High-performance Liquid Chromatographic Separation Research Articles

Choline chloride-L-lactic acid mixtures as solvents for extraction of Coumarin from cinnamon-containing foods

The determination of coumarin in generally available cinnamon-containing foods such as biscuits, cereals is important to ensure that the populations consuming these foods more frequently are not exposed to higher levels and are not at risk. This study describes the development of an analytical method for determination of coumarin in foods containing cinnamon. Solid-liquid phase extraction with conventional stirring-assisted procedure and ultrasound-assisted extraction (UAE) procedures were used to prepare sample extracts for liquid chromatography analysis. The green solvent, a mixture of choline chloride:L-lactic acid (1:4, mol/mol)/water (50:50 v/v), as a deep eutectic solvent-like mixture, was selected as an extraction solvent. The extraction conditions were optimized by using the design of the experiment strategy that resulted in values, temperature 47 °C, time 36 min, solvent volume 4 mL, sample amount 0.8 g, stirring speed 20 rpm for rotary stirring-assisted extraction procedure, and temperature 30 °C, time 5 min, solvent volume 3.5 mL, sample amount 0.8 g for UAE. High-performance liquid chromatography separation with a total analysis time of 12 min was performed with a stationary phase of octadecyl type and a gradient of mobile phase of methanol-1 % acetic acid. The methods were successfully validated to show the detection limit of 0.13 μg.mL−1, the linearity range of 0.4–50 µg.mL−1 (R2 = 0.999), recovery rates of more than 80 %, and the RSDs of less than 7 %. Rotary stirring-assisted extraction showed moderately higher recovery values than UAE, while lower RSD % was observed for UAE.

Chemical profiling, anticonvulsant and anxiolytic effects of the smoke constituents isolated from Leonotis leonurus (L.) R.Br.

Ethnopharmacological relevanceThe use of medicinal plants for central nervous system (CNS)-related ailments, such as epilepsy and anxiety, is prevalent in South Africa. Plants from the Lamiaceae family are commonly used for their therapeutic benefits. Leonotis leonurus (L.) R.Br. has been reported in ethnobotanical literature to have anticonvulsant and anxiolytic effects through the inhalation of pyrolysis products obtained by combustion of the aerial parts. Aim and objectivesTo explore the chemical profiles and CNS activity of the smoke extract and isolated constituents of L. leonurus in zebrafish larvae, through anticonvulsive and anxiolytic activity assays. Materials and methodsThe smoke extract of L. leonurus was obtained through the combustion of the aerial parts of the plant using a custom-built smoke recovery apparatus. The chemical profile of the smoke constituents was determined using Ultra-Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS). Targeted compounds were subjected to preparative High-Performance Liquid Chromatography for separation before structure elucidation using Nuclear Magnetic Resonance (NMR). The maximum tolerated concentrations, as well as the anxiolytic activity of the smoke extract were determined in five days post fertilisation zebrafish larvae. Reverse-thigmotaxis and locomotor activity of larvae in the light/dark transition assay were used to determine anxiolytic activity. Zebrafish larvae at six days post fertilisation (dpf) were subjected to several concentrations of the smoke constituents of L. leonurus. The baseline locomotor activity of the larvae was tracked for 30 min, prior to addition of pentylenetetrazole (PTZ) to induce seizure-like behaviour in the larvae, after which the locomotor activity of the larvae was once again tracked for an additional 30 min. ResultsThe UPLC-MS profiles of the smoke extract revealed the presence of two main compounds, leoleorin A and leoleorin B, which were targeted and isolated. Upon subjection to NMR spectroscopy for structure elucidation, the compounds were confirmed to be labdane diterpenoids. Both leoleorin A and leoleorin B, and the smoke extract displayed suppression of the PTZ induced seizure-like behaviour in zebrafish larvae. Under light and dark conditions, the smoke extract and compounds displayed potential anxiolytic activity at different concentrations. ConclusionOur results suggest that the smoke constituents of L. leonurus may exert anticonvulsant and anxiolytic effects which align with the traditional indications and the mode of administration.

