High-pressure Liquid Chromatography Research Articles

APPLICATION OF VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF METAXALONE AND DICLOFENAC POTASSIUM IN PLASMA

Objective: The present investigation demonstrates a simple, sensitive and accurate high-pressure liquid chromatographic (HPLC) method for simultaneous determination of metaxalone (MTX) and Diclofenac potassium (DIC) in plasma by using Valsartan (VSN) as internal standard. Methods: The chromatographic separation was achieved within 10 min by using methanol: potassium dihydrogen phosphate buffer pH 4.5 adjusted with orthophosphoric acid (60:40) as mobile phase on Altima Grace Smart C-18 column (5μ; 250×4.6 mm) at flow rate of 1.0 ml/min with injection volume 25µl. The drug was extracted from plasma by liquid-liquid extraction using methanol as a solvent. The retention times of drugs (MTX and DIC) and internal standard were found to be 5.83, 9.65 and 11.79 min, respectively. This method was validated as per United States Food and Drug Administration (US-FDA) guidelines. Results: The results of the validation parameters were found to be within the acceptance limits. The method was linear in the concentration range from 25-1000 ng/ml (r2= 0.9998) and the extraction recovery was found to be 77.06% for MTX and 78.37% for DIC. The lower limit of quantification was found to be 25ng/ml and the stability of recovered samples at different conditions were found to be more than 95% for both the drugs. Conclusion: The developed method possesses good selectivity specificity, there was no interference found in the plasma blanks at retention times of MTX and DIC. We found good correlation between the peak area and concentration of the drug under prescribed conditions. Furthermore, the method can also be used to estimate the pharmacokinetic parameters of MTX and DIC simultaneously.

Comparative pharmacokinetics of a single oral dose of meloxicam in the California sea lion (Zalophus californianus) and Pacific harbor seal (Phoca vitulina richardii).

Pharmacokinetics studies have investigated meloxicam, a non-steroidal anti-inflammatory drug, dosing strategies in a wide variety of non-domestic animals; however, there is no prior study examining well-founded dosing for pinnipeds. To develop dosing protocols, pharmacokinetic information is needed, with an examination of differences between pinniped species. Apparently, healthy California sea lions (Zalophus californianus: CSL; n = 13) and Pacific harbor seals (Phoca vitulina richardii: PHS; n = 17) that had completed rehabilitation were enrolled into a population-based pharmacokinetic study. Each animal was administered a single oral dose of meloxicam at 0.1 mg/kg, and two blood samples were collected from each animal at varying intervals during a 96-h study period. Plasma concentrations of meloxicam were determined by high-pressure liquid chromatography. Data were analyzed with nonlinear mixed effects modeling (Phoenix® NLME™, Certara, St. Louis, MO 63105, USA). The results indicated that in PHS, peak plasma concentration (Cmax) was 0.33 μg/mL with an elimination half-life (Ke t½) of 31.53 h. In CSL, Cmax was 0.17 μg/mL with Ke t½ of 32.71 h. All animals enrolled completed the study without outward adverse clinical signs. The elimination half-life was longer than previously recommended dosing intervals for pinnipeds; however, we cannot speculate in the optimum clinical dose from these results.

Early evidence of an archaeological dyed textile using scale-insects: The Cave of Skulls, Israel

The color red has held significant historical symbolism and meaning throughout history and can be produced from various sources, including plants and animals. Some of the most luxurious red dyes were derived from various species of scale insects, particularly those of the Coccoidae superfamily. Today, advanced analytical methods allow to trace the origins of dyes even after thousands of years, providing insights into the technological capabilities of the ancient world as well as past economic and social conditions. In our research on textiles discovered in the Cave of Skulls in the Judean Desert, we conducted dye analyses using High-Pressure Liquid Chromatography (HPLC) and identified one unique, red-dyed textile, which was dyed with scale insects. Our analysis enables to pinpoint the precise source of the red color in archaeological textiles down to the species level, and it can be determined with a high degree of certainty that the dye used was Kermes vermilio. Direct radiocarbon dating places this textile in the Middle Bronze Age (1954–1767 BCE), shedding light on the early use of scale-insect dyes in ancient textile production.

HPLC method for the determination of thymoquinone in growth cell medium.

Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 μM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

Utilizing plant-based biogenic volatile organic compounds (bVOCs) to detect aflatoxin in peanut plants, pods, and kernels

Mycotoxins and particularly aflatoxin are a concern for peanut growers, processors, and consumers. Aflatoxins are responsible for approximately 25 % of liver cancer cases worldwide, while also costing millions of dollars in lost revenue on an annual basis. Current detection techniques based on high pressure liquid chromatography (HPLC) are time, labor and cost intensive. Peanut product can be held multiple days before testing is complete, costing processors additional revenue loss. Over the past few years, The Georgia Tech Research Institute (GTRI) has been investigating the use of plant-based biogenic volatile organic compounds (bVOCs) for monitoring the status of peanut plants. Initially, GTRI utilized these bVOCs to monitor for heat/drought stress in peanut plants. They quickly saw very promising indications of the ability of bVOCs to monitor other parameters in these plants. More recently, the team has started to investigate the of use of bVOCs to monitor aflatoxin development in plants, pods, and kernels pre- and post-harvest. A field trial for detection of aflatoxin using bVOCs was conducted in August–September of 2020 where three test groups were prepared: plants treated with Aspergillus fungus; plants treated with Afla-Guard (biocontrol agent); plants not treated – acting as a control group. Plant-based bVOCs were collected from the plants before treatment, and once a week post treatment using Stir Bar Sorptive Extraction (SBSE) devices or Twisters®. Each Twister® was then analyzed via gas chromatography–mass spectrometry (GC/MS). Pods from tested plants were harvested and sent to GTRI where bVOCs were collected and analyzed using GC/MS. Several statistical analysis and machine learning techniques were applied to all the collected GC/MS data. It was found using only bVOCs, that Random Forest classification performed well for the analysis of the pod and kernel samples with an F1 score of 0.80. On the other hand, Linear Discriminate Analysis (LDA) was only able to correctly classify 50 % of plant-based samples solely on bVOCs alone, which may be due to training models developed using original labels assuming no cross contamination at the field level. These results indicate the potential for bVOC screening for aflatoxin as an important way to lower impacts to growers, shellers, and consumers.

Precise method to measure fungal and bacterial necromass using high pressure liquid chromatography with fluorescence detector adjusted to inorganic, organic and peat soils

Soil organic matter is the dominant pool of carbon (C) in terrestrial ecosystems. Recent advances in understanding of the mechanisms of C stabilization in the soil emphasize microbes as the main drivers. Special attention is placed on the accumulation of bacterial and fungal necromasses. This calls for development of fast and reliable methods to estimate microbial necromass in a various type of soils, including peat soils. Here we provide precise method to measure fungal and bacterial necromasses with high-pressure liquid chromatography-fluorescence detector (HPLC-FLD) and its comparison with gas chromatography method. Purity of the chromatographic peaks was confirmed with mass spectrometry. The HPLC-FLD method provides reliable results for mineral, organic and highly organic peat soils.

Method development and validation of Metformin HCL and Dapagliflozin by using RP-HPLC

AbstractThe prevalence of diabetes is increasing day by day as per a report by the year 2045, 1 out of every 8th individuals may suffer from diabetes. This research article focuses on developing and validating Metformin and Dapagliflozin in combination by using high-pressure liquid Chromatography (RP-HPLC). The validation of the method was followed as per the guidelines provided by the International Conference on Harmonization (ICH) and United States Pharmacopeia (USP). Separation of both drugs takes place in less than 4 min. This separation takes place using Phosphate buffer (pH 6.8) and acetonitrile in a 45:55 (v/v) ratio at a 1.0 mL min−1 flow rate. Furthermore, studies on both drugs were conducted by using the bulk and pharmaceutical dosage forms (Tablets). The developed method was accurate as drug recoveries in both cases of Metformin, and Dapagliflozin ranged between (100.8, 99.6, 98.8%) to (100.8, 99.3, and 101.5%) respectively having a concentration range of solutions between 70, 100 and 130 μg mL−1 dilution. The recommended method for simultaneous quantification of Metformin and Dapagliflozin was established and validated and no excipient interactions were found.

