Reversed-phase Liquid Chromatographic Method Research Articles

Development of liquid chromatographic and UV-visible spectrophotometric methods for determination of pKa values of folic acid antimetabolites

In this study, the values of ionization/protonation constants (pKa) of pemetrexed and raltitrexed, which are folic acid antimetabolites, were determined by reversed-phase liquid chromatography (RPLC) and UV-visible spectrophotometric methods at 25 °C. The pKass values were evaluated by retention time (tR) in binary acetonitrile-water mixtures with acetonitrile (ACN) contents of 13%, 15%, 17% and 20% (v/v) for the RPLC method and 13%, 15% and 17% (v/v) for the spectrophotometric method. The aqueous pKa values (pKaww) of the studied compounds were calculated from the pKass value using the macroscopic parameters (mole fraction and dielectric constant), which play an important role in solvent properties. In addition, the degree of ionization of pemetrexed and raltitrexed was calculated using the pKaww values.

A Quick and Accurate High Performance Liquid Chromatography (HPLC) Method to Determine the Amount of Trimethylamine in Fish Oil Softgels and Multivitamin Softgels Containing Fish Oil

Ocean fish cells contain trimethylamine oxide for osmoregulation. The odorless trimethylamine oxide reduces to trimethylamine by bacterial activity, resulting in a strong fishy odor. Therefore, it is important to find out whether trimethylamine levels in fish oil and multivitamin softgels containing fish oil will affect the quality of the finished products. A derivatization-based, reversed-phase high performance liquid chromatography (HPLC) method was developed using a Lichosphere 100 RP18 column, utilizing acetonitrile and water as the mobile phases. Experimental products were dissolved in ether, followed by a pH jump and buffer exchange, and finally derivatized with 9-fluorenylmethyl chloroformate. Our research has indicated that the level of trimethylamine in the products containing fish oil does not have a correlation with age and rancidity level of the products.

Stability-Indicating New RP-UPLC Method for Simultaneous Determination of a Quaternary Mixture of Paracetamol, Pseudoephedrine, Chlorpheniramine, and Sodium Benzoate in (Cold-Flu) Syrup Dosage Form.

A combination of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form is specified for the treatment of common cold and flu symptoms. The functional role of this study is to develop a novel, reliable, and selective stability-indicating reversed-phase ultra-performance liquid chromatography (RP-UPLC) method for simultaneous identification of a quaternary mixture of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form. The specific method is accomplished using an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm), 1.8 µm particle size with pore size 100 Å, utilizing a mixture of purified water-methanol-trifluoroacetic acid (72.5:27.5:1.5, v/v) as the mobile phase at a flow rate of 0.3 mL/min. The column void volume is 1.15 min. UPLC detection is adjusted at 205 nm using a photodiode array detector. Calibration curves are obtained in the linearity ranges: 25-500 µg/mL for paracetamol, 10-50 µg/mL for pseudoephedrine, 0.5-5 µg/mL for chlorpheniramine, and 3-30 µg/mL for sodium benzoate with a correlation coefficient > 0.9992. The mean recovery of the developed method is tested and shows good recovery results between 99-101%; selectivity and forced degradation studies are investigated as per the International Council for Harmonisation Guidelines and no interference is detected due to degradation peaks. The proposed stability-indicating UPLC method for simultaneous determination of the three drugs, paracetamol, pseudoephedrine, and chlorpheniramine, with a preservative sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form is successfully accomplished, developed, and validated, and can be easily used in the analysis of drugs in pure or dosage form. The novelty of the current research work lies in the development of the UPLC method for simultaneous determination of a quaternary mixture of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form.

