High-performance Chromatographic Method Research Articles

Validated chromatographic methods for determination of teriflunomide and investigation of its intrinsic stability

Two rapid, precise, and sensitive stability-indicating high performance chromatographic methods for the measurement of Teriflunomide in its degradation products’ existence were developed. These were RP-HPLC and HPTLC using UV detector. HPLC separation was accomplished utilizing Thermo BDS hypercil C18 column (250 × 4.6 mm, 5 μm) and acetonitrile: 0.03 M potassium dihydrogen phosphate: triethylamine (50:50:0.1%, by volume) as mobile phase at flow rate of 1mL/min. The separated peaks were detected at 250.0 nm. The densitometric approach was conducted utilizing HPTLC 60 F254 silica gel plates, and a developing system of benzene: ethanol: acetic acid (7.5:1:0.25, by volume) and detection was done at 250.0 nm. The developed approaches were evaluated regarding the International Conference on Harmonization (ICH) instructions. The calibration curves of both techniques were constructed with linearity ranges of (5-100) µg/mL and (2–10) µg /band, for HPLC and densitometric determination, consecutively. Teriflunomide was exposed to base and acid hydrolysis, oxidation using H2O2 and finally, thermal degradation as stated in ICH guidelines. The degradation product structures’ elucidation was achieved through LC-MS.

A Method for Simultaneous Quantification of Chlorine Dioxide and Hypochlorous Acid/hypochlorite and its Application to Disinfection Products

A novel high performance chromatographic method was devised to simultaneously quantify chlorine dioxide and hypochlorous acid/hypochlorite, leveraging their similar chemical properties but distinct toxicities. In this study, it consists of a two-step derivatization reaction: The first step is the reaction of hypochlorous acid/hypochlorite with 2,6-dimethylphenol in a water matrix to produce 4-chloro-2,6-dimethylphenol; the second step is a reaction in which iodine ions added after the first step react with chlorine dioxide to produce iodine and then react with 2,6-dimethylphenol to produce 4-iodo-2,6-dimethylphenol. It was confirmed that the concentration of 4-chloro-2,6-dimethylphenol produced was proportional to the total concentration of hypochlorous acid/hypochlorite, and the concentration of 4-iodo-2,6-dimethylphenol was proportional to the concentration of chlorine dioxide. With a detection limit of 0.011 mg/L for chlorine dioxide and 0.009 mg/L for hypochlorous acid/hypochlorite, the calibration curve showed excellent linearity (r2=0.999) in disinfectant solution. This sensitive and selective method is well-suited for the routine analysis of chlorine dioxide and hypochlorous acid/hypochlorite in water and disinfection byproducts.

Pentacyanidoferrate(III) complex of metformin provides a simple colorimetric test for this important anti-hypoglycemic medicament

Metformin (MF) is one of the most important medicaments in the market. It has been extensively employed in the treatment of type-2 diabetes, but its analytical detection is rather complicated, requiring, for instance, high-performance chromatographic methods. Here, we report that metformin reacts with the pentacyanidoferrate(II) complex, generating a deep red product in the presence of sodium percarbonate, Na2CO3·1.5H2O2. A colorimetric assay has been performed by measuring the absorption band at 520 nm, with a limit of detection (LOD) of 0.018 mmol/L. The reaction can also be probed by Raman spectroscopy, since the [FeIII(CN)5MF]3− complex, exhibits a strong resonance effect which enhances the vibrational peaks of the iron(III)-guanidine chromophore. The Raman spectral features are associated with the Fe(III)-NC-bonds and the metal-to-ligand charge-transfer transition, as confirmed by DFT calculations.