A Scoping Review on Carotenoid Profiling in Passiflora spp.: A Vast Avenue for Expanding the Knowledge on the Species.

The Passiflora genus is recognised for its ethnopharmacological, sensorial, and nutritional significance. Yet, the screening of its dietary and bioactive molecules has mainly targeted hydrophilic metabolites. Following the PRISMA-P protocol, this review assessed the current knowledge on carotenoid composition and analysis within Passiflora, examining 968 records from seven databases and including 17 studies focusing on carotenoid separation and identification in plant parts. Those publications originated in America and Asia. P. edulis was the most frequently examined species of a total of ten, while pulp was the most studied plant part (16 studies). Carotenoid analysis involved primarily high-performance liquid chromatography separation on C18 columns and detection using diode array detectors (64.71%). Most studies identified the provitamin A β-carotene and xanthophylls lutein and zeaxanthin, with their geometric configuration often neglected. Only one study described carotenoid esters. Besides the methodology's insufficient description, the lack of use of more accurate techniques and practices led to a high risk of bias in the carotenoid assignment in 17.65% of the articles. This review highlights the opportunity to broaden carotenoid studies to other species and parts within the diverse Passiflora genus, especially to wild, locally available fruits, which may have a strategic role in enhancing food diversity and security amidst climatic changes. Additionally, it urges the use of more accurate and efficient analytical methods based on green chemistry to better identify Passiflora carotenoids.

Comprehensive Review on Chiral Stationary Phases in Single-Column Simultaneous Chiral-Achiral HPLC Separation Methods.

This comprehensive review explores the utilization of chiral stationary phases (CSPs) in the context of single-column simultaneous chiral-achiral high-performance liquid chromatography (HPLC) separation methods. While CSPs have traditionally been pivotal for enantioselective drug analysis, contemporary CSPs often exhibit notable chemoselective properties. Consequently, there is a discernible trend towards the development of methodologies that enable simultaneous enantio- and chemoselective separations utilizing a single CSP-based chromatographic column. This review provides an exhaustive overview of reported HPLC methods in this domain, with a focus on four major CSP types: cyclodextrin-, glycopeptide antibiotic-, protein-, and polysaccharide-based CSPs. This article delves into the diverse applications of CSPs, encompassing various chromatographic modes such as normal phase (NP), reverse phase (RP), and polar organic (PO). This review critically discusses method development, emphasizing the additional chemoselective separation mechanisms of CSPs. It also explores possibilities for method optimization and development, concluding with future perspectives on this evolving field. Despite the inherent challenges in understanding the retention mechanisms involved in chemoselective separations, this review highlights promising trends and anticipates a growing number of simultaneous enantio- and chemoselective methods in pharmaceutical analyses, pharmacokinetic studies, and environmental sample determinations.

Moment Analysis Method for Measurement of Reaction Equilibrium and Rate Constants by Using High-Performance Liquid Chromatography.

The moment analysis method was developed for the determination of association equilibrium constant (KA) and association (ka) and dissociation (kd) rate constants of intermolecular interactions between solute and ligand molecules. They are accurately determined by using moment equations from elution peak profiles because they are measured by using high-performance liquid chromatography (HPLC) under preferable conditions that neither immobilization nor chemical modification (i.e., fluorescence labeling) of solute and ligand molecules is required. To demonstrate the effectiveness of the method, it was applied to the inclusion complex formation system between dibenzo-18-crown-6 (DB18C6) and alkaline earth metal cations, i.e., Mg2+, Ca2+, and Sr2+, as a concrete example. Because the diameter of the three metal cations is smaller than that of the inner cavity of DB18C6, the values of KA, ka, and kd were analytically determined by assuming the stoichiometry of 1:1 between DB18C6 and the metal cation. They reflected the influence of the difference in the size between the inner cavity of DB18C6 and the metal cations on the inclusion complex formation. It seems that the moment analysis method based on HPLC separation is effective for the multifaceted analysis of chemical reactions because some characteristics of the method are different from those of other conventional methods. It must contribute to the dissemination of an opportunity for the study of chemical reactions to many researchers because of the versatility of HPLC.