HPLC determination of Escitalopram in tablet dosage forms

Purpose: A simple, specific, precise, and accurate reversed phase liquid chromatographic (RP-LC) method has been developed for the determination of Escitalopram in tablet dosage form. Methods: The chromatographic separation was achieved on a LiChrosorb C18, 250 mm x 4.6 mm, 5 μm column at a detector wavelength of 270 nm and a flow rate of 1.0 ml/min. The mobile phase was composed of methanol, acetonitrile (70:30 v/v). The retention time of Escitalopram was 5.49 min. The method was validated for the parameters like specificity, linearity, precision, accuracy, limit of quantitation and limit of detection. Results: The method was found to be specific as no other peaks of impurities and excipients were observed. The square of correlation coefficient (R2) was 0.9999 while relative standard deviations were found to be <2.0%. Conclusion: The proposed RP-LC method can be applied for the routine analysis of commercially available formulations of Escitalopram.

Analytical method validation of alfuzosin hydrochloride and its organic impurities by using ultra performance liquid chromatography

A low cost ultra-high performance reverse phase liquid chromatography (RPUPLC) method has been proven for the investigation of alfuzosin hydrochloride in the existence of organic pollutants and decomposition products due to forced decomposition products. Attempts have been made to develop several methods for separating drugs from organic impurities. In addition, the best chromatographic separation is Waters Acquity HSS T3 C18, 100 mm × 2.1 mm, UV detection at particle size 1.8, 254 nm, and Perchloric acid (pH 3.5 with sodium hydroxide) with a mixture of organic solvents. This method was endorsed for specificity / selectivity, linearity / range, reproducibility, recovery, and reliability. They can be utilized for quality control during manufacturing and for assessing the stability of commercially available alfuzosin hydrochloride samples. With a gradient time of about 6.0 min and an equilibration time of about 2.0 min, it was possible to analyze more than 100 samples per day. In addition, the pH susceptibility analysis methods referred to in the United States Pharmacopeia and the European Pharmacopoeia were discussed. The Method was successfully validated as per ICH Q2R1 guideline.

Concentration, purification and quantification of milk protein residues following cleaning processes using a combination of SPE and RP-HPLC

Detection and quantification of milk protein residues can be of utmost importance for validation of cleaning process efficiency in removing even traces of residues as well as quality assurance and product safety. However, currently available assays cannot provide a combination of high sensitivity and a simultaneous quantification of the individual milk proteins. Furthermore, a low protein-to-protein-variability and high compatibility with other reagents such as residual cleaning agents (e.g. surfactants) cannot be ensured. Therefore, a new method was developed comprised of a pre-concentration of proteins by solid-phase extraction and optimisation of the sensitivity of an existing reversed-phase high performance liquid chromatography method for the separate quantification of bovine milk proteins κ-Casein, αS2-Casein, αS1-Casein, β-Casein, α-Lactalbumin, and β-Lactoglobulin. Hereby, solid-phase extraction enables robust and reproducible purification and concentration of protein residues with a high protein recovery rate and flexible adjustment of concentration factors. The increased sensitivity of the reversed-phase high performance liquid chromatography method was achieved by changes in the measurement wavelength and guanidine buffer concentration. This new method enables reproducible concentration, purification and quantification of protein concentrations below 7 ng mL−1 and thus can be used to detect milk protein residues in highly diluted aqueous systems.•Concentration, purification and quantification of milk protein residues with a high recovery rate of proteins (> 94%) and high reproducibility (coefficient of variation (CV) < 3.0%)•Flexible adjustment of sample volumes allows the utilisation of high concentration factors (≤ 500) without compromising the recovery rate of proteins (recovery rate of proteins decreases by 2.74% per 100 CF)

Development of a quick reverse phase liquid chromatographic method with photodiode-array detection for quantitative determination of chlorthalidone, metoprolol succinate and telmisartan in tablet formulation

Development of a quick reverse phase liquid chromatographic method with photodiode-array detection for quantitative determination of chlorthalidone, metoprolol succinate and telmisartan in tablet formulation

Reversed-Phase Liquid Chromatographic Internal Standard Method Using Losartan Potassium for Quantitative Estimation of Ivabradine Hydrochloride in Pharmaceutical Tablet Dosage Form and Plasma

