Liquid Chromatographic Method Research Articles

Validation of Analytical Method for Vitamin A in Bioadhesive Ocular Cationic Nanoemulsion Loaded into Thermosensitive Gel Using RP-HPLC

Various test methods have been previously documented for determining vitamin A levels in different dosage forms. This study specifically examines an isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method designed for the direct extraction of vitamin A. The objective is to validate an analytical method for quantifying vitamin A in bioadhesive cationic nanoemulsions incorporated into thermosensitive gels. The method employs isocratic RP-HPLC with a YMC-Triart C18 column (L1), dimensions of 4.6 mm × 250 nm, particle size of S-5 µm, and a UV detector at λ = 265 nm. The mobile phase consists of HPLC-grade methanol, acetonitrile, and n-hexane in a ratio of 46.5:46.5:7. Validation parameters were assessed including selectivity, linearity, accuracy, precision, limit of quantification (LOQ), and limit of detection (LOD). Correlation coefficients were determined with an R2 value of 0.9995 in the concentration range of 264–396 μg/mL (w/v). Recovery percentages ranged from 99.295% to 99.878%. Repeatability and intermediate precision relative standard deviations (RSD) were found to be 0.318% and 0.254%, respectively. The LOD was established at 2.018 μg/mL, and the LOQ was determined to be 6.114 μg/mL. The results affirm cost-effective and well-suited for the accurate measurement of vitamin A levels in bioadhesive thermosensitive gel formulations.

Simultaneous Estimation of Atazanavir & Ritonavir in API & Marketed Formulations by Using RP-HPLC

Development and validation of RP-HPLC method for simultaneous estimation of Atazanavir and Ritonavir in their combined tablet dosage form. Atazanavir and Ritonavir are antiviral agents used in treatment of HIV. A simple, precise, rapid, accurate and cost-effective high- performance liquid chromatography (HPLC) method was successfully developed and validated for simultaneous estimation of Atazanavir and Ritonavir in their combined tablet dosage form. The selected mobile phase was Methanol: Phosphate Buffer in proportion 65:35 v/v respectively. The optimized columns used are C18 column, Symmetry and Zodiac column. X bridge C18 (4.6×150 mm, 5 µm) particle size was found to be ideal as it gave good peak shape and resolution at 1ml/min flow for Atazanavir and Ritonavir. In this study, the validation of Atazanavir and Ritonavir in API and marketed formulations were performed keeping in accordance with the parameters like system suitability, specificity, linearity, accuracy, precision (reproducibility & repeatability), robustness. The developed stability indicating method is capable for determination of impurities of Atazanavir and Ritonavir in combined tablet dosage form as well as individual dosage forms also. The method has been successfully validated according to ICH guidelines and the results obtained by using RP – HPLC are rapid, accurate and precise. The proposed methods were successfully applied for the analysis of synthetic mixtures and pharmaceutical formulations of Atazanavir and Ritonavir.

Salivary glycated albumin could be as reliable a marker of glycemic control as blood glycated albumin in people with diabetes

AimsMeasurements of diabetes-related biomarkers are necessary to assess glycemic control. The commonly used finger-prick blood glucose and HbA1c measurements are invasive, as is blood glycated albumin (GA) measurement. Therefore, we developed a non-invasive method, namely, measurement of the salivary GA with high accuracy using a high-performance liquid chromatography (HPLC) method. MethodsWe recruited participants with diabetes mellitus admitted to The University of Tokyo Hospital. Blood and saliva samples were collected at three time points (fasting and 2-hour postprandial at admission and fasting at discharge). After partial purification using an antibody-based column, the samples were subjected to GA measurement by HPLC method. ResultsAmong the 56 participants with diabetes mellitus enrolled in the study, there was a correlation between the GA levels measured in blood and saliva at the three time points described above (n = 45, R2 = 0.985, P < 0.001; n = 48, R2 = 0.973, P < 0.001; n = 51, R2 = 0.979, P < 0.001). Multiple regression analysis revealed the associations were maintained even after adjustments for age, BMI, and nephropathy stage. ConclusionsThis exploratory research revealed that the salivary GA levels by this method were accurate and might be able to replace blood GA measurement. The home salivary GA measurement is expected to be developed that may reduce the burden and complications in people with diabetes mellitus and improve the quality of life.

