.

Elucidation of bioactivity of tricin released by thioacidolysis from wheat straw lignin

Tricin is a complex compound with chemical bonds to phenylpropane units of lignin in gramineous plants, and it is predominantly bound to lignin by β-O-4 ether bonds. Thioacidolysis cleaves the alkyl aryl ether bonds, which releases tricin from the tricin-lignin complex and maintains the natural structure of the tricin. In this study, milled wood lignin (MWL) was isolated from wheat straw by Bjӧrkman’s method, and the MWL was subjected to thioacidolysis to release tricin from the MWL. Medium-pressure preparative liquid chromatography was used for further purification. FT-IR and 1H-NMR analyses showed that the purified fraction was composed mainly of tricin-type flavonoids. The extracted tricin had strong scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, with a half maximal inhibitory concentration (IC50) of 51.9 mg/L. Drug sensitivity paper testing showed that the extracted tricin inhibited the growth of Escherichia coli. The bacteriostatic circle diameters of tricin from wheat straw MWL and quercetin standard were 9.17 and 7.52 mm, respectively. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the tricin from wheat straw MWL had possible inhibitory effect on lung cancer A549 cells, with a maximum inhibitory performance of 44.5%.

An open-source smart fraction collector for isocratic preparative liquid chromatography

Preparative liquid chromatography is a technique for separating complex samples or isolating pure compounds from complex extracts. It involves eluting samples through a packed column and selectively collecting or isolating the separated bands in a sequence of fractions. Depending on the column length and the sample complexity, a large number of fractions may be obtained, making fraction collection a laborious and time-consuming process. Manual fraction collection is also tedious, error-prone, less reproducible, and susceptible to contamination. Several commercial and lab-made solutions are available for automated fraction collection, but most systems do not synchronize with the instrument detector and collect fractions at fixed volumes or time intervals. We have assembled a low-cost Arduino-based smart fraction collector that can record the signal from the UV–vis detector of the chromatography instrument and enable the automated selective collection of the targeted bands. The system consists of a robot equipped with position sensors and a 3-way solenoid valve that switches the column effluent between the waste or collection positions. By proper programming, an Arduino board records the detector response and actuates the solenoid valve, the position sensors, and the stepper motors to collect the target chromatographic bands.

Compound-specific radiocarbon analysis of benzene polycarboxylic acids for source apportionment of polyaromatic organic matter in ambient aerosols

Polyaromatic organic matter (POM) is an important group of pollutants and light absorbers in ambient aerosols, which consists of a wide range of chemicals with fused benzene rings. POM in ambient aerosols is mainly derived from the incomplete combustion of fossil fuel and biomass. Source apportionment of POM is crucial for advising efficient mitigation of anthropogenic emissions, but that is a challenge due to the complicated composition of POM and ambient aerosols. Benzene polycarboxylic acids (BPCAs) have recently been introduced as molecular markers of atmospheric POM. Compound-specific radiocarbon analysis of BPCAs is expected to be a powerful tool for apportioning fossil sources and contemporary (i.e., biomass burning) sources of POM in ambient aerosols, yet this application is still lacking. We developed a method for radiocarbon analysis of BPCAs substituted with 3–6 carboxylic groups (B6CA, B5CA, B4CAs and B3CAs). BPCAs were isolated with preparative liquid chromatography with high recoveries (≥85%). The method is validated with reference materials with fossil and contemporary radiocarbon signatures. Successful radiocarbon analysis of BPCAs was achieved for these reference materials after correcting for the presence of average blanks (B6CA: 1.2 ± 0.2 μg, B5CA: 2.3 ± 0.6 μg, individual B4CAs: 2.7 ± 0.2 μg and individual B3CAs: 6.9 ± 0.7 μg). Source apportionment of POM based on radiocarbon analysis of BPCAs was performed for an urban dust reference material, as well as bulk aerosols and water-soluble, methanol-soluble fractions of ambient aerosols from a megacity of southern China. The results show varied contributions of fossil and contemporary sources among different POM fractions.

Identification of a Bitter Peptide Contributing to the Off-Flavor Attributes of Pea Protein Isolates

The aversive bitter taste of pea protein ingredients limits product acceptability. Compounds contributing to the bitter perception of pea protein isolates were investigated. Off-line multi-dimensional sensory-guided preparative liquid chromatography fractionation of a 10% aqueous PPI solution revealed one main bitter compound that was identified by Fourier transform ion cyclotron resonance mass spectrometry and de novo tandem mass spectrometry (MS/MS) sequencing as the 37 amino acid peptide PA1b from pea albumin and further confirmed by synthesis. Quantitative MS/MS analysis reported that the concentration of the bitter peptide was 129.3 mg/L, which was above the determined bitter sensory threshold value of 3.8 mg/L and in agreement with the perceived bitter taste of the sample.