A reversed-phase liquid chromatographic method for ivabradine hydrochloride using Diode Array Detector (DAD) and internal standard technique was developed and validated according to ICH and SWGTOX guidelines. The prime objective of this study was to develop a precise and accurate method that can be equally applicable to biological (plasma) as well as non-biological (active pharmaceutical ingredient and pharmaceutical tablets) matrices. Losartan potassium was used as an internal standard due to its easy availability. After liquid-liquid extraction using acetonitrile, the ivabradine hydrochloride and internal standard were chromatographed on Agilent 1200 series HPLC system equipped with DAD detector, auto-sampler and chemstation software. Analytical separation was achieved on Agilent C-18 (5µm, 25cm x 4.6mm) reversed-phase column at 30°C column oven temperature, 10μL injection volume and 286nm wavelength. Isocratic mobile phase system comprised of 60:40 v/v ratio of HPLC grade methanol and water adjusted to pH 6.8 using orthophosphoric acid was employed with 1mL/min flow rate. The method linear range was 0.025-3µg/mL (25-3000ng/mL) for pharmaceutical tablets and plasma with the coefficient of linearity ranged 0.997-0.999. Results for precision, accuracy, recovery, stability and matrix effect studies were within acceptable limits for both plasma and tablets. Method was successfully applied to the commercial tablet products and patient plasma samples to estimate the amount of ivabradine hydrochloride.

English

Tetracyclines are commonly used in livestock for the treatment of various diseases. Their residues above the maximum residual limit-MRL (200 μg kg-1) can cause health hazard issues not only in humans but also in animals. For this purpose, efforts were made to study distribution and depletion profile of Oxytetracycline in broiler chicken by ELISA and HPLC. Regarding this, an experiment was conducted on healthy chickens of average weight 600 ± 20 g. Six broiler chickens were treated with formulation (OXTRA L.A.) containing Oxytetracycline dehydrate @20 ppk solution (150 μL) equivalents to recommended dose 20 mg kg-1 through intramuscular route, while control birds were untreated. Treated chickens were slaughtered at an interval of 1, 8, 16, 32, 64 and 120 h. Different tissue samples including liver, kidney, thigh and chest muscles were collected and screened by ELISA (Cat No. 5091TC, Euro Proxima). Lowest detection limit (IC20) was calculated as 0.12 ng mL-1 and middle of the test (IC50) 0.5 ng mL-1 with recovery 75 to 86%. Reverse phase liquid chromatography (HPLC-UV) method was also standardized by using range of standards from 25 to 200 μg kg-1. Calibration curve showed good response with correlation coefficient (R2) 0.9727 and recovery 85 to 93%. Overall, withdrawal period (&lt; MRL) in all tissue samples was calculated as 117 h (4.8 days) by ELISA while 128 h (5 days) by HPLC-UV. Both validated methods can be further utilized to generate reliable data for food safety measures in Pakistan to enhance international trade. © 2021 Friends Science Publishers

Highly sensitive quantification of pemetrexed in human plasma using UPLC-MS/MS to support microdosing studies.

Pemetrexed is an antifolate drug approved for the treatment of non‐small‐cell lung cancer and mesothelioma. Assessing pemetrexed pharmacokinetics after administration of a microdose (100 μg) may facilitate drug–drug interaction and dose individualization studies with cytotoxic drugs, without causing harm to patients. Therefore, a highly sensitive bioanalytical assay is required.A reversed‐phase ultra‐high performance liquid chromatography method was developed to determine pemetrexed concentrations in human ethylenediaminetetraacetic acid–plasma after microdosing. [13C5]‐Pemetrexed was used as the internal standard. The sample preparation involved solid‐phase extraction from plasma. Detection was performed using MS/MS in a total run time of 9.5 min.The assay was validated over the concentration range of 0.0250–25.0 μg/L pemetrexed. The average accuracies for the assay in plasma were 96.5 and 96.5%, and the within‐day and between‐day precision in coefficients of variations was <8.8%. Extraction recovery was 59 ± 1 and 55 ± 5% for pemetrexed and its internal standard. Processed plasma samples were stable for 2 days in a cooled autosampler at 10°C.The assay was successfully applied in a pharmacokinetic curve, which was obtained as a part of an ongoing clinical microdosing study.