Determination of Aflatoxin M1 in raw cow milk, raw camel milk, and heat-processed milk in the North region of Iran

ABSTRACT In this study, 40 milk samples (10 raw cow milk, 10 camel milk, 10 pasteurised milk, and 10 ultra-high temperature milk) were randomly collected from Golestan Province (including two cities, Gorgan and Gonbad-e Kavus) from December 2021 to March 2022. Aflatoxin M1(AFM1) level was determined by the high-performance chromatography method. AFM1 was found in all samples. The mean concentration of AFM1 in raw cow milk, camel milk, pasteurised milk, and ultra-high temperature milk were 72.81 ± 2.18, 57.10 ± 1.86, 34.73 ± 1.34, and 49.36 ± 1.78 ng/kg, respectively. The highest level of AFM1 was found in raw cow milk (p < 0.05). According to the EU standard of AFM1 level, pasteurised milk is the safest type of milk in Golestan Province.

VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF JANUMET XR (SITAGLIPTIN/METFORMIN HCL EXTENDED-RELEASE FIXED DOSE COMBINATION (FDC) CONTENT IN BULK AND PHARMACEUTICAL DOSAGE FORM

A simple, accurate, precise and rapid stability indicating reverse phase High performance chromatography method was used for estimation of Sitagliptin/metformin HCl ER tablets in bulk and fixed-dose combination solid oral dosage form. The proposed analytical method has been validated for specificity, Linearity, Accuracy, Precision and Robustness. The chromatography was achieved in a Avantor, ACE C18 (Length 150 x Diameter 4.6mm Particle size 5µm) column with gradient flow. The optimal chromatographic condition consisted of mobile phase pH 3.5 at a flow rate of 1.2 mL/min, with a column temperature of 35°C, run time 17 minutes and detector wavelength of 210 nm (Sitagliptin&amp; Propyl gallate and Metformin HCl).

REVIEW OF HPLC METHODS FOR DETERMINATION OF AZITHROMYCIN IN DIFFERENT SAMPLES

Azithromycin is a semi-synthetic macrolide antibiotic of the azalide groups. It inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit of the bacterial 70S ribosome. It inhibits peptidyl transferase activity and interferes with amino acid translocation during the process of translation. Its effect may be bacteriostatic or bactericidal depending on the organism and the drug concentration. Azithromycin is one of the famous and important antibiotics agents and the determination methods of azithromycin in this article were tabulated with lots of chemical and instrumental methods that used in different parameters. Different high performance chromatographic methods have been reviewed in this paper. Keywords: Azithromycin, HPLC Citation: Taqi RMM, Hammoudy SR, Hamzah MJ. Review of HPLC methods for determination of azithromycin in different samples. Iraqi JMS. 2022; 20(1): 77-82. doi: 10.22578/IJMS.20.1.10

VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF TELMISARTAN AND HYDROCHLOROTHIAZIDE CONTENT IN BULK AND PHARMACEUTICAL DOSAGE FORM

A simple, accurate, precise and rapid stability indicating reverse phase High performance chromatography method was used for estimation of Telmisartan and Hydrochlorothiazide in bulk and fixed-dose combination solid oral dosage form. The proposed analytical method has been validated for specificity, Linearity, Accuracy, Precision and Robustness. The chromatography was achieved in a GL science, Inertsil C8 (Length 125x Diameter 4.0mm Particle size 5µm) column with gradient flow. The optimal chromatographic condition consisted of mobile phase pH 3.0 at a flow rate of 1.2mL/min, with a column temperature of 40°C, run time 14 minutes and detector wavelength of 270nm.

Novel Determination of Anti-Hypertensive Combination; Benidipine Hydrochloride and Nebivolol Hydrochloride by High Performance Chromatographic Method