Cyclic Voltammetric Behaviour and High-Performance Liquid Chromatography Amperometric Determination of Levamisole

The electrochemical oxidation of levamisole, a glassy carbon electrode, was investigated over the pH range 2.0–10.0. Cyclic voltammetric investigations showed a single oxidation process was recorded, with a peak potential (Ep) shown to be pH-dependent in the range 5.0–8.0; between pH 2.0 and pH 5.0, and above pH 8.0, the Ep was found to be independent of pH, indicating apparent pKa values of 5.0 and 8.0. Peak currents were found to increase with increasing pH values. This voltammetric oxidation process was found to be consistent with a two-electron, two-proton oxidation to the corresponding sulfoxide. Based on these findings, the development of a of method based on the high-performance liquid chromatography separation of levamisole, with electrochemical detection being used for its determination, was explored. The chromatographic conditions required for the separation of levamisole were first investigated and optimized using UV detection. The conditions were identified as a 150 mm × 4.6 mm, 5 µm C18 column with a mobile phase consisting of 50% methanol, and 50%, 50 mM, pH 8.0 phosphate buffer. The technique of hydrodynamic voltammetry was applied to optimize the applied potential required for the determination of levamisole, identified as +2.3 V versus a stainless-steel pseudo-reference counter-electrode. Under the optimized conditions, levamisole exhibited a linear response of 1.00–20 mg/L (R2 = 0.999), with a detection limit of 0.27 mg/L. The possibility of determining levamisole in artificial urine was shown to be possible via simple dilution in the mobile phase. Mean recoveries of 99.7%, and 94.6%, with associated coefficients of variation of 8.2% and 10.2%, respectively, were obtained for 1.25 µg/mL (n = 5) and 2.50 µg/mL (n = 5).

A Multi-step High-Performance Liquid Chromatography (HPLC) Technique for the Extraction and Purification of Medical-Grade Cyclosporine A

Cyclosporine A (CsA), a potent immunosuppressive drug, is used to treat autoimmune diseases and prevent organ transplant recipients from rejecting their new organs. Maintaining CsA's uniformity and purity is essential to maintaining its therapeutic efficacy and safety. Impurities and contaminants in CsA formulations can exacerbate side effects and patient toxicity. Purification processes can improve the safety profile of the drug and reduce the likelihood of adverse effects by eliminating impurities. Using High-performance liquid chromatography (HPLC) methods, CsA has been isolated and refined from crude in recent years. However, after the procedure, the CsA purity was not at its best. To solve this issue, this work employs a multi-step HPLC method to increase purity levels to above 95%. When exploring the HPLC detection conditions of CsA, we found that the method from the Chinese pharmacopoeia: acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430 : 520 : 50 : 1) was a better mobile phase solution, our further research, including the substitution of tert-butyl methyl ether with other buffer solvents, showed no significant improvement. At the same time, we established that the key factor affecting its separation was temperature. When the temperature is lower than 70 ℃, the HPLC separation effect worsens, the retention time increases, and the peak width becomes longer. Finally, the utilization of acetonitrile-water-tert-butyl methyl ether-phosphoric acid (430 : 520 : 50 : 1) as the mobile phase with a column temperature of 70 °C, petroleum ether: acetic acid Ethyl ester (70 : 30) as the mobile phase, and filling the φ 40 mm × 500 mm chromatography column using a 40~60 μm silica gel so that a 10 : 1 height-to-diameter ratio of the packed part is obtained, under normal temperature conditions, resulted in an average cyclosporine A yield of 80.1%. The corresponding average purity was 97.2%, of which the first isolation yield was 82.3%, and the purity was 98.1%.