Analysis of pharmaceutical drug oligomers by selective comprehensive two-dimensional liquid chromatography-high resolution mass spectrometry

The presence of oligomeric impurities in drugs is often overlooked and less studied due to conventional small molecule analytical methods which are often not capable of capturing these oligomers. In the present work, the oligomer species of a lipophilic active pharmaceutical ingredient (API) containing an azetidine ring was investigated. No separation was observed by reversed-phase liquid chromatography (RPLC) methods, and only a partial separation of oligomer peaks was achieved by a size exclusion chromatography (SEC) method. To improve the resolution of the different oligomers species and understand the root cause of the formation of the oligomers, a selective comprehensive two-dimensional liquid chromatography (2D-LC) method was developed by coupling SEC to RPLC. The selective comprehensive SEC × RPLC method allowed the separation of 16 species and evidenced four main groups of oligomer impurities. The contour plots of 3 API lots helped to visualize the oligomer profiles and quickly compare the difference between these lots. Finally, the oligomer peaks separated by 2D-LC were identified by high-resolution mass spectrometry (HRMS) using a Q Exactive mass spectrometer. The developed 2D-LC/HRMS workflow provides a fast and generic screening approach to quickly examine and visualize the oligomeric impurities in API materials, and direct the impurity control strategy during process development.

Separation of chiral and achiral impurities in paroxetine hydrochloride in a single run using supercritical fluid chromatography with a polysaccharide stationary phase

Separating paroxetine hydrochloride and its impurities using conventional reversed-phase liquid chromatography (RPLC) is challenging due to their highly similar structures. In the present study, a rapid, simple, sensitive and environmentally friendly method was developed for the determination of chiral and achiral impurities in raw materials of paroxetine hydrochloride using chiral supercritical fluid chromatography (SFC). The impacts of chiral stationary phases (CSPs), mobile phases, column temperature and back pressure on the retention and separation of analytes were comprehensively evaluated. After method optimization, a satisfying result was obtained on a cellulose tris-(3-chloro-4-methylphenylcarbamate) stationary phase in 4.0 min using 70% CO2 and 20 mM ammonium acetate in 30% methanol as the mobile phase. Molecular docking was further performed to understand the interactions between the analytes and CSP. The results suggested that hydrogen bonding and π–π interactions were the dominant interactions. The affinity given by the software was in good agreement with the elution order and free energy (△G) values obtained from van’t Hoff equations. The results of molecular docking also provide insights into the different retentions of N-methylparoxetine at different temperatures. The results of method validation revealed that the method was sensitive with a limit of detection of approximately 0.05 μg·mL−1 (corresponding to approximately 0.005% paroxetine hydrochloride in the sample solution). The relative standard deviations (RSDs) of precision and intra-assay precision were all less than 2.0%, and the recoveries of the method were 93.8~105.3% with RSDs less than 3.0%. The chiral and achiral RPLC methods included in the Chinese pharmacopoeia and the SFC method proposed in this study were simultaneously used to determine the impurity content in the raw materials of paroxetine hydrochloride. The results showed that impurities that cannot be detected by the reference method can be accurately quantified using the SFC method. In addition, the SFC method has advantages in terms of throughput, analysis cost and simplicity. This study can provide a reference for further research of impurities in paroxetine hydrochloride and promote the application of chiral SFC in the rapid separation of structurally similar compounds.