This research work was designed to establish and validate a novel stability indicating RP-HPLC method for the combined determination of Benidipine hydrochloride (BHE) and Nebivolol hydrochloride (NHE) in bulk and tablets, dependent on ICH guidelines.The assay method to analyse BHE and NHE was optimized with isocratic elution using acetonitrile: 0.1M acetate buffer (45:55, pH 5.1), Lichrospher ODS RP-18 column and flow pace of 1 ml/min. Total time for single run was 14 min. The injection quantity was 20μl, and was detected at 249nm. The method was verified on a concentration series of 1.25-10μg/ml (NHE) and 1.0-10μg/ml (BHE) for precision, accuracy and linearity. The LOD values were 0.059µg/ml and 0.028µg/ml for NHE and BHE, respectively. The LOQ values were 0.196µg/ml for NHE and 0.094µg/ml for BHE. The recovery percentages were 98.60-100.11% (BHE) and 98.94-101.50% (NHE) with relative standard deviation 0.250-0.694% (BHE) and 0.183-0.400% (NHE). The method was also observed to be efficient, and was sufficiently specific to measure BHE and NHE in the presence of stress-produced degradation products.

Determination of Kresoxim-Methyl in Water and in Grapes by High-Performance Liquid Chromatography (HPLC) Using Photochemical-Induced Fluorescence and Dispersive Liquid-Liquid Microextraction (DLLME)

A high-performance chromatographic method was developed to determine the fungicide kresoxim-methyl. Off-line photochemical derivatization was used to induce the formation of a stable and fluorescent product since the fungicide does not present natural fluorescence. Intense fluorescence at 370/430 nm was achieved by treating the analyte in solution at pH 6 to ultraviolet light for 45 s. The chromatographic conditions included isocratic elution with 50/50% (v/v) acetonitrile/water and the photochemical product appeared at a retention time of 7.2 min. The short and long term stabilities of the photoproduct were evaluated and variation of less than 5% was achieved. The limits of detection in water samples and in grapes samples were 0.019 mg kg−1 and 0.065 mg kg−1 of kresoxim- methyl residue, respectively. The linear response covered three orders of magnitude up to 10.6 mg kg−1 of kresoxim-methyl. The robustness was evaluated through a Box–Behnken experimental design showing the insignificance of all factors and their interactions. The potential interference of tebuconazole for the determination of kresoxim-methyl was studied. The use of the dispersive liquid-liquid microextraction (DLLME) allowed recoveries between 80% and 101% depending on concentration with the minimum generation of waste products.

Validation of a high-performance liquid chromatographic method for the simultaneous assessment of vitamin D isoforms

Background: Asthma is one of the common conditions clinicians encounter in children. Various studies report the association of poor vitamin D (Vit D) status of mothers during pregnancy, with an increased risk of asthma in their children, especially in HIV-positive mothers. Simultaneous measurement of Vit D metabolites, rather than measuring total Vit D, would improve assessment of Vit D metabolism in these children. This study aimed to validate a high-performance liquid chromatographic (HPLC) method using ultraviolet detection for the measurement of five Vit D isoforms (25 OHD2 and D3; 1,25 [OH]2D2 and D3 and 24,25 [OH]2D) simultaneously. Methodology: Serum samples from 80 paediatric patients with asthma were subjected to solid phase extraction and 4-phenyl-1,2,4-triazole-3,5-dione-derivatisation. Chromatographic separation was achieved with a pentafluoro-phenylpropyl (PFPP) analytical column. Total 25 OH Vit D was measured on an automated immunochemistry analyser for comparison purposes. Results and discussion: Good linearity was achieved for 25 OHD2, 25 OHD3 and 24,25 (OH)2D with r2 ≥ 0.99, with limits of detection ≤ 5.0 ng/ml and lower limits of quantitation ≤ 10.0 ng/m. The 1,25 (OH)2D metabolites, present in the blood at pg/ml levels, could not be quantified within the required ranges. Comparing total 25 OH Vit D immunoassay and HPLC results showed a poor correlation (r2 = 0.073), possibly due to cross-reactivity of the antibodies in the immunoassay with other Vit D isoforms. Conclusion: This method provides a precise and accurate, with a potential for high throughput, determination of Vit D isoforms in the routine laboratory.