Hederifolic acids A-D, hepta and hexasaccharides from the resin glycosides of Ipomoea hederifolia

Scarlet morning glory, Ipomoea hederifolia L. (Convolvulaceae), is an ornamental vine native to the Americas with oxytocic, cytotoxic, antipsychotic, anti-inflammatory, antioxidant, and antimicrobial properties. A chemical study of the glycosidic acids from the resin glycosides contained in the aerial parts was carried out, through their isolation as peracetylated derivatives, by recycling preparative liquid chromatography. Structure elucidation was performed by HR-MS in accordance with NMR. Four peracetylated derivatives of glycosidic acids, named hederifolic acids A-D, were identified as heptaglycosides and hexaglycosides linked to 3S,12S-dihydroxyheptadecanoic acid or 12 S-hydroxyheptadecanoic acid. Consequently, hederifolic acids B and D were found to be dehydroxylated homologs at C-3 of the fatty acid aglycones of hederifolic acids A and C, respectively.

Gastrointestinal digestion fates of lipids based on pH-stat and Caco-2 cells models: Superior bioavailability of triglyceride monomer derived from silkworm pupae oil

Silkworm pupae oil (SPO) was regarded as a good alternative to alpha-linolenic acid supplements, which contain a variety of triacylglyceride with high nutritional value. To investigate the connections between triacylglyceride composition and lipid gastrointestinal digestive absorption profiles, isolation and bioavailability of triacylglyceride monomers from silkworm pupae oil was analyzed. In this study, two triacylglyceride monomers rich in α-linolenic acid were separated by preparative liquid chromatography, namely LnLnLn (1,2,3- α-linolenic acid triacylglyceride) and LnLnL (1,2- α-linolenic acid, 3- linoleic acid triacylglyceride). The in vitro digestion in the pH-stat model results showed the digestive rate of LnLnLn was 0.4101 ± 0.0089 s−1, which was significantly higher than that of SPO (0.2494 ± 0.0108 s−1) (p < 0.05). The in vitro absorption in the Caco-2 cells model results showed the absorptivity of LnLnLn and LnLnL were 6.40 ± 0.19% and 6.06 ± 0.54%, which were significantly higher than SPO (5.43 ± 0.51%) (p < 0.05). In addition, the mRNA expression levels of lipid metabolism-related proteins provide evidence for the absorption superiority of LnLnLn and LnLnL. The present work provides novel structural lipids for rapid and efficient supplementation of α-linolenic acid.

Separation and structural characterization of unknown impurity in vancomycin by two-dimensional preparative liquid chromatography, LC-MS and NMR

Vancomycin is an effective antibiotic used for the treatment of Gram-positive bacterial infections. During the analysis of vancomycin, an unknown impurity at the level of 0.5% was detected by high-performance liquid chromatography (HPLC). To characterize the structure of the impurity, a new two-dimensional preparative liquid chromatography (2D-Prep-LC) method was developed to separate the impurity from the vancomycin sample. After further analysis including liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the structure of the unknown impurity was identified as a vancomycin analog in which the N-Methyl-leucine residue on the side chain is replaced by an N-methylmethionine residue. In this study, we established a reliable and efficient method for separating and identifying vancomycin impurities, which will provide a valuable contribution to the field of pharmaceutical analysis and quality control.

Modeling a pesticide remediation strategy for preparative liquid chromatography using high-performance liquid chromatography

