High-performance Liquid Chromatography Column Research Articles

Comparison of HPLC, HPTLC, and In Silico Lipophilicity Parameters Determined for 5-Heterocyclic 2-(2,4-Dihydroxyphenyl)-1,3,4-thiadiazoles

The 5-heterocyclic 2-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles were obtained as potential biologically active compounds. Lipophilicity is one of the most important physicochemical properties of compounds and was already taken into account during the drug candidates design and development. The lipophilicity of compounds was determined using the computational (log P) and chromatography (log kw, RMw) methods. The experimental ones included the reverse-phase column high performance liquid chromatography RP (HPLC) with C8, C18, phosphatidylcholine (IAM), and cholesterol stationary phases and the thin layer chromatography (RP-HPTLC) with C8 and C18 stationary phases and various organic modifiers under the isocratic conditions. Descriptive statistics, correlation, and PCA analyses were used to compare the obtained results. For lipophilicity estimation of the tested compounds by HPTLC, dioxane and MeOH seem to be particularly beneficial as organic modifiers. The chromatographic lipophilicity parameters log kw (RMw) were well correlated and highly redundant (85%) compared with those calculated. Most compounds possess lipophilicity parameters within the recommended range for drug candidates.

Cross-column density functional theory-based quantitative structure-retention relationship model development powered by machine learning.

Quantitative structure-retention relationship (QSRR) modeling has emerged as an efficient alternative to predict analyte retention times using molecular descriptors. However, most reported QSRR models are column-specific, requiring separate models for each high-performance liquid chromatography (HPLC) system. This study evaluates the potential of machine learning (ML) algorithms and quantum mechanical (QM) descriptors to develop QSRR models that can predict retention times across three different reversed-phase HPLC columns under varying conditions. Four machine learning methods-partial least squares (PLS) regression, ridge regression (RR), random forest (RF), and gradient boosting (GB)-were compared on a dataset of 360 retention times for 15 aromatic analytes. Molecular descriptors were calculated using density functional theory (DFT). Column characteristics like particle size and pore size and experimental conditions like temperature and gradient time were additionally used as descriptors. Results showed that the GB-QSRR model demonstrated the best predictive performance, with Q2 of 0.989 and root mean square error of prediction (RMSEP) of 0.749min on the test set. Feature analysis revealed that solvation energy (SE), HOMO-LUMO energy gap (∆E HOMO-LUMO), total dipole moment (Mtot), and global hardness (η) are among the most influential predictors for retention time prediction, indicating the significance of electrostatic interactions and hydrophobicity. Our findings underscore the efficiency of ensemble methods, GB and RF models employing non-linear learners, in capturing local variations in retention times across diverse experimental setups. This study emphasizes the potential of cross-column QSRR modeling and highlights the utility of ML models in optimizing chromatographic analysis.

Cytotoxic Sulfoquinovosyl Glycerols from the Seaweed Sargassum Angustifolium from Persian Gulf.

Seaweeds are an important source of marine organisms that produce a lot of bioactive compounds. In this research, the seaweed Sargassum angustifolium was collected from Bushehr province of Persian Gulf of Iran. The seaweed was extracted by methanol:ethyl acetate (1:1) using maceration method. The compounds were isolated with different column chromatography and HPLC(High Performance Liquid Chromatography) by silica gel and hexane:ethyl acetate as mobile phase. The isolated compounds were elucidated structurally by various 1 and 2 D-NMR and MS spectra. Besides the cytotoxicity test was done against HeLa using standard MTT assay and normal cells. It afforded four known sulfoquinovosyl diacylglycerides and fucosterol. Compounds 1-5 showed cytotoxic effects against HeLa and HUVEC cell lines, with IC50 values of 12.2 ± 2.3, 25.8 ± 3.7, 14.9 ± 2.6, 9.8 ± 1.2 μM, and 5.6 ± 1.2, respectively.