Investigation of the Cholesterol Biosynthesis by Heart-Cut Liquid Chromatography and Mass Spectrometric Detection

The biosynthesis and homeostasis of cholesterol are essential for cellular function. Cholesterol is a major lipid with multiple roles in membrane stability, signaling, or as a precursor for other molecules. Because of the structural similarity of the sterols involved in the biosynthesis, their accurate identification and quantification is still challenging. Moreover, the huge difference in the concentration of cholesterol and its precursors can cause interferences during the detection. To overcome these problems, a heart-cut liquid chromatographic method was developed by evaluating 38 different columns to achieve optimal separation. The method efficiently separates all sterol biosynthesis intermediates, with detection limits in the low nmol/L-range and an upper limit of quantification of 9 mmol/L for cholesterol by using triple quadrupole mass spectrometric detection. Investigation of lung carcinoma cells treated with statins demonstrated the capability to detect a biological response, showing inhibition of sterol synthesis. This technique offers a robust tool for studying cholesterol biosynthesis and its role in disease.

Implications of crystal disorder on the solid-state stability of Olanzapine

Mechanical perturbations of drug during solid pharmaceutical processing like milling can often generate crystal disorder posing serious implications to drug's stability. While physical changes like amorphization, recrystallization, polymorphism of the disordered drugs are extensively studied and reported in the literature, the propensities and inter-dependencies of recrystallization and degradation propensities of disordered drugs have seldom received deep attention. Previous investigations from our lab have explored some of these interplays, aiming to develop predictive stability models. As a follow-up, the implication of crystal disorder on the oxidative instability of Olanzapine (OLA) during accelerated storage is investigated in this work. Cryo-milling OLA at varied time intervals generated different extents of crystal disorder. The milled samples were characterized using calorimetry and infrared (IR) spectroscopy to examine the physical state, while their degradation was evaluated using ultra-performance liquid chromatographic methods. An X-ray amorphous OLA sample was generated by melt-cooling, and used as an amorphous reference. The crystallinity of the cryo-milled samples was quantified using a partial least square regression model based on ATR-FTIR spectroscopic data. The cryo-milled samples were exposed to different accelerated stability conditions along with crystalline (unmilled) and quench cooled (amorphous) samples, serving as controls. At periodic intervals, samples were removed from the stability storage, and analyzed using ATR-FTIR and UPLC methods to quantify the crystallinity- and degradation extents. A positive relation was witnessed between the initial degree of crystallinity and degradation kinetics of the disordered OLA samples during stability storage indicating a strong dependency of degradation on the disorder contents for such disordered solids. The results obtained in this study can potentially explain consequences of inter-batch variations of drugs during stability storage, in addition to enabling de-risking strategies towards eliminating solid drug instabilities in product development.

Development, validation and application of a high-performance liquid chromatography method for the detection of ibuprofen in elephant plasma

A simple and accurate method for the determination of ibuprofen concentrations in elephant plasma was developed and validated using reverse phase high-performance liquid chromatography (HPLC). Utilizing a liquid-liquid extraction with ethyl acetate, methanol, and phosphoric acid, samples were separated on an XBridge C18 column. The mobile phase consisted of 0.005 M ammonium phosphate and acetonitrile (40:60, v/v). Both ibuprofen and the internal standard flurbiprofen were quantitated using an ultraviolet (UV) detector set at 214 nm. The lower limit of quantification (LLOQ) was 0.05 μg/mL in 100 μL of plasma. The average recovery was over 95% while the intra-assay variability ranged from 0.8 to 9.9% and the inter-assay variability ranged from 2.7 to 9.8%. The method was used successfully in a therapeutic drug monitoring program for elephants and could be used in future pharmacokinetic studies.

Regulatory compliance of taurine containing food products marketed in India using a validated method: A pilot study

Taurine is an important functional food ingredient found in infant food, nutritional supplements and energy drinks. Concerns about the safety and quality of these products have prompted enhanced regulatory interest and enforcement. This study aimed to investigate regulatory compliance of taurine, in selected food products available in the Indian market. To assess compliance, a liquid chromatography method based on post-column derivatization with ninhydrin for the determination of taurine was validated for selectivity, accuracy, precision, linearity range, limit of detection, recovery and analysis of certified reference material. Excellent sensitivity, reproducibility and selectivity were achieved. The limit of detection and limit of quantification for the method was 0.0014 mg/100 mL and 0.0044 mg/100 mL, respectively. The overall percentage recoveries ranged from 95 % to 98 %. A total of 26 different products were analysed for their taurine content and compared with the label claim. Box-plots revealed wide variation in the taurine content. The determined concentrations of taurine ranged from 21.69 to 36.80 mg/100 g for infant foods with 7 out of the 9 samples analysed in the permitted tolerance limit of 20 % of the stated claim. Taurine content varied from 195.7 to 445.8 mg/100 mL for energy drinks with the percentage difference between obtained and declared values ranging from 4.15 to 11.46. All the 9 dietary supplements analyzed were found to contain taurine (20.48–68.62 mg/100 g), even though it was not listed as an ingredient. Our results confirm the efficacy of the proposed method in ensuring compliance with taurine in food products, underlining its importance for risk assessors and regulators.

