Sensitive Reversed-phase High-performance Liquid Chromatography Research Articles

Implementing analytical quality by design in reversed phase-high performance liquid chromatography for simultaneous estimation of teriflunomide and quercetin: Applicability in dual drug loaded topical microemulsion

The current study reports the development of a novel, robust, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of teriflunomide (TFD) and quercetin (QCN) in dual drug-loaded microemulsion. TFD and QCN have been reported to exhibit anti-inflammatory effects by targeting various inflammatory mediators involved in the pathogenesis of rheumatoid arthritis (RA). This analytical method employs a risk-based approach and follows the principles of analytical quality by design (AQbD). The study involved preliminary screening trials and systematic risk analysis to identify critical method attributes, that significantly impact the critical quality attributes (CQAs). For the optimization of method,a face centered central composite design was utilized, and 16 experimental runs were performed using design expert software version 7.0.0. Chromatographic conditions were carefully optimized using AQbD, falling within the design space and consisting of ammonium acetate buffer (pH 3.5) and acetonitrile (60:40, % v/v) with a flow rate of 1.0 mL/min. An analytical Hibar Lichospher 100 RP-18e column (250 × 4.6 mm, 5 µm) was employed, with detection wavelength of 280 nm and 367 nm for TFD and QCN, respectively. The optimized method underwent validation according to ICH guidelines and demonstrating applicability for evaluating topical microemulsion.

VALIDATED STABILITY INDICATING RP-HPLC METHOD FOR QUANTIFICATION OF SPARSENTAN IN PHARMACEUTICAL DOSAGE FORM: A 2023 USFDAAPPROVED DRUG

The quantitative analysis of Sparsentan in the pharmaceutical dosage form has been accomplished through the development and validation of a simple rapid, precise, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method. Using a Zorbax SB 18 (150 x 4.6 mm, 3.5 µm) column and a mobile phase containing acetonitrile and potassium dihydrogen phosphate pH-3.0 adjusted with diluted orthophosphoric acid in a ratio of 40:60% v/v, Sparsentan was separated chromatographically using a Waters Alliance-e2695. The flow rate was 1.0 mL/min, and a photodiode array detector (PDA) operating at room temperature was used for absorption detection at 287 nm. For Sparsentan, the theoretical plate count was greater than 2000 and the tailing factor was less than 2, respectively. All measurements have a relative standard deviation of less than 2.0 for their peak areas. The suggested approach was verified in compliance with ICH Q2(R1) standards. The approach was determined to be an easy, affordable, appropriate, robust, precise, and accurate way for the quantitative study of Sparsentan.

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.

Regulatory Perspective Reverse Engineering Analysis of the Mast Cell Stabilizer and the Histamine Receptor Antagonist (Olopatadine HCl): Instrumental and Classical Methods for Multiple Formulations.

This study evaluates the unknown qualitative (Q1) and quantitative (Q2) formulas for nasal spray and ophthalmic solution formulations of olopatadine HCl by classical and instrumental techniques to match the generic formula with reference-listed drugs to avoid clinical study. Reverse engineering of olopatadine HCl nasal spray 0.6% and ophthalmic solution 0.1, 0.2% formulations was accurately quantified using a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. Both formulations possess similar components, namely ethylenediaminetetraacetic acid (EDTA), benzalkonium chloride (BKC), sodium chloride (NaCl), and dibasic sodium phosphate (DSP). These components were qualitatively and quantitatively determined using the HPLC, osmometry, and titration techniques. With derivatization techniques, EDTA, BKC, and DSP were determined by ion-interaction chromatography. NaCl in the formulation was quantified by measuring the osmolality and using the subtraction method. A titration method was also used. All the employed methods were linear, accurate, precise, and specific. The correlation coefficient was >0.999 for all components in all the methods. The recovery results ranged from 99.1 to 99.7% for EDTA, 99.1-99.4% for BKC, 99.8-100.8% for DSP, and 99.7-100.1% for NaCl. The obtained % relative standard deviation for precision was 0.9% for EDTA, 0.6% for BKC, 0.9% for DSP, and 1.34% for NaCl. The specificity of the methods in the presence of other components, diluent, and the mobile phase was confirmed, and the analytes were specific.