BackgroundCannabis sativa L. also known as industrial hemp, is primarily cultivated as source material for cannabinoids cannabidiol (CBD) and ∆9-tetrahydrocannabinol (∆9-THC). Pesticide contamination during plant growth is a common issue in the cannabis industry which can render plant biomass and products made from contaminated material unusable. Remediation strategies to ensure safety compliance are vital to the industry, and special consideration should be given to methods that are non-destructive to concomitant cannabinoids. Preparative liquid chromatography (PLC) is an attractive strategy for remediating pesticide contaminants while also facilitating targeted isolation cannabinoids in cannabis biomass.MethodsThe present study evaluated the benchtop-scale suitability of pesticide remediation by liquid chromatographic eluent fractionation, by comparing retention times of 11 pesticides relative to 26 cannabinoids. The ten pesticides evaluated for retention times are clothianidin, imidacloprid, piperonyl butoxide, pyrethrins (I/II mixture), diuron, permethrin, boscalid, carbaryl, spinosyn A, and myclobutanil. Analytes were separated prior to quantification on an Agilent Infinity II 1260 high performance liquid chromatography with diode array detection (HPLC-DAD). The detection wavelengths used were 208, 220, 230, and 240 nm. Primary studies were performed using an Agilent InfinityLab Poroshell 120 EC-C18 3.0 × 50 mm column with 2.7 μm particle diameter, using a binary gradient. Preliminary studies on Phenomenex Luna 10 μm C18 PREP stationary phase were performed using a 150 × 4.6 mm column.ResultsThe retention times of standards and cannabis matrices were evaluated. The matrices used were raw cannabis flower, ethanol crude extract, CO2 crude extract, distillate, distillation mother liquors, and distillation bottoms. The pesticides clothianidin, imidacloprid, carbaryl, diuron, spinosyn A, and myclobutanil eluted in the first 3.6 min, and all cannabinoids (except for 7-OH-CBD) eluted in the final 12.6 min of the 19-minute gradient for all matrices evaluated. The elution times of 7-OH-CBD and boscalid were 3.44 and 3.55 min, respectively.Discussion7-OH-CBD is a metabolite of CBD and was not observed in the cannabis matrices evaluated. Thus, the present method is suitable for separating 7/11 pesticides and 25/26 cannabinoids tested in the six cannabis matrices tested. 7-OH-CBD, pyrethrins I and II (RTA: 6.8 min, RTB: 10.5 min), permethrin (RTA: 11.9 min, RTB: 12.2 min), and piperonyl butoxide (RTA: 8.3 min, RTB: 11.7 min), will require additional fractionation or purification steps.ConclusionsThe benchtop method was demonstrated have congruent elution profiles using preparative-scale stationary phase. The resolution of pesticides from cannabinoids in this method indicates that eluent fractionation is a highly attractive industrial solution for pesticide remediation of contaminated cannabis materials and targeted isolation of cannabinoids.

Ultrasonic sample introduction combined with flame assisted thermal ionization: Pretreatment-free direct mass spectrometry analysis for fraction collecting tubes of preparative liquid chromatography

Ultrasonic sample introduction combined with flame assisted thermal ionization mass spectrometry (USI-FATI-MS) was developed to monitor the fractions of preparative liquid chromatography. Recently, ultrasound-based sample introduction techniques have achieved great advance in the field of high-throughput analysis. However, it is still a challenge to directly apply these existing techniques to the analysis of macro volume samples (mL level). In this work, ultrasonic sample introduction combined with flame assisted thermal ionization was used for pretreatment-free direct mass spectrometry analysis of micro to macro volume samples (μL-mL level). Utilizing this unique design of ultrasonic sample introduction, liquid sample in the container can be quickly atomized to the gas phase without contact. Then, due to the flame assisted thermal ionization source, desolvation and ionization of the sample droplets will occur immediately. USI-FATI-MS has shown excellent sensitivity, repeatability and great compatibility to solvents and compounds with a wide range of polarity. As a proof of concept, USI-FATI-MS has been applied for rapid monitoring and identification of purified synthetic and natural products in fractions.

Trapping array-based preparative two-dimensional liquid chromatography for the purification of four components in tobacco leaves

Two-dimensional liquid chromatography (2D-LC) has gained increased attention because of its high peak capacity for separating complex samples. However, preparative 2D-LC aimed at isolating compounds is significantly different compared with one-dimensional liquid chromatography (1D-LC) in terms of method development and system configuration; thus, it is less developed than its analytical counterpart. The use of 2D-LC in large-scale product preparation has rarely been reported. Hence, a preparative 2D-LC system was developed in this study. The system was composed of one set of preparative LC modules as a separation system, with a dilution pump, switch valves, and trap column array as the interface, to enable the simultaneous isolation of several compounds. Tobacco was used as a sample, and the developed system was applied to isolate nicotine, chlorogenic acid, rutin, and solanesol. The chromatographic conditions were developed by investigating the trapping efficiency of different types of trap column packings, and chromatographic behaviors under different overload conditions. The four compounds were isolated in one 2D-LC run with high purity. The developed system features low cost because it employs medium-pressure isolation, excellent automation owing to its use of an online column switch, high stability, and capability for large-scale production. The isolation of chemicals from tobacco leaves as pharmaceutical raw materials could aid in the development of the tobacco industry and promote the local agricultural economy.