Selective quantitation of co-formulated ternary mixture in the presence of potential impurities by liquid chromatographic methods

Two high performance chromatographic methods were developed and validated for the simultaneous determination of Ambroxol, Guaifenesin and Theophylline in pharmaceutical dosage forms and in the presence of Guaiacol and Caffeine as the officially stated impurities. These were a reversed phase liquid and a thin layer chromatographic methods. The liquid chromatographic separation was achieved using Inertsil ODS-3 C18 column (4.6 mm × 250 mm, 5 μm). Gradient elution was performed using a mixture of solvent A (0.05 M ammonium acetate, pH 3, adjusted with glacial acetic acid) and solvent B (methanol), at a flow rate of 1.0 mL/min. The separated peaks were detected at 260.0 nm. The thin layer chromatography was performed using HPTLC 60 F254 silica gel plates, mobile phase was consisting of ethyl acetate: methanol: acetic acid (10:0.5:1, v/v/v) and detection was performed at 254.0 nm. Validation of the developed methods was achieved according to International Conference on Harmonization (ICH) guidelines. The proposed methods were fast, accurate, precise, and sensitive. Hence, they could be employed for routine quality control of the ternary mixture in capsule and syrup dosage forms.

Application of Ion Mobility Mass Spectrometry in Lipidomics.

Lipids play significant roles in biological system, and the study of lipid metabolisms may provide a new insight into the diagnosis and pathophysiology of diseases. Recent developments in high-resolution mass spectrometry techniques combined with high-performance chromatographic methods provide deep insight into lipid analysis. Addition of ion mobility mass spectrometry orthogonal to LC-MS analysis workflow enhances separation of complex lipids, improve isomers resolution, and intensify confidence in lipid identification and characterization. In this chapter, we describe the principle of travelling wave ion mobility mass spectrometry (TWIMS) and its applications in untargeted LC-MS analysis for characterizing the structural diversity and complexity of lipid species in biological samples.

Stability-indicating high-performance thin-layer chromatographic method for the estimation of antipsychotic drug combination clozapine and aripiprazole

A sensitive and precise high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous estimation of clozapine and aripiprazole in combination. The method employed HPTLC aluminum plates pre-coated with silica gel 60 F254 as the stationary phase, while the solvent system was toluene‒methanol‒ethyl acetate‒ammonia (6.5:2.5:1:0.1, v/v). The RF values were observed to be 0.43 and 0.60 for clozapine and aripiprazole, respectively. Densitometric analysis was carried out in absorbance mode at 218 nm. The method was linear in the range of 200–1600 ng per band for clozapine and 100-800 ng per band for aripiprazole. The stress degradation study was performed, and it was found that clozapine was susceptible to acid hydrolysis, base hydrolysis, and photolytic degradation study. Aripiprazole was susceptible to oxidative stress degradation study. The method was validated and applied successfully for the estimation of both drugs in the synthetic mixture.

Optimization and Validation of Reversed Phase High Performance Chromatography Method for Rapid Estimation of Theaflavin

The manuscript deals with the optimization of simple, rapid, sensitive and economic HPLC method for estimation of Theaflavin in Camellia sinensis extract and Theaflavin loaded nanoformulation. The preliminary screening was performed employing fractional factorial (25-1) design with mobile phase pH, flow rate, column temperature, injection volume, and acetonitrile concentration as independent variables while tailing factor as the dependent variable. The separation was achieved using the isocratic system as mobile phase, flow rate of 1 ml/min, and ultraviolet detection at 275 nm. Upon further evaluation, the method revealed a high degree of linearity, accuracy, precision, sensitivity and robustness, while, forced degradation studies indicated stability of Theaflavin at pH 3-6 in an air tight, light-protected container. The method when applied towards estimation of Theaflavin in C. sinensis extract and Theaflavin loaded nanoformulation, indicated no significant change in retention time. Thus, a HPLC method for rapid estimation of Theaflavin with an understanding of influencing variables for enhancing the method performance was developed.

