Mobile Phase Flow Rate Research Articles

OpenPrep: Sensor Event-driven Open Column Chromatographic Sample Preparation System.

We propose a sensor event-driven, open-column chromatographic sample preparation system─OpenPrep. This system replaces the problematic flow selector valve with a noncontact built-in sensor, eliminating carryover and clogging issues, which are commonly encountered in pump-driven chromatographic systems. The innovative valveless column-flow design, combined with a compact gantry dispenser and motion stage-based fraction collector, reduces the sample flow path to a disposable component with a postcolumn dead volume of only 0.5 mL. This configuration is superior to conventional dynamic flow systems owing to its ability to minimize sample consumption, eluent loss, and cleaning waste generation. Thus, OpenPrep can be ideal for preventing cross-contamination. Owing to the rapid flow-transfer and stop approach, OpenPrep achieves higher reagent delivery accuracy and precision compared to ISO 8655-2 piston-operated pipettes, by minimizing liquid residue on the dispensing tip. Sensor-driven flow control ensures precise eluent collection and automatic regulation of the mobile-phase flow rate through an online calibration algorithm, meeting all the requirements for accurate and reproducible column flow control. OpenPrep features an open architecture for both hardware and software, offering high flexibility for analyzing samples with considerable variations in radioactivity and matrix compositions, such as radioactive waste. The proof-of-concept implementation of a multistage sequential separation strategy, using five resin columns to separate Re, Sr, Fe, and Ni from a single sample, demonstrates that OpenPrep provides enhanced flexibility and reconfigurability, improved operational efficiency, and cost-effective customization. Excellent chemical yields were obtained for Re, Sr, and Ni in the range of 83-97%, with less than 2.6% relative standard deviation at a flow rate three times faster than the gravity method. Additionally, improved recovery performance of Fe was achieved by applying a slower flow rate compared to gravity flow.

Quality by Design Approach Driven Development and Validation of Reverse Phase‐High‐Performance Liquid Chromatography Methodology for Dual Quantification of Aspirin and Rivaroxaban

ABSTRACTClinical trials have shown that combining a modest dose of Rivaroxaban (RIV) with Aspirin (ASP) is more effective than ASP alone in reducing cardiovascular mortality, myocardial infarction, and stroke. A high‐performance liquid chromatography method developed using Quality by Design (QbD) effectively estimates ASP and RIV for routine pharmaceutical analysis. The Plackett‐Burman design assessed key factors including organic phase volume, mobile phase pH, flow rate, temperature, and injection volume using an Ishikawa fish‐bone diagram for risk assessment. These parameters were then optimized via Box‐Behnken design in 15 trials. The method meets the International Council For Harmonization standards with a mobile phase ratio of 45:55 (v/v) acetonitrile: buffer (pH 3, adjusted with orthophosphoric acid), a flow rate of 1 mL/min, a column temperature of 30°C, and an injection volume of 20 µL. A Phenomenex Luna C18 column (250 × 4.6 mm, 5 µm particle size) with detection at 251 nm ultra‐violet wavelength. The retention times of 4.54 min for ASP and 5.81 min for RIV were obtained. Validation confirmed linear ranges: 40–200 µg/mL for ASP and 2–10 µg/mL for RIV, with excellent recovery rates (ASP: 99.30%–101.9%; RIV: 98.52%–101.84%). Developed using QbD principles, the method proved specific, accurate, precise, sensitive, and robust for routine quality control of both compounds in pharmaceutical formulations.