RP-HPLC-Based Bioanalytical Approach for Simultaneous Quantitation of Cinnarizine and Domperidone in Rat Plasma

An accurate, precise and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) bioanalytical approach was developed for the simultaneous estimation of cinnarizine (CIN) and domperidone (DOM) in rat plasma using irbesartan (IRB) as an internal standard (IS). The proposed RP-HPLC approach was validated as per the latest ICH M10 guidelines. The analytes (CIN and DOM) and IS were extracted from plasma samples using the protein precipitation strategy. Chromatographic separation is accomplished by a C18 SunfireTM (5 µm, 250 mm × 4.6 mm) analytical column, using an isocratic mobile phase consisting of acetonitrile-methanol in 30:70 proportions at a flow rate of 1 mL/min. The detection of all three constituents was recorded at a wavelength of 270 nm with a UV detector. DOM, CIN and IS were eluted at 3.2, 4.5 and 6.1 min, respectively, utilizing a total run time of 10 min. The lower limit of quantification (LLOQ) was 5 ng/mL for CIN and DOM in rat plasma. The proposed RP-HPLC approach was linear in the 5–200 ng/mL range for CIN and DOM. The recovery of the method was greater than 95%, and the relative uncertainty was less than 2%, indicating that the proposed bioanalytical approach was accurate and precise. The limit of detection was established as 1.1 ng/mL for CIN and 1.7 ng/mL for DOM. The created approach was found to be robust and passed all validation criteria; thus, the proposed RP-HPLC approach can be employed successfully for the simultaneous assessment of CIN and DOM in rat plasma.

Can Celecoxib Assay in Preclinical Studies Be Improved?

Celecoxib, a cyclooxygenase-2 inhibitor (COX-2), is attracting considerable interest owing to its potential anticancer activity. The repurposing strategy of this drug, however, requires preclinical assessment involving the use of increasingly improved analytical methods. In this work, a rapid, accurate, precise, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the quantification of celecoxib in five mouse matrices (plasma, brain, spleen, liver, and kidney). Chromatographic separation was achieved within 8 min on a reversed-phase C18 column at 35 °C using a mixture of acetonitrile and 2% (v/v) acetic acid (50:50) as mobile phase, at a flow rate of 0.6 mL/min. Celecoxib and curcumin, as the internal standard, were analyzed at 425 nm and 250 nm, respectively. Linearity was observed (r2 ≥ 0.996) in the concentration ranges selected for celecoxib. Overall precision was below 14.9%, and accuracy was between −14.9% and 13.2%. The acceptance criteria specified in FDA and EMA guidelines were met. Celecoxib was reproducibly recovered (≥84%) and showed stability in all biological matrices at room temperature for 24 h. The method was then effectively applied for the quantification of celecoxib to understand in vivo biodistribution following its intraperitoneal administration in mice.

An improved and sensitive method for nitrotyrosine estimation by reverse phase-high performance liquid chromatography

Reactive nitrogen species such as peroxynitrite anion (ONOO-), nitrogen oxide radical (NO.) may lead to nitrotyrosine formation by oxidative nitration of tyrosine. Nitrotyrosine is considered as an important marker to estimate the severity of biological oxidative damage. Therefore, in the present study, we evaluated a reverse phase-high performance liquid chromatography (RP-HPLC) method to improve the sensitivity and specificity of the nitrotyrosine estimation. The mobile phase used in the present study was methanol (pH 4.7) and 0.1% phosphoric acid (60:40%). The standard serial dilutions were injected onto the HPLC column (C-18) at a flow rate of 1 ml/min mobile phase with a time window of 6 min. The results were monitored on the Diode Array detector at wavelength 210 nm. The evaluation and quantification of output signals were made on the Chromelion software version 6.80 which controls the whole liquid chromatography system. The same standard serial dilutions were monitored spectrophotometrically at 210nm. We found the standard curves were linear over the concentration range of 0.3 to 20 ng/ml for both RP-HPLC with UV detector (r2 = 0.997) and spectrophotometric (r2 = 0.999) methods. In spectrophotometric method, only four standard points were detected out of seven and represented on a straight line, this shows the spectrophotometric method was unable to measure less than 2.5 ng/ml concentration. On the other hand, in RP-HPLC method, all the seven standards were lying on a straight line and HPLC machine can detect up to 1ng/ml or 1pg/µl. The present study established the highly specific, sensitive, accurate and cost-effective RP-HPLC with UV detector method for nitrotyrosine estimation.

