Liquid Chromatography Methodology Research Articles

Optimized reversed phase liquid chromatography methodology for the determination of vonoprazan fumarate impurities: Towards Six Sigma quality standards and sustainability assessment

Vonoprazan fumarate (VPZ), a potent potassium-competitive acid blocker, holds great promise as a therapeutic option for addressing acid-related disorders. This study introduces a refined reversed phase liquid chromatography methodology tailored for the comprehensive analysis of eight related substances, including starting materials, byproducts, and degradants within VPZ. Our approach integrates response surface methodology and tolerance analysis to achieve six sigma quality standards in chromatographic performance. By embedding specifications into the optimization process, we ensure robustness during method development. Chromatographic separation was executed using an XSelect CSH Phenyl-Hexyl column under stepped gradient conditions, employing a mobile phase comprising 0.1 % trifluoroacetic acid aqueous solution and acetonitrile. The flow rate was maintained at 1.3 mL/min, with UV absorbance at 252 nm, and a column temperature set at 25 °C. To evaluate the stability indicating ability of the method, forced degradation studies were conducted. Importantly, identified degradants did not interfere with the accurate quantification of VPZ and its associated impurities. Validation of the method was achieved through accuracy profiles. A greenness assessment was conducted using National Environmental Methods Index (NEMI), carbon footprint analysis, Analytical Greenness Calculator (AGREE), and Complementary Green Analytical Procedure Index (Complex GAPI). Additionally, blueness and whiteness assessments were conducted using the Blue Applicability Grade Index (BAGI) and Red-Green-Blue 12 (RGB 12) algorithms, respectively. The proposed method exhibited a green profile in NEMI and Complex GAPI. The carbon footprint was calculated at 0.055 kg CO2 equivalent per sample. The AGREE score was 0.67, BAGI was 80.0, and the whiteness score from the RGB12 algorithm was 83.5.This methodological framework holds promise for utilization in process development and quality assurance of VPZ in bulk drug manufacturing, particularly in the absence of official monographs within recognized compendia.

A UPLC Method for Simultaneous Estimation of Trastuzumab and Hyaluronidase in Bulk and Pharmaceutical Dosage Form

Objective: The proposed research aimed at establishing a quick, accurate, and exact ultra-performance liquid chromatography (UPLC) methodology for contemporaneously analyzing trastuzumab and hyaluronidas in bulk and pharmaceutical formulation, with a focus on the stability-indicating properties of the assay. Method: Using a Water X-Bridge C18 column (50 mm x 4.6 mm x 2.5 μ) and a mobile phase of ACN: Buffer (pH 2.5) [50% v/v], pushed at 0.5 mL/min, compounds were separated chromatographically. An ultraviolet (UV) detector fixed at 260 nm was employed to identify the isolated compounds. Results: The findings showed that 1.3529 and 2.404 minutes were optimal for separating Trastuzumab and Hyaluronidase, correspondingly. The current procedure has been verified in accordance with ICH standards Q2 R1, and stability-indicating tests have been performed in accordance with ICH standards Q1A R2. Both the intra- and inter-day precisions were determined to be satisfactory. The suggested approach showed linearity between 30 to 180 μg/mL of trastuzumab and between 12.50-75μg/ mL of hyaluronidase. It was determined that the limit of detection (LoD) and limit of quantitation (LoQ) for trastuzumab were 0.36 and 1.2 μg/mL, correspondingly, whereas those for hyaluronidase were 0.15 and 0.5 μg/mL. The approach was shown to have a recovery rate around 98.6 to 99.7%. Conclusion: The suggested approach successfully separated the chemical compounds from their by-products. Therefore, trastuzumab and hyaluronidase routine evaluation and stability-indicating tests were both effectively implemented using the approach.

A voltammetric method coupled with chemometrics for determination of a ternary antiparkinson mixture in its dosage form: greenness assessment

An electroanalytical methodology was developed by direct differential pulse voltammetric (DPV) measurement of Levodopa (LD), Carbidopa (CD) and Entacapone (ENT) mixture using bare glassy carbon electrode (GCE) in Britton Robinson (BR) buffer (pH = 2.0). A multivariate calibration model was then applied to the exported preprocessed voltammetric data using partial least square (PLS) as a chemometric tool. Additionally, the model was cross-validated and the number of latent variables (LVs) were determined to produce a reliable model for simultaneous quantitation of the three drugs either in their synthetic mixtures or in their marketed pharmaceutical formulation with high accuracy and precision. Data preprocessing was used to tackle the problem of lacking bi-linearity which is commonly found in electrochemical data. The proposed chemometric model was able to provide fast and reliable technique for quantitative determination of antiparkinson drugs in their dosage forms. This was successfully achieved by utilizing sixteen mixtures as calibration set and nine mixtures as validation set. The percent recoveries for LD, CD and ENT were found to be 100.05% ± 1.28%, 100.04% ± 0.53% and 99.99% ± 1.25%, respectively. The obtained results of the proposed method were statistically compared to those of a previously reported High Performance Liquid Chromatography (HPLC) methodology. Finally, the presented analytical method strongly supports green analytical chemistry regarding the minimization of potentially dangerous chemicals and solvents, as well as reducing energy utilization and waste generation.