Targeted isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata using affinity ultrafiltration combined with preparative liquid chromatography

Saussurea obvallata (S. obvallata) is widely used in Qinghai-Tibet Plateau with high medicinal and edible values of reducing inflammation. But, the individual components and mechanisms of action still ill-defined. In this work, an integrated method using affinity ultrafiltration combined with preparative liquid chromatography was developed to identify and separate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata. The sample was pretreated using on-line medium pressure liquid chromatography to yield the active fraction. Then, the potential COX-2 ligands were screened out using affinity ultrafiltration from the targeted fraction and the identified compounds were isolated via preparative liquid chromatography. As a result, four main compounds, coniferin (1), syringin (2), roseoside (3) and grasshopper ketone (4) were targeted isolated with IC50 values of 12.34 ± 1.81, 4.04 ± 0.43, 13.91 ± 2.46 and 7.97 ± 1.21 µM, respectively. Results of this work demonstrated that the developed strategy was effective for the targeted separation of COX-2 inhibitors from natural product extracts.

Isolation and purification of arsenolipids from natural marine sources for use in speciation and toxicological studies

Environmental context Arsenic exists in different chemical forms in our environment, among others as arsenolipids that are predominately found in appreciable quantities in various marine organisms (seafood). Their origin, biosynthetic pathway, toxicity and their potential biological role are not fully understood. We report purification strategies for environmental and human health relevant arsenolipids to broaden and support innovative arsenolipid research.Rational This study aims to isolate and purify common arsenolipids found in various seafood from commercially available marine sources to provide an alternative approach to the challenging chemical synthesis.Methodology Arsenic containing fatty acids and arsenic containing hydrocarbons were purified from salmon oil, and the edible dried seaweed Wakame was used for the purification of di-acyl arsenosugar phospholipids and arsenic containing hydrocarbons. The developed purification protocols apply and combine straightforward analytical techniques by means of chemical hydrolysis, solid–liquid extraction, solvent-partitioning, solid-phase extraction and preparative liquid chromatography. Elemental mass spectrometry and HPLC/mass spectrometry were used for the development of the purification protocols and the characterisation of purified natural extracts of arsenolipids.Results The salmon oil (20.0 kg) and Wakame (0.50 kg) samples contained 12.8 and 3.19 mg of lipid-soluble arsenic, respectively. The final purified extracts of arsenic fatty acids and arsenic hydrocarbons obtained from salmon oil contained 0.52 mg As in 85 mg of total mass and 2.19 mg As in 28 mg of total mass, respectively. Purification of di-acyl arsenosugar phospholipids and arsenic containing hydrocarbons from Wakame returned extracts containing 0.345 and 0.237 mg As in ≤20 mg of total mass, respectively.Discussion In a natural product approach, arsenic containing fatty acids, arsenic containing hydrocarbons and di-acyl arsenosugar phospholipids have been purified in a multi-stage process from fish oil and algae samples. In the final purified extracts, around 90% or more of the arsenic was present as mixtures of arsenolipids of their respective arsenolipid classes in only minute amounts of total mass. Application of the purified mixtures in toxicological and speciation studies will benefit the future investigation of arsenolipids.

Antimicrobial activity of flavonoids glycosides and pyrrolizidine alkaloids from propolis of Scaptotrigona aff. postica