Antiviral action of aqueous extracts of propolis from Scaptotrigona aff. postica (Hymenoptera; Apidae) against Zica, Chikungunya, and Mayaro virus

The limited availability of antivirals for new highly pathogenic strains of virus has become a serious public health. Therefore, news products against these pathogens has become an urgent necessity. Among the multiple sources for news antibiotics and antivirals, insect exudates or their products has become an increasingly frequent option. Insects emerged 350 million years ago and have showed a high adaptability and resistance to the most varied biomes. Their survival for so long, in such different environments, is an indication that they have a very efficient protection against environmental infections, despite not having a developed immune system like mammals. Since the ancient civilizations, the products obtained from the bee have been of great pharmacological importance, being used as antimicrobial, anti-inflammatory, antitumor and several other functions. Investigations of biological activity of propolis have been carried out, mainly in the species Apis mellifera, and its product have showed activity against some important viruses. However, for the Meliponini species, known as stingless bees, there are few studies, either on their chemical composition or on their biological activities. The importance of studying these bees is because they come from regions with native forests, and therefore with many species of plants not yet studied, in addition to which they are regions still free of pesticides, which guarantees a greater fidelity of the obtained data. Previous studies by our group with crude hydroalcoholic extract of propolis demonstrated an intense antiviral activity against Herpes, influenza, and rubella viruses. In this work, we chose to use aqueous extracts, which eliminates the presence of other compounds besides those originally present in propolis, in addition to extracting substances different from those obtained in alcoholic extracts. Therefore, this study aimed to identify, isolate and characterize compounds with antiviral effects from aqueous propolis extracts from Scaptotrigona aff postica, in emerging viruses such as zicavirus, chikungunya, and mayaro virus. The evaluation of the antiviral activity of the crude and purified material was performed by reducing infectious foci in VERO cell cultures. The results obtained with crude propolis, indicate a high reduction of zica virus (64×) and mayaro (128×) when was used 10% v/v of propolis. The reduction of chikungunya virus was of 256 fold, even when was used 5% v/v of propolis. The chemical characterization of the compounds present in the extracts was performed by high-pressure liquid chromatography. Through the purification of propolis by HPLC and mass spectrometry, it was possible to identify and isolate a peak with antiviral activity. This substance showed activity against all viruses tested. When purified fraction was used, the reduction observed was of 16 fold for zicavirus, 32 fold for mayaro virus and 512 fold for chikungunya virus. Likewise, it was observed that the antiviral response was concentration dependent, being more intense when propolis was added 2 h after the viral infection. Now we are carrying out the chemical characterization of the purified compounds that showed antiviral action.

Morpho- and Chemotyping of Holopelagic Sargassum Species Causing Massive Strandings in the Caribbean Region

The specific identification of three major morphotypes of the tropical holopelagic Sargassum species causing massive strandings on the African and Caribbean coastlines was attempted by morphological characterisation as well as quantitative and qualitative analyses of several metabolites. Of the 25 morphological variables studied on 208 samples from the North Atlantic Ocean, 22 were used to establish a dichotomous identification key, allowing without any doubt the identification of each morphotype based on their morphological criteria alone. We also attempted to differentiate morphotypes using chemical fingerprintings (HR-MAS NMR) and markers by analysing pigment level and composition using High Pressure Liquid Chromatography, terpene profiles by Thin Layer Chromatography, phenolic compound levels by the Folin-Ciocalteu assay and structures by 2D Nuclear Magnetic Resonance spectroscopy, and fatty acid composition by Gas Chromatography. While pigment level and composition, terpene profiles, and phenolic contents were not discriminating, quantification of eight fatty acids enabled the differentiation of the three morphotypes. Furthermore, phlorotannin purification permitted their structural characterisation allowing discrimination between the three morphotypes. Our study highlights the potential of the free fatty acid profile and phlorotannin structure as good chemomarkers in order to discriminate between the three morphotypes of holopelagic Sargassum.

СООТНОШЕНИЕ H2S/ГОМОЦИСТЕИН КАК ПОТЕНЦИАЛЬНЫЙ БИОМАРКЕР ПРОГНОЗИРОВАНИЯ ПРОГРЕССИРОВАНИЯ АРТЕРИАЛЬНОЙ ГИПЕРТЕНЗИИ У ДЕТЕЙ