Scientifc Validation and Chromatographic Method Development for the Estimation of Piperine in Herbal Formulation Babbularishta: A Quality Control Strategy for Critical Quality Attributes

Background: Babbularishta is an Ayurvedic medicine in liquid form used for the treatment of phthisis, skin diseases, urinary disorders,, and breathing problems. It is also beneficial in cases of blood disorders, acne, dermatitis, hemoptysis, lung injury, asthma, persistent cough, whooping cough, etc. Objective: The functional role of this study is to develop a simple, novel, precise, accurate, reliable, and selective RP-HPLC method for the estimation of piperine content in herbal drug formulations (Babbularista). Methods: A traditional approach to method development could fail to meet the desired separation. Consideration of the current regulatory requirement for analytical method development and RP-HPLC method for routine analysis of piperine in the herbal formulation has been optimized using an analytical Quality by Design (QbD) approach. Three laboratory formulations and three marketed preparations of Babularishta were investigated for organoleptic parameters, physicochemical parameters, and validation. The mobile phase composition (methanol and water % as A), pH (B), and flow rate (mL/min as C) were selected as independent variable,. In contrast, retention time (min. as Y2), area (AU as Y1) is selected as dependent variables. Results: The fingerprinting method was developed and optimized by using the AQbD approach. Piperine content was found to be in Piper nigrum (Marica) 3.01±0.01527 % w/w and in Piper longum (Pipali) 2.74±0.01154% w/w in Babbularista. Conclusion: An attempt has been made to develop quality control parameters of Babbularista by developing a suitable and sensitive HPLC methods for the quantitation of piperine in herbal drug formulations (Babbularista). The developed method was found to be simple, accurate, precise,, and economical for the estimation of piperine in herbal formulations.

Development and Validation of HSPiP- and Optimization-Assisted Method to Analyze Tolterodine Tartrate in Pharmacokinetic Study

A new approach was applied for the development of a precise, simple, and economic analytical process for the accurate analysis of tolterodine tartrate (TOT) in its bulk and tablet using HSPiP- and quality by design (QbD)-assisted methods. The HSPiP program predicted several solvents and their right ratios for the mobile phase, followed by simulating the experimental solubility data in various predicted solvents. QbD was used to identify the impact of the composition and the mobile phase flow rate on the peak area and retention time. TOT was estimated using an Agilent TC C18 column employing an optimized mobile phase. The HSPiP shortened the solvent selection time with high reliability, whereas QbD identified critical factors. The optimized composition and process variables were used to develop an analytical method for TOT estimation. Various analytical validation parameters were estimated with constructed linearity of 5–30 μg/mL and a percent recovery yield value of 100.36%. To ensure the reliability of the optimized method, we estimated validation parameters (linearity, specificity, precision, accuracy, robustness, and ruggedness) to comply with the ICH guidelines. Considering the high recovery yield, good regression coefficient, low detection limit, and low noise ratio, the optimized method was accurate and precise with a high degree of specificity, rapid process, and reproducibility for the quantitative estimation of tolterodine from both oral analytes (I and II). The validated method was implemented for pharmacokinetic study in rats for quantitative estimation of the analytes with high accuracy, sensitivity, and reproducibility.

THE INDUSTRIAL IMPORTANCE OF TECHNOLOGY TRANSFER FOR ANALYTICAL METHOD DEVELOPMENT AND VALIDATION–APPLICATION TO VILAZODONE HYDROCHLORIDE DOSAGE FORM

Objective: The research aims to create a fast, economical, and efficient tech transfer method for analyzing bulk materials and pharmaceutical dosage forms. Methods: The analysis was performed using an Acquity UPLC BEH C18 analytical column (130 Å, 1.7 µm, 3 mm x 100 mm) with detection of the analyte at 238 nm. The mobile phase flow rate was optimized at 0.3 ml/min, and an injection volume of 0.5 µl** was used. The isocratic mobile phase consisted of a blend of buffer (0.1% OPA) and acetonitrile at a ratio of 40:60% v/v. Results: All validation parameters of the analytical method were acceptable. The analyte eluted at 3.08 min with satisfactory theoretical plates and a tailing factor. The recovery of the analyte was 99-100%, and the method's precision was 0.59 and 0.72% RSD. The technique showed no interference, indicating specificity. The linearity was demonstrated over the 20-60 µg/ml range, with a regression coefficient of 0.999. Conclusion: The developed method is cost-effective, specific with minimal interference, and time-saving. It also reduces solvent consumption for quality control analysis of vilazodone hydrochloride samples. The method demonstrates good linearity, robustness, and accuracy.