Simultaneous Identification and Quantification of Phenylalanine, Glycine and Lysine amino acids and conjugated heterocyclic amines in Maillard chemical model system using RP-HPLC-DAD

In this study, we developed a simple reversed-phase high-performance liquid chromatography (RP-HPLC) methodology for simultaneous identification and quantification of three types of mix: (1) phenylalanine and PHIP; (2) glycine and MeIQx; (3) lysine and 4,8 DiMeIQx in Maillard chemical model systems. In addition, we investigated the effects of different conditions on decreasing amino acids and heterocyclic amines (HCA) formation in chemical model systems. The results show that the developed methods used to separate the proposed mixes gave a relative standard deviation (RSD)% of less than 2% of peak area and retention time, thus proving the robustness and high reproducibility of the proposed method. The relationship between concentration and peak areas is linear with high statistical significance (p<0.001). The residual and slope of linear regression give a limit of detection LoD) and (LoQ) of phenylalanine and PHIP were (2.92- 0.029 ppm),(9.73- 0.098 ppm),respectively, which indicate the high sensitivity of the developed method. Moreover, the findings illustrate that a water ratio of 20% leads to a decrease in phenylalanine versus the largest amount of PHIP, and phenylalanine is the most active amino acid with a decreasing percentage up to 99%.

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

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

Incidences of aflatoxin contaminations in ingredients, feed and products of poultry from two regions in Ghana

Commercial poultry feed required for proper growth of birds and egg production contains essential nutrients with maize as key component. Inadequately dried maize is prone to aflatoxin contamination and therefore when used in feed formulation for poultry may compromise the safety of the feeds and poultry products. This study investigated the levels of aflatoxins in feed ingredients, feed and poultry products sampled from Eastern and Greater -Accra regions of Ghana. The aflatoxin levels of B1, B2, G1, and G2 were determined using a High-Performance Liquid Chromatography (HPLC) methodology. Main feed ingredients used were fishmeal, cotton seed cake, soya meal, rice, wheat and maize bran as well as maize grains. There was correlation between the level of aflatoxins and moisture content in poultry feed ingredients. All the poultry feeds (100 %) analysed showed the presence of aflatoxins with total aflatoxins recorded ranging from 5.32 to 29.88 μg/kg. Maize samples of the poultry feeds, from all two regions, revealed maize to be a major contributor to the overall total aflatoxin contents found in the feed. Five (5) out of ten (10) communities investigated in the two (2) regions where the poultry feeds were examined recorded total aflatoxin levels in maize above the Ghana Standard Authority (GSA) specification of 20 μg/kg. Aflatoxins G1 and G2 were not detected in all samples of chicken meat and eggs. Total aflatoxin levels recorded for all chicken meat and eggs were below GSA specification of 5 μg/kg; implying that these products were safe for consumption.

Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs(>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

Quantitative Analysis of Bioactive Compounds in Commercial Teas: Profiling Catechin Alkaloids, Phenolic Acids, and Flavonols Using Targeted Statistical Approaches.

Tea, an extensively consumed and globally popular beverage, has diverse chemical compositions that ascertain its quality and categorization. In this investigation, we formulated an analytical and quantification approach employing reversed-phase ultra-high-performance liquid chromatography (UHPLC) methodology coupled with diode-array detection (DAD) to precisely quantify 20 principal constituents within 121 tea samples spanning 6 distinct variants. The constituents include alkaloids, catechins, flavonols, and phenolic acids. Our findings delineate that the variances in chemical constitution across dissimilar tea types predominantly hinge upon the intricacies of their processing protocols. Notably, green and yellow teas evinced elevated concentrations of total chemical moieties vis à vis other tea classifications. Remarkably divergent levels of alkaloids, catechins, flavonols, and phenolic acids were ascertained among the disparate tea classifications. By leveraging random forest analysis, we ascertained gallocatechin, epigallocatechin gallate, and epicatechin gallate as pivotal biomarkers for effective tea classification within the principal cadre of tea catechins. Our outcomes distinctly underscore substantial dissimilarities in the specific compounds inherent to varying tea categories, as ascertained via the devised and duly validated approach. The implications of this compositional elucidation serve as a pertinent benchmark for the comprehensive assessment and classification of tea specimens.

