Rapid Reversed-phase High-performance Liquid Chromatography Research Articles

Rapid Reversed-Phase High-Performance Liquid Chromatography Profiling of Serotonin Receptor Ligands and their Related Compounds

Rapid Reversed-Phase High-Performance Liquid Chromatography Profiling of Serotonin Receptor Ligands and their Related Compounds

Development and validation of a green RP-HPLC method for detection and quantitation of meropenem trihydrate in the bulk: A comparison with HPTLC method

This research introduces an eco-friendly green Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) method for detecting and quantifying meropenem trihydrate. Traditional methods use buffered solutions, gradient mobile phases, and longer retention times, but this method offers a rapid RP-HPLC technique with a C8 column and isocratic mobile phase (60% methanol, 40% ultra-pure water), eliminating the need for buffers and acetonitrile. It features a short 2.1-minute retention time and a high r² value of 0.9995. It delivers accuracy (101.1-102.3%) and precision (RSD ≤ 2%), coupled with low LOD and LOQ values of 1.72 and 5.20 µg/ml. Aqueous dilution simplifies sample preparation, reducing degradation and interference. The method is compared with an HPTLC method, showing an extended linear range (6.25-200 µg/ml for HPLC, 7.81-62.5 µg/ml for TLC) and high sensitivity, making it significant for meropenem trihydrate quality control in bulk and dosage forms.

A stability indicating method development and validation of a rapid and sensitive RP-HPLC method for Nintedanib and its application in quantification of nanostructured lipid carriers

Background: Nintedanib (NTB) is a multiple tyrosine kinase inhibitor, been investigated for many disease conditions like idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD) and non-small cell lung cancer (NSCLC). NTB is available as oral capsule formulation, but its ability to detect degradants formed through oxidative, photolytic and hydrolytic processes makes it difficult to quantify. In the current work, a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated. Methods: The developed method is simple, precise, reproducible, stable and accurate. The inherent stability of NTB was evaluated using the proposed analytical method approach and force degradation studies were carried out. NTB was separated chromatographically on the Shimadzu C18 column as stationary phase (250 ×4.6 mm, 5 µm) using an isocratic elution method with 0.1% v/v triethyl amine (TEA) in HPLC grade water and acetonitrile (ACN) in the ratio 35:65% v/v. The mobile phase was pumped at a constant flow rate of 1.0 ml/min, and the eluent was detected at 390 nm wavelength. Results: NTB was eluted at 6.77±0.00 min of retention time (tR) with a correlation coefficient of 0.999, the developed method was linear in the concentration range of 0.5 µg/ml to 4.5 µg/ml. The recovery rate was found to be in the range of 99.391±0.468% for 1.5 µg/ml concentration. Six replicate standards were determined to have an % RSD of 0.04. Conclusion: The formulation excipients didn't interfere with the determination of NTB, demonstrating the specificity of the developed method. The proposed approach of the analytical method developed can be used to quantify the amount of NTB present in bulk drugs and pharmaceutical formulations.

A Validated RP-HPLC Method Development and Validation for The Simultaneous Estimation of Nebivolol and Valsartan in Bulk Drug and Pharmaceutical Dosage Form

A novel, simple, accurate and rapid Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed and validated for the determination of Nebivolol and Valsartan in bulk and tablet dosage form. Gradient elution chromatography has been developed on C18 column (5µm, 4.6mm* 250mm) with a mobile phase consist of pH 4 acetonitrile,water and Methanol (25:35:40) with a flow rate of 1ml/min at detection of 270 nm wavelength. The retention time for Nebivolol and Valsartan was found to be 4.59 and 10.2 respectively. Chromatography parameters were validated as per ICH guidelines and the method can be applicable for routine quantitative analysis of drugs in combined dosage form.