Determination of Polyphenols in Cigar Tobacco by HPLC

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

Method Development for Analysis of Cyclodextrin Inclusion Complexes with HPLC

Cyclodextrin inclusion complexes have been widely used in the pharmaceutical industry to improve drug solubility and bioavailability. Analysis of these complexes using HPLC requires the development of appropriate and validated methods. To analyze and compare various HPLC methods used for the analysis of cyclodextrin inclusion complexes, and to identify challenges and trends in method development. A literature review was conducted of articles published in the last 10 years, focusing on HPLC methods for the analysis of cyclodextrin inclusion complexes. Parameters compared included column type, mobile phase composition, flow rate, and detection method. The majority of studies used C18 columns with varying lengths and particle sizes. The composition of the mobile phase varied, reflecting the diversity of properties of the complexes analyzed. The flow rate was generally 1.0 mL/min, with the exception of the HPLC/MS method. Detection methods included UV-Vis, PDA, fluorescence, and mass spectrometry. The main challenges included the dynamic equilibrium of the complexes and stability during analysis HPLC method development for the analysis of cyclodextrin inclusion complexes requires optimization of various parameters to overcome specific challenges. Future trends are towards the use of advanced technologies such as UHPLC and high-resolution mass detection.

UHPLC method for simultaneous assessment of pharmacokinetic parameters of co-administered voriconazole and omeprazole using rat plasma

Voriconazole (VRC) is a first-line therapeutic agent for the treatment of invasive fungal infections, whereas omeprazole (OMZ) is a commonly used acid suppressant; however, the two drugs are often used in combination in clinical practice. The aim of this research was to investigate how the co-administration of OMZ and VRC affects the pharmacokinetic characteristics of VRC in rats. A new ultra high-performance liquid chromatography (UHPLC) analytical method was developed and validated for simultaneous analysis of co-administered drugs using VRC and OMZ. A Shim-pack GIST-HP C18 column with 0.1 M triethylamine:acetonitrile (70:30, v/v) as the mobile phase (flow rate: 0.3 mL/min) was used, and UV detection was performed at 240 nm. Liquid–liquid extraction of plasma samples was carried out using dichloromethane. The bioanalytical method was linear over a range of VRC concentrations (100–2000 ng/mL) and OMZ (50–10,000 ng/mL) concentrations, and exhibited both accuracy and precision within the acceptable respective ranges. The average extraction recoveries for VRC and OMZ in plasma were 94.88 % and 82.76 %, respectively. Additionally, the method was successfully applied in vivo to estimate the pharmacokinetic features of VRC in the plasma of rats receiving gavage with low and high doses of OMZ. In conclusion, using a new UHPLC method, we determined that co-administration of OMZ substantially decreased the bioavailability of VRC in rats. Potentially significant drug interactions should be considered in patients receiving the combination of OMZ and VRC.

Development and Validation RP-HPLC Method for Estimation of Antidiabetic Drugs in Pharmaceutical Dosage Form

This study details the development and validation of an RP-HPLC method for estimating Canagliflozin and Metformin in pharmaceutical dosage forms. Canagliflozin, an SGLT2 inhibitor, and Metformin, an antihyperglycemic agent, were obtained from Arti Drug Pvt. Ltd. Marketed formulation Invokamet was sourced locally. Chromatographic separation was achieved on a C18 column (250 x 4.6 mm, 5 µm) using a mobile phase of Methanol: Water (80:20) with 0.1% ortho phosphoric acid at a 1.0 ml/min flow rate and detection at 254 nm. The retention times for Canagliflozin and Metformin were 8.183 min and 3.633 min, respectively. Method validation parameters included system suitability, accuracy, precision, and robustness. System suitability tests confirmed adequate resolution and repeatability. Accuracy was evaluated through recovery studies at 80%, 100%, and 120% concentration levels, yielding satisfactory results. Precision was demonstrated with intra-day and inter-day % RSD values below 2. Robustness was assessed by varying mobile phase composition and flow rate, showing consistent retention times and tailing factors. The method proved selective, accurate, precise, and robust, with a short run time, facilitating rapid quantification of samples. It is suitable for routine quality control analysis of Canagliflozin and Metformin in pharmaceutical formulations

Central Composite Design Expert-Supported RP-HPLC Optimization and Quantitative Evaluation of Efonidipine Hydrochloride Ethanolate & Chlorthalidone in Tablet.