Validation of a Rapid and Sensitive Reversed-Phase Liquid Chromatography Method and Force Degradation Study of Synthesized (E)-3-(2,4,6-tri-Methoxyphenyl)-1-phenylpro-2 ene-1-one

A stability-indicating RP-HPLC method was developed and validated for the determination of the estimation of newly synthesized (E)-3-(2,4,6-tri-methoxyphenyl)-1-phenylpro-2 ene-1-one in API form by using C18 column Phenomenix (250 mm x4.6 mm,5µm), with a mobile phase consisting of a acetonitrile and methanol (40:60 v/v) with flow rate of 0.7 mL/min. The detection was carried out at 370 nm and retention time (Rt) of (E)-3-(2, 4, 6- tri-methoxyphenyl)-1-phenylpro-2 ene-1-one was found to be 2.55 min. Linearity was observed from the concentration range of 10-50µg/mL (n=5), (coefficient of determination R2 was 0.997) with the equation of y= 0.495x + 0.111. The method was repeatable with a relative standard deviation (RSD) of 0.16 to 1.98 %. The newly developed method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. (E)-3-(2,4,6-tri-methoxyphenyl)-1-phenylpro-2 ene-1-one was subjected to different stress conditions as per ICH guidelines like acidic alkaline, oxidative, thermal and the results showed that it was more sensitive towards oxidative.

STABILITY INDICATING RP-HPLC METHOD FOR SEPARATION AND QUANTIFICATION OF RELATED SUBSTANCES OF TICLOPIDINE IN BULK AND PHARMACEUTICAL FORMULATIONS

A simple, specific, accurate and stable reverse phase liquid chromatographic method was developed for the simultaneous determination of ticlopidine and its related impurities A and B in bulk drug and tablet dosage forms. The analysis has been performed on XTerra C18 column (250 mm×4.6 mm; 5 µ id) and mobile phase containing of methanol and pH 6.8 phosphate buffer in the ratio of 80:20 (V/V). The detection was carried at 228 nm with a flow rate of 1.0 mL/min. The retention times were found to be 8.9, 5.98 and 4.62 min for ticlopidine, impurities A and B, respectively. The method was validated according to ICH guidelines. The method was validated for specificity, precision, linearity, accuracy and robustness. The linearity range of 50-200 µg/mL for ticlopidine and 0.5-2.0 µg/mL for impurity A and B. The recoveries of ticlopidine and impurities were found to be within the range of 98-102 and the % RSD in each spiked level was found to be less than 2. The stress degradation studies confirmed that the method was effectively separate the degradation products and impurities formed in the stress studies and hence the method was found to be stability indicating method. The method can effectively quantify the standard drug ticlopidine and its impurities A and B in bulk drug and pharmaceutical formulations.

Bioanalytical Method Development and Validation for the Determination of Favipiravir in Spiked Human Plasma by using RP-HPLC

A precise, simple and reproducible reverse phase liquid chromatography (RP-HPLC) method was developed and validated for determination of Favipiravir by using Carbamazepine as internal standard in spiked human plasma. A chromatographic separation was accomplished with Cromasil C18 (250mm x 4.6ID, Particle size: 5 micron) column using mobile phase consists of methanol: water in the ratio (35:65, %v/v), at pH 3.0 with binary gradient system-maintained flow rate at 0.8ml/min. The detection wavelength of drug sample was at 225 nm. Extraction was done by using ethyl acetate as extracting solvent. The retention time of Favipiravir was found to be 6.62 min. The method was found to be linear in the concentration range of 0.2-3.2 µg/ml. Limit of quantitation (LOQ) value was found to be 0.72. The intra- and inter day precision and accuracy lies within the specified range. The recovery studies were found to be in the range of 97.6 to 100.2%. %Relative standard deviation (RSD) was found to be in the range of 0.07-2.80%. All parameters were found to be validated from spiked human plasma. The proposed RP-HPLC method is highly accurate and rapid for the determination of favipiravir in human plasma and can be applied for pharmacokinetic studies and Therapeutic drug monitoring.