Stingless bees, or simply meliponines, are a large group of bees (about 550 described species), comprising the tribe Meliponini. They belong in the family Apidae, and are closely related to Apis mellifera. Scaptotrigona affinis postica Latreille, 1807 from northeast of Brazil is popularly known as “tubi” in Maranhão State. Flavones di-C-glycosides, and the pyrrolizidine alkaloid 3-methoxy-7-(2-methylbutyryl)-9-echimidinyl retronecine (7) were reported previously in propolis from S. postica and detected only in samples collected in October, November and December. The presence of pyrrolizidine alkaloids and their N-oxides were reported in honey and pollen. In the present study, the preparative liquid chromatography in reversed-phase column from ethanolic extract of propolis allowed the separation of fractions 40AEP and 40 MEP, which were analyzed by LC-MS. Many pyrrolizidine alkaloids, among them, lithosenine (14), lithosenine arabinoside (19), 7-angeloyl-9-(2,3- dihydroxybutyryl) retronecine (1), 7-(2- methylbutyryl) retronecine (3), 9-sarracinoyl retronecine (13) and viridinatine (8), besides the flavonoids schaftoside (15), aromadendrin-7-O-methyl ether (12), 7- methoxy-5,6,3’,4’,5’,6’-hexahydroxy-flavone-3-O-glucuronide (11), mangiferin (10) and mangiferin-O-methyl ether (17) were detected in these fractions. Fraction 40MEP showed antibacterial effect against gram positive and gram negative bacterial pathogens with MIC ranging from 62.5 μg/mL to 200 μg/mL, while fractions 40AEP and 40MEP showed antimicrobial activity against gram negative bacteria, including Escherichia coli D31- streptomycin resistant. The cells were treated with 40MEP and 40AEP at a concentration of 200 μg/mL and were not observed statistical difference between treated and untreated cells. HIGHLIGHTS Propolis from Scaptotrigona aff. postica bees exhibited antimicrobial activity against Escherichia coli D31- streptomycin resistant. Preparative liquid chromatography in reversed-phase column allowed the separation of fractions rich in alkaloids and flavonoids. Pyrrolizidine alkaloids and flavonoids were detected as the main constituents. Flavones di-C-glycosides and flavones C-glycosides were reported in propolis. Cell viability demonstrated that pyrrolizidine alkaloids did not cause cytotoxicity in epithelial cells.

Procedure for chromatography using mobile phase with high elution strength to recover tacrolimus from the fermentation broth

A simple, accurate, cost effective and precise method of reverse phase - high performance preparative liquid chromatography was developed to maximize recovery and yield of tacrolimus and to bring down the concentration of analog impurities with relative retention time (RRT) 0.94 and 1.2 less than 0.5%, the two impurities that affect the assay of the product. For the high performance preparative liquid chromatography, Kromacil-C8, 13 μm column consisting of 10*250mm I.D. was used with the flow rate of 3.925 ml/min and eluent was monitored at 210 nm. In isocratic mode, mobile phase containing KH2PO4 Buffer: Acetonitrile: Butanol of varying ratios was used for the resolution study. The retention time was analyzed and gradient run was carried out for the mobile phase ratio that gives maximum resolution. The gradient run was carried out for two different trials with mobile phase in two different lanes. Loading trials were carried out for the trials which gave maximum resolution. The samples were analyzed and the yield on load was calculated for all the runs. The gradient run with gradient time of 200 mins and mobile phase ratio 22.2:77.8 to 28.8:71.2 acetonitrile:butanol:buffer gave 82% yield on the basis of load which was found to be maximum among all the trials carried out. Adsorption isotherm parameters were determined for the same.

Less Configuration and More Dimensionality: Preparative Heart-Cut Multidimensional Liquid Chromatography Based on Trapping Arrays.

The resolving power of multiple dimensional liquid chromatography (mD-LC) is multiplicative as it adds dimensions. However, the issue in creating a preparative mD-LC system is that the higher the dimensionality, the more complicated the system configuration. Thus, we presented a new configuration of preparative mD-LC using one set of LC modules and trapping array-based multiple heart-cut interfaces. A preparative two-dimensional liquid chromatography (2D-LC) separation of herbal medicine formulation produced 40 compounds with a purity of >90%. During the separation process, the interface stores the fractions and allocates positions for the fractions from a different dimension; LC draws the fraction from the interface, makes nD separation, and sends isolated fractions to the interface. By repeating this process, we achieved variable dimensionality of LC separations. We also presented a preparative 3D-LC separation of herbal medicines to validate the principle of "less configuration and more dimensionality". Thus, we can explore the higher dimensional preparative separations. The developed preparative mD-LC displayed exceptional power in the isolation of various compounds and has great potential in the application of natural products.

Rapid separation of lactucin and lactucopicrin from Cichorium glandulosum by medium-pressure preparative liquid chromatography and quantitative analyses by high-performance thin-layer chromatography

Rapid separation of lactucin and lactucopicrin from Cichorium glandulosum by medium-pressure preparative liquid chromatography and quantitative analyses by high-performance thin-layer chromatography

Urea complexation combined with rapid preparative reversed-phase liquid chromatography to separate α-linolenic acid from perilla seed oil: Purity, yield, and oxidation stability

