On-line High-performance Liquid Research Articles

Screening and characterization of potential anti-gout components from Polygonum cuspidatum by integration off-line two-dimensional liquid chromatography-mass spectrometry with affinity ultrafiltration and on-line HPLC-ABTS

Polygonum cuspidatum (P. cuspidatum) is a traditional herbal medicine with a long history and proven efficacy in treating gout. However, due to the complexity of composition and extensive content distribution, the substance basis of its anti-gout effectiveness is still unclear. A strategy was proposed via integrating off-line two-dimensional liquid chromatography (2D-LC) and targeted rapid screening technology based on ultrafiltration-liquid chromatography-mass spectrometry (UF-LC/MS) and on-line high-performance liquid chromatography-2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (HPLC-ABTS) to accomplish high coverage and high throughput screening of anti-gout components from P. cuspidatum. As a result, twenty components were screened from P. cuspidatum extract with both xanthine oxidase (XOD) inhibitory activity and free radical scavenging activity, then were preliminarily identified by high-resolution electrospray ionization-quadrupole-time-of-flight mass spectrometer (ESI-Q-TOF/MS). The screened results were verified by the in vitro assays. Meanwhile, molecular docking further elucidated that the screened bioactive ingredients had favourable binding capabilities with XOD. The performance of this study can achieve high efficiency and high coverage screening of the anti-gout components from P. cuspidatum, which provides methodology and strategy support for the rapid screening of bioactive ingredients from complex medicinal plants.

Realization of digital twin for dynamic control towardsample variation of ion exchange chromatography in antibody separation.

Digital twin (DT) is a virtual and digital representation of physical objects or processes. In this paper, this concept is applied to dynamic control of the collection window in the ion exchange chromatography (IEC) towardsample variations. A possible structure of a feedforward model-based control DT system was proposed. Initially, a precise IEC mechanistic model was established through experiments, model fitting, and validation. The average root mean square error (RMSE) of fitting and validation was 8.1% and 7.4%, respectively. Then a model-based gradient optimization was performed, resulting in a 70.0% yield with a remarkable 11.2% increase. Subsequently, the DT was established by systematically integrating the model, chromatography system, online high-performance liquid chromatography, and a server computer. The DT was validated under varying load conditions. The results demonstrated that the DT could offer an accurate control with acidic variants proportion and yield difference of less than 2% compared to the offline analysis. The embedding mechanistic model also showed a positive predictive performance with an average RMSE of 11.7% during theDT test under >10% sample variation. Practical scenario tests indicated that tightening the control target could further enhance the DT robustness, achieving over 98% success rate with an average yield of 72.7%. The results demonstrated that the constructed DT could accurately mimic real-world situations and perform an automated and flexible pooling in IEC. Additionally, a detailed methodology for applying DT was summarized.

Online LC-ESI-MS/MS comparative analysis of N/O-glycopatterns in human colostrum from different ethnic groups in Northwest China

Human milk oligosaccharides, including free oligosaccharides and glycoconjugates, exert a key role in neonatal health and development. Changes in free oligosaccharides of milk from different ethnic groups have been documented. In this study, human milk was collected from Han, Hui, and Tibetan populations in northwest China, and differences in N/O-glycome among these three ethnic groups were systematically compared using online high-performance liquid chromatography-tandem mass spectrometry. Among the 63 detected N-glycans, 35 showed significant differences between the three ethnic groups (p < 0.05). Among the 70 detected O-glycans, four neutral O-glycans and six acidic O-glycans exhibited significant differences among the three ethnic groups (p < 0.05), with six acidic O-glycans reported for the first time. Overall, the extent of milk N/O-glycosylation was higher in the Han population than in the Hui or Tibetan groups. This trend was particularly pronounced for the main sialylated N/O-glycans. Except for sulfated O-glycans, which were higher in the milk from Tibetan mothers, the other types of N/O-glycans were present in similar proportions across all ethnic groups. Understanding the composition of N/O-glycans in human milk can help research on the structure-function relationship of glycans.

