Liquid Chromatography Methodology Research Articles

HPLC Method Validated for Quantification of Fluconazole Co-Encapsulated with Propolis Within Chitosan Nanoparticles.

The online version contains supplementary material available at 10.1007/s12088-021-00954-2.

A sensitive and practical ELISA for analyzing naringenin in pummelo and herb samples

Naringenin, a flavonoid compound found in pummelo, is a key biological active compound in some traditional Chinese medicines, including Citri reticulatae pericarpium, Citri reticulatae pericarpium viride, Aurantii fructus immaturus, and Aurantii fructus. These Chinese medicinal preparations are the peels or immature fruits of certain citrus species. Aiming at detecting naringenin in complex matrices such as pummelo and traditional Chinese medicines, we put forward a sensitive and practical indirect competitive enzyme-linked immunosorbent assay (icELISA) based on anti-naringenin monoclonal antibodies (anti-Nar-mAbs). The median inhibitory concentration (IC50) was 4.43 ng/mL, and the working range was 1.15–15.81 ng/mL. The findings of the icELISA for the analysis of naringenin in pummelo and herb samples had a good correlation with the ultra performance liquid chromatography (UPLC) methodology and showed good accuracy and reproducibility. These data demonstrated that the developed icELISA is reliable, accurate, and suitable for detecting naringenin in pummelo and traditional Chinese medicines.

Implementation of Analytical Quality-by-Design for Developing a Robust HPLC Method for Quantitative Estimation of Voriconazole: Application in Drug Formulations

The current studies endeavour to develop and validate a simple, sensitive and robust reversed- phase (RP) high-performance liquid chromatography (HPLC) methodology for quantitative estimation of voriconazole (VRC), following systematic principles of Analytical Quality-by-Design (AQbD). A comprehensive risk assessment plan, followed by factor screening studies employing Taguchi design, was executed to select “vital few” critical method parameters, influencing critical analytical attributes. Subsequently, response surface optimisation studies were conducted on the identified critical method parameters, viz., mobile phase ratio and flow rate, with chosen critical analytical attributes, viz., peak area, theoretical plate count, retention time and peak tailing, employing a Central Composite Design. The design space, demarcating the optimal method operating conditions, was identified using graphical optimisation and subsequently validated employing Monte Carlo simulations. The optimal solution consisted of acetonitrile and acetic acid solution (0.05 %, pH 4) as mobile phase (50:50 v/v), flow rate of 1 mL/ min with a detection wavelength of 256 nm, exhibiting linearity between 0.1 and 50 μg/mL in methanol and Hanks balanced salt solution. Validation studies confirmed precision (97.78-103.42 %), accuracy (98.99-102.34 %), and suitability of the developed analytical method for effective quantification of VRC with high sensitivity (LOD: 0.031 μg/mL; LOQ: 0.093 μg/mL). The validated analytical technique was successfully ratified for quantification of VRC in drug nanoformulations too. In a nutshell, the application of AQbD and Monte Carlo simulations aided in developing a robust analytical method for the estimation of VRC per se as well as in drug nanoformulations.

Mycotoxins-Biomonitoring and Human Exposure.

Mycotoxins are secondary metabolites produced by fungal species that commonly have a toxic effect on human and animal health. Different foodstuff can be contaminated and are considered the major source of human exposure to mycotoxins, but occupational and environmental exposure can also significantly contribute to this problem. This review aims to provide a short overview of the occurrence of toxigenic fungi and regulated mycotoxins in foods and workplaces, following the current literature and data presented in scientific papers. Biomonitoring of mycotoxins in plasma, serum, urine, and blood samples has become a common method for determining the exposure to different mycotoxins. Novel techniques are more and more precise and accurate and are aiming toward the simultaneous determination of multiple mycotoxins in one analysis. Application of liquid chromatography (LC) methodologies, coupled with tandem mass spectrometry (MS/MS) or high-resolution mass spectrometry (HRMS) has become a common and most reliable method for determining the exposure to mycotoxins. Numerous references confirm the importance of mycotoxin biomonitoring to assess the exposure for humans and animals. The objectives of this paper were to review the general approaches to biomonitoring of different mycotoxins and the occurrence of toxigenic fungi and their mycotoxins, using recent literature sources.