Background. The H2S/homocysteine ratio is a potential biomarker for predicting the progression of cardiovascular diseases. The purpose. To establish the features of changes in the H2S/homocysteine ratio in children with arterial hypertension. Material and methods. A total of 111 children aged 14 to 18 years were examined. Based on the results of 24-hour blood pressure monitoring (n=81), two groups were formed: group 1 (n=51) – children with arterial hypertension (AH), group 2 (n=30) – children with high-normal blood pressure (HNBP). Group 3 included 30 healthy children. Plasma homocysteine level was determined by high-pressure liquid chromatography [Naumov A.V. et al., 2010]. The level of endogenous hydrogen sulfide was assessed by spectrophotometric method based on the reaction between sulfide anion and an acidic solution of the reagent N, N-dimethyl-p-phenylenediamine hydrochloride [Norris Eric J., 2011]. Results. A significant increase in plasma homocysteine concentration was revealed in patients with AH and HNBP compared with healthy children. The plasma levels of hydrogen sulfide in children with AH were significantly lower than in the comparison group. Hypertensive children in comparison to healthy children showed significantly lower plasma H2S/homocysteine ratio. Correlations have been established between the H2S/homocysteine ratio and plasma levels of homocysteine and hydrogen sulfide. Conclusions. Hypertensive children have a lower H2S/homocysteine ratio, which is due to the presence of hyperhomocysteinemia and reduced H2S levels. The H2S/homocysteine ratio can be used as a potential marker for predicting the progression of AH.

Exploration of Calocybe indica mushroom phenolic acid-kidney bean protein complex: Functional properties, amino acid profiles, in-vitro digestibility, and application in vegan product development

The study evaluates the interaction between Calocybe indica mushroom polyphenols (phenolic acid) and kidney bean protein (KBPM), aiming to enhance vegan food quality. The mushrooms exhibited a carbohydrate content of 3.65%, an antioxidant activity of 55.04 ± 0.17%, and a phenolic content of 4.86 mg GAE/g. Caffeic and cinnamic acids were identified through high-pressure liquid chromatography. Various concentrations of KBPM were tested at phenolic acid concentrations of 0.025, 0.050, 0.1, 0.2, 0.4, 0.8, and 1%, among these, KBPM 0.2 demonstrated the highest binding efficiency of 99.40 ± 0.05%. Notably, this complex improved the protein's functional properties, such as solubility by 11.43%, water and oil holding capacities by 10.62% and 22.04%, and emulsion capacity and stability by 3.69% and 5.83%, respectively, compared to the native protein. The protein-phenolic acid complex also enhanced thermal stability, surface charge, amino acid content, and reduced particle size compared to native protein. These enhancements also improved protein digestibility and sensory attributes in a fruit-based smoothie.

Separation of full, empty, and partial adeno-associated virus capsids via anion-exchange chromatography with continuous recycling and accumulation

The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.

Fractionation and characterization of Dunaliella microalgal biomass and extraction of carotenoids

This paper presents findings on the application of two fractionation protocols of Dunaliella microalgal biomass with the aim to separate and recover the lipid, pigment and protein fraction, leaving as “residue” the fraction that contains carbohydrates. The first protocol (LPC) is based on the initial extraction of lipids, followed by extraction of proteins, while in the second protocol (CLP), the pigments are first extracted followed by lipids extraction. With the LPC protocol the recovered lipid fraction was 34 and 32 wt%, while the recovered proteinic fraction was 46 and 50 wt% for the two samples under study, i.e. 1120 (D. granulata) and 1220 (D. minutissima). The CLP protocol led to the initial extraction of the pigment fraction with a percentage of 10–15 wt% while the subsequently recovered lipidic fraction was 16–24 wt%. The initial biomass was characterized for its moisture (<0.7 wt% for both samples), ash (approx. 45 wt% for both samples), organic matter, elemental composition and higher heating value (HHV) which was 23 and 30 MJ/kg (organic matter based-OMB). The composition of the lipid fraction was analyzed via esterification and gas chromatography (GC-FID) showing C16 and C18 as the predominant acids in the samples. The residue/carbohydrate fraction was analyzed via acid hydrolysis and analysis by high pressure liquid chromatography (HPLC) showing about 20 wt% (-OMB) content of carbohydrates. The high ash content of the biomass was found to be co-extracted during the fractionation protocols at a percentage of ≈49 % in the lipidic fraction and 45 % in the proteinic fraction. Furthermore, emphasis was given on pigment extraction and recovery using various solvents and quantification methods. Measurements of β-carotene, carotenoids, and chlorophyll content through UV and HPLC analyses is presented with chlorophyl and carotenoid content being ≈12 wt% and ≈9 wt% for samples 1120 and 1220 (-OMB). Additionally, a comparison between three solvents (THF, dichloromethane, acetone) was performed regarding their ability to extract the pigments from the microalgal biomass showing that acetone is capable of extracting pigments when concentration of the pigments is not high. The comparison of the HPLC with the UV method, suggested that UV spectroscopy is a reliable and rapid alternative for pigments determination, while HPLC enables identification and quantitative determination of specific pigments and chlorophyll species. The study provides insights for different fractionation protocols applied in two not yet fully studied Dunaliella species as well as the chlorophyl and carotenoid content.