An analytical framework combining online high-performance liquid chromatography methodologies and biological properties of different extracts of Leonurus cardiaca.

Little or no information is available concerning online high-performance liquid chromatography (HPLC) antioxidants and the antibiofilm effect of Leonurus cardiaca. Five distinct extractions of methanolic, ethyl acetate, dichloromethane, hexane, and water were obtained from L. cardiaca. In the online-HPLC-antioxidant analysis of all examined samples, rosmarinic acid emerged as the primary antioxidant, registering concentrations ranging from 6 to 15ppm at wavelengths of 517 and 734nm. Notably, the water extract exhibited robust antioxidant activity In vitro. Regarding acetylcholinesterase and butrylcholinesterase inhibition, the n-hexane extract exhibited superior inhibition with values of 3.08 and 5.83 galanthamine equivalent, respectively. Except for the water extract, all tested extracts (at a concentration of 20μg/mL) exhibited substantial inhibitory activity against biofilm formation, in many cases superior to 80%, and reached even 94.52% againstEscherichia coli. Although less vigorous, the extracts also acted against the mature biofilm (inhibition up 76.50% againstStaphylococcus aureus). They could work against the metabolism inside an immature and mature biofilm, with inhibition percentages up to 93.18% (vs.Pseudomonas aeruginosa) and 76.50% (vs.Acinetobacter baumannii), respectively. Considering its significant antioxidants, enzyme inhibition, and antimicrobial activity, L. cardiaca emerges as a promising candidate for therapeutic potential.

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.

Target Discovery of Flavonoids from Elymus nutans Griseb Using Medium- and High-Pressure Liquid Chromatography Combined with Online High-Performance Liquid Chromatography–1,1-diphenyl-2-picrylhydrazyl Detection

Forage-based nutrients constitute the main forage value of forage grass. Elymus nutans Griseb possesses a wide ecological adaptability, enhanced crude protein content, good palatability, and excellent genes. Herein, employing medium- (MPLC) and high-pressure liquid chromatography (HPLC), along with online HPLC-DPPH (OHD)-based identification, two primary radical scavenging compounds were extracted and identified from the methanolic extract of Elymus nutans Griseb. With a starting material of 300 g of Elymus nutans Griseb, 5.95 g of the target DPPH suppressors fraction (Fr6) was separated following one cycle of MCI GEL® CHP20P medium pressure liquid chromatography. A Kromasil 100-5-Phenyl column was subsequently employed for further purification of the fraction, which yielded 432.16 mg of Fr62 (7.26% recovery) and 489.01 mg of Fr63 (8.22% recovery). The target compounds were then assessed based on their structure and purity, and two compounds (salcolin A and tricin) were extracted with &gt; 95% purity. This newly designed procedure was highly effective for the targeted flavonoids, and high-purity radical scavenger extraction from forage extracts. This methodology can potentially provide a scientific basis for their quality evaluation.

A study on the impact of harvesting operations on the mineral oil contamination of olive oils

During the 2020–21 olive oil campaign, the contribution of harvesting operations to mineral oil saturated (MOSH) and aromatic hydrocarbon (MOAH) contamination was studied. Oils extracted from hand-picked olives (15 different olive groves) generally had background MOSH (<2.7 mg/kg), and no quantifiable MOAH. In 40% of the cases, an important contamination increase was observed after harvesting operations. Except for one sample (325.8 and 111.0 mg/kg of MOSH and MOAH, respectively), other samples reached 4.3–33.7 mg/kg of MOSH and 1.1–11.3 mg/kg of MOAH. Accidental leaks of lubricants and/or contact with lubricated mechanical parts, were identified as important sources of contamination. Chromatographic traces obtained by on-line high-performance liquid chromatography (HPLC)-gas chromatography (GC)-flame ionization detection (FID) allowed for source identification. A comprehensive two-dimensional gas chromatographic platform (GC × GC) with parallel FID/MS detection was implemented for confirmation and to attempt the characterization of the contaminations. Good harvesting practices are suggested to minimize contamination risks.