Separation and quantification of organic‐related impurities of anti‐histamine drug hydroxyzine in pharmaceutical dosage forms using stability‐indicating high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and high‐resolution mass spectrometry techniques

AbstractA simple and robust high‐performance liquid chromatography (HPLC) method was developed for organic impurities of hydroxyzine hydrochloride in pharmaceuticals. The developed method was designed to estimate all organic impurities of hydroxyzine. The HPLC separation was achieved using C18 column (150 × 3.9 mm, 5 μm) along with a binary gradient consisting of mobile phases A (0.1%, trifluoroacetic acid in purified water) and B (0.05%, trifluoroacetic acid in acetonitrile), a flow rate of 0.7–mL/min, a column temperature of 30°C and a sample temperature of 25°C. The detection wavelength used was 230 nm for the estimation of impurity‐A, impurity‐B, and all unspecified impurities and degradation products, whereas impurity‐C was quantitated using 254 nm. The stability‐indicating property of the developed HPLC technique was assessed using stress testing conditions of hydrolysis, oxidation, thermal, photo‐light, and humidity. The validation study was performed for the limit of detection and limit of quantification, linearity, and recoveries were 0.03%, 0.05%, and 0.1132–2.9920 μg/mL (R2 > 0.999), and 84.09%–109.74%, respectively. The proposed method is highly suitable for the determination of assay, organic impurities, and degradation products of the hydroxyzine. The chemical structure of degradation product 1 (hydroxyzine N‐Oxide) and degradation product 2 (O‐Acetyl hydroxyzine) were identified with the supporting data of LC‐mass spectrometry (LC‐MS) and high‐resolution MS.

Capillary electrokinetic chromatography for chiral separation of potential SARS-CoV-2 3CL protease inhibitors.

In this work, a capillary electrophoresis method was developed as a quality control tool to determine the enantiomeric purity of a series of five chiral compounds evaluated as potential severe acute respiratory syndrome coronavirus 2 3CL protease inhibitors. The first cyclodextrin tested, that is, highly sulfated-β-cyclodextrin, at 6% (m/v) in a 25mM phosphate buffer, using a capillary dynamically coated with polyethylene oxide, at an applied voltage of 15kV and a temperature of 25°C, was found to successfully separate the five derivatives. The limits of detection and quantification were calculated together with the greenness score of the method in order to evaluate the method in terms of analytical and environmental performance. In addition, it is noteworthy that simultaneously high-performance liquid chromatography separation of the enantiomers of the same compounds with two different columns, the amylose tris(3,5-dimethylphenylcarbamate)-coated and the cellulose tris(3,5-dichlorophenylcarbamate)-immobilized on silica stationary phases, was studied. Neither the former stationary phase nor the latter was able to separate all derivatives in a mobile phase consisting of n-heptane/propan-2-ol 80/20 (v/v).

A facile creation of hydrophilic-lipophilic-balanced covalent organic frameworks with mixed-mode retention mechanisms for multiple applications in liquid chromatography

Currently, high-performance liquid chromatographic separation materials that can provide multiple retention mechanisms are highly favored. Covalent organic frameworks (COFs) have undoubtedly demonstrated promising application prospects in chromatographic separation on account of excellent stability and porosity. Nevertheless, the utilization of COFs as liquid chromatographic fillers is still in the early stages of development. Especially, developing powerful COF@SiO2 with versatile separation capabilities still poses certain difficulties. Herein, a hydrophilic-lipophilic-balanced imine-linked COF was synthesized and in-situ growth on the amino-modified silica microsphere to obtain a multifunctional TAPT-TP-COF@SiO2 composite material. The synergistic effect of multiple functional groups including hydroxyl, amino, aldehyde, triazine and aromatic ring in the TAPT-TP-COF provided multiple retention mechanisms. The selection of 2,4,6-triformylphloroglucinol (TP) as the aldehyde counterpart improved the hydrophilic effect of the developed COF-based separation material, which enabled the effective separation of hydrophilic analytes (nucleosides, sulfonamides and vitamins). The novel TAPT-TP-COF@SiO2 column displayed typical reverse-phase and hydrophilic interaction chromatographic retention behavior. The successful applications demonstrated that TAPT-TP-COF@SiO2 had enormous potential for efficient liquid chromatographic analysis.