Developed rapid and simple RP-HPLC method for simultaneous separation and quantification of bovine milk protein fractions and their genetic variants

The aim was to develop a reliable rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine the main bovine milk protein fractions, including their genetic variants. Compared to the previous studies, our method is able to separate the main protein fractions within 20 min of total run time. The method validation consisted of testing repeatability, reproducibility linearity, repeatability, and accuracy. The procedure was developed using raw individual, bulk, and commercially available heat-treated cow milk samples. The RSD of peak areas ranged from 1.43 to 3.16% within analytical day and from 3.29 to 6.70% across analytical days. The method can be applied to investigate both raw and heat-treated milk samples.

Development and Analytical Method Validation for Simultaneous Estimation of Evoglptin Tartrate and Metformin Hydrochloride in Combine Dosage Form

Metformin hydrochloride is a diabetes medication that is used orally. Evogliptin tartrate is a dipeptidyl peptidase-4 (DPP-4) inhibitor which has been authorized as a new and powerful treatment in the process of reducing glucagon-like peptide 1 (GLP-1) deficiency while increasing insulin production and decreasing glucagon levels. The current study created a simple, highly exact, and rapid reverse phase high-performance liquid chromatography (RP-HPLC) approach for the simultaneous measurement of metformin hydrochloride and evogliptin tartrate in their combined dose form. High-performance liquid chromatography (HPLC), was performed on a phenomenex Luna C18 column with a mobile phase of water 25: acetonitrile 75 (25:75) at a 1.2 mL/min flow rate, with detection at 210 and 230 nm for metformin hydrochloride.and evogliptin tartrate. The validation method comprised of several variables like linearity, accuracy, selectivity, precision, robustness and specificity. The retention time of the metformin hydrochloride and evogliptin tartrate was 2.009 and 2.956 minutes, respectively. The method procedure linear responses of metformin hydrochloride and evogliptin tartrate was found to be 1 and 0.997, respectively. The percent relative standard deviation was less than 2% for intraday variation/precision, interday variation/precision, intermediate ruggedness, and robustness. The projected technique produced excellent results for many dependent variables. The projected method showed outstanding results with respect to different dependent and independent variables like limit of detection and limit of quantitation, suitability of system, linearity range etc., linearity, accuracy, precision, and specificity. The results were within the acceptance criteria given in ICH guideline.

A simple and rapid RP-HPLC method for the assessment of cobalamin (vitamin B12) intilapia and snoek fishes

This study developed a rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method equipped with a UV-Vis detector. The aquaponics system?s tilapia and marine snoek fishes were extracted using an autoclaving process and enzymatic treatment. The technique enabled the separation and quantification of cobalamin present in these fishes extracts. Phenomenex Luna® 5 μm C18 (2) 100 A LC-column (150 × 4.6 mm) was used as a stationary phase, while the mobile phase consisted of methanol and phosphoric acid in a ratio of 35:65 (v/v), respectively. The developed method was revealed to be rapid (a retention time of less than 5.0 min), linear (R2 = 0.9988), sensitive (limits of detection and quantification showed to be 0.0004 and 0.0011 μg/mL, respectively), precise (percentage relative standard deviation of 0.14% to 0.76%), and accurate (percentage mean recovery of 87.44 ± 0.33% to 97.08 ± 0.74%). The quantified concentrations of this vitamin in extracts of tilapia and snoek fishes were found to be 08.95 ± 0.35 and 14.97 ± 0.04 μg/ mL, respectively. Therefore, we suggested that the developed RP-HPLC technique could be applicable for quality control evaluation in the food and pharmaceutical industries. Besides, the method could be vital for diagnostic analysis in clinical laboratories.

RP-HPLC method development and validation for simultaneous estimation of efonidipine hydrochloride ethanolate and telmisartan in their synthetic mixture