Central composite design based RP-HPLC method optimization for the synchronized analysis of Efonidipine Hydrochloride Ethanolate (EFE) and Chlorthalidone (CHL) in tablet. The effective separation was performed using Inertsil ODS C18 column (250 × 4.6mm, 5μm), PDA detector with 0.05M KH2PO4 Buffer (pH4.5): Acetonitrile (40:60%v/v) mobile phase. Independent variables were investigated include the concentration of KH2PO4 (X1) and flow rate of mobile phase (X2). Based on responses obtained (retention time, resolution and tailing factor), the optimum condition selected was X1 = 40% and X2 = 1ml/min. Optimized HPLC condition was validated by assessing validation parameters and it meets the acceptance criteria set by ICH. The linear calibration curve was found to be in the quantity range 6.25-18.75 and 20-60μg/ml Assay of drugs was 100.94 and 100.06% for CHL and EFE. The validated RP-HPLC-PDA method can be used for routine analysis of EFE and CHL in tablet.

A Quality by Design (QbD) driven gradient high performance liquid chromatography method development for the simultaneous estimation of dasatinib and nilotinib in lipid nanocarriers

Tyrosine kinase inhibitors (TKIs) are effective as a targeted treatment for chronic myeloid leukemia (CML), which can selectively suppress BCR-ABL1 kinase activity. CML therapy with TKIs combination has been supported by in-vitro, in-vivo, and patient-based data where the nilotinib-dasatinib co-administration has exerted superior anticancer efficacy with greater cellular uptake, less resistance to chemotherapy, and no additive adverse events encountered. Therefore, it is essential to develop a suitable analytical method for the simultaneous estimation of these drugs in the developed novel lipid nanocarriers like liposomes. Design of Experiment (DoE) has been implemented as a tool of QbD to systematically investigate the relation between the HPLC method attributes and analytical responses, i.e., chromatographic detection, quantification, and peak properties for dasatinib and nilotinib. An Ishikawa diagram is constructed to delineate possible influencing variables to the analytical performances. Afterward, 4 factors 2 level full factorial design (FFD) was employed to model and identify the main effects and interaction effects between the factors selected after the initial risk assessment. The suggested design space for optimized chromatographic conditions by QbD analysis is linear within the selected range of drug concentrations, accurate and precise, sensitive, and robust according to the ICH guidelines. The optimal method is comprised of a 1 mL/min flow rate of mobile phase (ACN and 20 mM KH2PO4 of pH 7.00) in gradient mode at 25 °C column temperature for 20 μL sample injection volume and detection wavelength fixed at 297 nm. Most importantly, this novel HPLC method is simple and selective enough to evaluate dasatinib and nilotinib content in the lipid nanocarriers.

Development of a Validated RP-HPLC Method Using Full Factorial Design for the Analysis of Ramipril

The primary objective of this study is to develop and optimize a simple, novel, reproducible, and efficient RP-HPLC method using quality by design (QbD) approach for the routine analysis of ramipril. The chromatographic separation was carried out by C18 column with an isocratic elution of a mobile phase of 65:35 (%v/v) acetonitrile: water at a flow rate of 0.9 mL/min. The detection was done at a wavelength of 210 nm using a photo-diode array plus (PDA+) detector. A 32 full-factorial design was employed for the development of analytical method using Design Expert® software in which the mobile phase composition and flow rate were taken as independent variables and the retention time (RT), tailing factor (TF) and theoretical plate count (TP) were chosen as responses of the study. Statistically significant models were obtained for the development of the method (p<0.05). The empirical responses perfectly fitted to that of predicted, with an error within the tolerance of ± 2%. This indicates that the model efficiently identified the optimum levels of independent variables, i.e. the composition of mobile phase and its flow rate, to get the desirable responses. The validation of the developed method followed the ICH Q2 (R1) guidelines, demonstrating that the method is robust and well-suited for the routine analysis of ramipril in active pharmaceutical ingredients and in drug products. Dhaka Univ. J. Pharm. Sci. 23(1): 93-102, 2024 (June)