Characterization and Isolation of Piperamides from Piper nigrum Cultivated in Costa Rica

The piperamides profile of Piper nigrum cultivated in Costa Rica was studied using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry (UPLC-ESI-QTOF-HRMS) on enriched-piperamides extracts. A total of 31 different piperamides were identified, 24 of them with a methylenedioxyphenyl moiety, including piperine and nine other compounds with the characteristic piperidine ring, as well as guineensine, retrofractamide B, and eight other piperamides with an N-isobutyl group. In addition, piperyline and two other compounds with a pyrrolidine ring, as well as piperflaviflorine B, holding a N-2-methylbutyl chain, were characterized. In turn, pellitorine and six other piperamides exhibiting a long olefinic chain instead of the methylenedioxyphenyl group were also tentatively identified. In addition, quantification was performed using UPLC coupled with a diode array detector (UPLC-DAD), with 15 piperamides being quantified, including piperine, piperyline, piperanine, and piperloguminine with values within the range of previous reports, while results obtained for guineensine (276.5–421.0 mg/100 g dry material) and pellitorine (414.4–725.0 mg/100 g dry material) were higher than those reported in the literature. Additionally, preparative and semi-preparative high-performance liquid chromatography (HPLC) separations allowed to isolate, besides piperine, four other piperamides, which were identified through HRMS, 1H and 13C nuclear magnetic resonance (NMR). These included retrofractamide B, guineensine, pellitorine, and (2E,4E,12Z)-N-isobutyl-octadeca-2,4,12-trienamide, with yields of 134.0 mg/100 g dry material, 209.7 mg/100 g dry material, 361.8 mg/100 g dry material and 467.0 mg/100 g dry material, respectively, with all these values higher than those reported in previous studies in the literature. The findings constitute the first report of such a number and diversity of compounds in P. nigrum cultivated in Costa Rica.

Simultaneously characterization of multiple constituents in Valeriana jatamansi Jones using an online supercritical fluid extraction-high-performance liquid chromatography/supercritical fluid chromatography-mass spectrometry system.

Valeriana jatamansi Jones is a commonly used traditional Chinese medicine, boasting rich effective compositions with versatile chemical structures and wide polarity, including iridoids, chlorogenic acid, and flavonoids. Previous reports indicate that conventional high-performance liquid chromatography (HPLC) analytical methods have proven inefficient performance in comprehensively characterizing components in Valeriana jatamansi. In the present study, a hybrid online analytical platform combining supercritical fluid extraction with both conventional HPLC separation (reverse phase) and supercritical fluid chromatography (normal phase) has been established and validated. This system can provide online extraction with two different chromatographic separation modes to increase separation ability and has been connected to a mass spectrometer to acquire high-resolution mass spectrometry data. Then, the online platform was applied to screening components in Valeriana jatamansi. A total of 117 compounds were identified, including five lignans, 18 organic acids, six flavonoids, and 88 iridoids. Thirty-three compounds were reported from Valeriana jatamansi for the first time. These results enrich our understanding of the components of Valeriana jatamansi and prove that the developed online platform in this study is a robust approach for accelerating working efficiency in comprehensively analyzing complicated samples.