A Novel, selective, accurate and rapid Reversed Phase High Performance Liquid Chromatographic (RPHPLC) method for the analysis of Efonidipine Hydrochloride Ethanolate and Telmisartan in binary mixture has been developed and validated. The chromatographic system consisted of a Phenomenex Kinetex ® 5µ C18 Size: 150 * 4.6mm column and the separation was achieved by using ambient temperature with a mobile phase containing mobile Phase Acetonitrile:25mM Phosphate Buffer pH 4.9 (45:55). The samples were monitored at 253 nm for detection at a flow rate of 1.0 mL/min and the retention time was about 7.77 and 4.10 mins for Efonidipine Hydrochloride Ehanolate and Telmisartan respectively. The calibration curve was linear over the concentration range 5-30 and 10-60 ?g/mL for Efonidipine Hydrochloride Ehanolate and Telmisartan respectively. The proposed method is accurate in the range of 99.75% - 100.10% recovery and precise (%RSD of intraday variation and % RSD of inter day variation were found to be within the acceptance criteria). Therefore, this method can be used as a more convenient and efficient option for the analysis of Efonidipine Hydrochloride Ehanolate and Telmisartan in Quality control laboratory.

A rapid RP-HPLC stability-indicating method development and validation of moxifloxacin hydrochloride-related substances in finished dosage forms.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the identification and quantification of moxifloxacin hydrochloride-related substances in finished dosage forms. Chromatographic separation was achieved on an Agilent C18 column (150 × 4.6 mm, 5 μm) with the mobile phase (0.01 M potassium dihydrogen orthophosphate as buffer and methanol in the ratio of 70:30) eluted in isocratic mode. The HPLC flow rate was 1.0 mL min-1 , and peaks were monitored at 230 nm using a photodiode array (PDA) detector. The column temperature was kept constant at 30°C, and the injection volume was 10 μL. The run time of the method was 16 min. The method was validated as per the International Conference on Harmonization (ICH) guidelines. Linearity was recorded at various concentrations ranging from 0.25 to 1.5 μg mL-1 for all the moxifloxacin impurities. Linearity, regression value, recovery, %relative standard deviation (RSD) of method precision values were found within the acceptance limits. The method for related substances (RS) in moxifloxacin was found to be specific, linear, accurate, precise, rugged, and robust. The validated method was suitable for the quantification of the RSs in moxifloxacin drug products. The method was applied in quality control lab for the analysis of moxifloxacin impurities in stability analysis.

Rapid Reverse-phase High-performance Liquid Chromatography Estimation of Methotrexate in Bulk, Pharmaceutical Preparation and in Spiked Plasma Samples

Rapid Reverse-phase High-performance Liquid Chromatography Estimation of Methotrexate in Bulk, Pharmaceutical Preparation and in Spiked Plasma Samples

A simple and rapid RP-HPLC method for the assessment of cobalamin (vitamin B12) in tilapia and snoek fishes.

This study developed a rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method equipped with a UV-Vis detector. The aquaponics system›s tilapia and marine snoek fishes were extracted using an autoclaving process and enzymatic treatment. The technique enabled the separation and quantification of cobalamin present in these fishes extracts. Phenomenex Luna® 5 μm C18 (2) 100 A LC-column (150 × 4.6 mm) was used as a stationary phase, while the mobile phase consisted of methanol and phosphoric acid in a ratio of 35:65 (v/v), respectively. The developed method was revealed to be rapid (a retention time of less than 5.0 min), linear (R2 = 0.9988), sensitive (limits of detection and quantification showed to be 0.0004 and 0.0011 μg/mL, respectively), precise (percentage relative standard deviation of 0.14% to 0.76%), and accurate (percentage mean recovery of 87.44 ± 0.33% to 97.08 ± 0.74%). The quantified concentrations of this vitamin in extracts of tilapia and snoek fishes were found to be 08.95 ± 0.35 and 14.97 ± 0.04 μg/mL, respectively. Therefore, we suggested that the developed RP-HPLC technique could be applicable for quality control evaluation in the food and pharmaceutical industries. Besides, the method could be vital for diagnostic analysis in clinical laboratories.