Amino acids profile of Two Chinese nuts (Juglans sigillata and Castanea mollissima ) in local markets of Kurdistan region/Iraq using HPLC

Aim: current study was aimed to study the amino acid contents of two Chinese nuts, walnuts (Juglans sigillata Dode.) and chestnuts (Castanea mollissima Blume.) from the local market of the Kurdistan region / Iraq. Methodology: Walnut fruits (Juglans sigillata) and chestnut fruits (Castanea mollissima) were collected from local market in the Kurdistan region / Iraq, samples were collected, dried and ground. one milgram of each sample was weighed, then dissolved in 10 ml High Performance Liquid Chromatography (HPLC) methanol, the sample was shaken and agitated in an ultrasonic bath for 10 minutes , then concentrated by evaporating the solvent with a stream of liquid N2 until it reached 1 ml, filtrated the liquid sample on deposable sep-pak cartridge filter ( supleco Co, Ltd),then 20 ml were injected on HPLC column . Results: firstly current study can identified thirteen amino acids from chestnuts but there are only eight amino acids were identified from walnuts. Secondly, there are big different between them in the percentage of amino acids where valine is appear in the highest percentage which record 69.43 %, while it recorded 6.32% in chestnut fruit , While threonine showed the highest percentage in chestnuts arrived to 54.09% compared to only 3.72% in walnuts fruits.

Separation of undeuterated and partially deuterated enantioisotopologues of some amphetamine derivatives on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography

The different behavior of enantiomers of chiral compounds in non-isotropic environments (among them in living organism) is well known. On the other hand, the importance of a kinetic isotope effect in the biomedical field has become evident during past few decades. Thus, separation of both, enantiomers and isotopologues is now critical. Only very few published studies have attempted the simultaneous separation of enantioisotopologues. In this article we report baseline separation of partially deuterated isotopologues of a few amphetamine derivatives in high-performance liquid chromatography (HPLC) using achiral columns. In addition, the simultaneous separations of enantiomers and isotopologues (i.e. enantioisotopologues) were attempted on polysaccharide-based chiral columns. For several compounds the isotope effect was tunable and could be switched from a “normal” to “inverse” by making changes to the mobile-phase composition. A stronger isotope effect was observed in acetonitrile-containing mobile phases compared to methanol-containing ones with both chiral and achiral columns. In a separation system where both “normal” and “inverse” isotope effects were observed the “normal” isotope effect was favored in polar organic solvents while increasing content of the aqueous component in the reversed-phase (RP) mobile phase favored an “inverse” isotope effect. This observation indicates that polar, hydrogen bonding-type noncovalent interactions are involved in the “normal” isotope effect, while apolar hydrophobic-type interactions are mostly responsible for the “inverse” isotope effect.

Analysis of Gizzerosine in Foodstuffs by HPLC Involving Pre-column Derivatization with o-Phthaldialdehyde.

Gizzerosine [2-amino-9-(4-imidazolyl)-7-azanonanoic acid] is a toxic amino acid formed from histamine and lysine at high temperatures, and may be present in foodstuffs (e.g., fishmeal and meat-bone meal) for animals including cats and dogs. Here we developed a simple, rapid, sensitive, specific, and automated method for the analysis of gizzerosine in foodstuffs by high-performance liquid chromatography (HPLC) involving pre-column derivatization with o-phthaldialdehyde (OPA) in the presence of N-acetylcysteine (instead of the usual 2-mercaptoethanol or ethanethiol reagent). OPA reacted immediately (within 1 min) with gizzerosine in an autosampler at room temperatures (e.g., 20-25°C), and their derivative was directly injected into the HPLC column. The highly fluorescent gizzerosine-OPA derivative was well separated from the OPA derivatives of all natural amino acids known to be present in physiological fluids (e.g., plasma), proteins and foodstuffs, and was detected at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total time for chromatographic separation (including column regeneration) was 20 min per sample rather than 40 min and longer in previous HPLC methods. The detection limit for gizzerosine was at least 6 pmol/ml in an assay solution (HPLC vial) or at least0.09pmol per injection into the HPLC column. The analysis of gizzerosine was linear between 1 and 100pmol per injection. When gizzerosine was extracted from foodstuffs, its detection limit was at least 875pmol/g foodstuff or at least0.21 mg/kg foodstuff. Our routine HPLC technique does not require any cleanup of samples or the OPA derivatization products (including the OPA-gizzerosine adduct), and is applicable for the analysis of gizzerosine in bothfoodstuffs and animal tissues.