Development and optimization of high-performance extraction chromatography method for separation of rare earth elements

The effectiveness of commonly used extractants for chromatographic separation of rare earth elements (REEs) was compared. Columns loaded with similar molar concentrations of tributyl phosphate (TBP), di-(2-ethylhexyl) phosphoric acid (HDEHP), and N-Methyl-N, N, N-tri-octyl-ammonium chloride (Aliquat-336), with mineral acid as eluent were evaluated. Retention factors were determined, and separation efficiency was assessed based on the resolution data of the REEs acquired under the same elution conditions for each column. HDEHP demonstrated the best separation efficiency for the entire REE series (mean Rs = 2.76), followed by TBP (mean Rs = 1.52), while Aliquat-336 exhibited the lowest performance (mean Rs = 1.42). The HDEHP-coated column was then used to optimize the extraction chromatographic separation of the REEs. The primary challenge was to completely elute the heavy REEs (Tb – Lu) while maintaining adequate separation of the light REEs (La – Gd) within a reasonably short time. The stepwise gradient elution procedure improved the resolution between adjacent REEs, allowing the complete separation of the entire REE series within 25 minutes. Better separation efficiency for light REEs was achieved at higher column temperatures and a mobile phase flow rate of 1.5 mL/min in the tested domain of 20–60 °C, and 0.5–2.0 mL/min, respectively, resulting in plate heights (H) ranging from 0.011 to 0.027 mm.

Stability indicating RP-HPLC-UV method for quantitative estimation of isomer and other organic impurities of atopic dermatitis drug abrocitinib in drug substance and pharmaceutical dosage form using quality by design (QbD) approach

A modest, reliable, robust, stability-indicating RP-HPLC-UV method was developed and validated following ICH Q2(R2) for the separation and quantification of isomer and other organic impurities of Abrocitinib (Janus kinase inhibitor). Quality by design (QbD) approach was used to achieve the desired specifications. The chromatographic separation was achieved for impurities method of Abrocitinib within 35 min of chromatographic run time on Agilent ZORBAX Eclipse plus C18 150*4.6 mm, 3.5 µm column with gradient elution mode: time (min)/B (%): 0.01/0, 2.5/15, 8.0/20, 15.0/40, 20.0/50, 25.0/50, 25.1/0, 35.0/0 with mobile phase-A (MP-A): 10 mmol/L CH3COONH4 and acetonitrile with the ratio of 900:100 (v/v), mobile phase-B (MP-B): 10 mmol/L CH3COONH4 and acetonitrile with the ratio of 100: 900 (v/v). The detection wavelength, mobile phase flow rate, column temperature, and injection volume were used at 287 nm, 1.0 mL/min, 40 °C, and 20 µl respectively. Utilizing forced degradation and mass balance investigations, the new method’s stability-indicating properties were assessed. The developed method was validated as per ICH and found to be specific, precise, rugged, robust, accurate, linear, and with a limit of quantification values,0.0359 µg/mL for the impurity IMP-A, 0.0192 µg/mL for the impurity IMP-B, 0.0322 µg/mL for the impurity IMP-C and 0.0201 µg/mL for abrocitinib.

An Analytical Approach by Tri-Combination of Gradient UFLC, Response Surface Methodology and Green Chemistry Principle for Simultaneous Quantification of Azelnidipine and Chlorthalidone in Rabbit Plasma.