Occurrence of common plastic additives and contaminants in mussel samples: Validation of analytical method based on matrix solid-phase dispersion

A new matrix solid-phase dispersion (MSPD) extraction methodology, combined with high-performance liquid chromatography equipped with a diode-array detector, was developed and validated for the simultaneous determination of 10 compounds in mussels from Galician Rias (Spain). These pollutants are compounds commonly used for plastic production as additives, as well as common plastic contaminants. The compounds selected were bisphenol-A, bisphenol-F, bisphenol-S, nonylphenol-9, nonylphenol, diethyl phthalate, dibutyl phthalate, di-2-ethylhexyl phthalate, dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethane, and dichlorodiphenyldichloroethylene. The parameters affecting the MSPD extraction efficiency such as the type of sorbent, mass sample-sorbent ratio, and extraction solvent were optimised. The proposed method provided satisfactory quantitative recoveries (80–100%), with relative standard deviations lower than 7%. In all cases, the matrix-matched calibration curves were linear in the concentration range of 0.32–120.00 µg/kg, with quantification limits of 0.25–16.20 µg/kg. The novel developed MSPD-high-performance liquid chromatography methodology provided good sensitivity, accuracy, and repeatability for quality control analysis in mussels.

Sialic acids expression in newborn rat lungs: implications for pulmonary developmental biology

Mammalian lung development proceeds during the postnatal period and continues throughout life. Intricate tubular systems of airways and vessels lined by epithelial cells are developed during this process. All cells, and particularly epithelial cells, carry an array of glycans on their surfaces. N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic (Neu5Gc) acids, two most frequently-occurring sialic acid residues, are essential determinants during development and in the homeostasis of cells and organisms. However, systematic data about the presence of cell surface sialic acids in the postnatal lung and their content is still scarce. In the present study, we addressed the histochemical localization of Neu5Ac > Neu5Gc in 0-day-old rat lungs. Furthermore, both residues were separated, identified and quantified in lung membranes isolated from 0-day-old rat lungs using high-performance liquid chromatography (HPLC) methodologies. Finally, we compared these results with those previously reported by us for adult rat lungs. The Neu5Ac > Neu5Gc residues were located on the surface of ciliated and non-ciliated cells and the median values for both residues in the purified lung membranes of newborn rats were 5.365 and 1.935 μg/mg prot., respectively. Comparing these results with those reported for the adults, it was possible to observe a significant difference between the levels of Neu5Ac and Neu5Gc (p < 0.001). A more substantial change was found for the case of Neu5Ac. The preponderance of Neu5Ac and its expressive increase during the postnatal development points towards a more prominent role of this residue. Bearing in mind that sialic acids are negatively charged molecules, the high content of Neu5Ac could contribute to the formation of an anion “shield” and have a role in pulmonary development and physiology.

Reactivity Ratios Estimation of the Free‐Radical Polymerization of Itaconic Acid and N‐Vinyl‐2‐Pyrrolidone by the Error‐in‐Variables Methodology

AbstractDesigned copolymers require an understanding of the relative reactivity ratios of the monomer pair and a proper regression technique that can extract this data from the experiments. An implementation of the error‐in‐variables method previously describes in the literature is written and made available for use, along with an high‐performance liquid chromatography (HPLC) methodology for acquiring the kinetic data for the regression analysis of the itaconic acid and N‐vinyl‐2‐pyrrolidone (IA/NVP) free‐radical copolymerization system. From the data, it is observed that higher initial mole fractions of IA in the reaction medium change the reaction kinetics, limiting the validity of the Mayo–Lewis model to a narrow range of initial mole fractions of IA. This effect is not observed at molar fractions below 10%, enabling the parameters to be estimated. The performance of the developed algorithm is satisfactory, and within less than 5% error when applied to data found in the literature. The parameters estimated by the algorithm for the IA/NVP system are r1 = 0.3046 and r2 = 0.0170, with IA as compound 1. A rather large deviation is observed for r1 and this is attributed to measurement errors associated with the low IA concentrations and high initial reaction rates.