A novel RP-HPLC method development and validation for simultaneous quantification of gefitinib and resveratrol in polymeric hybrid lipid nanoparticles and glioma cells

Resveratrol and Gefitinib are adjunct therapies for various cancers; however, both have been limited by low solubility, low cellular uptake, and bioavailability issues. As a result, this research aimed to develop an accurate, precise, selective, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine both compounds in nanoformulation and Glioma cells. The phenomenex luna C8 column, a mobile phase (80: 20 ratios of acetonitrile: 200 mM ammonium acetate) with a flow rate of 1 mL. min−1, 40 ± 0.2 °C as a column temperature, and the injection volume was 20 μl were selected as optimized chromatographic conditions. Retention time (RT) of resveratrol (1.80 min) and gefitinib (2.56 min) were identified using an optimized analytical method and detected at 345 nm (isosbestic point). The approach was proven to be specific for resveratrol and gefitinib analysis in the existence of PHLNPs, precise (RSD 2 %), and accurate (>90 %). The simultaneous analytical method was successfully developed to identify percentage drug entrapment efficiency (% DEE), % drug loading (% DL) of resveratrol and gefitinib in PHLNPs, and secondary estimates of in-vitro drug release profile and percentage cellular uptake studies. The in-vitro results revealed that the developed analytical method could simultaneously detect and quantify these drugs in other nanoformulations and in-vivo studies.

Simple and Sensitive RP-HPLC and UV Spectroscopic Methods for the Determination of Remogliflozin Etabonate in Pure and Pharmaceutical Formulations

Simple, novel and selective reverse phase-high performance liquid chromatography (RP-HPLC) and ultraviolet (UV) spectroscopic methods have been developed and optimized for the determination of remogliflozin etabonate (RMZ) in bulk and dosage forms. In the HPLC method, the principal peak and internal standard peak were eluted separately at different retention times (RT) with the chromatographic conditions such as, mobile phase consisting of 0.02 M ammonium acetate buffer (pH was adjusted to 4.0 by 1.0 M ortho phosphoric acid), acetonitrile and tetrahydrofuran in the ratio 50:45:05, respectively (v/v) and the stationary phase used was C18, 5 μm, 4.6 mm x 250 mm kromasil column. The flow rate was 2.0 mL min-1, sample injection volume was 10 μL, and the wavelength of detection was fixed at 228 nm. In case UV spectroscopic method, the RMZ was diluted with pure ethanol. The RMZ showed a maximum absorbance at 228 nm. Hence throughout analysis 228 nm was used for the determination of RMZ. The RT of RMZ and internal standard, atorvastatin (ATST) were 6.2 min and 7.0 min, respectively. The resolution between the peaks was found to be more than 2.0. The total run time was fixed at 10 min. The linearity range for RP-HPLC method was found to be 10 μg mL-1 to 50 μg mL-1, at a fixed concentration of ATST. The linearity range for the UV spectroscopic method was found to be in the range of 100 to 250 μg mL-1. Regression coefficients (R2) were found above 0.999 for both of the techniques. The limit of detection and quantification for RMZ were found to be 1.0 μg mL-1 and 3.5 μg mL-1 respectively, in RP-HPLC method and 10.0 μg mL-1 and 40 μg mL-1, respectively, in UV spectroscopic method. The developed methods were found to be simple, accurate, reproducible, and precise. The RMZ can be analyzed in dual techniques, i.e., chromatographic and UV spectroscopic methods for its routine analysis.

Development and validation of UV chromatographic method for quantification of copper and copper nanoparticles in different matrices and pharmaceutical products

Background The applications of Cu and CuNPs based on the earth-abundant and inexpensive Cu metal have generated a great deal of interest in recent years, including medical applications. A novel, specific, precise, accurate and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method with UV detection has been developed and validated to quantify copper (Cu) and copper nanoparticles (CuNPs) in different biological matrices and pharmaceutical products. Methods The developed method has been validated for linearity, precision, sensitivity, specificity and accuracy. Cu concentration was detected in pharmaceutical products without an extraction process. Moreover, liver, serum and muscle tissues were used as biological matrices. High Cu recovery in biological samples was afforded by using citric acid as a green chelating agent, exact extraction time and pH adjustment. Cu pharmaceutical and biological samples were eluted by acetonitrile: ammonium acetate (50 mM) with 0.5 mg/ml EDTA (30:70 v:v) as an isocratic mobile phase. EDTA reacted with Cu ions forming a Cu-EDTA coloured complex, separated through the C18 column and detected by UV at 310 nm. Results The developed method was specific with a short retention time of 4.95 min. It achieved high recovery from 100.3% to 109.9% in pharmaceutical samples and 96.8–105.7% in biological samples. The precision RSD percentage was less than two. The method was sensitive by achieving low detection limits (DL) and quantification limits (QL). Conclusion The validated method was efficient and economical for detecting Cu and CuNPs by readily available chemicals as EDTA and Citric acid with C18 column, which present the best results on RP-HPLC.

