Target-Guided Isolation and Purification of Antioxidants from Urtica laetevirens Maxim. by HSCCC Combined with Online DPPH-HPLC Analysis.

Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-ß-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by 1H NMR spectroscopy.

Endogenous n-Alkanes in Vegetable Oils: Validation of a Rapid Offline SPE-GC-FID Method, Comparison with Online LC-GC-FID and Potential for Olive Oil Quality Control.

The potential of endogenous n-alkane profiling for the assessment of extra virgin olive oils (EVOO) adulteration (blends with cheaper vegetable oils) has been studied by relatively few authors. Analytical methods used for this purpose often involve tedious and solvent-intensive sample preparation prior to analytical determination, making them unattractive. A rapid and solvent-sparing offline solid phase extraction (SPE) gas chromatography (GC) flame ionization detection (FID) method for the determination of endogenous n-alkanes in vegetable oils was, therefore, optimized and validated. The optimized method demonstrated good performance characteristics in terms of linearity (R2 > 0.999), recovery (on average 94%), and repeatability (residual standard deviation, RSD < 11.9%). The results were comparable to those obtained with online high-performance liquid chromatography (HPLC)-GC- FID ( RSD < 5.1%). As an example of an application to prove the potentiality of endogenous n-alkanes in revealing frauds, the data set obtained from 16 EVOO, 9 avocado oils (AVO), and 13 sunflower oils (SFO), purchased from the market, was subjected to statistical analysis and principal component analysis. Two powerful indices, namely (n-C29 + n-C31)/(n-C25 + n-C26) and n-C29/n-C25, were found to reveal the addition of 2% SFO in EVOO and 5% AVO in EVOO, respectively. Further studies are needed to confirm the validity of these promising indices.

Combining Sprague-Dawley rat uterus cell membrane chromatography with HPLC/MS to screen active components from Leonurus artemisia.

CONTEXT: Leonurus artemisia (Lour.) S.Y.Hu (Lamiaceae) (YiMuCao in Chinese) is a traditional Chinese medicine. Leonurus artemisia has been shown to have many pharmacological effects such as increasing uterine contraction amplitude, and tension, but the active components are still unknown. OBJECTIVE: The objective of this study is to determine active components of L. Artemisia that are responsible for the biological activity using HPLC and cell membrane-based system. MATERIALS AND METHODS: The whole L. artemisia ethanol extract and its eight fractions were screened using Sprague-Dawley rat uterus cell membrane chromatography (CMC) combined with the HPLC/MS system. Oxytocin was used to investigate the activity of CMC column. The effect of active components screened from L. artemisia was studied by tension measurement of isolated rat uterine strips in vitro at a dose of 10(-7)-10(-4 )mol/L with oxytocin as a control. RESULTS: The acetone extract showed obvious activity when compared with the eight extracts of L. artemisia. From the acetone extract, in the negative ionization mode, the active compound was identified as genkwanin, with a molecular weight of 283. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions at a dose from 10(-7) to 10(-4 )mol/L. The EC50 value was 4.86 ± 4.21 µmol/L for genkwanin and 4.30 ± 3.65 µmol/L for oxytocin on the contractile amplitude of uterine strips isolated from rats. DISCUSSION AND CONCLUSION: Genkwanin was identified as the active compound in L. artemisia by this method. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions. Genkwanin may be used to uterine inertia and may have an effect on postpartum hemorrhage.

Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution.

Small-angle X-ray scattering (SAXS) measurements of proteins in solution are becoming increasingly popular with biochemists and structural biologists owing to the presence of dedicated high-throughput beamlines at synchrotron sources. As part of the ESRF Upgrade program a dedicated instrument for performing SAXS from biological macromolecules in solution (BioSAXS) has been installed at the renovated BM29 location. The optics hutch has been equipped with new optical components of which the two principal elements are a fixed-exit double multilayer monochromator and a 1.1 m-long toroidal mirror. These new dedicated optics give improved beam characteristics (compared with the previous set-up on ID14-3) regarding the energy tunability, flux and focusing at the detector plane leading to reduced parasitic scattering and an extended s-range. User experiments on the beamline have been successfully carried out since June 2012. A description of the new BioSAXS beamline and the set-up characteristics are presented together with examples of obtained data.