Development and validation of simplified RP-HPLC method for quantification of Darunavir in commercial tablets

Darunavir (DRV) is a peptidic protease inhibitor class of antiretroviral drug which is regularly included in the antiretroviral therapy with several other drugs to treat Human immunodeficiency virus (HIV) patients to avoid those developing Acquired Immune Deficiency Syndrome (AIDS). In this research paper, an accurate, precise, reproducible, simple and rapid Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for quantifying DRV in the tablet dosage form (Danavir 600). The liquid chromatography was carried out on a Phenomenex C18 analytical column with dimensions of 250 × 4.6 mm, 5 µm using a mixture of acetonitrile (ACN) and water in the ratio of 30:70 v/v as mobile phase. The sample was injected in a volume of 20 µl and determined at 268 nm with a flow rate of 1 ml/min (ACN:water). The retention time of standard and sample drug was found to be 3.16 and 3.12 min respectively. The linearity in the calibration curve was achieved for DRV in the concentration range of 40–200 µg/ml and the regression coefficient (R2) was found to be 0.982. The percentage recovery of DRV was obtained in the range of 98.10–103.65% indicated the great accuracy of the current method. Reproducibility and recovery studies with % relative standard deviation (%RSD) with intra and inter-day precision was found to be less than 2%, which proven a developed method was reproducible. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.31 and 0.96 µg/ml respectively. The method was validated as per the International Conference on Harmonization (ICH) guidelines and can be strongly adopted for routine analysis of DRV since it is the most reliable and rapid approach. Even though a lot of work has been performed for estimating DRV in dosage forms, this study was found to be under green chemistry with the rapid and economical condition. Thus, the statistical validation of the data showed that the proposed method can be applied for the estimation of DRV in commercial tablets.

Bioanalytical Method Development and Validation for Determination of Sulfasalazine in Rabbit Plasma by HPLC-UV

In present study, an advanced, simple and a rapid reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of sulfasalazine in rabbit plasma. Sulfasalazine was separated using Chromatopak C-18 basic peerless (250 mm × 4.6 mm, 5μ) column in an isocratic mode using mobile phase consisting of the mixture of 10mM Ammonium acetate pH adjusted to 4.5 and acetonitrile (70:30 v/v) with a flow rate of about 1.0 mL/min at ambient temperature. An ultra-violet detection of sulfasalazine and the internal standard was carried out at 362 nm. Both sulfasalazine and internal standard (IS, 4-hydroxy benzoate) were extracted from plasma matrices with high efficiency using a simple protein precipitation method. The method was found to be highly selective with no carryover effects. Linearity of sulfasalazine was found with the range of 2.5-100 μg/mL with the value of r2 > 0.995 a correlation coefficient. At all three quality control levels, developed bioanalytical method was found as repeatable and reproducible as well. The average recoveries of sulfasalazine from plasma were in the range of 95.59-97.16%. The bioanalytical samples showed good and acceptable stability of sulfasalazine solution at different storage, packaging and handling conditions. Hence, in conclusion, the validated and developed HPLC-UV method could be effectively utilized for determination of sulfasalazine in pharmacokinetic studies involving novel formulations.

Method Development and Validation of Metoprolol and Felodipine by using RP-HPLC in Bulk and Pharmaceutical Dosage Form

The main objective of this research is to develop and validate a simple, specific, precise, sensitive, cost effective and rapid Reversed-Phase High-Performance Liquid Chromatographic (RP-HPLC) method for simultaneous quantification of Felodipine and Metoprolol in bulk and pharmaceutical dosage forms. The separation of the analytes were carried out on a X-bridge phenyl column with a moving phase composed of 0.1 % Tri ethyl amine: acetonitrile (30:70 v/v) delivered at a stream of 1.0 ml/min, and separation has been observed by UV detector, at a detection wavelength of 235 nm. This method was proven to be linear over a concentration limit of 10-150 µg/ml for Metoprolol, 2-30 µg/ml for Felodipine with correlation coefficient of 0.999. The retention time of Metoprolol and Felodipine were 2.936, 4.535 minutes respectively. To separate Metoprolol and Felodipine peaks a run time of 8 min. was used. The validation results were in good agreement with acceptable limits. RSD values which are less than 2.0 % indicating the accuracy and precision of this method. Hence it was evident that the proposed method was said to be a suitable one for the regular analysis and quality control of pharmaceutical preparations which contain these active drugs either individually or in combination.