Establishment of a Rapid and Highly Sensitive Reverse-phase High-performance Liquid Chromatography Based Analytical Assay Method for Duvelisib

Establishment of a Rapid and Highly Sensitive Reverse-phase High-performance Liquid Chromatography Based Analytical Assay Method for Duvelisib

Development and validation of RP-HPLC method for estimation of brexpiprazole in its bulk and tablet dosage form using Quality by Design approach

BackgroundA new, sensitive, suitable, clear, accurate, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of brexpiprazole in bulk drug and tablet formulation was developed and validated in this research. Surface methodology was used to optimize the data, with a three-level Box-Behnken design. Methanol concentration in the mobile phase, flow rate, and pH were chosen as the three variables. The separation was performed using an HPLC method with a UV detector and Openlab EZchrom program, as well as a Water spherisorb C18 column (100 mm × 4.6; 5m). Acetonitrile was pumped at a flow rate of 1.0 mL/min with a 10 mM phosphate buffer balanced to a pH of 2.50.05 by diluted OPA (65:35% v/v) and detected at 216 nm.ResultThe developed RP-HPLC method yielded a suitable retention time for brexpiprazole of 4.22 min, which was optimized using the Design Expert-12 software. The linearity of the established method was verified with a correlation coefficient (r2) of 0.999 over the concentration range of 5.05–75.75 g/mL. For API and formulation, the percent assay was 99.46% and 100.91%, respectively. The percentage RSD for the method’s precision was found to be less than 2.0%. The percentage recoveries were discovered to be between 99.38 and 101.07%. 0.64 μg/mL and 1.95 μg/mL were found to be the LOD and LOQ, respectively.ConclusionThe developed and validated RP-HPLC system takes less time and can be used in the industry for routine quality control/analysis of bulk drug and marketed brexpiprazole products.Graphical abstract

Optimization and Validation of a Facile RP-HPLC Method for Determination of Betrixaban and Lercanidipine in Pharmaceutical and Biological Matrices.

A simple, accurate, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method was established for determination of a novel non-vitamin K antagonist oral anticoagulant drug, betrixaban, and its co-administered calcium-channel blocker drug, lercanidipine, in pharmaceutical formulations and biological fluids. The proposed HPLC method was the first chromatographic method applied to estimate this mixture in a short chromatographic run (<6min), high resolution between betrixaban/lercanidipine (Rs = 7.12) and acceptable values of limit of detection (LOD), limit of quantification (LOQ) and percentage of relative standard deviation (%RSD). The chromatographic separation was performed on a cyano column using a mobile phase composed of acetonitrile:methanol:water (35:35:30, v/v/v) containing 0.2% orthophosphoric acid adjusted to pH3.2 by triethylamine, programmed with a flow rate of 1mL/min with UV detection at 240nm. The proposed method showed linearity over the concentration ranges of (0.20-20.0μg/mL) and (0.25-25.0μg/mL) for betrixaban and lercanidipine, respectively. All validation parameters met the acceptance criteria according to ICH guidelines in terms of linearity, LOD, LOQ, accuracy, precision, robustness, specificity and system suitability. The method was applied to assay the studied analytes in their pharmaceutical formulations with high % recovery (98-102%) and low %RSD (<1.5). Furthermore, the proposed method was successfully applied for the determination of betrixaban in spiked human plasma.

Development and Validation of a New RP-HPLC Analytical Method for the Simultaneous Determination of Luliconazole and Clobetasol Propionate in Synthetic Mixture