An Integrated Strategy for Investigating Antioxidants from Ribes himalense Royle ex Decne and Their Potential Target Proteins.

Natural products have been used extensively around the world for many years as therapeutic, prophylactic, and health-promotive agents. Ribes himalense Royle ex Decne, a plant used in traditional Tibetan medicine, has been demonstrated to have significant antioxidant and anti-inflammatory properties. However, the material basis of its medicinal effects has not been sufficiently explored. In this study, we established an integrated strategy by online HPLC-1,1-diphenyl-2-picrylhydrazyl, medium-pressure liquid chromatography, and HPLC to achieve online detection and separation of antioxidants in Ribes himalense extracts. Finally, four antioxidants with quercetin as the parent nucleus were obtained, namely, Quercetin-3-O-ß-D-glucopyranoside-7-O-α-L-rhamnopyranoside, Quercetin-3-O-ß-D-xylopyranosyl(1-2)-ß-D-glucopyranoside, Quercetin-3-O-ß-D-glucopyranoside, and Quercetin-3-O-ß-D-galactoside. Until now, the four antioxidants in Ribes himalense have not been reported in other literatures. Meanwhile, the free-radical-scavenging ability of them was evaluated by DPPH assay, and potential antioxidant target proteins were explored using molecular docking. In conclusion, this research provides insights into the active compounds in Ribes himalense which will facilitate the advancement of deeper studies on it. Moreover, such an integrated chromatographic strategy could be a strong driver for more efficient and scientific use of other natural products in the food and pharmaceutical industries.

The Mineral Oil Hydrocarbon Paradox in Olive Pomace Oils.

The aim of this work was to understand the actual content of mineral oil hydrocarbons (MOH) in olive pomace oil in order to contribute to the monitoring requested by EFSA for the food groups making a relevant impact on human background exposure. Such information will complement both the information inferred from the limits established by the EU and the interpretation of the coming toxicological risk assessment. At the same time, the origin of such a group of compounds is discussed. From the raw material to the commercial product, olive pomace oils were sampled and analyzed at different points and/or conditions. Through the ultimate online HPLC-GC-FID system, we gathered information on the MOH concentrations and molecular mass profiles (C-fractions), and through GCxGC-TOF/MS, we identified the key structures that prove the innocuousness of the mineral oil aromatic hydrocarbon (MOAH) fraction. Our approaches provided chromatographic signals on the C10-C50 range, rendering 33-205 mg/kg mineral oil saturated hydrocarbon (MOSH) and 2-55 mg/kg MOAH in the commercial product. The results confirmed that the C25-C35 cut is the main fraction to which humans are exposed via olive pomace oil, showing concentrations highly dependent on the extraction process. Moreover, the identification of the main MOAH groups showed that in olive pomace oil, mainly 1- and 2-ring species were present, being virtually free of the carcinogenic 3-7 ring aromatics.

Dual control system for comprehensive evaluation of quality consistency of herbal preparations.

This study was undertaken to establish an integrated quality control method based on the fingerprint of different testing instruments and a four-dimensional antioxidant activity profile. Firstly, the comprehensive ratio quantitative fingerprint method was used to rapidly calculate the similarity of GC fingerprint, UV-vis quantized fingerprint, HPLC fingerprint and ATR-FT-IR quantized fingerprint. In addition, a comprehensive evaluation strategy was proposed using the variation coefficient weighting algorithm, incorporating four complementary inputs, to achieve highly accurate analysis. At the same time, multi-markers assay by monolinear method was used for the first time for the quantitative analysis of multiple components. Based on this, the fingerprint-efficacy relationship was investigated, and substances that might be biologically active and the wavelength range of their distribution were predicted using the online-HPLC-DPPH-FIA method in conjunction with a PLS model built from individual and combined data matrices. The results showed that all 20 batches of samples were within 1-3 grades, with good quality consistency. According to index E, the samples' overall antioxidant capacity was also highly correlated with the year of production. The four-dimensional antioxidant activity profile also led to the conclusion that variations in antioxidant characteristics were caused by differences in the concentration and volatility of particular bioactive chemicals. Finally, mid-level data fusion produced better classification results than a single technique, further demonstrating the synergistic effects that may occur when the four types of data were combined. This study demonstrated that the combination of GC, UV-vis, ATR-FT-IR and HPLC can be used for the consistency control of herbal preparations and can elucidate the potential in predicting the antioxidant capacity of herbal preparations.