Facile analysis of rice bran oil to compare free unsaturated fatty acid compositions of parental and hybrid rice lines.

Rice bran oil (RBO) has been a popular choice of cooking oil in several Asian countries for decades, and the interest in RBO is fast growing in Western countries due to the high levels of hearty unsaturated fats and other components beneficial to health. Further knowledge of unsaturated fatty acid content and composition in rice lines will assist in improving the quality of rice bran processing by allowing robust extraction of rice bran for oil production. The studies focused on the RBO composition of rice lines with beneficial genotypes are scarce. Accordingly, we investigated the total bran lipid content and composition of three of the most abundant, healthy, unsaturated fatty acids that freely exist in RBO: oleic, linoleic, and α-linolenic acids in nine parental lines (two male sterile lines and seven male lines) and seven hybrid rice lines, by utilizing an efficacious organic extraction to collect RBO and by developing a user-friendly reverse-phase high-performance liquid chromatography (HPLC) methodology. Our results showed that the hybrid lines had the highest oil content (F ratio = 7.2017, p value = 0.0019), while the male lines had the highest levels of two of the three free unsaturated fatty acids analyzed (linoleic acid, mg and oleic acid, mg). Oil weight was negatively correlated with α-linolenic acid (r = -0.6535, p value <0.0001). All three free unsaturated fatty acids were positively correlated. Our samples' natural variation in lipid content suggests that some rice lines are more suitable for oil production.

HPLC analysis of β-caryophyllene in copaiba oleoresin: Development, validation and applicability

Copaiba oil (CO) is an oleoresin containing resinous acids, comprising mainly of diterpenes, and volatile compounds, comprising of sesquiterpenes. CO has been used for many years as a therapeutic agent and cosmetic, being the β-caryophyllene (CAR) one of the main sesquiterpene markers found in CO samples. During the last years, some analytical methods have been developed for analysis of sesquiterpenes like CAR from CO. However, these methods are based on gas chromatography, and requiring additional steps, such as derivatization or extraction of the essential fraction of the CO for sesquiterpenes analysis. Liquid chromatography methodologies have been proposed only for analysis of diterpenes. Therefore, the aim of this study was to develop a high-performance liquid chromatography (HPLC) assay for CAR analysis in CO samples (Copaifera reticulata Ducke) and in emulsion systems containing CO. The HPLC system suitability was determined through the capacity factor, repeatability, relative retention, resolution, tailing factor, theoretical plate number and the height of the theoretical plate; where the method developed showed the efficiency for separation of CO compounds. The method was validated displaying specificity, linearity, precision, accuracy, and robustness. Moreover, it showed to be of utmost importance to analyze CO in emulsion systems, displaying versatile and applicability.

Experimental and computational studies of enantioseparation of three profen enantiomers with a focus on quantification of the enantiomeric impurities present in the corresponding enantiopure S-profen drugs

A rapid reversed-phase high performance liquid chromatographic (HPLC) methodology for chiral separation of three profen compounds has been developed and then applied to enantiomeric impurity testing of their corresponding enantiopure drugs. The assay is specific, allowing quantitation of the enantiomeric impurities at levels of 0.0078%, 0.0105%, and 0.0416% relative to S-ibuprofen, S-naproxen, and S-ketoprofen, respectively. In order to gain a better insight into the chiral recognition mechanisms of chiral profens on an FLM Chiral NQ(2)-RH column, molecular docking studies were carried out using AutoDock 4.0 software. It was found that hydrogen bonding, hydrophobic interactions, and π-π stacking were all involved in stereoselective interactions, and the calculated binding energy (BE) obtained reflected the binding strength of each enantiomer interacting with a chiral selector. The higher the BE value, the harder it was to elute the corresponding enantiomer, which also accorded with the enantiomer elution order observed in the actual enantiomeric separation. Additionally, thermodynamic analysis revealed that the enantioseparation process at 15–40°C was driven mainly by entropic contributions. The methodology was further validated according to the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q2 (R1) and proved to be sensitive, linear, precise, and accurate for determining R-profen impurities in three commercially available single-enantiomer S-profen drugs. As expected, in the case of products acquired in actual pharmacies, the levels of all of the monitored impurities were found to be lower than the allowable impurity limits.

Front Cover: Generic reversed‐phase ultra‐high‐pressure liquid chromatography methodology developed by using computer‐assisted modeling for streamlined performance evaluation of a wide range of stationary phase columns

DOI: 10.1002/sscp.202200002 A generic probe methodology was developed using in silico chromatography modeling tools. The separation of a standard multicomponent mixture was optimized on multiple columns/mobile phases. An optimal space across the overlay of different resolution maps is identified and used as a generic probe condition under which the components of the standard mixture will be resolved on all columns/mobile phases combinations. The resulting generic probe is used as a framework for testing column robustness and stability to ensure a straightforward method development and transfer workflow.