The Creation and Validation of a New High-Performance Liquid Chromatography Method for Analyzing Pyridostigmine Bromide and its Degradation Products in a Pharmaceutical Formulation

The Creation and Validation of a New High-Performance Liquid Chromatography Method for Analyzing Pyridostigmine Bromide and its Degradation Products in a Pharmaceutical Formulation

Validation of a High-Performance Liquid Chromatography Method for the Online Determination of Phthalates in Water at a Trace Level

Validation of a High-Performance Liquid Chromatography Method for the Online Determination of Phthalates in Water at a Trace Level

Evaluation of Microbial Phospholipase and Lipase Activity Through the Chromatographic Analysis of Crude, Degummed, and Transesterified Soybean Oil for Biodiesel Production.

The present study aimed at synthesizing fatty acid methyl esters in a combined enzymatic method by applying degumming and transesterification of soybean oil. A soluble lipase from Serratia sp. W3 and a recombinant phosphatidylcholine-preferring phospholipase C (PC-PLC) from Bacillus thuringiensis were used in a consecutive manner for phosphorus removal and conversion into methyl esters. By applying 1% of recombinant PC-PLC almost 83% of phosphorus was removed (final content of 21.01mg/kg). Moreover, a sensitive and selective high-performance liquid chromatography method coupled to tandem mass spectrometry was applied to obtain a comprehensive lipid profile for the simultaneous evaluation of phospholipids removal and diacylglycerol (DAG) increase. A significant increase for all the monitored DAG species, up to 138.42%, was observed by using the enzymatic degumming, in comparison to the crude sample, resulting in an increased oil yield. Serratia sp. W3 lipase was identified as a suitable biocatalyst for biodiesel production, converting efficiently the acylglycerols. The results regarding the physical-chemical characteristics show that the cetane level, density and pour point of the obtained biodiesel are close to current regulation requirements. These findings highlight the potential of a two-step process implementation, based on the combination of lipase and phospholipase, as a suitable alternative for biodiesel production.

Rapid Characterization of the Potential Active of Sinomenine in Rats by Ultra-High-Performance Liquid Chromatography-Quadrupole-Exactive Orbitrap Mass Spectrometry and Molecular Docking.

Sinomenium acutum (Thunb.) Rehd. et Wils is widely used in the treatment of rheumatoid arthritis, with its alkaloid compound sinomenine (SIN) being renowned for its significant anti-inflammatory properties. However, despite its widespread application, the in vivo anti-inflammatory mechanisms and metabolic pathways of SIN remain incompletely understood. This study established a rapid and reliable method based on an ultra-high-performance liquid chromatography method coupled with Quadrupole-Exactive Orbitrap mass spectrometry and molecular docking to identify and characterize SIN and 69 metabolites in rat plasma, urine, and feces, revealing primary metabolic pathways of hydroxylation, demethylation, sulfation, and glucuronidation. Molecular docking results revealed that phase I reactions, including dedimethylation, demethylation, dehydrogenation, and dihydroxylation, along with their composite reactions, were pivotal in influencing SIN's in vivo anti-inflammatory activity. M28, M36, and M59 are potentially the most anti-inflammatory active metabolites of SIN in vivo. This comprehensive analysis unveils SIN's metabolic pathways, offering insights into its biological processes and suggesting a novel approach for exploring active drug constituents. These findings pave the way for further understanding SIN's anti-inflammatory mechanisms, contributing significantly to the development of new therapeutic strategies.

Individualized medication of venetoclax based on therapeutic drug monitoring in Chinese acute myeloid leukemia patients using an HPLC method.