Preparative isolation of antioxidative furanocoumarins from Dracocephalum heterophyllum and their potential action target.

Traditional Tibetan medicine has extensively documented the health benefits of Dracocephalum heterophyllum. However, there are few reports on the chemical composition of furanocoumarins, probably because of their complicated isolation and purification procedures. In this study, four antioxidative furanocoumarins were isolated from Dracocephalum heterophyllum by medium- and high-pressure liquid chromatography in combination with on-line high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl recognition. Crude samples were sequentially pretreated by medium-pressure liquid chromatography using silica gel, MCI GEL CHP20P, and diol as stationary phases, whereas on-line high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl system was used to recognize antioxidant peaks in target fractions. Thereafter, the antioxidative peaks were separated and purified through high-pressure liquid chromatography to obtain four furanocoumarins with purities greater than 95%; namely isodemethylfuropinarine, demethylfuropinarine, alloimperatorin, and alloisoimperatorin. Finally, the antioxidant capacity of the isolated furanocoumarins was determined using in vitro experiments (1,1-diphenyl-2-picrylhydrazyl assays, molecular docking, and cellular validation) and it was concluded that nuclear factor erythroid 2-related factor 2 protein is a potential target of these compounds for their antioxidation effects. Thus, the proposed methodology exhibits excellent efficacy for the preparative isolation of high-purity antioxidative furanocoumarins from extracts of Dracocephalum heterophyllum and it can be efficiently utilized for isolating antioxidants from other natural products.

Mineral oil contamination in basil pesto from the Italian market: Ingredient contribution and market survey

Mineral oil hydrocarbons (MOH) are complex mixtures of saturated hydrocarbons (MOSH) which bioaccumulate in human tissues, and aromatic hydrocarbons (MOAH) which include genotoxic and carcinogenic substances. This work aimed to investigate these emerging food contaminants in basil pesto from the Italian market, and ingredient contribution to the final product contamination. Twelve market samples and 4 additional samples (and related ingredients) produced in a pilot plant, were analyzed by on-line high-performance liquid chromatography (HPLC)- gas chromatography (GC), preceded by matrix-tailored sample preparation. Method performance was good with recoveries from 94% to 109%, residual standard deviations (RSD) less than 10%, and a limit of quantitation (LOQ) of 0.5 mg/kg (for total MOAH). Vegetable oils, followed by cashews, which showed a similar contamination profile to foods transported in jute bags, were the major contributors to contamination, while cheese and basil contribution was negligible. Sunflower oil alone accounted for more than 80–85% of the total contamination. Samples from the Italian market had an average of 5.6 and 0.6 mg/kg of MOSH and MOAH, respectively, and a contamination profile that confirmed that the main contributors to total contamination were sunflower oil and cashews. Mitigation actions should be directed toward careful control of these two ingredients.

Assignation of inorganic mercury and methylmercury mass fractions in a soil matrix certified reference material by two analytical methodologies based on species-specific isotope dilution mass spectrometry and chromatographic separation.

Assignment of inorganic mercury and methyl mercury mass fractions at an ultratrace level in soil certified reference material EnvCRM 03 with a complex matrix composition was undertaken. Inorganic mercury and methyl mercury contents by species-specific isotope dilution inductively coupled plasma mass spectrometry with on-line high-performance liquid chromatography or with classical off-line chromatography were established. Different extraction protocols: sequential extraction ((1) H2 SO4 /KBr/CuSO4 ; (2) dichloromethane; (3) Na2 S2 O3 ) or one-step extraction (diluted HCl) in solid-liquid systems were verified. Sequential extraction allowed quantification and separation of inorganic mercury and methyl mercury on HPLC column in one chromatographic run and were found to be (316 ± 10) μg/kg (U = 3.2%, k = 2) and (0.53 ± 0.02) μg/kg (U = 3.8%, k = 2), respectively. Extraction by diluted HCl and application of classical off-line chromatography led to the separation of methyl mercury from predominant inorganic mercury form and was found to be (0.54 ± 0.03) μg/kg (U = 5.4%, k = 2). To the best-obtained literature knowledge, there was no available soil material aimed for speciation analysis of inorganic mercury and methyl mercury so far. Both developed analytical methodologies were found to be equally sensitive and could be successfully applied for mercury species determination in samples with the complex matrix.