Comprehensive Multidimensional Liquid Chromatography-Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody.

Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium- and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level.

Multiattribute Monitoring of Antibody Charge Variants by Cation-Exchange Chromatography Coupled to Native Mass Spectrometry.

The aim of this study was to characterize the product variants of a therapeutic T-cell bispecific humanized monoclonal antibody (TCB Mab, ∼200 kDa, asymmetric) and to develop an online cation-exchange chromatography native electrospray mass spectrometry method (CEC-UV-MS) for direct TCB Mab charge variant monitoring during bioprocess and formulation development. For the identification and functional evaluation of the diverse and complex TCB Mab charge variants, offline fractionation combined with comprehensive analytical testing was applied. The offline fractionation of abundant product variant peaks enabled identification of coeluting acid charge variants such as asparagine deamidation, primary and secondary Fab glycosylation (with and without sialic acid), and the presence of O-glycosylation in the G4S-linker region. Consequently, a new nonconsensus N-glycosylation motif (N-338-FG) in the heavy chain CDR region was discovered. Functional evaluation by cell-based potency testing demonstrated a clear and negative impact of both asparagine deamidations, whereas the O-glycosylation did not affect the TCB Mab biological activity. We established an online native CEC-UV-MS method, with an ammonium acetate buffer and pH gradient, to directly monitor the TCB Mab charge variants. All abundant chemical degradations and post-translational amino acid modifications already identified by offline fraction experiments and liquid chromatography mass spectrometry peptide mapping could also be monitored by the online CEC-UV-MS method. The herein reported online native CEC-UV-MS methodology represents a complementary or even alternative approach for multiattribute monitoring of biologics, offering multiple benefits, including increased throughput and reduced sample handling and intact protein information in the near-native state.

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium- and high- pressure liquid chromatography combined with online high performance liquid chromatography-1,1-diphenyl-2- picrylhydrazyl detection.

Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELⓇ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.

Haas in grilled meat: Determination using an advanced lab-on-a-chip flat electromembrane extraction coupled with on-line HPLC.

In this research, a new design of channels in a lab-on-a-chip device with flat electromembrane extraction (LOC-FLEME) was fabricated. The latter microfluidic device was successfully used for the determination of 2-amino-3-methyl imidazo[4-5-f]quinolone (IQ), 2-amino-3, 8-dimethlylimidazo[4, 5-f]quinolone (MeIQ), 2-amino-3,4- dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4, 5-b] pyridine (PhIP) in grilled meat, by on-line coupling of LOC-FLEME to an HPLC system. Important parameters in extraction process were optimized. The calibration curve was linear over the range of 5-1000 ng g-1 with a correlation coefficient higher than 0.9991. The relative recoveries were between 95 and 98% at three concentration levels. The relative standard deviations were 4.1 to 6.0%. The limits of detection were 1.4, 0.9, 1.7 and 1.3 ng g-1 for PhIP, IQ, MeIQ and MeIQx, respectively. Sums of 4HAAs concentrations for different grilled meat samples were 2.75-6.17 ng g-1.

Screening Method for Argan Oil Adulteration with Vegetable Oils: An Online HPLC Assay with Postcolumn Detection Utilizing Chemometric Multidata Analysis.

This study is focused on examining the tocopherol isomers (α-, γ-, and δ-) fingerprinting by online RP-HPLC analysis with post column detection using CUPRAC (cupric reducing antioxidant capacity) methodology for argan oil authenticity. The proposed online assay was validated with good precision, reproducibility, and linearity. Sixteen argan oil samples (100% pure-certified and other commercial argan oils), possible adulterating vegetable oils (i.e., olive, sunflower, corn, and soya oils), and virgin argan oil blended with olive, sunflower, corn, and soya oils at levels of 5%, 10%, 15%, and 20% were analyzed. Spectrophotometric CUPRAC, DPPH, and ABTS assays were applied. Discrimination of fraudulent argan oils from virgin samples was performed by utilizing orthogonal partial least-squares discriminant analysis (OPLS-DA) regression modeling with good sensitivity and specificity. We suggested [γ-toc/α-toc] value as a new first screening adulteration factor (AF) that could be used to assess fraudulent argan oil samples. The distinct decrement in AF value was observed by the increase of adulteration rate. The AF values for virgin argan oils were ranged from 11.8 (lower limit) to 18.6 (upper limit). The presence of ß-sitosterol detected in commercial argan oils (with AF values out of limit values) was evaluated as fraudulent which was in accordance with the proposed assay. Our method enabled the detection of argan oil samples at adulteration levels of >5% in the case of sunflower, olive, and soya oils, >15% in the case of corn oil. This method may be an alternative and specific assay for the authentication and quality detection of commercial argan oils.