NOVEL VALIDATED REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF GLUCOSAMINE, DIACEREIN, AND METHYL SULFONYL METHANE IN MICRO SAMPLE RAT PLASMA AND ITS APPLICATION TO PHARMACOKINETIC AND DISSOLUTION STUDIES

Objective: The main objective of this research is to develop and validate a simple, specific, precise, sensitive, cost-effective, and rapid reversed-phase high-performance liquid chromatography method for simultaneous quantification of glucosamine (GLU), diacerein (DIA) and methyl sulfonyl methane in bulk and pharmaceutical dosage forms, and micro-sample of rat plasma using ultraviolet (UV) detection, to perform the studies of drug dissolution from tablets.
 Methods: Sprague-Dawley rats were used for pharmacokinetic study after intravenous administration of the drug samples at dose 5 mg/kg. The drug samples were extracted by liquid-liquid extraction technique using acetonitrile, which also acted as a deproteinization agent. The separation of the analyte was carried out on a phenomena C18 column with a mobile phase composed of 0.1 % orthophosphoric acid:acetonitrile (80:20 v/v) delivered at a flow rate of 1.0 ml/min, and separation has been monitored by a UV detector, at detection of the wavelength of 285 nm.
 Results: This method was proven to be linear over a concentration range of 30–450 μg/ml for GLU, 2–30 μg/ml for DIA, and 10–150 μg/ml for methyl sulfonyl methane with a correlation coefficient of 0.999. The retention time of GLU, DIA, and methyl sulfonyl methane were 2.89, 6.32, and 9.87 min, respectively. Recovery of the drugs was found to be in the range of 98.0–102.0%. Validation results were found to be satisfactory and the method applicable for bulk and formulation analysis. Hence, it was evident that the proposed method was said to be a suitable one for the regular analysis and quality control of pharmaceutical preparations which contain these active drugs either individually or in combination.
 Conclusion: The validation results were in good agreement with acceptable limits. Relative standard deviation values which are less than 2.0% are indicating the accuracy and precision of this method. The usefulness of the method is that the common chromatographic conditions have been adopted for assay, dissolution, and pharmacokinetic studies. This developed method showed reliable, precise, and accurate results under optimized conditions.

Development and Validation of RP-HPLC Method for Estimation of Amlodipine Besylate and Celecoxib in Pharmaceutical Formulation

A simple, precise, accurate, and rapid reverse phase-high performance liquid chromatography (RP-HPLC) method with UV-Visible detector has been developed and subsequently validated for the simultaneous determination of amlodipine besylate(AML) and celecoxib(CEL) in their combined tablet dosage form. The separation was based on the use of a Flowrosil C18 analytical column (250 × 4.6 mm, i.d., 5 µm). The mobile phase consisted of a mixture of 80 volumes of acetonitrile and 20 volumes of water. The chromatography was performed by isocratic elution at a flow rate of 1 mL/min. Analytes were detected at 250 nm, with linear calibration curves at concentration ranges of 2-12 µg/ml and 50-300 µg/ml for AML and CEL respectively. The retention time of AML and CEL were 1.98 and 3.15 min respectively. The recoveries obtained were 99.46‒101.36% for AML, 99.57‒101.42% and 99.96–100.87 % for CEL. The method was validated according to International conference of harmonisation guidelines in terms of accuracy, precision, specificity, robustness, limits of detection and quantitation, and other aspects of analytical validation. The developed method was applied successfully for HPLC analysis of commercial pharmaceutical products including AML and CEL. Keywords: Amlodipine besylate; Celecoxib; RP-HPLC.

A Quantitative, Sensitive and Rapid Validated Analytical RP-HPLC Method for the Estimation of Dapagliflozin in Bulk and Pharmaceutical Dosage Formulations