A synergistic chemometric combination for whiteness and greenness assessed HPLC-DAD assay of aqueous extracts of ivy and thyme and potassium sorbate in a syrup formula

A stability-indicating fit-for-purpose method has been developed for co-determination of hederacoside C (HC), thymol (TL), and potassium sorbate (PS) combined in a syrup dosage form. Carvacrol (CL) was separated from thyme extract (THY) with demonstrated identity at high confidence. This method was based on a reversed-phase (RP) high performance liquid chromatographic (HPLC) separation of the cited analytes. The HPLC separation was performed on a RP Zorbax Eclipse XDB-C18 analytical column [250 × 4.6 mm, 5 µm (80 Å)] and SecurityGuard C18 guard column (4 × 3 mm, 5 µm) with gradient elution system of HPLC far UV grade acetonitrile and 20 mM aqueous potassium dihydrogen phosphate buffer adjusted to pH 2.1 as the mobile phase at a flow rate of 2.5 mL min−1 and column oven was set at 50 °C. Quantitation was achieved with the photodiode array detection (DAD) with interferent peak suppression. Target Measurement Uncertainty (TMU) was determined by misclassification rates approach employing 100,000 Monte Carlo simulations (MCS). The bias and imprecision standard deviation were combined as a total analytical error (TAE) and compared via an enhanced approach linked to product specifications, process variation, and shelf-life variability for proving a fit-for-purpose analytical procedure. The robustness of the method was checked using supersaturated design constructed from Plackett-Burman design (PB-SSD) which was statistically analysed via the very recent approach of autovalidation using self-validated ensemble modelling (SVEM). The calibration graphs for each analyte were rectilinear in the range of 42.12–560.00, 3.37–47.15 and 0.51–7.07 µg mL−1 for PS, HC, and TL, respectively. The proposed HPLC-DAD method was successfully applied for the determination of the investigated analytes in a syrup formula. Moreover, the whiteness and greenness of the procedure were assessed via the most recently cited approaches.

Tandem-Column High Performance Liquid Chromatography Separation of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)

Tandem-Column High Performance Liquid Chromatography Separation of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)

Advances in Extraction Techniques for Beer Flavor Compounds

Owing to the unique chemical properties exhibited by beer flavor compounds, different extraction methods have been utilized to extract these compounds from the sample matrix. Carbonyl compounds, which significantly contribute to flavor instability in beer, pose challenges in detection due to their low concentrations and reactivity. Consequently, the analysis of beer flavor compounds has focused on improving sensitivity and specificity through techniques that minimize sample preparation requirements and reduce interactions between factors involved in the analysis. Notably, extraction techniques such as headspace solid-phase microextraction (HS-SPME), stir bar sorptive extraction (SBSE), and gas diffusion microextraction (GDME) have been successfully applied to the analysis of carbonyl compounds in alcoholic beverages, including beer. Derivatization agents like 2,4-dinitrophenylhydrazine (DNPH) and O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) enhance the volatility and stability of analytes, facilitating their separation and detection in gas chromatography and high-performance liquid chromatography. Chromatographic separation methods, particularly gas chromatography and liquid chromatography, are extensively employed to identify and quantify aroma/flavor compounds in various foodstuffs, including beer. This review provides a comprehensive overview of extraction techniques and chromatographic methods used in the analysis of beer compounds.

Prevalence Of Diabetes And Association Of Fasting And Postprandial Glucose With Hba1c, 6-Year Study In Reference Laboratory

Background
 Prevalence of diabetes is estimated in the Indian population using hemoglobin A1c (HbA1c) data collected over the period of 6 years, in the Metropolis Reference Laboratory Mumbai. HbA1c values with gender and age structure was correlated during fasting and postprandial glucose for the non-diabetic, pre-diabetic, and diabetic populations in terms of frequency and percentile.
 Method
 Retrospective analysis of HbA1c data obtained in a referral laboratory based in Mumbai over a span of 6 years from 2015 to 2020. A total of 2,94,264 cases were divided in six age groups i.e. 15-20Y, 21-30Y, 31-40, 41-50, 51-60, and >60Y. High-performance liquid chromatographic separation and quantification of HbA1c in blood with gender and age were done. HbA1c values were also correlated with glucose fasting and postprandial in the studied population.
 Results
 Prevalence of diabetes (HbA1C >6.5) was 57.60% in the studied population. The incidence and prevalence of HbA1c in the diabetic range were highest in the age group of >60 years. Males showed a higher prevalence of diabetes than females, while females are found to be more pre-diabetic. A significant (p>0.001) positive linear correlation was found between HbA1c with fasting and postprandial glucose, respectively.
 Conclusion
 The age group of >60Y showed a maximum diabetic prevalence. Gender disparity was recorded more in males than females having higher HbA1c. The sensitivity of HbA1c is comparable with fasting and postprandial glucose. However, HbA1c cannot replace plasma glucose estimation and could be used in conjunction with fasting and postprandial glucose for proper diagnosis and disease management.