Online HPLC–HRMS Platform: The Next-Generation Process Analytical Technology Tool for Real-Time Monitoring of Antibody Quality Attributes in Biopharmaceutical Processes

Online monitoring of critical quality attributes (CQAs) directly within the bioreactor can provide the basis for advanced processing of therapeutics production, including automated real-time monitoring, feedback control process intensification, smart manufacturing, and real-time release testing. This paper presents recent developments in online high performance liquid chromatography–high-resolution mass spectrometry (HPLC–HRMS) platforms as a promising process analytical technology (PAT) tool for real-time monitoring of antibody quality attributes (QAs) in biopharmaceutical processes. This technology can be used to monitor multiple CQAs and process parameters during cell culture production, enabling real-time decisions.

Three new dihydroflavonols with free radical scavenging activity from Ribes himalense Royle ex Decne

Ribes himalense Royle ex Decne, a small shrub, is widely used as a Tibetan medicine in Chinese folk. In this study, three novel 1,1-diphenyl-2-picrylhydrazyl inhibitors named Rihimaside A, Rihimaside B, and Rihimaside C, as well as one known 1,1-diphenyl-2-picrylhydrazyl inhibitor, dihydromyricetin, were isolated from the leaves and stems of Ribes himalense Royle ex Decne using online high performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl activity screening system combined with medium and high-pressure liquid chromatography. All four 1,1-diphenyl-2-picrylhydrazyl inhibitors are dihydroflavonols. The 1,1-diphenyl-2-picrylhydrazyl scavenging activity and IC50 values of three novel 1,1-diphenyl-2-picrylhydrazyl inhibitors were determined using 1,1-diphenyl-2-picrylhydrazyl methods. Rihimaside A, Rihimaside B, and Rihimaside C exhibited IC50 values of 9.58 μg/mL, 12.57 μg/mL and 387 μg/mL, respectively.

A Method to Separate Two Main Antioxidants from Lepidium latifolium L. Extracts Using Online Medium Pressure Chromatography Tower and Two-Dimensional Inversion/Hydrophobic Interaction Chromatography Based on Online HPLC-DPPH Assay

Free radicals, including 1,1-diphenyl-2-picrylhydrazyl, mediate oxidative stress to cause many chronic diseases (including cardiovascular diseases, diabetes and cancer). The extract of traditional Tibetan medicine Lepidium latifolium L. (L. latifolium) was reported to have free radical inhibition ability. Therefore, a system method was established to separate the ethanol extract of L. latifolium to prepare two main antioxidant compounds. First of all, silica gel and a medium-pressure liquid chromatography tower were used for pre-treatment of the ethanol extract of L. latifolium to obtain the main antioxidant active component fraction 4 through online high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl (HPLC-DPPH) assay. Then, fraction 4-1 was obtained by one-dimensional preparation using Megres C18 chromatographic column, and two active compounds with IC50 values 59.9 and 71.3 μg/mL were obtained by two-dimensional preparation using Click XIon chromatographic column. Through the study of the chemical components and separation methods of L. latifolium, the combination of HPLC-DPPH assay and two-dimensional preparative liquid chromatography was realized, providing a reference for the separation of active compounds from L. latifolium.

Electrooxidation of Formamidine Disulfide Simultaneously Investigated by On-Line High Performance Liquid Chromatography and Cyclic Voltammetry.