SPECTROPHOTOMETRIC AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHODS FOR SIMULTANEOUS DETERMINATION OF ATORVASTATIN AND PIOGLITAZONE IN COMBINED TABLET DOSAGE FORM

Simple, accurate, precise, and sensitive ultraviolet spectrophotometric and reversed-phase high-performance liquid chromatographic (RP-HPLC) methods for simultaneous estimation of Atorvastatin (ATOR) and Pioglitazone (PIO) in combined tablet dosage form have been developed and validated. The spectroscopic methods employs formation and solving of simultaneous equation at 247 nm and 267 nm as 2 wavelengths for estimation of ATOR and PIO respectively (method 1) While method 2 involves formation of Q- absorbance equation at 233 nm (isoabsorptive point) and 267 nm (I» max of Pioglitazone) with methanol as solvent. Beer’s law is obeyed in the concentration range of 5.0–50.0 mcg/mL for ATOR and PIO, respectively. The RP-HPLC method uses HPLC system with a Phenomenex Luna C18 (5 mm x 25cm x 4.6mm i.d) using Methanol, acetonitrile and potassium dihydrogen phosphate buffer, pH 2.5 adjusted with orthophosphoric acid (60:20:20 v/v) at a flow rate of 1.0 mL/min at ambient temperature as the mobile phase. The detection was carried out using an ultraviolet detector set at 233 nm. For the HPLC method, Beer’s law is obeyed in the concentration range of 5.0-50.0 µg/ml for ATOR and PIO, respectively. LOD values for ATOR and PIO were found to be 57.12µg/ml and 12.01 µg /ml respectively. All the methods have been successfully applied for the analysis of the drugs in a pharmaceutical formulation. Results of analysis were validated statistically and by recovery studies.1-4

Quantitative Determination and Validation of Ivabradine-HCl in Pharmaceutical Formulation and Rabbit Plasma by High Performan ce Liquid Chromatography Method

Background: The objective of this work was to develop and validate a rapid and effective high performance chromatographic method for the analysis of Ivabradine-HCl. Keywords: Ivabradine-HCl, HPLC, validation, acetonitrile, plasma, mobile phase.

Pteridine determination in human serum with special emphasis on HPLC methods with fluorimetric detection

Abstract Conjugated and unconjugated pteridines and their derivatives are important cofactors in cellular metabolism. Hence, the amount of unconjugated pteridines in biological fluids has been found to be modified as a result of several disorders. It is necessary to note that while for the control of pteridines in urine samples there are numerous reference data, the literature referred to for the analysis of these analytes in serum/plasma is scarce. In biological fluids, pteridines can exist in different oxidation states, and these compounds can be classified into two groups according to: (a) oxidized or aromatic pteridines and (b) reduced pteridines. Oxidized pteridines yield a strong fluorescence signal, whereas reduced pteridines present a low quantum yield of fluorescence. In order to enable the analysis of the reduced forms, several preoxidation procedures to generate aromatic rings have been established. Also, stabilization of the reduced forms by the addition of reducing agents has been widely reported. The objective of this paper is to show possibilities and different approaches in the analysis of pteridines in serum samples. We have mainly focused on the description of the current situation in the application of high-performance chromatography methods with fluorimetric detection.

Optimization and validation of high-performance chromatographic condition for simultaneous determination of adapalene and benzoyl peroxide by response surface methodology.

The aim of this study was to develop a simple and reliable high-performance chromatographic (HPLC) method for simultaneous analysis of adapalene and benzoyl peroxide in pharmaceutical formulation by response surface methodology (RSM). An optimized mobile phase composed of acetonitrile, tetrahydrofuran and water containing 0.1% acetic acid at a ratio of 25:50:25 by volume was successfully predicted by using RSM. An isocratic separation was achieved by using the condition. Furthermore, the analytical method was validated in terms of specificity, linearity, accuracy and precision in a range of 80% to 120% of the expected concentration. Finally, the method was successfully applied to the analysis of a commercial product.