Comprehensive and sensitive validation of the method and determination of measurement uncertainty for simultaneous specification of aflatoxin B1, B2, G1 and G2 in nuts

In this study, a comprehensive and sensitive validation study of the legally used method was conducted to simultaneously determine the levels of aflatoxin B1, B2, G1, and G2 in nuts. The applied method has been extensively evaluated in terms of in-laboratory detection limit and quantification limit, linearity, selectivity, precision, recovery, accuracy, adequacy, and measurement uncertainty. Levels of aflatoxins were examined by a validated immunoaffinity column-high performance liquid chromatography-post column derivatization-fluorescence detection (IAC-HPLC-PCA-FLD) analytical method. Detection limits (LOD) for aflatoxin G2, G1, B2, and B1 were found to be 0.05 μg/kg, 0.13 μg/kg, 0.06 μg/kg, and 0.15 μg/kg, respectively. The regression coefficient (R2) was greater than 0.999 according to constructed calibration curves for all aflatoxin components. Thus, the curves exhibited satisfactory linearity within the analyzed range. The combined standard uncertainty values for precision (RSD%) studies were below 10%. It showed a robust and reliable sensitivity with <10% RSD with the test procedure. Average recovery values were detected as 67-84% for hazelnut and 60-89% pistachio. All aflatoxin components in hazelnut paste, which is the proficiency test material, were determined within an appropriate |Z|-score range (≤ 2). The relative expanded uncertainty of measurement was 0.80 μg/kg for AFB1 and AFG1, 0.83 μg/kg for AFB2 and AFG2, and 0.78 μg/kg for total AFs. The results showed that the approved method for the investigation and monitoring of aflatoxins in hazelnuts and pistachios is highly sensitive, robust, reliable, selective, and reproducible. Validation parameters can be successfully used in routine analysis to monitor aflatoxins in nuts for compliance with current regulations.

Development of simple purification method for oligonucleotides synthesized using phosphoramidite for 5′-end modification as capping reagent

Oligonucleotide therapeutics are generally prepared via solid-phase synthesis, and many impurities derived from the synthesis process are present in crude oligonucleotides. Among the various impurities typical of oligonucleotide synthesis, shortmers, which lack a few residues from the desired oligonucleotide therapeutics, are difficult to remove using conventional high-performance liquid chromatography (HPLC) purification because they have similar structures and physical properties to the desired oligonucleotide. In this study, we developed a simple purification method for oligonucleotide therapeutics. In our strategy, the retention of shortmers on the reversed-phase HPLC column was increased by using a highly lipophilic phosphoramidite, which is a reagent for 5′-end modification of oligonucleotides, instead of acetic anhydride during solid-phase synthesis, and only the target oligonucleotide could be eluted from a reversed-phase HPLC pretreatment column.

The Effect of Particle Monodispersity in HPLC Column Performance

Many developments in silica particles used in liquid chromatography have been well documented over the years. The move from irregular silica to spherical silica, the decrease in particle size from &gt;5µm particle in HPLC to sub-2-µm particle size in UHPLC, the improved silica purity of type B silica over that of type A silica, and more recently the adoption of superficially porous particles versus traditional fully porous particles. One area of development that has been discussed less and is still open to debate is the particle size distribution (PSD) of these chromatographic materials. In this article, we discuss the move towards improved monodispersity of silica particles for use in high performance liquid chromatography (HPLC) and how the use of monodisperse particles can impact the resulting chromatographic parameters such as reduced plate height and column impedance. On a practical level we review how the reduction of PSD impacts efficiency, backpressure and sensitivity.

Simultaneous Analysis of Hydrophobic Atractylenolides, Atractylon and Hydrophilic Sugars in Bai-Zhu Using a High-Performance Liquid Chromatography Column Tandem Technique.

An analytical method was established using high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) with -C18 and -NH2 column tandem for the simultaneous determination of hydrophobic atractylenolide I, II, III, atractylone and hydrophilic compounds glucose, fructose and sucrose in the dried rhizome of Atractylodes macrocephala Koidz (a natural raw material for health foods, Bai-Zhu aka. in Chinese). The method combines the different separation capabilities of reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. It can provides a new choice for the simultaneous determination of hydrophilic and hydrophobic compounds in traditional Chinese medicines and health foods. It provided a reference method for the quality control of Bai-Zhu. The results showed that the linear correlation coefficients of the established column tandem chromatographic method were all greater than 0.9990, the relative standard deviation was 0.1-2.8%, and the average recovery was 96.7-103.1%. The contents of atractylenolide I, II, III, atractylone, fructose, glucose, and sucrose in 17 batches of Baizhu were 172.3-759.8 μg/g, 201.4-612.8 μg/g, 160.3-534.2 μg/g, 541.4-8723.1 μg/g, 6.9-89.7 mg/g, 0.7-7.9 mg/g, and 1.2-21.0 mg/g, respectively.