Aim: To develop new selective and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) approach for the quantification of antifungal drug Luliconazole integrate with corticosteroid drug Clobetasol Propionate in a synthetic mixture.&#x0D; Methods: The method was validated to achieve International Conference Harmonization (ICH) requirements. Chromatographic separation was carried out by isocratic technique on a reversed-phase Inertsil C18 column (5 µm, 250mm x 4.6mm i.d with the mixture of Acetonitrile: Water pH adjusted with H3PO4 (60: 40) and UV detection at 264 nm. The compounds were eluted at a flow rate of 1.0 mL/min with an injection volume of 20μL.&#x0D; Results: The calibration curves were linear (r2 &gt; 0.999) over the concentration range 10-200 μg/mLfor Luliconazole and 5-100 μg/mL for Clobetasol Propionate. The average retention times for Luliconazole and Clobetasol Propionate were 3.16 and 6.94 min, respectively. The % RSD for the proposed method was found to be less than %2. The % recovery was found to between 99.22-99.48% for the developed method.&#x0D; Conclusion: The developed method is simple, rapid, precise, and accurate and hence was successfully applied for the determination of Luliconazole and Clobetasol Propionate in a synthetic mixture.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ESTIMATION METHOD FOR TRANSDERMAL DIFFUSION STUDY OF GRANISETRON

Objective: The study is aimed to develop a simple, isocratic and sensitive reversed-phase high-performance liquid chromatography method for the analysis of granisetron during transdermal permeation studies through porcine ear epithelium.&#x0D; Methods: A reversed-phase (C18) column was used with ultraviolet detection at 210 nm. Selected mobile phase contained 25% (v/v) of acetonitrile and 75% (v/v) of 0.25 mM potassium dihydrogen orthophosphate solution pH adjusted to 3.0 using 1% orthophosphoric acid solution.&#x0D; Results: Calibration curve showed good linearity over 0.1–30 μg/mL concentration range of porcine ear permeates in 80% ethanol and 20% water (blank permeates). The applicability of the method was demonstrated by the analysis of diffusion study samples of granisetron through porcine ear epithelium and the steady-state permeability flux (J) from ethanolic solution was found to be 4.707 µg/square cm/h.&#x0D; Conclusion: The developed method can be used in the analysis of diffusion study samples of granisetron transdermal formulations through the porcine ear epithelium.

Design of Experiment (DoE)-Approach Based RP-HPLC Analytical Method Development and Validation for Estimation of Efavirenz in Bulk and Formulations

Present study reports design of experiment (DoE) based development and validation of a simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for estimation of efavirenz (EFZ), a non-nucleotide reverse transcriptase inhibitor (NNRTs), used in the treatment of acquired immunodeficiency syndrome (AIDS). Plackett-Burman design was explored to screen the critical method variables (CMVs) for the RP-HPLC method. A response surface Box-Behnken design was employed to optimize the screened CMVs which affect the analytical responses (ARs) of RP-HPLC method. Using the optimized CMVs the HPLC method was developed and validated according to International Conference on Harmonization (ICH) guidelines. EFZ in marketed formulation was estimated using the validated method. Acetonitrile proportion, pH of the phosphate buffer and mobile phase flow rate were the CMVs and retention time and number of theoretical plates were the ARs for the study. The optimized chromatographic parameters were acetonitrile proportion in mobile phase: 51.17%v/v, pH of phosphate buffer: 4.04 and flow rate: 1.25mL/min. Use of these optimized parameters resulted in retention time of 11.031min and 9,498.787 number of theoretical plates as ARs of the HPLC method. The method was further validated in harmony with current ICH guidelines Q2 (R1). The method was capable of the successful estimation of EFZ in marketed formulation. The study depicts successful development and validation of a simple RP-HPLC method of EFZ using DoE approach.

Reverse-phase High-Performance Liquid Chromatography Method Development and Validation for Estimation of Glimepiride in Bulk and Tablet Dosage Form

The present work demonstrates a simple, rapid, precise, specific, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method for analyzing glimepiride in pure and tablet forms. The present method was developed using a C18 column 150 × 4.6 mm, with 5 μm, and packing L1 maintained at a temperature of 30°C. The mobile phase was prepared by dissolving 0.5 gram of monobasic sodium phosphate in 500 mL of distilled water, pH of the solution adjusted to 2.1 to 2.7 with 10% phosphoric acid, and added 500 mL of acetonitrile. The mobile phase was pumped in the highperformance liquid chromatography (HPLC) system at a flow rate of 1 mL/min, and separation was carried out at 228 nm, using an ultraviolet (UV) detector. The chromatographic separation was achieved with peak retention time (RT) at about 9.30 minutes, and the method was found to be linear over a concentration range of 40 to 140 μg/mL. The specificity of the method represented no interference of the excipients during the analysis, and stability testing after 24 hours also showed that the method is suitable and specific. The accuracy was between 99.93 to 99.96%, with limit of detection (LOD) and limit of quantitation (LOQ) being 0.354 μg/mL, 1.18 μg/mL, respectively. Satisfactory results were found for precision and robustness parameters during the development and validation stage for the analytical method. The proposed method was also adopted for the analysis of glimepiride tablets to improve the overall quality control. Using this method, symmetric peak shape was obtained with reasonable retention time. The retention time of glimepiride for six repetitions is 9.3 ± 0.1 minutes; the run time is 21 minutes. The proposed RP-HPLC method is a modification of the United States Pharmacopeia (USP) method, and it was found to be valid for glimepiride within concentration ranges 40 to 140 μg/mL, using C18 analytical columns, and isocratic elution with UV detection, and at 1 mL/min of flow rate.