Comparative study of Scleromitrion diffusum and Oldenlandia corymbosa: Microscopy, TLC, HPLC, and antioxidant activity.

Quality control of herbal medicines is crucial, especially the role of herbal drug identification. This is essential for preventing the misuse of herbs, which can affect efficacy or cause toxicity. Scleromitrion diffusum is a common herb, yet it is often mistaken for Oldenlandia corymbosa. This study analyzed the morphology, microscopy, thin-layer chromatography (TLC), and high-pressure liquid chromatography (HPLC) using two markers, asperuloside and scandoside methyl ester, to distinguish between S. diffusum and O. corymbosa with the analysis included 10 samples of S. diffusum and 10 samples of O. corymbosa collected from the Taiwan market. By quantifying the total polyphenols and flavonoids, we investigated the antioxidant activity, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging effect, 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) scavenging effect, and reducing power to further elucidate the biological effects of the two herbs. The results of this study revealed notable differences in microscopy and suggested a TLC method for distinguishing between the two herbs in the market. In HPLC, the ratios of asperuloside and scandoside methyl ester differed between the two herbs. S. diffusum contained a higher asperuloside content. In contrast, O. corymbosa contained higher concentrations of scandoside methyl esters. With more total polyphenols and flavonoids in S. diffusum than those in O. corymbosa, the antioxidant activity of S. diffusum was superior to that of O. corymbosa. This study provides a comprehensive understanding for the identification and quality evaluation of S. diffusum in the market. RESEARCH HIGHLIGHTS: The study consolidates and clarifies the morphological and microscopic differences between Scleromitrion diffusum and Oldenlandia corymbosa - a common adulterant species of S. diffusum on the Taiwan markets. Using Asperuloside and Scandoside methyl ester as two chemical markers, the study proposes a TLC method for rapidly testing S. diffusum and O. corymbosa on the market. Through HPLC analysis, our results showed that S. diffusum and O. corymbosa had a clear difference in the ratio of two markers, Asperuloside and Scandoside methyl ester: Asperuloside/Scandoside methyl ester in S. diffusum is higher than that in O. corymbosa. Through phytochemicals contents, including total phenols content, flavonoids content, and antioxidant activity, including DPPH, ABTS•+ scavenging activity, and reducing power, S. diffusum showed slightly higher levels of phenols and flavonoids as well as a better antioxidant activity than O. corymbosa.

Unraveling the multi-faceted role of Rosmarinus officinalis L. (rosemary) and diosmetin in managing gut motility

Ethnopharmacological relevanceRosmarinus officinalis L. (Rosemary) is a popular herb with reported effectiveness against diarrhea, anxiety and constipation, albeit with limited pharmacological evidence. Aim of the studyThe current study was aimed at evaluating the therapeutic potential, possible pharmacological mechanisms of action and active constituents of hydro-ethanolic extract of rosemary (Rs.Cr), as potential anti-diarrheal, laxative and anxiolytic agent. MethodRs.Cr was analyzed through reverse-phase high pressure liquid chromatography (RP-HPLC). Laxative, antidiarrheal, and anxiolytic activities were assessed using in vivo models. Spasmogenic and spasmolytic mechanisms were studied on isolated guinea pig ileum and rabbit jejunum tissues, respectively. Possible role of diosmetin, one of the active constituents of Rs.Cr was also evaluated. ResultsRP-HPLC analysis revealed presence of diosmetin, rutin and apigenin in Rs.Cr. Laxative effect was seen at low doses, which was partially reversed in atropinized mice. The spasmogenic mechanism was mediated by cholinergic and histaminergic receptors stimulation. At higher doses, antidiarrheal activity was evident, with reduction in gastrointestinal motility and secretions using charcoal meal and enteropooling assays, respectively. Rs.Cr also showed dose-dependent anxiolytic effect. The antispasmodic mechanisms were mediated by anti-muscarinic and K+ channel opening-like effect (predominant KATP-dependent). Diosmetin exhibited antidiarrheal and antispasmodic activities, but spasmogenic effect was not seen. ConclusionRosemary leaves have dual antidiarrheal and laxative effects, and as well as anxiolytic activity. In addition, the possible modulation of muscarinic and histaminergic receptors, and KATP channels show it as potential herb to be explored for irritable bowel syndrome. Diosmetin is possibly one of its constituents that contributes to its antidiarrheal activity.