DoE-Based Analytical Quality Risk Management for Enhanced AQbD Approach to Economical and Eco-Friendly RP-HPLC Method for Synchronous Estimation of Multiple FDC Products of Antihypertensive Drugs.

According to the literature review, numerous chromatographic methods have been published for estimation of fixed-dose combination products of telmisartan but no reverse-phase high-pressure liquid chromatographic (RP-HPLC) method has been published yet for synchronous estimation of fixed-dose combination (FDC) products of telmisartan to save time, cost and solvent for analysis. Hence, an economical and eco-friendly RP-HPLC method has been developed for synchronous estimation of multiple FDC products of antihypertensive drugs using the quality risk management (QRM) and DoE-based enhanced analytical quality by design approach. The analytical-QRM was started with the identification of potential method risk parameters followed by their risk assessment by risk priority number ranking and filtering. The identified critical method parameters were optimized using the DoE-based central composite design. The method operable design range was navigated and the control strategy was framed for control and mitigation of risk throughout the life-cycle of the developed method. The method was developed using Shimpack Octadecyl silane (ODS) C18 column and acetonitrile-1.0%v/v triethylamine in water (pH6.0; 45 + 55, %v/v). The developed method was validated as per the International Council for Harmonization Q2 (R1) guideline. The developed method was applied for the analysis of seven different antihypertensive dosage forms. The developed RP-HPLC method can be used as an eco-friendly, robust and economical alternative analytical tool to several published methods for estimation of FDC products of antihypertensive drugs in the pharmaceutical industry.

Sweeping-micellar electrokinetic chromatography versus reversed phase liquid chromatography for the determination of coumarin in curry

Coumarin is a phytotoxin found in the popular spice cinnamon, which is used to flavor many Asian curry dishes. In this work, we developed and compared the analytical performance of reversed-phase liquid chromatography (RP-LC) and sweeping-micellar electrokinetic chromatography (MEKC) methods for the determination of coumarin in complex curry (gravy) samples. Using a matrix matched sample (curry after solvent extraction with methanol and diluted with 100 mM phosphoric acid), the intra-day and inter-day repeatability of retention/migration time and (corrected) peak area for both methods were acceptable (%RSD (n=6) ≤ 5%). The linear range and limit of quantitation (LOQ) were an order of magnitude better in RP-LC (RP-LC linear range = 0.11-108 mg/kg, LOQ = 0.11 mg/kg) (Sweeping-MEKC linear range = 2.16-216 mg/kg, LOQ = 2.16 mg/kg). However, the limit of detection (S/N=3) and LOQ in sweeping-MEKC was 0.65 mg/kg and 2.16 mg/kg, which were sufficient to report the levels of coumarin ≥ the European limit of 2 mg/kg in foods. During the analysis of 25 curry samples, relatively similar results for sweeping-MEKC and RP-LC were obtained for 6 samples that contained coumarin >LOQ of sweeping-MEKC. Interferences in RP-LC lead to significant overestimation of coumarin levels in 3 samples. Coumarin levels above the EU limit was found in 6 curry samples using the more selective sweeping-MEKC. This work should also raise public awareness on the presence of potentially high levels of coumarin in some foods.

Validated RP-HPLC Method for the Estimation of Amiloride and Hydrochlorothiazide in Combined Tablet Dosage Form