The electro‐oxidation of formamidine disulfide, an important sulfur‐containing compound, was simultaneously investigated with on‐line high‐performance liquid chromatography and cyclic voltammetry. Using a home‐made microporous sampler located at the electrode interface, the solution on the electrode surface was in situ sampled and analyzed. The electrochemical scanning was synchronously performed, which allowed the electro‐oxidation products to be detected at a given potential. The main products on the surface of platinum electrode were found to be thiourea, formamidine sulfinic acid, cyanamide, and elemental sulfur. Forced convection arising from the sampling played an important role in the electrochemical oxidation. The extraction of electrode surface solution promoted the renewal of reactant and its intermediates, which induced the change of cyclic voltammetry curve. The forced convection also contributed to the redox peak current of the species on the cyclic voltammetry curves through the change of concentration of reactant and its intermediates. This technique can help to explore the reaction mechanism of complex electrochemical reactions.

Chemical Constituents of the Leaves of Diospyros kaki (Persimmon).

Diospyros kaki (persimmon) leaves have long been utilized as traditional medicine for the treatment of ischemic stroke, angina, and hypertension and as a healthy beverage and cosmetic for anti-aging. This study aimed to isolate as many compounds as possible from an ethanol extract of the persimmon leaves to identify the biologically active compounds. The antioxidative effect of the ethyl acetate layer from the ethanol extract of the persimmon leaves was demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and online high-performance liquid chromatography-2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (HPLC-ABTS) analysis. A new flavonoid, kaempferol-3-O-β-d-2″-coumaroylgalactoside (1), and a new natural compound, kaempferol-3-O-β-d-2″-feruloylglucoside (3) were isolated from the ethyl acetate layer, along with 25 previously known compounds, including fourteen flavonoids, one ionone, two coumarins, seven triterpenoids, and one acetophenone. Their structures were determined by the interpretation of spectrometric and spectroscopic data. All isolated compounds were rapidly evaluated using an online HPLC-ABTS assay, and of these, compounds 4–8, 11, 13, 15, and 16 clearly showed antioxidative effects. The amount of these compounds was 0.3–0.65% of the extract.

Simultaneous determination of saturated and aromatic hydrocarbons in soil by on-line high performance liquid chromatography-gas chromatography

为加强对土壤中石油烃类污染物的风险管控,生态环境部已将石油烃类列为土壤中的重点监测项目。石油烃源于石油与合成油,是涵盖一定碳数范围的碳氢化合物,主要分为饱和烃和芳香烃两大类。芳香烃通常是高度烷基化的单环、双环与多环芳烃,其对人和动物的毒性较饱和烃大很多,因此,仅仅测定土壤中总石油烃含量难以准确评估其环境毒性。目前环境领域的标准方法尚未区分土壤中饱和烃和芳香烃。该研究针对土壤样品的基质干扰特点,对样品的提取和净化环节进行了优化,并且应用高效液相色谱-气相色谱在线联用(HPLC-GC)技术,建立了同时测定土壤中饱和烃和芳香烃的方法。其中,提取方法选择正己烷-乙醇(1:1, v/v)以固液比1:4常温振荡提取1 h,然后水洗去除乙醇,取正己烷层提取液净化;净化方法选择自制硅胶柱,以正己烷-二氯甲烷(8:2, v/v)洗脱;洗脱液经浓缩注入HPLC-GC分析,以内标法同时测定试液中的饱和烃和芳香烃,方法的定量限为0.4 mg/kg。该方法经过土壤石油烃标准物(SQC-116)验证,测定值在证书提供的可信区间内,相对误差(RE)为10.6%,相对标准偏差(RSD)为1.4%,说明方法准确可靠且精密度达到分析要求。最后,该文采用建立的方法检测了北京地区的5个土壤样品,结果表明:5个样品均含有饱和烃(C10~C40),其含量范围为3.3~32.1 mg/kg;其中4个样品中检出芳香烃(C10~C40),其含量范围为0.8~4.3 mg/kg;此外,通过谱图分析还可以初步判别烃类物质的污染来源。

Survey of mineral oil hydrocarbons in Chinese commercial complementary foods for infants and young children