The analysis of saturated and aromatic mineral oil hydrocarbons in dry foods and from recycled paperboard packages by online HPLC-GC-FID.

The purpose of this study was to determine the concentrations of mineral oil hydrocarbons in dry foodstuffs packed in recycled paperboard, which were imported from different foreign countries to Germany. After collection, mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in dry foodstuffs and recycled paperboard were analysed using online coupled high-performance liquid chromatography-gas chromatography-flame ionisation detection (online HPLC-GC-FID) far before the end of the shelf life of the samples. Our results showed that recycled paperboard has MOAH content higher than that of dry foodstuffs. The proportion of MOAH within total mineral oil hydrocarbons was determined to be 7-45% in dry foodstuffs and 4-48% in paperboard. In addition, 29% of the products were found to contain over 1.00 mg/kg MOAH, with a maximum of 2.72 mg/kg in oatmeal. White colour recycled paperboard contained lower amounts of MOSH and MOAH than that of brown and grey colour recycled paperboard. The MOSH concentration in dry foodstuffs ranged from 0.11 to 21.92 mg/kg (˂C25 hydrocarbons), which may be an indication of rapid migration. The lowest determined MOSH concentrations (˂C25 hydrocarbons) were found in sea salt and soda samples, even when their paperboard contained high mineral oil hydrocarbons. Our three samples in packages containing internal bags (for complete barriers) were found to have low mineral oil concentration due to reduced migration through plastic (acrylate-coated polypropylene). However, one sample, a 'crispy' product with an internal bag, contained the extreme amount of 21.92 mg/kg. Differences in contaminants observed in both dry foodstuffs and recycled paperboard may have been due to the different packaging and production techniques of the different countries. In addition, 8 of 24 dry foodstuff samples contained MOSH concentrations frequently exceeding the 2.0 mg/kg limit for MOSH C20-C35.

Differences in the metabolic profiles and antioxidant activities of wild and cultivated black soybeans evaluated by correlation analysis.

Wild soybeans are considered a potential resource for soybean domestication and an important source of genetic diversity for soybean crop improvement. Understanding metabolite-caused bioactivity differences between cultivated and wild soybeans is essential for designing a soybean with enhanced nutritional traits. In this study, the non-targeted metabolic profiling of 26 soybean varieties, 15 wild black soybeans (WBS) and 11 cultivated black soybeans (CBS), using liquid chromatography-mass spectrometry (LC-MS) in combination with multivariate analysis revealed significant differences in 25 differential metabolites. Among these, the soyasaponins Ab and Bb were found to be characteristic metabolites expressed more substantially in CBS than in WBS. Three different antioxidant assays and correlation analysis identified major and minor antioxidants that contributed to WBS having an antioxidant activity 4- to 8-fold stronger than that of CBS. Epicatechin, procyanidin B2, and cyanidin-3-O-glucoside were identified by both association analysis and the online LC-ABTS radical scavenging assay as being major antioxidants.

Screening of antioxidant activity and volatile compounds composition of Chamerion angustifolium (L.) Holub ecotypes grown in Lithuania.

Since biological activity of medicinal plants is dependent on cultivation area, climatic conditions, developmental stage, genetic modifications and other factors, it is important to study flora present in different growing sites and geographical zones. This study was focused on screening of antioxidant activity of C. angustifolium harvested in six different locations in Lithuania. The total contents of phenolic compounds, flavonoids and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated by spectrophotometric methods. A correlation between radical scavenging activity and total phenolic compounds content was observed (correlation coefficient 0.98). HPLC with online post-column DPPH radical scavenging reaction detection was used for the separation of extracts. Oenothein B, rutin and one unidentified compound were predominant. Volatile compounds were analysed using solid-phase microextraction coupled with gas chromatography-mass spectrometry. Based on the analysis of volatiles, all samples were classified into two chemotypes: (I) with predominant α- and ß-caryophyllenes and (II) with predominant anethole.