Nucleoside Analogs: A Review of Its Source and Separation Processes.

Nucleoside analogs play a crucial role in the production of high-value antitumor and antimicrobial drugs. Currently, nucleoside analogs are mainly obtained through nucleic acid degradation, chemical synthesis, and biotransformation. However, these methods face several challenges, such as low concentration of the main product, the presence of complex matrices, and the generation of numerous by-products that significantly limit the development of new drugs and their pharmacological studies. Therefore, this work aims to summarize the universal separation methods of nucleoside analogs, including crystallization, high-performance liquid chromatography (HPLC), column chromatography, solvent extraction, and adsorption. The review also explores the application of molecular imprinting techniques (MITs) in enhancing the identification of the separation process. It compares existing studies reported on adsorbents of molecularly imprinted polymers (MIPs) for the separation of nucleoside analogs. The development of new methods for selective separation and purification of nucleosides is vital to improving the efficiency and quality of nucleoside production. It enables us to obtain nucleoside products that are essential for the development of antitumor and antiviral drugs. Additionally, these methods possess immense potential in the prevention and control of serious diseases, offering significant economic, social, and scientific benefits to the fields of environment, biomedical research, and clinical therapeutics.

Chemical Constituents from the Roots of Jasminum sambac (L.) Ait. and their Cytotoxicity to the Cancer Cell Lines.

The roots of J. sambac is the Traditional Chinese Medicine (TCM) with analgesic and anesthetic effects. However, relatively fewer studies on the chemical compositions and the biological activities of the roots of J. sambac have been carried out till now. We studied the chemical compositions of the roots of J. sambac planted in Fujian Province to discover new compounds from this TCM to develop new drugs or drug candidates. This work aims to find the new compounds from the roots of Jasminum sambac (L.) Ait. (J. sambac) for the development of new drugs or drug candidates. The dichloromethane (DCM) extract was selected to isolate over silica gel column chromatography to obtain different polar fractions. Several similar fractions were combined according to Thin Layer Chemotherapy (TLC) or High-Performance Liquid Chromatography (HPLC) analysis. The combined fractions were reisolated by silica gel column chromatography, preparative TLC or HPLC to obtain nine pure compounds (1-9). The purity of the isolated compounds was detected by HPLC, and their structures were determined by 1D, 2D NMR, and HRESIMS analysis. The in vitro anticancer activity was evaluated using Cell Counting Kit-8 (CCK8) method. Nine compounds were isolated in this work. Compounds (1-3) are new compounds, while compounds (4-9) were isolated for the first time from the roots of J. sambac. Their structures were elucidated by 1D, 2D NMR, and HRESIMS analysis. The biological evaluation showed that compound 7 exhibited potent cytotoxic efficacy against MCF-7 cell lines with IC50 values of 148.3 μM for 24 hs and 35.94 μM for 48 hs, respectively; compound 1 displayed significant cytotoxic potential against MCF-7 cell lines with IC50 value of 38.5 μM for 24 hs; while compound 3 and 4 displayed potent cytotoxic effects against MCF-7 cell lines with IC50 values of 161.1 μM and 243.7 μM for 48 hs, respectively. We discovered new compounds from the roots of J. sambac. and several compounds exhibited potent cytotoxity to MCF-7 cell lines. This work encourages us to further study the chemical constituents and their biological activities from the roots of J. sambac.