Development and validation of HPLC methodology for quantitative estimation of Efinaconazole in topical pharmaceutical formulation prepared in‐house for the treatment of onychomycosis

AbstractThe purpose of this research work was to design, develop, and optimize new high‐performance liquid chromatography method for quantitative estimation of Efinaconazole in topical pharmaceutical formulation developed in‐house for the treatment of onychomycosis. Based on the outcomes of preliminary investigation, optimized high‐performance liquid chromatography conditions were reverse phase column (Luna C18) as stationary material, with simple isocratic mobile phase comprising mixture of water and acetonitrile (20:80 v/v), flow rate 1 mL/min, column oven temperature 30°C, injection volume 10 µL, detection wavelength of 230 nm, and total run time of 10 min. The optimized method was successfully validated as per International Council for Harmonization guideline Q2(R1), and statistical analysis proved that the method is specific (peak purity ≥ 0.999), accurate (99.21–99.63% w/w), precise (% relative standard deviation &lt; 2.0), and sensitive (limit of detection = 9.26 µg and limit of quantitation = 28.05 µg). As a result, this research work provides a new and simple high‐performance liquid chromatography methodology with optimized chromatographic parameters for quantitation of Efinaconazole as a routine assay purpose, contributing to the technological and scientific domain and guaranteeing a safe and effective use of Efinaconazole for the treatment of onychomycosis.

Human Biomonitoring of Mycotoxins in Blood, Plasma and Serum in Recent Years: A Review.

This manuscript reviews the state-of-the-art regarding human biological monitoring (HBM) of mycotoxins in plasma, serum and blood samples. After a comprehensive and systematic literature review, with a focus on the last five years, several aspects were analyzed and summarized: (a) the biomarkers analyzed and their encountered levels, (b) the analytical methodologies developed and (c) the relationship between biomarker levels and some illnesses. In the literature reviewed, aflatoxin B1-lysine (AFB1-lys) and ochratoxin A (OTA) in plasma and serum were the most widely studied mycotoxin biomarkers for HBM. Regarding analytical methodologies, a clear increase in the development of methods for the simultaneous determination of multiple mycotoxins has been observed. For this purpose, the use of liquid chromatography (LC) methodologies, especially when coupled with tandem mass spectrometry (MS/MS) or high resolution mass spectrometry (HRMS) has grown. A high percentage of the samples analyzed for OTA or aflatoxin B1 (mostly as AFB1-lys) in the reviewed papers were positive, demonstrating human exposure to mycotoxins. This review confirms the importance of mycotoxin human biomonitoring and highlights the important challenges that should be faced, such as the inclusion of other mycotoxins in HBM programs, the need to increase knowledge of mycotoxin metabolism and toxicokinetics, and the need for reference materials and new methodologies for treating samples. In addition, guidelines are required for analytical method validation, as well as equations to establish the relationship between human fluid levels and mycotoxin intake.

Dissipation of six neonicotinoids in cotton by high performance liquid chromatography and quechers methodology

Dissipation of six neonicotinoids in cotton by high performance liquid chromatography and quechers methodology

Development of a rapid HPLC method for quantification of the copper tetramibi tetraflourborate in a kit for preparation of Technetium 99 m Sestamibi Injection.

Radiopharmaceuticals are radioactive compounds that can be used for diagnostic and therapeutic purposes. Technetium (99mTc) Sestamibi is the most commercialized radiopharmaceutical in the world. It includes a coordination complex consisting of the radioisotope 99 m technetium bound to six copper tetramibi tetrafluorborate ligands, and is mainly used to image the myocardium via scintigraphy. As radiopharmaceuticals are regarded as drugs, they are subject to the same regulations; therefore, the objective of this study was to develop a quantification method for the active pharmaceutical ingredient before their complexation with the radioisotope by employing high-performance liquid chromatography (HPLC) methodology. A simple and efficient method (retention time = 2.5 min) was developed and validated for copper tetramibi tetrafluorborate in the final product using a buffer and organic solvent mixtures (ACN-methanol-ammonium sulfate buffer) and a C18 column. The analytical protocol was fast, taking around 30 min until evaluation of results. The validation parameters were evaluated with satisfactory results: in terms of linearity r > 0.99 (160-240 μg/mL) and no deviation was observed. The RSD of precision was <5%, and an average recovery of 99% was observed for accuracy. The proposed method was thus considered adequate for routine analysis in the pharmaceutical industries.