Association of Candidate Single-Nucleotide Polymorphism Genotypes With Plasma and Skin Carotenoid Concentrations in Adults Provided a Lycopene-Rich Juice

BackgroundCarotenoids are fat-soluble phytochemicals with biological roles, including ultraviolet protective functions in skin. Spectroscopic skin carotenoid measurements can also serve as a noninvasive biomarker for carotenoid consumption. Single-nucleotide polymorphisms (SNPs) in metabolic genes are associated with human plasma carotenoid concentrations; however, their relationships with skin carotenoid concentrations are unknown. ObjectivesThe objective of this study was to determine the relationship between 13 candidate SNPs with skin and plasma carotenoid concentrations before and after a carotenoid-rich tomato juice intervention. MethodsIn this randomized, controlled trial, participants (n = 80) were provided with lycopene-rich vegetable juice providing low (13.1 mg), medium (23.9 mg), and high (31.0 mg) daily total carotenoid doses for 8 wk. Plasma carotenoid concentrations were measured by high-pressure liquid chromatography, and skin carotenoid score was assessed by reflection spectroscopy (Veggie Meter) at baseline and the end-of-study time point. Thirteen candidate SNPs in 5 genes (BCO1, CD36, SCARB1, SETD7, and ABCA1) were genotyped from blood using PCR-based assays. Mixed models tested the effects of the intervention, study time point, interaction between intervention and study time point, and SNP genotype on skin and plasma carotenoids throughout the study. Baseline carotenoid intake, body mass index, gender, and age are covariates in all models. ResultsThe genotype of CD36 rs1527479 (P = 0.0490) was significantly associated with skin carotenoid concentrations when baseline and the final week of the intervention were evaluated. Genotypes for BCO1 rs7500996 (P = 0.0067) and CD36 rs1527479 (P = 0.0018) were significant predictors of skin carotenoid concentrations in a combined SNP model. ConclusionsThese novel associations between SNPs and skin carotenoid concentrations expand on the understanding of how genetic variation affects interindividual variation in skin carotenoid phenotypes in humans.This trial was registered at clinicaltrials.gov as NCT03202043.

Repeated High-Dose Vitamin A Supplements, Standard of Care for Treating Xerophthalmia, Leads to Hypervitaminosis A in Piglets

BackgroundVitamin A (VA) deficiency and excess negatively affect development, growth, and bone health. The World Health Organization’s standard of care for xerophthalmia due to VA deficiency, is 3 high-dose VA supplements of 50,000–200,000 IU, based on age, which may cause hypervitaminosis A in some individuals. ObjectivesThis study measured VA status following 3 VA doses in 2 piglet studies. MethodsIn Study 1, 5 groups of piglets (n = 10/group) were weaned 10 d postbirth to VA-free feed and orally administered 0; 25,000; 50,000; 100,000; or 200,000 IU VA ester on days 0, 1, and 7. On days 14 and 15, the piglets underwent the modified relative dose–response (MRDR) test for VA deficiency, and were killed. Tissues were collected for high-pressure liquid chromatography analysis. Study 2 used the same design in 3 groups (n = 13/group) weaned at 16 d and administered 0; 25,000; and 200,000 IU doses. ResultsIn Study 1 (final weight: 3.6 ± 0.7 kg), liver VA concentration was hypervitaminotic in 40%, 90%, and 100% of 50,000; 100,000; and 200,000 IU groups, respectively. The 25,000 IU group was 100% adequate, and the placebo group was 40% deficient. In Study 2 (final weight: 8.7 ± 0.8 kg), where 200,000 IU could be prescribed to infants with a similar body weight, 31% of the piglets were hypervitaminotic, the 25,000 IU group was 100% VA adequate, and the placebo group was 100% deficient. The MRDR test measured deficiency in 50% and 70% of the placebo group in each study but had 3 false positives among hypervitaminotic piglets in Study 1. ConclusionsRepeated high-dose VA may cause hypervitaminosis, indicating dose sizes may need reduction. The MRDR resulted in false positives in a hypervitaminotic state during malnutrition and should be paired with serum retinyl ester evaluation to enhance VA status assessment in populations with overlapping interventions.