ABSTRACT Recently, mineral oil hydrocarbons (MOH) in various foods have raised significant concern, especially for infants and young children due to their potential adverse health effects. Two fractions can be distinguished by certain analytical techniques, mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). The toxicological profile of MOSH and MOAH differs greatly. The toxicity of MOSH is linked with long-term accumulation of some hydrocarbons. MOAH with three to seven, non- or simple-alkylated, aromatic rings may be mutagenic and carcinogenic. However, data on the occurrence of mineral oils in commercial complementary foods for infants and young children are lacking in China. In the present study, 100 commercial food samples were collected, including 26 pureed or paste canned foods, 21 high-protein ground cereal foods (rice flour), 25 raw cereal foods (noodles), and 28 cereal-based molar sticks and biscuits. The content of MOSH and MOAH in those samples was determined by optimised sample preparation methods combined with on-line high-performance liquid chromatography coupled with gas chromatography and flame ionisation detector (HPLC-GC-FID), with a limit of quantification of 0.5 mg/kg. The results indicated that there were no MOAH detected in any of the foods, but MOSH and polyolefin oligomeric saturated hydrocarbons (POSH) existed in most of the food samples, at <0.5–23.68 mg/kg. Moreover, the data and chromatograms of the MOSH and POSH also indicated that these contaminants were closely correlated to their ingredients and manufacturers. The current study provides basic data to understand MOH exposure and consequent health impact.

Research Progress of Analysis of Mineral Oil Hydrocarbons using On-line High Performance Liquid Chromatography Coupled with Gas Chromatography

Abstract In the past decades, the application of on-line high performance liquid chromatography coupled with gas chromatography (HPLC-GC) has been rapidly developed for the quantitative analysis of mineral oil hydrocarbons (MOH). MOH are derived from petroleum and synthetic fuels, and cover a large range of boiling points, mainly consisting of mineral oil saturated hydrocarbons (MOSH) and aromatic hydrocarbons (MOAH). MOH can migrate into food and environment through a variety of pathways, consequently impose an adverse impact on human health. However, the analysis of MOH is challenging due to the limited selectivity and sensitivity of hydrogen flame ionization detector (FID). On-line HPLC-GC combines the high separation efficiency of HPLC with the equal-carbon-response detection of GC-FID, which solves the problems associated with selectivity and sensitivity of MOH analysis. The innovation of the retention gap, Y-piece interface, and solvent vapor exit (SVE) with which the HPLC-GC is equipped enables large-volume injection into GC-FID. By allowing the elution fractions from HPLC to be completely transferred into the GC, the analytical sensitivity of the MOH is enhanced by more than two orders of magnitude, and the contaminants introduced are minimized during the analytical procedure, ensuring the reproducibility and accuracy of the MOH analysis. In this paper, the research history of the interfaces used in on-line HPLC-GC and the matching conditions between HPLC and GC were described in detail, and the sample preparation methods for the removal of interferences and the enrichment of MOH based on HPLC-GC were reviewed. In addition, the limitations of HPLC-GC were summarized, and the future applications of this technology and the trends in the development of MOH analysis were prospected.

Preparative isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Ribes himalense using medium-pressure and two-dimensional reversed-phase/reversed-phase liquid chromatography guided by an online HPLC-1, 1-diphenyl-2-picrylhydrazyl assay.

The lack of suitable chromatographic purification methods makes it a challenge to effectively isolate the chemical components of traditional Tibetan medicines. Ribes himalense is a rarely studied Tibetan medicine, reputed to have free radical-scavenging effects. In the present work, we utilized it as a model herb to highlight an approach for the separation of 1,1-diphenyl-2-picrylhydrazyl inhibitors via medium-pressure chromatography and two-dimensional reversed-phase/reversed-phase interaction liquid chromatography under the guidance of an online high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl assay. Finally, we obtained two free radical inhibitors (>95% purity) from the R. himalense extract. This is the first report of the rapid isolation of these free radical inhibitors from R. himalense. This method can be useful in quality standard assessment and further pharmacological activity research, and may be used as a reference for the composition research of various natural products.

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.