Vitamin C in camu-camu [Myrciaria dubia (H.B.K.) McVaugh]: evaluation of extraction and analytical methods

Camu-camu, a typical Amazonian fruit, is known for the high vitamin C content of the peel and pulp. As vitamin C is widely used in the food, pharmaceutical, and cosmetics industries, it is of interest to study new sources, extraction techniques, and analytical methods for the identification and quantification of this compound. Here, evaluation was made of extraction and quantification methods, as well as the differences in vitamin C content according to the origin and part of the camu-camu fruit. The extraction techniques studied were pressurized liquid extraction (PLE), acid extraction, and maceration. The analytical methods evaluated were titrimetry and chromatography. Camu-camu samples were obtained from different regions, and the peel and pulp were studied separately. Acid extraction using sulfuric acid as solvent provided the highest vitamin C yields, while PLE, as a completely clean technique, proved to be a promising alternative for the recovery of ascorbic acid (L-AA). The application of an ultra-high performance liquid chromatography methodology (UHPLC-DAD) enabled the fast identification and quantification of L-AA and dehydroascorbic acid (DHAA), with high resolution, sensitivity, and specificity. The results obtained using the chromatographic and titration methods were not significantly different (p < 0.05), indicating that titrimetry is useful for routine analyses. L-AA and DHAA were found in the peel, but only L-AA was found in the pulp. The variation of vitamin C content among the lots could be explained by the edaphoclimatic conditions. The combination of a clean extraction technique and a fast analytical method is a promising approach for the determination of vitamin C in food products.

High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants.

Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g., differentiation, host defense and metabolic programs) but also contribute to the development of a wide variety of pathological conditions (e.g., cancer and Alzheimer’s disease). In particular, DNA methylation represents very stable epigenetic modification of cytosine bases that is strongly associated with a reduction in gene activity. Although High Performance Liquid Chromatography (HPLC) methodologies have been used to resolve methylated cytosine from unmodified cytosine bases, these represent only two of the five major cytosine analogs in the cell. Moreover, failure to resolve these other cytosine analogs might affect an accurate description of the cytosine methylation status in cells. In this present study, we determined the HPLC conditions required to separate the five cytosine analogs of the methylation/demethylation pathway. This methodology not only provides a means to analyze cytosine methylation as a whole, but it could also be used to more accurately calculate the methylation ratio from biological samples.

Recent Analytical Techniques Advances in the Carotenoids and Their Derivatives Determination in Various Matrixes.

In the present perspective, different approaches to the carotenoids analysis will be discussed providing a brief overview of the most advanced both monodimensional and bidimensional liquid chromatographic methodologies applied to the carotenoids analysis, followed by a discussion on the recents advanced supercritical fluid chromatography × liquid chromatography bidimensional approach with photodiode-array and mass spectrometry detection. Moreover a discussion on the online supercritical fluid extraction-supercritical fluid chromatography with tandem mass spectrometry detection applied to the determination of carotenoids and apocarotenoids will also be provided.

24-Hour Pharmacokinetic Relationships for Vancomycin and Novel Urinary Biomarkers of Acute Kidney Injury.