The present study is aimed to develop a linear, precise, and accurate RP-HPLC (Reverse Phase High-Performance Liquid Chromatography) method for the determination of dapagliflozin in the formulation. The method was accomplished on a C18 column (250×4.6mm; 5µm), & Samples were eluted using acetonitrile: water (40:60%v/v) delivered at a flow rate of 1.0ml/min with a chromatographic run time of 10 min. The eluents were observed utilizing a UV detector with a wavelength set at 277nm. The method that was developed resulted in the retention of dapagliflozin at 7.029minutes. Dapagliflozin through current method has shown linearity (r2 > 0.999) over the concentration range of 1-16 µg/ml. The percentage recovery was observed to be within the limits of 98-102%, demonstrating the accuracy of the method. Limit of detection (LOD) and limit of quantification (LOQ) were qualified at 0.049µg/ml and 0.1485µg/ml, respectively. A Linear, precise, accurate, simple, and rapid RP-HPLC method has been developed and validated for the evaluation of dapagliflozin in bulk drug and tablet dosage forms (5mg &10mg) according to ICH Q2(R1) rules. Additionally, the proposed method could be of use in quality control tests of dapagliflozin in pharmaceutical industries.

HPLC method development for estimation of pitavastatin in liquid dosage form

For the determination of pitavastatin calcium nanoemulsion liquid dosage form a simple, sensitive, reliable and rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated. 5 μ particle size column in isocratic mode at 25ºC temperature. The sample was injected through an injector valve with a 10 μl, sample loop. Phosphate buffer (pH 6.4): Methanol (50:50 v/v) was used as a mobile phase with a flow rate of 1 ml per min at 286 nm wavelength. A calibration graph was plotted range between 25-200 μg/ml with the correlation coefficient of 0.999 which showed linearity. Validation studies revealed the method is specific, rapid, reliable, and reproducible. The validity of the method, degradation studies were carried out using the same optimum conditions. Therefore the proposed method is reliable, rapid, precise and selective and may be used for the quantitative analysis of nanoemulsion liquid dosage form of pitavastatin calcium.

METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF LAMIVUDINE AND ZIDOVUDINE IN TABLET BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The objective of the study was to develop and validate reverse-phase high-performance liquid chromatography (RP-HPLC) method and apply method to tablet dosage form.
 Methods: A simple, rapid, economical, precise, and accurate RP-HPLC method for simultaneous estimation of lamivudine and zidovudine in their combined dosage form has been developed.
 Results: A RP-HPLC method was developed for the simultaneous estimation of lamivudine and zidovudine. In their combined dosage form has been developed. The separation was achieved by LC-C18 column (150 mm ×4.6 mm, 5 μm) and water: methanol (65:35v/v) as mobile phase, at a flow rate of 0.8 ml/min. Detection was carried out at 272 nm. Retention time of lamivudine and zidovudine was found to be 3.007 min and 4.647, respectively. The method has been validated for linearity, accuracy, and precision. The assay method was found to be linear from 50% to 150% for lamivudine and zidovudine.
 Conclusion: Developed method was found to be accurate, precise, and rapid for simultaneous estimation of lamivudine and zidovudine in their combined dosage form.

Fast Screening Methods for the Analysis of Topical Drug Products

Considering the recent regulatory requirements, the overall importance of in vitro release testing (IVRT) methods regarding topical product development is undeniable, especially when addressing particulate systems. For each IVRT study, several hundreds of samples are generated. Therefore, developing rapid reversed-phase high-performance liquid chromatography (RP-HPLC) methods, able to provide a real-time drug analysis of IVRT samples, is a priority. In this study, eight topical complex drug products exhibiting distinct physicochemical profiles were considered. RP-HPLC methods were developed and fully validated. Chromatographic separations were achieved on a XBridgeTM C18 (5 µm particle size, 150 mm × 2.1 mm), or alternatively on a LiChrospher® 100 RP-18 (5 µm particle size, 125 mm × 4.6 mm) at 30 °C, under isocratic conditions using UV detection at specific wavelengths. According to the physicochemical characteristics of each drug, different mobile phases were selected. Irrespective of the drug (hydrocortisone, etofenamate, bifonazole, clotrimazole, acyclovir, tioconazole, clobetasol, and diclofenac) and formulation, retention time values did not exceed 6.5 min. All methods were linear, specific, precise, and accurate at the intraday and interday levels, robust, and stable. These were successfully applied to establish product-specific IVRT profiles, thus providing a key database useful for topical pharmaceutical manufacturers.