Guilty by dissociation: Part A: Development of a rapid Ultra-High Performance Liquid Chromatography (UHPLC)-MS/MS methodology for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS)

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (tG = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Generic reversed‐phase ultra‐high‐pressure liquid chromatography methodology developed by using computer‐assisted modeling for streamlined performance evaluation of a wide range of stationary phase columns

AbstractSelection of column and mobile phase eluent/buffer is a crucial step in chromatographic method development. The introduction of new column technologies requires a continuous performance evaluation enabling the deployment of improved chromatographic screening workflows. Consequently, the development of a universal method that could be used across different column chemistries and mobile phase pH ranges would be highlighted as advantageous. Herein, we introduce a single generic probe developed via software‐based modeling that delivers excellent results for the separation of a standard multicomponent mixture across a wide range of stationary phases under different pH and temperature conditions. This approach has been applied to interrogate the performance and stability of multiple columns (C18, Cyano, Phenyl Hexyl, PFP, Polar‐embeded, and other stationary phases) using an automated ultra‐high‐pressure liquid chromatography screening system illustrating its practicality and effectiveness.

Development of a Generic Ultra-High-Pressure Gradient Liquid-Chromatography Methodology for the Analysis of Residual Multi-Class Antibiotics in Food Products

Development of a Generic Ultra-High-Pressure Gradient Liquid-Chromatography Methodology for the Analysis of Residual Multi-Class Antibiotics in Food Products

Efficient Synthesis of Potential Impurities in Levonadifloxacin (WCK 771).

Levonadifloxacin (WCK 771) is a novel broad-spectrum anti-methicillin-resistant Staphylococcus aureus (MRSA) agent recently launched in India. Five process impurities and one degradation impurity were synthesized as reference standards for their quantification by high-performance liquid chromatography (HPLC) methodology in drug substance and drug product. These compounds are not easily commercially available. The synthesis and characterization of these impurities are discussed in detail.

A Rapid HPLC-UV Protocol Coupled to Chemometric Analysis for the Determination of the Major Phenolic Constituents and Tocopherol Content in Almonds and the Discrimination of the Geographical Origin.

Reversed phase-high-pressure liquid chromatographic methodologies equipped with UV detector (RP-HPLC-UV) were developed for the determination of phenolic compounds and tocopherols in almonds. Nineteen samples of Texas almonds originating from USA and Greece were analyzed and 7 phenolic acids, 7 flavonoids, and tocopherols (−α, −β + γ) were determined. The analytical methodologies were validated and presented excellent linearity (r2 > 0.99), high recoveries over the range between 83.1 (syringic acid) to 95.5% (ferulic acid) for within-day assay (n = 6), and between 90.2 (diosmin) to 103.4% (rosmarinic acid) for between-day assay (n = 3 × 3), for phenolic compounds, and between 95.1 and 100.4% for within-day assay (n = 6), and between 93.2–96.2% for between-day assay (n = 3 × 3) for tocopherols. The analytes were further quantified, and the results were analyzed by principal component analysis (PCA), and agglomerative hierarchical clustering (AHC) to investigate potential differences between the bioactive content of almonds and the geographical origin. A decision tree (DT) was developed for the prediction of the geographical origin of almonds proposing a characteristic marker with a concentration threshold, proving to be a promising and reliable tool for the guarantee of the authenticity of the almonds.

Non-mercaptalbumin is significantly associated with the coronary plaque burden and the severity of coronary artery disease

Human non-mercaptalbumin (HNA), oxidized form of serum albumin, has been reported as a useful marker in oxidative stress-related diseases; however, few reports have examined the association between HNA and the severity of coronary artery disease (CAD). The present study evaluated whether the HNA fraction is correlated with coronary artery stenosis in 140 patients considered to have a high risk of CAD or who were suspected of having acute coronary syndrome. The severity of CAD was defined by the number of stenotic coronary vessels and a severity score system (the Gensini score). HNA measurements were performed using our newly established high-performance liquid chromatography methodology. The results had shown that HNA was significantly increased in patients with three-vessel disease, compared with those without CAD or with single-vessel disease (p = 0.025), and was positively correlated with the Gensini score (ρ = 0.421, p < 0.001). A multivariate analysis showed that the number of stenotic vessels was an independent and significant factor associated with HNA (ρ = 1.246, p = 0.012). A logistic regression analysis showed that HNA was a strong predictor of multivessel CAD (odds ratio, 1.12; 95% confidence interval, 1.020–1.229; p = 0.017). These findings indicate that the measurement of HNA could be clinically practical for predicting the severity of coronary artery stenosis.

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.