Synthesis of hierarchically porous zirconium-based metal-organic framework@silica core-shell stationary phase through etching strategy for liquid chromatography

Metal organic frameworks (MOFs) show promise to be employed as stationary phase for high performance liquid chromatography (HPLC), however, the microporous structures of MOFs seriously restrict the diffusion and mass transfer of solute molecules, leading to a low column efficiency. In this paper, the fabrication of hierarchically porous UiO-66@SiO2 (HP- UiO-66@SiO2) core-shell microspheres via H2O2 etching has been proposed as a viable approach to enhance the separation performance of MOFs-based columns for HPLC. Through the direct treatment of the preliminary prepared UiO-66@SiO2 microspheres with H2O2 etching, HP-UiO-66@SiO2 core-shell microspheres were successfully synthesized with an enlarged pore size of up to 9 nm, facilitating efficient mass transfer in chromatographic separation. The prepared HP-UiO-66@SiO2 core-shell microspheres were then explored as stationary phase in HPLC to separate the nonpolar alkyl benzene homologues, the polar aromatic alcohol homologues and the xylene isomers. The results indicated that the baseline separations of these solutes were achieved successfully with narrow peak width and higher resolution than the UiO-66@SiO2 column. The HP-UiO-66@SiO2 column exhibited superior separation performance, reaching a maximum plate number of 134,459/m for fluorene, and showing good reproducibility. As a result, this template-free approach suggests that the fabrication of hierarchically porous MOFs@silica core-shell microspheres is a successful approach to enhance the column efficiency of MOFs-based columns in HPLC.

TAMOF-1 as a Versatile and Predictable Chiral Stationary Phase for the Resolution of Racemic Mixtures.

Metal-organic frameworks (MOFs) have become promising materials for multiple applications due to their controlled dimensionality and tunable properties. The incorporation of chirality into their frameworks opens new strategies for chiral separation, a key technology in the pharmaceutical industry as each enantiomer of a racemic drug must be isolated. Here, we describe the use of a combination of computational modeling and experiments to demonstrate that high-performance liquid chromatography (HPLC) columns packed with TAMOF-1 as the chiral stationary phase are efficient, versatile, robust, and reusable with a wide array of mobile phases (polar and non-polar). As proof of concept, in this article, we report the resolution with TAMOF-1 HPLC columns of nine racemic mixtures with different molecular sizes, geometries, and functional groups. Initial in silico studies allowed us to predict plausible separations in chiral compounds from different families, including terpenes, calcium channel blockers, or P-stereogenic compounds. The experimental data confirmed the validity of the models and the robust performance of TAMOF-1 columns. The added value of in silico screening is an unprecedented achievement in chiral chromatography.

Development of a liquid chromatographic method for enantioseparation of Eflornithine using (S)-α-ethyl benzylamine as a chiral derivatizing agent

ABSTRACT Eflornithine (2-fluoromethyl-DL-ornithine) is a potent and irreversible inhibitor that selectively targets ornithine decarboxylase, a key enzyme in the polyamine biosynthesis pathway. Due to the lack of chromophoric moiety in the eflornithine structure, its detection via UV detector is difficult. Thus, direct high-performance liquid chromatographic (HPLC) separation of enantiomers of Eflornithine is not feasible, and pre-column derivatization is required for its determination through chiral HPLC columns. We developed an indirect HPLC method using (S)-α-ethyl benzylamine as a chiral derivatizing agent for the enantioseparation of Eflornithine. The diastereomers thus produced were then separated via LiChrospher C18 column (5 μm particle size, L × I. D. 25 cm × 4.6 mm). The mobile phase used was a mixture of acetonitrile and 0.1% aq. TFA and varied in linear gradients of 30–70% of acetonitrile for 30 min run at a flow rate of 1.0 mL min−1 and UV detection at 320 nm. The separation parameters were optimized by altering the mobile phase composition and flow rate. The findings revealed that the chromatographic separation was accomplished within 15 min, with resolution values greater than 4.5 for Eflornithine enantiomers. The detection and quantitation limits were 9.26 ng mL− 1 and 18.52 ng mL− 1 for Eflornithine enantiomers.