A novel cannabinoid in hemp: Isolation by flash chromatography and identification by liquid chromatography high resolution mass spectrometry

A novel and major cannabinoid (epicannabidiol hydrate) present in hemp plants and oils was isolated and characterized by a combination of flash chromatography and liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS). This novel cannabinoid differs from cannabidiol (CBD) by the absence of a double bond in the terpene ring and the presence of an additional hydroxyl group in the same terpene ring. The isolation procedure involved the use of normal phase chromatography with a silica gel preparative column, followed by reversed phase chromatography with a C18 preparative column. In this way, other major cannabinoids present in the samples, such as cannabidiol and Δ9-tetrahydrocannabinol, were separated and the focus was placed on the novel cannabinoid compound. Exact accurate masses were obtained for the compound of interest at m/z = 333.2424 in positive ion mode and m/z = 331.2279 in negative ion mode. Additional MS-MS analysis in negative ion mode revealed the position of the additional hydroxyl group in the molecule. Finally, the structural characterization was corroborated with 1H NMR and 13C NMR analysis, thus verifying the exact chemical structure of this novel cannabinoid, which has not previously been reported in hemp samples.

HPLC based Phytochemicals Analysis of Phyllanthus emblica (Indian Gooseberry/Amla): A mini Review

High-Pressure Liquid Chromatography (HPLC) is an analytical tool extensively used for the scientific analysis of components in a mixture. Several reports attribute its high analytical potential to bioactive components from different medicinal plants. Hepatic disorders, which have been a major threat to public health for decades, affect cells, tissues, structure, or liver function. The damage caused by this can be triggered by biological causes, autoimmune diseases, excessive alcohol consumption, or the action of different compounds, for instance, few medicines. Phyllanthus emblica Linn. is a therapeutic plant that has been used to treat liver disorders in Asia for many decades now. It is mentioned in the Ayurvedic scriptures for its medicinal value. This review focuses on the intricacies of HPLC-based extraction and analysis of medicinally important phytochemicals, notably for hepatic disorders from the P. emblica plant. This will be useful for future phytochemical analysts working with medicinal plants.

РАЗРАБОТКА СОСТАВА И ТЕХНОЛОГИИ ГЛАЗНЫХ КАПЕЛЬ АНЕСТЕЗИРУЮЩЕГО ДЕЙСТВИЯ

In modern ophthalmological practice, the use of local anesthetics for surface anesthesia of the eyeball becomes an integral stage in preparing the patient for diagnostic studies, therapeutic manipulations, surgical and laser interventions. The share of regional pain management methods is steadily increasing. However, at the same time, the list of requirements that local anesthetics must meet is also growing. According to them, the drug that combines high anesthetic activity and minimal toxicity should be considered the best. The local anesthetic anilocaine hydrochloride, developed according to the author's method in PGFA, exhibits high surface anesthetic activity with relatively low toxicity, and also has anti-inflammatory and moderate antimicrobial activity, which distinguishes it very favorably from the local anesthetics used and is an alternative to the use of expensive imported drugs. The purpose of this study is to develop the composition and technology of anesthetic eye drops. Materials and methods. The pharmaceutical substance anilocaine hydrochloride was used as a pharmacologically active component. The determination of the hydrogen index was carried out by the potentiometric method in accordance with the requirements of the OFS "Ionometry". To study the stability of eye drops, characterized by the indicator "Related impurities", a high-pressure liquid chromatograph "ShimadzuLCProminence" (Japan) was used. Statistical processing of the results of chemical studies was carried out according to generally accepted methods using standard EXCEL computer programs. Results and discussion. As a result of the conducted research, the composition of anesthetic eye drops based on anilocaine hydrochloride has been substantiated and developed. A technology for manufacturing eye drops has been developed to ensure the sterility of the resulting form. Conclusion. Based on technological and microbiological studies, the composition and technology of locally anesthetic eye drops based on anilocaine hydrochloride for surface (drip) anesthesia of the eyeball has been developed.