The aim of this work is to build up a rapid, exact, precise and accurate reverse phase liquid chromatographic method for the simultaneous analysis of amiloride and hydrochlorothiazide in tablet dose structure. The chromatographic strategy was normalized utilizing Hypersil ODS coulmn (250×4.6mm, 5μm molecule size) with UV detection at 210nm and flow rate of 1ml/min. The mobile phase includes phosphate buffer (pH acclimated to 2.5 with dilute Ortho Phosphoric acid) and acetonitrile in the proportion of 60:40 v/v. The linearity of proposed technique was found in the range of 5-30μg/ml (R²=0.999) for amiloride and 50-300μg/ml (R²=0.999) for Hydrochlorothiazide appropriately. The limit of detection (LOD) was discovered to be 0.10μg/ml and 0.40μg/ml for Amiloride and Hydrochlorothiazide appropriately. The limit of quantitation (LOQ) was discovered to be 0.30μg/ml and 1.20μg/ml for Amiloride and Hydrochlorothiazide separately. The retention times of Amiloride and Hydrochlorothiazide were found to be 3.258min and 2.383min separately. The technique was truly recommended and %RSD was found to be under 2 demonstrating high degree of exactness and accuracy. Subsequently proposed strategy can be effectively evaluated for the simultaneous estimation of Amiloride and Hydrochlorothiazide in promoted formulations.

Analytical Method Development and Validation for Simultaneous Determination of Simvastatin and Mupirocin Using Reverse-Phase High-pressure Liquid Chromatographic Method

This study was aimed to develop and validate the use of reverse-phase high pressure liquid chromatographic method for the estimation of simvastatin (SIM) and mupirocin (MUP) simultaneously. The chromatographic method developed is optimized for flow rate with the column, solvent, and buffer used and mobile phase ratio, molarity, and pH. The validation of the optimized method and the forced degradation studies of both drugs (under acidic, alkaline, oxidation, heat, light, and neutral conditions) were conducted following the The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Kromasil C18 column (250 mm × 4.6 mm, 5 µm) with ultraviolet detection at 224 nm and acetonitrile/phosphate buffer (30 mM, 70:30 v/v, pH 3.5; adjustment done using orthophosphoric acid) as mobile phase at a flow rate of 1.1 mL/min were observed to provide a good resolution for MUP and SIM at retention times of 2.32±0.008 and 13.55±0.254 min, respectively, with high accuracy (percent recovery was 99.69±0.82 for MUP and 101.10±0.02 for SIM) and linearity in the range of 5-30 µg/mL (r2: 0.9969 for MUP and r2: 0.9959 for SIM). The diagnostic limit and the lower limit of determination were 0.771±0.234 and 2.338±0.246 μg/mL for MUP and 0.595±0.282 and 1.803±0.334 µg/mL for SIM, respectively. The validated method was used to understand the degradation behavior of both drugs after the forced degradation studies. The analytical method developed is determined to be specific, sensitive, precise, and accurate for the estimation of MUP and SIM simultaneously in the combined dosage form.

Validated RP-HPLC method for the estimation of Amiloride and hydrochlorothiazide in combined tablet dosage form

The aim of this work is to build up a fast, exact, precise and touchy reverse phase liquid chromatographic method for the synchronous assessment of amiloride and hydrochlorothiazide in tablet dose structure. The chromatographic strategy was normalized utilizing Hypersil ODS segment (250×4.6mm, 5μm molecule size) with UV discovery at 210nm and stream pace of 1ml/min. The portable stage includes phosphate buffer (pH acclimated to 2.5 with dilute Ortho Phosphoric acid) and acetonitrile in the proportion of 60:40 v/v. The linearity of proposed technique was examined in the scope of 5-30μg/ml (R²=0.999) for amiloride and 50-300μg/ml (R²=0.999) for Hydrochlorothiazide appropriately. The limit of detection (LOD) was discovered to be 0.10μg/ml and 0.40μg/ml for Amiloride and Hydrochlorothiazide appropriately. The limit of quantitation (LOQ) was discovered to be 0.30μg/ml and 1.20μg/ml for Amiloride and Hydrochlorothiazide separately. The retention times of Amiloride and Hydrochlorothiazide were found to be 3.258min and 2.383min separately. The technique was truly recommended and %RSD was found to be under 2 demonstrating serious level of exactness and accuracy. Subsequently proposed strategy can be effectively evaluated for the synchronous assessment of Amiloride and Hydrochlorothiazide in promoted formulations.