Vancomycin has been associated with acute kidney injury in preclinical and clinical settings; however, the precise exposure profiles associated with vancomycin-induced acute kidney injury have not been defined. We sought to determine pharmacokinetic/pharmacodynamics indices associated with the development of acute kidney injury using sensitive urinary biomarkers. Male Sprague-Dawley rats received clinical-grade vancomycin or normal saline as an intraperitoneal injection. Total daily doses between 0 and 400 mg/kg of body weight were administered as a single dose or 2 divided doses over a 24-h period. At least five rats were utilized for each dosing protocol. A maximum of 8 plasma samples per rat were obtained, and urine was collected over the 24-h period. Kidney injury molecule-1 (KIM-1), clusterin, osteopontin, cystatin C, and neutrophil gelatinase-associated lipocalin levels were determined using Milliplex multianalyte profiling rat kidney panels. Vancomycin plasma concentrations were determined via a validated high-performance liquid chromatography methodology. Pharmacokinetic analyses were conducted using the Pmetrics package for R. Bayesian maximal a posteriori concentrations were generated and utilized to calculate the 24-h area under the concentration-time curve (AUC), the maximum concentration (Cmax), and the minimum concentration. Spearman's rank correlation coefficient (rs ) was used to assess the correlations between exposure parameters, biomarkers, and histopathological damage. Forty-seven rats contributed pharmacokinetic and toxicodynamic data. KIM-1 was the only urinary biomarker that correlated with both composite histopathological damage (rs = 0.348, P = 0.017) and proximal tubule damage (rs = 0.342, P = 0.019). The vancomycin AUC and Cmax were most predictive of increases in KIM-1 levels (rs = 0.438 and P = 0.002 for AUC and rs = 0.451 and P = 0.002 for Cmax). Novel urinary biomarkers demonstrate that kidney injury can occur within 24 h of vancomycin exposure as a function of either AUC or Cmax.

Occurrence of Chiral Bioactive Compounds in the Aquatic Environment: A Review

In recent decades, the presence of micropollutants in the environment has been extensively studied due to their high frequency of occurrence, persistence and possible adverse effects to exposed organisms. Concerning chiral micropollutants in the environment, enantiomers are frequently ignored and enantiomeric composition often neglected. However, enantioselective toxicity is well recognized, highlighting the need to include enantioselectivity in environmental risk assessment. Additionally, the information about enantiomeric fraction (EF) is crucial since it gives insights about: (i) environmental fate (i.e., occurrence, distribution, removal processes and (bio)degradation); (ii) illicit discharges; (iii) consumption pattern (e.g., illicit drugs, pharmaceuticals used as recreational drugs, illicit use of pesticides); and (iv) enantioselective toxicological effects. Thus, the purpose of this paper is to provide a comprehensive review about the enantioselective occurrence of chiral bioactive compounds in aquatic environmental matrices. These include pharmaceuticals, illicit drugs, pesticides, polychlorinated biphenyls (PCBs) and polycyclic musks (PCMs). Most frequently analytical methods used for separation of enantiomers were liquid chromatography and gas chromatography methodologies using both indirect (enantiomerically pure derivatizing reagents) and direct methods (chiral stationary phases). The occurrence of these chiral micropollutants in the environment is reviewed and future challenges are outlined.

Antimutagenic evaluation of traditional medicinal plants from South America Peumus boldus and Cryptocarya alba using Drosophila melanogaster

ABSTRACTPeumus boldus Mol. (“Boldo”) and Cryptocarya alba Mol. Looser (“Peumo”) are medicinal shrubs with wide geographical distribution in South America. Their leaves and fruits are commonly used in traditional medicine because they exhibit natural medicinal properties for treatment of liver disorders and rheumatism. However, there are no apparent data regarding potential protective effects on cellular genetic components. In order to examine potential mutagenic and/or antimutagenic effects of these medicinal plants, the Drosophila melanogaster (D. melanogaster) wing-spot test was employed. This assay detects a wide range of mutational events, including point mutations, deletions, certain types of chromosomal aberrations (nondisjunction), and mitotic recombination. Qualitative and quantitative analyses of phenolic and anthocyanin compounds were carried out using biochemical and high-performance liquid chromatography methodologies. In addition, the antioxidant capacity of P. boldus and C. alba leaf extracts was also analyzed. P. boldus and C. alba extracts did not induce significant mutagenic effects in the D. melanogaster model. However, simultaneous treatment of extracts concurrently with the mutagen ethyl methane sulphonate showed a decrease of mutant spots in somatic cells of D. melanogaster, indicating desmutagenic effects in this in vivo model. Flavonoids and anthocyanins were detected predominantly in the extracts, and these compounds exerted significant antioxidant capacity. The observed antimutagenic effects may be related to the presence of phytochemicals with high antioxidant capacity, such as flavonoids and antohocyanins, in the extracts.