The aim of this study was to establish a simple and sensitive high-performance liquid chromatography method for therapeutic drug monitoring of venetoclax (VEN) and optimize regimens. The analysis required the extraction of a 50 μl plasma sample and the precipitation of proteins using acetonitrile extraction. The chromatographic method employed a mobile phase of acetonitrile: 0.5% KH 2 PO 4 (pH 3.5) (60/40, v/v) on a Diamond C 18 (4.6 mm × 250 mm, 5 μm) column at a flow rate of 1.0 ml/min. The quantitative method was validated based on standards described in 'Bioanalytical Method Validation: Guidance for Industry' published by the US Food and Drug Administration (FDA). The calibration curve was linear ( R2 = 0.9998) over the range of 75-4800 ng/ml, with limits of quantification of 25 ng/ml. The coefficients of intraday and interday validation, specificity, recovery, and stability all met the criteria of FDA guidance. The method was successfully applied to analyze VEN concentrations in 30 cases of acute myeloid leukemia patients. The peak concentration ( Cmax ) was 1881.19 ± 756.61 ng/ml, while the trough concentration ( Cmin ) was 1212.69 ± 767.92 ng/ml in acute myeloid leukemia patients. Our study establishes a simple, precise, and sensitive high-performance liquid chromatography method for monitoring VEN and confirms its applicability for therapeutic drug monitoring of VEN in hematological cancers.

Study on determination of cynarin in dietary supplement containing artichoke by high performance liquid chromatography

There has been no announcement on the standardization and testing of cynarin in dietary supplements containing artichokes in Vietnam. The high-performance liquid chromatography (HPLC) method was developed to determine cynarin in dietary supplements containing artichokes. The sample was extracted twice with 80% methanol solvent and ultrasonicated at 50&amp;deg;C for 20 minutes. Centrifuge at 6000 rpm for 5 minutes, filter, and inject into HPLC system with XBridge C18 column (250 mm x 4,6 mm, 5 &amp;micro;m) and corresponding pre-column, mobile phase consisting of two solvents phosphoric acid 0,1% and acetonitrile eluted according to gradient program, flow rate 1mL/min, detection wavelength 322nm. The calibration curves for cynarin had R2 = 1, and the validation parameters met AOAC standards. The method detection limits and the limit of quantification were 3.00 mg/kg and 10.0 mg/kg, respectively. The validated method was applied to determine cynarin in 15 dietary supplement samples. The content of cynarin varied depending on the manufacturer

STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF AMLODIPINE AND LOSARTAN POTASSIUM BY UPLC

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Amlodipine and Losartan Potassium, in its pure form as well as in tablet dosage form. Chromatography was carried out on Acquity BEH-shield RP18 UPLC column (3.0 mm × 100) mm, particle size Column using a mixture of Acetonitrile and Acetate buffer (pH-4.3) (35:65% v/v) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 238nm. The retention time of the Amlodipine and Losartan Potassium was found to be 2.179, 3.610 ±0.02min respectively. The method produce linear responses in the concentration range of 20-60µg/ml of Amlodipine and 10-30µg/ml of Losartan Potassium respectively. The method precision for the determination of assay was below 2.0% RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

Improved Reverse Phase Liquid Chromatography Method for Iodine Value of Oils

Improved Reverse Phase Liquid Chromatography Method for Iodine Value of Oils

A sustainable RP-HPLC method for concurrent estimation of capecitabine and celecoxib in liposomal formulation: Greenness and whiteness appraisal.

Liposomes have been reported for combination therapy due to their ability to carry both hydrophilic and lipophilic drugs together. The current investigation aims to develop a novel, eco-friendly, and sustainable reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of capecitabine and celecoxib co-encapsulated in liposomes. The method reported herein uses a C18 column (4.6 × 250 mm2, 5 μm) and a mobile phase consisting of water, and acetonitrile/methanol in a ratio of 60:40, containing 0.1% formic acid in both the phases. The flow rate is maintained at 1 mL/min, with an injection volume of 10 μL in the gradient mode. Detection is set at λmax = 240 nm for capecitabine and 252 nm for celecoxib. The developed liposomes are mono-disperse with a surface potential of -6.93 mV. The average size of the liposomes is 142 nm. The percentage entrapment efficiency for capecitabine is 52.39 ± 0.94%, and for celecoxib, it is 77.13 ± 0.74%. The Analytical Greenness Metric of 0.61 and Analytical Eco-Scale Score of 75 signify the greenness of the developed method. Also, the Red-Green-Blue model shows excellent whiteness, with a score of 83.2. Thus, the developed method offers a reliable, accurate, precise, buffer-free, and environment-friendly RP-HPLC approach for the simultaneous analysis of capecitabine and celecoxib co-encapsulated in liposomes.