In this study, a sustainable and eco-friendly method is developed to quantify azelnidipine and chlorthalidone in rabbit plasma by gradient liquid chromatography based on green chemistry principle and analytical quality by design. The separation was achieved on a Shim pack C18 (25 cm × 5 cm × 4.6 μm) column with L1 packing. The mobile phase compromised of ethanol and 50-Mm ammonium acetate buffer (pH.6) at flow rate of 0.6 mL/min with 25-min runtime. The resolution and asymmetric factor were identified as critical analytical attributes (CAAs). The screening studies employing Control Noise Experimentation revealed that mobile phase pH, flow rate and ethanol concentration at 6 and 15 min significantly affected the CAAs method. The critical method parameters were optimized using Central Composition design. Chromatogram showed peak of the drugs at retention time of 9.03 min for chlorthalidone and 16.83 min for azelnidipine. The greenness score of the analytical method was found to be 1876.43 using analytical method greenness score calculator. The validation of the developed method was done which showed linearity at the range of 16-520 ng/mL, with R2 of 0.9992 and 0.9996 for azelnidipine and chlorthalidone, respectively, furthermore accuracy, precision, recovery and stability studies are carried out.

Development of UHPLC-Q-Exactive Orbitrap/MS Technique for Determination of Proanthocyanidins (PAs) Monomer Composition Content in Persimmon.

The main units of persimmon proanthocyanidins (PAs) are composed of flavan-3-ols including epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG). Precise quantification of GCG is challenging due to its trace amounts in persimmon. In this study, to establish the optimal UHPLC-Q-Exactive Orbitrap/MS technique for the determination of PAs monomer composition in persimmon fruit flesh of different astringency types, mass spectrometry and chromatographic conditions were optimized. The results showed that when operating in negative ion mode, using a T3 chromatographic column (a type of C18 column with high-strength silica), acetonitrile as the organic phase, a 0.1% mobile phase acid content, and a mobile phase flow rate of 0.2 mL/min, the chromatographic peak shape and resolution of the PAs monomer composition improved. Additionally, there was no tailing phenomenon observed in the chromatographic peaks. At the same time, the intra-day and inter-day precision, stability, and recovery of the procedure were good. The relative standard deviation (RSD) of stability was less than 5%. The intra-day precision was in the range of 1.14% to 2.36%, and the inter-day precision ranged from 1.03% to 2.92%, both of which were less than 5%. The recovery rate ranged from 94.43% to 98.59% with an RSD less than 5%. The results showed that the UHPLC-Q-Exactive Orbitrap/MS technique established in this study can not only be used for the quantification of EGCG and GCG in persimmon fruit flesh but also be suitable for analyzing other PAs monomer compositions, providing robust support for the related research on persimmon PAs.

Resin screening and process optimization for erythritol mother liquor chromatographic separation

In order to improve the utilization value of the erythritol mother liquor, the separation and purification of the erythritol mother liquor was selected in this study. The selected chromatographic separation programme for erythritol crystallizing mother liquor is as follows: Firstly, erythritol is resolved from mannitol and arabitol with DTF-01Ca (Suqing Group) resin and then mannitol is resolved from arabitol with 99Ca/320 (Dowex) resin. At the same time, the chromatographic conditions of the DTF-01Ca (Suqing Group) and 99Ca/320 (Dowex) resins were optimized, resulting in an optimal separation temperature and mobile phase flow rate of 70 °C, 10 ml/min. On this basis, a single-column chromatographic model was used to calculate the TD model parameter ( N ) and the mass transfer coefficient ( k m ) of the separation of erythritol mother liquor by DTF-01Ca (Suqing Group) and 99Ca/320 (Dowex) resins. The adsorption isotherms, TD model parameter ( N ) and the mass transfer coefficient ( k m ) provides data references for the design and operation of the simulated moving beds (SMB) separation system for the industrial-scale separation of erythritol crystallizing mother liquor.

RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF CURCUMIN AND RESVERATROL IN NANO-MICELLE: DUAL DRUG DUAL FORM SIMULTANEOUS ESTIMATION

Objective: To develop a reverse-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous estimation of conjugated form of Curcumin (CCMN) and free form of Resveratrol (RSV) in nano-micelle. Methods: The conjugation of lipophilic CCMN and hydrophilic Chitosan (CHT) through succinyl linker produce amphipathic molecule that can self-assemble into RSV solution to form micelle. Here RSV exists in micelle core as free form and CCMN with micelle backbone as conjugated form. So it required to estimate conjugated drug and free drug simultaneously from nano-micelle. We developed a RP-HPLC method, utilized C18 column, follow flow rate of mobile phase 1.0 ml/min, which consist of acetonitrile with water (0.5% Ortho Phosphoric acid, pH 4.6) in the ratio of 1:1 for 20 min. Injection volume was 10μl and column temperature 25 ℃. Isosbestic detection of both drugs was at 254 nm. Results: The retention time of RSV and CCMN were at 8.15 min and 11.41 min respectively, completely distinguished sharp peak of CCMN and RSV developed with resolution 7.360±0.117, wide range of linearity with correlation coefficient value (R2) of CCMN and RSV were 0.99987 and 0.99992 respectively and recovery value of CCMN and RSV were 100.041±0.22 % and 100.041±0.21 % respectively. The RSD (relative standard deviation) for accuracy, precision and robustness of the method was found to be less than 2%. Conclusion: The develop method for simultaneous estimation of conjugated CCMN and free form of RSV in the nano-micelle formulation was consider to be accurate, precise, robust and sensitive.

RSM-CCD COUPLED RP-HPLC OPTIMIZATION AND QUANTITATIVE EVALUATION OF ANTIHYPERTENSIVE DRUGS IN TABLET

Central composite design (CCD) based RP-HPLC method optimization for the synchronized analysis of Efonidipine Hydrochloride Ethanolate (EFE) and Chlorthalidone (CHL) in Tablet. The effective separation was performed using Inertsil ODS C18 column (250 x 4.6 mm,5μm), PDA detector with 0.05M KH2PO4 Buffer (pH 4.5): Acetonitrile (40:60%v/v) mobile phase. Independent variables were investigated include the concentration of KH2PO4 (X1) and flow rate of mobile phase (X2). Based on responses obtained (retention time, resolution and tailing factor), the optimum condition selected was X1 = 40% and X2 = 1 ml.ml-1. Optimized HPLC condition was validated by assessing validation parameters and it meet the acceptance criteria set by ICH. It was showed linear calibration curve in the quantity range 6.25-18.75 μg.ml-1 and 20-60 μg.ml-1. % for CHL & EFE. Assay of drugs was 100.94% and 100.06% for CHL & EFE. The validated RP-HPLC-PDA method can be used for routine analysis of EFE and CHL in tablet

Development of Response Surface Approach for Determination of Paracetamol, Chlorpheniramine Maleate, Caffeine and Ascorbic Acid by Green HPLC Method: A Desirability-Based Optimization.

Design of experiment is an efficient and cost-effective tool to optimize the chromatographic separation of a multicomponent mixture. The central composite design was conducted to develop and optimize a green high performance liquid chromatography (HPLC) method for simultaneous quantitation of a quaternary mixture of paracetamol, chlorpheniramine maleate, caffeine and ascorbic acid in their pharmaceutical dosage form as well as the determination of their dissolution profile. A five-level three-factor model was performed to investigate the effect of mobile phase composition, pH and flow rate on enhanced resolution and short run time. Analysis was performed using a Kinitex EVO C18 column and a mobile phase composed of methanol: 0.02M phosphate buffer pH3.3 (34:66, v/v) at 1.0mL/min using photodiode array detection. Optimum chromatographic separation was achieved in <6min with a desirability of 0.999. Linearity was achieved over a range of 1.00-300.00, 1.00-50.00, 2.00-50.00 and 2.00-100.00μg/mL for paracetamol, chlorpheniramine maleate, caffeine and ascorbic acid, respectively, with a limit of detection (<0.1μg/mL). The greenness profile was evaluated using the analytical eco-scale and Analytical GREEnness Metric Approach with values of 81 and 0.77, respectively.