Development and Validation of Liquid Chromatography (RP-HPLC) Methodology for Estimation of Efonidipine HCl Ethanolate (EFD)

A Reversed Phase High Performance Liquid Chromatographic (RP-HPLC) method using symmetry C18, 5.0 mm column was developed for the determination of Efonidipine Hydrochloride Ethanolate (EFD). The mobile phase acetonitrile and water ratio was selected 85: 15 via flow rate were 0.8 mL/min and elution was monitored at 254 nm. Response was a linear function of concentration over the range 20-140 μg/ml (R2=0.9994) and the limits of detection was 681.83 ng/ml. The limit of quantification was 2.06 μg/ml. The coefficient of variation for intra-assay and inter-assay precision was less than or equal to 1.5% and the accuracy was 104.0-105.0% and method was validated accordance with International Conference on Harmonization (ICH) guidelines to check content uniformity. In this lieu, a simple and rapid with good accuracy precision validated method is developed which is applicable in quality-estimation, in-future.

Nutritionally Important Pigments in Purslane (Portulaca oleracea) Differ between Cultivars and in Response to Nitrogen

Purslane (Portulaca oleracea) is a succulent weedy annual in much of the United States. In other parts of the world, purslane is grown as a specialty crop, valued for its nutritional quality. As a leafy crop, purslane contributes carotenoid phytochemicals in the typical Mediterranean diet. Nitrogen (N) influences plant growth and alters pigment composition and accumulation. However, little is known about the impact N fertility may have on pigment concentrations in purslane shoot tissues. The objective of this study was to evaluate the influence of N fertility levels on biomass and concentrations of nutritionally important carotenoid and chlorophyll pigments in purslane. Green Leaf and Golden Leaf purslane cultivars were grown in nutrient solution culture under N concentrations of 13, 26, 52, or 105 mg·L−1. Plants were harvested at 45 days after planting (DAP), and measured for concentrations of shoot pigments using high-performance liquid chromatography (HPLC) methodology. There was no influence of N treatment concentration on purslane shoot tissue fresh weight (FW) accumulation. Nitrogen treatment significantly influenced shoot tissue β-carotene (BC), lutein (LUT), neoxanthin (NEO), total carotenoids, chlorophyll a, chlorophyll b, total chlorophyll, and the chlorophyll a to b ratio in purslane shoot tissues. Concentrations of LUT, NEO, violaxanthin (VIO), chlorophyll b, total xanthophyll cycle pigments, and the chlorophyll a to b ratio differed between the purslane cultivars. Increases in N concentrations acted to increase concentrations of nutritionally important shoot tissue carotenoid pigments in only the Green Leaf purslane cultivar. Therefore, N fertility management and cultivar selection should be considered when producing purslane as a nutritious specialty vegetable crop.

Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry

Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (−7≤cLogP≤2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC. The test mixtures were separated on a total of twelve different column chemistries at three different temperatures, using CO2-methanol-based mobile phases containing a variety of polar additives. Chromatographic performance was evaluated with a particular emphasis on peak capacity, overall resolution, peak distribution and repeatability. The results suggest that a new generation of stationary phases, specifically designed for improved robustness in mixed CO2-methanol mobile phases, can improve peak shape, peak capacity and resolution for all classes of polar analytes. A significant enhancement in chromatographic performance was observed for these urinary metabolites on the majority of the stationary phases when polar additives such as ammonium salts (formate, acetate and hydroxide) were included in the organic modifier, and the use of water or alkylamine additives was found to be beneficial for specific subsets of polar analytes. The utility of these findings was confirmed by the separation of a mixture of polar metabolites in human urine using an optimised 7min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.