Liquid Chromatographic Method Development and Validation Using RP-UPLC for Identification and Quantification of Phenazopyridine Hydrochloride Tablets to Reduce the Time and Cost: A Promising Green Technique

Liquid Chromatographic Method Development and Validation Using RP-UPLC for Identification and Quantification of Phenazopyridine Hydrochloride Tablets to Reduce the Time and Cost: A Promising Green Technique

Analytical Quality by Design (AQbD) Principles in Development and Validation of Stability‐Indicating RP‐HPLC Method for Simultaneous Estimation of Diltiazem HCl and Eugenol in Nanoethosomes

ABSTRACTThe study developed and validated a robust reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method for a simultaneous estimation of Diltiazem HCl and Eugenol in nanoethosomes using analytical quality by design (AQbD) principles. The optimized method utilized a mobile phase of methanol and 0.1% Orthophosphoric acid (OPA) in a 50:50 % v/v ratio, with a flow rate of 1.0 mL/min and isocratic elution on a Phenomenex Luna C‐18 column (5 µm, 150 mm × 4.6 mm) at 30°C. This setup resulted in retention times of 6.838 min for Diltiazem HCl and 23.135 min for Eugenol. Validation followed International Council for Harmonization (ICH) Q2 (R1) guidelines, demonstrating excellent linearity with correlation coefficients of 0.9982 for Diltiazem HCl and 0.9991 for Eugenol across the 5–25 µg/mL range. The limits of detection (LOD) were 1.341 µg/mL for Diltiazem HCl and 0.960 µg/mL for Eugenol, with limits of quantification (LOQ) at 4.065 and 2.912 µg/mL, respectively. Stability testing under acidic, alkaline, oxidative, thermal, and photolytic conditions adhered to ICH Q1A (R2) and Q1B guidelines. This AQbD‐based method is effective for routine analysis of Diltiazem HCl and Eugenol in bulk and pharmaceutical dosage forms, fully complying with ICH standards.

Determination of Polyphenols in Cigar Tobacco by HPLC

A high performance liquid chromatography (HPLC) method was developed for the determination of neochlorogenic acid, cryptochlorogenic acid and caffeic acid in cigar tobacco. Firstly, the extraction method, extraction liquid concentration and dosage, column temperature and mobile phase flow rate of polyphenols in cigar tobacco were discussed. Then the detection limit, recovery rate, precision and standard curve of three polyphenols were tested. Finally, tobacco samples were tested. The results showed that the optimal extraction conditions were: ultrasonic extraction, extraction time was 30 min, methanol volume concentration was 50%, dosage was 20 mL, the optimal detection conditions were: column temperature was 30 ℃, flow rate was 1.0 mL/min, detection wavelength was 340 nm. The contents of neochlorogenic acid, cryptochlorogenic acid and caffeic acid were in the range of 0~0.202 mg/g, 0~0.331 mg/g and 0.050~0.073 mg/g, respectively, and the linear correlation coefficients of the three polyphenols were ≥0.9991, indicating that the linear correlation of the method was good, and the detection limit was ≤0.066 mg/L. The precision was ≤0.427%, and the recovery was 95.13~96.39%. The results showed that the method had good precision, high accuracy, short analysis time and high detection efficiency, and could be used as an effective method for the detection of polyphenols in cigar tobacco leaves.

RP-HPLC-PDA-Based Simultaneous Estimation of Ramipril and Hydrochlorothiazide in Combined Dosage Forms: A Validated Approach

A rapid, highly sensitive High-Performance Liquid Chromatographic method has been developed for the simultaneous determination of Ramipril (RMP) and Hydrochlorothiazide (HCT) in bulk drug and in tablets. Ramipril (RMP) and Hydrochlorothiazide (HCT) were eluted from an Eclipse plus C18 (250 mm × 4.6 mm I.D.) with particle size 5 µm reversed phase column with a mobile phase consisting of acetonitrile and potassium dihydrogen ortho-phosphate pH 3 (40: 60 v/v) at a flow rate of 1 mL/min with UV detection at 210 nm. The retention time for RMP and HCT was found to be 3.31 ± 0.02 min and 6.64 ± 0.02 min, respectively. The linear response (r2 &lt;0.99) was observed in the range of 2 - 12 μg/mL for RMP and 4 - 24 μg/mL of HCT with low values of limits of detection (LOD) and quantification (LOQ) for both drugs. The method shows good recoveries and intra and inter-day relative standard deviations were less than 1.0%. Validation parameters as specificity, accuracy, ruggedness and robustness were also determined. The proposed method provides accurate and precise quality control tool for routine simultaneous analysis of Ramipril and Hydrochlorothiazide in bulk and in tablet dosage form.