Perilla seed oil (PSO) is a major source of α-linolenic acid (ALA). In order to better separate ALA from PSO and to improve its purity and yield, a purification method for ALA that combines urea complexation (UC) with rapid preparative reversed-phase liquid chromatography (RP-RPLC) has been studied. Within the selected ranges of conditions, four one-factor experiments were found to have significant effects on the purity and yield of ALA from PSO. A response surface methodology (RSM) has been applied to optimize UC for separating and purifying ALA from PSO, which yielded optimal parameters of 95% ethanol/urea (w/w) = 5:1, urea/esterified fatty acids (E-FAs) (w/w) = 2:1, complexation time 16.3 h, and complexation temperature 17 °C. The optimal purity and optimal yield of the one-time purification ALA (OTP-ALA) obtained by UC thereby reached 86.977 ± 0.082% and 34.696 ± 0.948 %, respectively. Two-time purification conditions of OTP-ALA by RP-RPLC were also determined, and the purity of the two-time purification ALA (TTP-ALA) was increased to 99.200 ± 0.061%, corresponding to a yield of 53.888 ± 0.904 %. The effect of different added amounts of α-tocopherol on the oxidation stability of TTP-ALA has also been studied. Accelerated oxidation tests showed that the addition of α-tocopherol effectively inhibited the oxidation of TTP-ALA by reducing the peroxide value (PV). of ALA separation and purification in PSO (triglycerides refers to the collective term for all fatty acid glycerides in PSO). • An improved method for purification of ALA from perilla seed oil was developed. • RSM has been used to optimize the separation of ALA from perilla seed oil. • The combined use of the two methods results in ALA purity exceeding 99.2 %. • Add an appropriate amount of α-tocopherol improved the oxidation stability of ALA.

Metabolomic and transcriptomic exploration of the uric acid-reducing flavonoids biosynthetic pathways in the fruit of Actinidia arguta Sieb. Zucc.

Flavonoids from Actinidia arguta Sieb. Zucc. can reduce uric acid in mice. However, the molecular basis of its biosynthesis is still unclear. In this paper, we used a combination of extensively targeted metabolomics and transcriptomics analysis to determine the types and differences of flavonoids in the fruit ripening period (August to September) of two main cultivated varieties in northern China. The ethanol extract was prepared, and the potential flavonoids of Chrysin (Flavone1), Rutin (Flavone2), and Daidzein (Flavone3) in Actinidia arguta Sieb. Zucc. were separated and purified by HPD600 macroporous adsorption resin and preparative liquid chromatography. The structure was identified by MS-HPLC, and the serum uric acid index of male Kunming mice was determined by an animal model test.125 flavonoids and 50 differentially regulated genes were identified. The contents of UA (uric acid), BUN (urea nitrogen), Cr (creatinine), and GAPDH in mouse serum and mouse liver glycogen decreased or increased in varying degrees. This paper reveals the biosynthetic pathway of uric acid-reducing flavonoids in the fruit of Actinidia arguta Sieb. Zucc., a major cultivar in northern China, provides valuable information for the development of food and drug homologous functional foods.

Integrative assessment of amino acid nitrogen isotopic composition in biological tissue samples determined by GC/C/IRMS, LC × EA/IRMS, and LC × GC/C/IRMS

AbstractCompound‐specific isotope analysis of nitrogen (δ15N) in amino acids (CSIA‐AA) has significantly contributed to environmental sciences such as anthropology, biogeochemistry, and ecology. Several methods exist for determining δ15N of amino acids (AAs). Although these methods have their own strength and weakness, they have not been intercalibrated yet, especially for biological samples with matrices. To address this issue, we systematically compared AA δ15N values among three methods using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), preparative liquid chromatography (LC) separation followed by elemental analyzer/IRMS (LC × EA/IRMS), and LC separation followed by GC/C/IRMS (LC × GC/C/IRMS). The δ15N values of glutamic acid (δ15NGlu) and phenylalanine (δ15NPhe) in fish muscle, two crucial AAs for estimating the trophic positions (TPs) of organisms, were compared among methods. Although a significant difference in fish muscle δ15NGlu values was found among the three analytical methods, their δ15NGlu and δ15NPhe values were fairly consistent between all pairs of methods (n = 8, R2 = 0.9968 for GC/C/IRMS vs. LC × GC/C/IRMS; 0.9936 for LC × EA/IRMS vs. LC × GC/C/IRMS; and 0.9912 for GC/C/IRMS vs. LC × EA/IRMS), which resulted in similar TP estimates among the methods. Thus, the results provide empirical validation that the CSIA‐AA is comparable among different methods in interdisciplinary research fields. We also highlighted some critical features of each of the three analytical methods that can be used as a guideline for future CSIA‐AA research.