Green method for determination of four anti-viral drugs using micellar liquid chromatography: Application to dosage form analysis

A micellar liquid chromatographic (MLC) method was developed to separate and analyze four anti-viral drugs. The method utilized a Thermo Hypersil ODS C18 HPLC column with an isocratic elution mode employing a mobile phase consisting of sodium dodecyl sulfate (0.15 M, pH 3), isopropanol and triethylamine (79.7:20:0.3, v/v/v). The Design Expert 8® software was used to optimize four factors simultaneously and the developed quadratic D-optimal model could achieve maximum resolution between peaks while minimizing the run time. The method was found linear over the concentration ranges of 10–100 μg/mL for sofosbuvir, daclatasvir, and 20–200 μg/mL for ledipasvir and velpatasvir, and successfully applied to the analysis of sofosbuvir and velpatasvir in tablets. The MLC method was compared with a previously reported high-performance liquid chromatography (HPLC) method using the analytical GREEnness metric approach (AGREE), and was found to exhibit greener performance due to the use of safer reagents. The results indicate the high potential of micellar liquid chromatography in the separation of quaternary mixtures with the aid of response surface methodologies.

Column Watch: Methods for the Passivation of HPLC Instruments and Columns

Metal-analyte interaction is a type of nonspecific adsorption (NSA) that occurs mainly between acidic analytes and active adsorptive sites present in the metal surfaces of the fluidic path of any chromatographic system. NSA represents a chromatographic challenge since it may lead to poor peak shape, low recovery, and poor performance from the high performance liquid chromatography (HPLC) system. As a result, many solutions that circumvent and mitigate this type of secondary interaction have been published or commercialized in the last few years. Such solutions encompass swapping stainless steel components for non-reactive polymers, “biocompatible” metals or alloys, the addition of additives into the mobile phase, pretreating the system with large amounts of sample or strong acids, and the use of coated stainless steel materials. This article explores these solutions in more detail, particularly the recent adaptation and benefits of coated metal surfaces in LC.

Determination of Related Substances in Promethazine Hydrochloride and Dextromethorphan Hydrobromide in Oral Solution by RP-HPLC Method.

The goal of this study is to provide a single, widely applicable high-performance liquid chromatographic (HPLC) technique for the determination of related substances in multicomponent oral solution of promethazine hydrochloride and dextromethorphan hydrobromide. For the assessment of impurities of promethazine hydrochloride and dextromethorphan hydrobromide in oral solution, a unique, sensitive, quick, stability-indicating gradient HPLC technique has been created. For chromatographic separation, an Agilent Eclipse XDB-C18, 250mm × 4.6mm, 5μm column was used with a buffered mobile phase consisting of a mixture of potassium dihydrogen phosphate pH3.0:acetonitrile (80:20) v/v as mobile phase A and potassium dihydrogen phosphate pH3.0:acetonitrile:methanol (10:10:80) v/v/v as mobile phase B. The separation was performed at a flow rate of 1.2mL/min and a detection wavelength of 224nm. The temperature of the column oven was regulated at 40°C. With good sensitivity and resolution, all compounds were effectively separated on a reverse-phase HPLC column. Acid, base, photolytic, thermal, oxidative and humidity stress conditions significantly degraded dextromethorphan hydrobromide and promethazine hydrochloride. The developed technique was validated according to the criteria of the International Conference on Harmonization for all validation parameters such as specificity, accuracy, linearity, precision, the limit of detection, the limit of quantitation and robustness.

Reducing pressure and increasing peak capacity by incorporating an adaptable flow stream splitting platform to high-performance liquid chromatography columns: A study on C18 silica-based monoliths.

In this study, an adaptable end-column platform was fitted to a commercially available monolith, which enabled the column to be fitted with a flow-splitting device. A variety of flow-splitting adapters could be incorporated into the platform, and in this study, a radial flow stream splitter was utilized. The advantage of the radial flow stream spitter was that it overcame issues relating bed density variations that could cause bands to distort in the radial cross-section of the column. Using propylbenzene as a test standard in isocratic elution mode, height equivalent to a theoretical plate curves were constructed across ten flow rates, and it was found that the column efficiency improved by as much as 73%. Furthermore, the dual outlet flow splitter enabled a very substantial reduction in column back pressure, with the decrease being consistently between 20 to 30% depending on the column length. Additionally, sensitivity increased by 45%, consistent with the observed increase in efficiency. The adaptable end-column platform could be retrofitted to almost any commercial column with the expectation of gaining efficiency, sensitivity, and reducing back pressure.

Effect-Directed Profiling of Akebia quinata and Clitoria ternatea via High-Performance Thin-Layer Chromatography, Planar Assays and High-Resolution Mass Spectrometry.

Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, β-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.