Development and Validation of Reverse Phase High Performance Chromatography Method for Determination of Olanzapine in Microsample Rat Plasma: Application to Preclinical Pharmacokinetic Study

Purpose: To develop a sensitive and validated reverse phase-high performance liquid chromatographic (RP-HPLC) method for quantification of olanzapine in micro-sample of rat plasma using UV detection.Methods: A single oral dose of olanzapine (7 mg/kg) was given to overnight fasted rats (n = 6). Rat plasma samples containing the drug were extracted by liquid-liquid extraction using a combination of dichloromethane: n-hexane (80:20). A reverse phase chromatographic column C18 hypersil-BDS was used for chromatographic separation with a mobile phase consisting of 50 mM phosphate buffer pH 5.5, acetonitrile and methanol (50:30:20, v/v/v) pumped at a flow rate of 1.2 ml/min. Olanzapine was measured using ultraviolet (UV) detection at 214 nm. The method was validated for precision and accuracy.Results: Separation of compounds of interest was not affected by endogenous interference. Good linearity within the concentration range of 1 - 500 ng/ml in rat plasma was obtained with coefficient of regression (r2) of 0.9986. Liquid-liquid extraction produced comparable recovery to solid phase extraction. Retention time of olanzapine and internal standard (fluoxetine) was 5.0 and 13.4 min, respectively. Lowest limit of quantification (LLOQ) was 1 ng/ml while inter-day and intra-day precision was &lt; 12.5 and 5.1 %, respectively. Accuracy of the method was between 94 and 105 % and the variation of results between two analysts was not significant (p = 0.626). Mean maximum plasma concentration (Cmax) of olanzapine was 412.7 ng/ml, time to attain maximum plasma concentration (tmax) was 1 h and half life (t½) was 2.54 h.Conclusion: The proposed method has been successfully validated for precision and accuracy that are within the limits of U.S. Food and Drug Administration (FDA)’s guidance for bioanalyitcal assay validation. The method was successfully applied to preclinical pharmacokinetic analysis of olanzapine in rats.Keywords: Olanzapine, Antipsychotic, Pharmacokinetics, Rat, Plasma, Bioanalytical assay Olanzapine, Antipsychotic, Pharmacokinetics, Rat, Plasma, Bioanalytical assay

Novel RP-HPLC Method for Simultaneous Estimation of Doxofylline and Sertraline in Bulk and Tablet Dosage Form

The objective of this study was to de evelop a simple, rapid, accurate and precise isocratic sta tability indicating reversed-phase high-performance liquidid chromatographic method for simultaneous estimation n of Doxofylline and Sertraline in tablet dosage form. Thhe separation method was carried out using Std XDB C1 18 column (150 x 4.6 mm; 5μ).The mobile phase used wa as a mixture of Acetonitrile and Potassium Di Hydrogen PPhosphate buffer in the ratio of 45: 55(v/v) delivered at an isocratic flow rate of 0.8mL/min. Column temperatu ture was 30°c and eluents were monitored at 231nm usin ng Waters 2695 HPLC instrument equipped with the w waters 2998 PDA detector and Empower-2 software for ddata collecting and processing. With the optimized meth thod, the retention times of Doxofylline and Sertraline we ere found to be 2.06 min and 4.14 min respectively, with h theoretical plate count and asymmetry as per the ICH lim mits. The method has shown a good linearity in the conce entration range of 100–600 μg/ml for Doxofylline and 122.5–75μg/ml for Sertraline with regression coefficient (R2) of 0.999 and 0.997.The percentage assays were foun nd to be 99.11 and 96.44 for Doxofylline and Sertraline. The method was found to be accurate (with percentage m mean recoveries 99.71 for Doxofylline, and 99.10 for Serrtraline), precise, robust, reliable, specific and stable. Th he proposed method was validated in accordance with ICHH guidelines and hence this method can be successfullyy used for quantitative analysis of Doxofylline and Se Sertraline in tablet formulations.