A Review on Moxifloxacin

Moxifloxacin is an antibiotic. It can be given by Various route like mouth, injection into a vein and as an eye drop. In this review we studied about the side effect, mechanism, dosage, and precaution. The Analytical method like the determination of high-performance liquid coronarography method, chromatographic Separation, Simple and sensitive isocratic RP-HPLC method was developed for various combinations with Moxifloxacin as per referred articles.

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.

A novel pillar[3]trianglimine macrocycle with a deep cavity used as a chiral selector to prepare a chiral stationary phase by thiol-ene click reaction for enantioseparation in high-performance liquid chromatography.

A chiral pillar[3]trianglimine (C60 H72 N6 O6 ) with a deep cavity has been developed as a chiral selector and bonded to thiolated silica by thiol-ene click reaction to fabricate a novel chiral stationary phase for enantioseparation in high-performance liquid chromatography. The enantioseparation performance of the fabricated chiral stationary phase has been evaluated by separating various racemic compounds, including alcohols, esters, amines, ketones, amino acids, and epoxides, in both normal-phase and reversed-phase elution modes. In total, 14 and 17 racemates have been effectively separated in these two separation modes, respectively. In comparison with two widely used chiral columns (Chiralcel OD-H and Chiralpak AD-H), our novel chiral stationary phase offered good chiral separation complementarity, separating some of the tested racemates that could not be separated or were only partially separated on these two commercial columns. The influences of analyte mass, mobile phase composition, and column temperature on chiral separation have been investigated. Good repeatability, stability, and column-to-column reproducibility of the chiral stationary phase for enantioseparation have been observed. After the fabricated column had been eluted up to 400 times, the relative standard deviations (n=5) of resolution (Rs) and retention time of the separated analytes were<0.39% and<0.20%, respectively. The relative standard deviations (n=3) of Rs and retention time for column-to-column reproducibility were<4.6% and<5.2%, respectively. This study demonstrated that the new chiral stationary phase has great prospects for chiral separation in high-performance liquid chromatography.

Antioxidant peptides generated from chicken feet protein hydrolysates.

As major industrial poultry by-products, chicken feet are considered as notable sources of several bioactive molecules. The current work covers the processing of chicken feet proteins as substrates to be hydrolysed by combinations of three commercial enzymes (Alcalase®, Flavourzyme® and Protana® Prime) during different hydrolysis periods and the evaluation of the identified peptides having antioxidant activity after simulated gastrointestinal digestion. Enzymatic hydrolysis with Alcalase® and Protana® Prime combination for 4 h resulted in the highest activities. Reversed-phase high-performance liquid chromatographic separation of the purified hydrolysate yielded three active fractions that were further identified by nano-liquid chromatography-tandem mass spectrometry. The bioactivities of over 230 identified peptide sequences were estimated after simulated gastrointestinal digestion, and those peptides with the highest chance of exerting antioxidant activity were selected to be further synthesised and tested. In this sense, the synthesised dipeptides CF and GY showed the highest antioxidant capacity. CF presented IC50 values of 69.63 and 145.41 μmol L-1 in 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assays, respectively. In contrast, GY IC50 values were 15.27 and 10.06 μmol L-1 in ABTS and ORAC assays, respectively. Significant differences (P < 0.05) were registered between peptides in the same antioxidant assays. Overall, the findings emphasised the favourable impact of enzymatic hydrolysis with the obtaining of antioxidant peptides from poultry by-products that could be evaluated as a safe and economical source to retard oxidation in food systems. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.