A sensitive RP-HPLC method for estimation of artemether from polymeric nanoparticles after pre-column acid treatment using UV-visible detector

Artemether; a sesquiterpene lactone is widely used for the treatment of malaria as artemisinin-based combination therapy (ACT). The present work involves the development and validation of sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantification of artemether (ART) in polymeric nanoparticles. ART was transformed to α, β-unsaturated decalones by pre-column acid treatment to enhance the sensitivity of chromophoric group lacking ART for quantification by HPLC-UV. Waters Spherisorb® 5 µm ODS(C18) column (4.6*250 mm) with gradient elution by mobile phase comprising of ACN and PBS (10 mM; pH 6.0) was used to separate acid-treated ART. The analysis was carried at λmax of 253 nm with 20 min and 20 µL run time and injection volume, respectively. The method was found to be linear in the concentration range of 0.5–10 µg mL−1 with 0.09 µg mL−1 and 0.27 µg mL−1 as LOD and LOQ respectively. Further, the method was also found to be specific for ART in presence of blank polymeric nanoparticles, accurate (% average recovery rate 101.7 ± 1.68%), precise (RSD <2%), and robust. The method was successfully used to determine % entrapment efficiency and in vitro release of ART-loaded polymeric nanoparticles with HPLC using a UV-visible detector.

DEVELOPMENT AND VALIDATION OF REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF OLANZAPINE AND ARIPIPRAZOLE IN SYNTHETIC MIXTURES

Objective: A simple, rapid, accurate, precise, specific, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for simultaneous estimation of olanzapine (OLZ) and aripiprazole (APR) in synthetic mixtures.&#x0D; Methods: The stationary phase used for chromatographic separation was Phenomenex C18 column (250 mm × 4.6 mm i.d, particle size 5 μm) and mobile phase used for separation was methanol: Phosphate buffer (pH 3) taken in ratio of 75:25 %v/v. The flow rate was used 1.0 ml/min at room temperature and drugs detected at 240 nm with injection volume 20 μL.&#x0D; Results: The retention time for OLZ and APR was found to be 4.231 and 6.523 min, respectively. The linearity was performed using a concentration range of 0.5–3.0 for both drugs. The correlation coefficient was found to be 0.999 for OLZ and APR. The % purity of both the drug was found to be 98–102%. The proposed RP-HPLC method has been validated, according to International council on harmonization Q2 (B) guidelines.&#x0D; Conclusion: There was no interference of any diluents and excipients in the determination of drugs from synthetic mixture. Hence, the developed method can be used for routine quality control analysis.

Novel Application of Pentabromobenzyl Column for Simultaneous Determination of Eight Antifungal Drugs Using High-performance Liquid Chromatography.

A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as an analytical method for the quantitative determination of eight antifungal drugs in spiked human plasma has been described optimized and validated. The analyzed compounds were voriconazole (VOR), luliconazole (LUL), clotrimazole (CLO), tioconazole (TIO), posaconazole (POS), ketoconazole (KET), sertaconazole (SER) and terconazole (TER). The separation of the analyzed compounds was conducted using a novel pentabromobenzyl column known as COSMOSIL PBB-R (150 mm × 4.6 mm I.D., particle size 5 μm). The analysis of the studied drugs was determined within 14 min using a diode array detector and the mobile phase consisted of: 10 mM potassium dihydrogen phosphate buffer (pH 2.1): Methanol (2: 98 v/v). A linear response was observed for all compounds in the range of concentration studied. Sample preparation was done through liquid-liquid extraction using diethyl ether. This proposed method was validated in terms of linearity, limit of quantification, limit of detection, accuracy, precision and selectivity. The method was successfully applied for the determination of these drugs in their pharmaceutical formulations and in human plasma samples.