Analysis of saturated and aromatic hydrocarbons migrating from a polyolefin-based hot-melt adhesive into food.

Hot-melt adhesives are widely utilised to glue cardboard boxes used as food packaging material. They have to comply with the requirements of Article 3 of the European Framework Regulation for food contact materials (1935/2004). The hot melt raw materials analysed mainly consisted of paraffinic waxes, hydrocarbon resins and polyolefins. The hydrocarbon resins, functioning as tackifiers, were the predominant source of hydrocarbons of sufficient volatility to migrate into dry foods: the 18 hydrocarbon resins analysed contained 8.2-118 g kg(-1) saturated and up to 59 g kg(-1) aromatic hydrocarbons eluted from GC between n-C16 and n-C24, substantially more than the paraffinic waxes and the polyolefins. These tackfier resins, especially the oligomers ≤ C24, have been characterised structurally by GC×GC-MS and (1)H-NMR spectroscopy. Migration into food was estimated using a simulating system with polenta as food simulant, which was verified by the analysis of a commercial risotto rice sample packed in a virgin fibre folding box sealed with a hot melt. About 0.5-1.5% of the potentially migrating substances (between n-C16 and n-C24) of a hot melt were found to be transferred into food under storage conditions, which can result in a food contamination in the order of 1 mg kg(-1) food (depending on the amount of potentially migrating substances from the hot melt, the hot melt surface, amount of food, contact time etc.). Migrates from hot melts are easily mistaken for mineral oil hydrocarbons from recycled cardboard.

Online screening of nitric oxide scavengers in natural products using high performance liquid chromatography coupled with tandem diode array and fluorescence detection.

Nitric oxide (NO) is an important cellular signaling molecule with extensive physiological and pathophysiological effects. NO scavengers have the potential to treat inflammation, septic shock and other related diseases, and numerous examples have been chemically synthesized or isolated from natural products. The chemical diversity of natural products, however, means that a huge effort is necessary to efficiently screen and identify bioactive compounds, especially NO scavengers. In this article, we propose an effective analytical method to screen for NO scavengers in three natural products using an online system that couples high performance liquid chromatography with tandem diode array and fluorescence detection (HPLC-DAD-FLD). Eighteen compounds from radix of Scutellaria baicalensis Georgi and green tea displayed significant NO scavenging activity whereas components of Pueraria lobata (Willd.) Ohwi had no discernable activity. The structures of the active compounds were elucidated using Agilent Accurate-Mass Q-TOF LC/MS system. Preliminary analysis of structure-activity relationships indicated that, in flavonoids, a 2,3-double bond and a 3-H atom or a 3-OH group are essential for activity. In tannins, poly-hydroxyl groups are important for NO scavenging activity. Method validation indicated that the newly developed method is both reliable and repeatable. The online method that we present provides a simple, rapid and effective way to identify and characterize NO scavengers present in natural products.

Radical-scavenging compounds from olive tree (Olea europaea L.) wood.

The purpose of this study was to complete knowledge on the chemical composition and radical-scavenging activity of olive tree wood. Two new monoterpene glycosides, (-)-oleuropeic acid 6'-O-α-D-glucopyranosyl ester (6a) and (-)-perillic acid 1'-O-ß-D-primeverosyl ester (8), together with the known compounds (-)-oleuropeic acid (1), (-)-olivil (2), the aldehydic form of oleuropein aglycone (3), (+)-1-hydroxypinoresinol 1-O-ß-D-glucopyranoside (4), (-)-oleuropeic acid 1'-O-ß-D-glucopyranosyl ester (5), (-)-oleuropeic acid 6'-O-ß-D-glucopyranosyl ester (6b), and (-)-olivil 4-O-ß-D-glucopyranoside (7) were isolated from an ethyl acetate extract. The radical scavengers found (2-4 and 7) were detected and isolated with the help of the online HPLC-DAD-DPPH/ABTS technique. Compounds 2-4 and 7 displayed a higher antioxidative effect against the free radical DPPH than the reference BHT and lower than hydroxytyrosol, whereas compounds 1, 5, 6a, 6b, and 8 showed no activity.