Method Development For Simultaneous Estimation Research Articles

HPLC profiling for the simultaneous estimation of antidiabetic compounds from Tradescantia pallida

Diabetes is a long-term metabolic disease epitomized by postprandial hyperglycemia. The prolonged use of synthetic drugs renders distinct side effects, necessitating the development of safe and cost-effective substitutes. The aim of the current study is to isolate, evaluate the antidiabetic potential and HPLC method development for simultaneous estimation of antidiabetic compounds from the leaves of Tradescantia pallida. The leaves were extracted, fractionated and subjected to column chromatography. The isolated compounds' antidiabetic potential was evaluated using α-amylase and glycosylation of hemoglobin assays. The study employed molecular docking to scrutinize interactions between antidiabetic compounds and human α-amylase and hemoglobin protein. Prime MM-GBSA calculations determined binding energies of ligand–protein complexes. Further analysis of morin and catechin involved exploring dynamic and thermodynamic constraints through molecular dynamics simulations under specific biological conditions. A rapid HPLC method was developed and validated for the simultaneous estimation of isolated compounds. The column chromatography culminated in the isolation of four antidiabetic compounds (syringic acid, catechin, p-coumaric acid and morin). The in vitro analyses revealed that morin and catechin exhibited 72.67 % and 78 % α-amylase inhibition and 67 % and 71.66 % inhibition of hemoglobin glycosylation, respectively. In silico studies substantiated the in vitro assay, confirming the stability of catechin and morin complexes via root mean square deviation analysis. Interactions, encompassing hydrophilic, hydrophobic, water bridges, and ionic interactions, identified key residues involved in these processes. The validated HPLC method exhibited excellent correlation coefficients ranged from 0.9909 to 0.9997. The antidiabetic compounds were quantified from the extract in the range of 0.072 – 0.160 µg/mL. The study concluded that the isolated compounds from Tradescantia pallida have remarkable antidiabetic activity, and the developed method can be successfully used for the identification and quantification of phenolic compounds in Tradescantia pallida and other plant-derived matrix.

RP-HPLC Method Development for Simultaneous Estimation of Brimonidine Tartrate and Timolol Maleate in Ophthalmic Preparation

Introduction: A simple, precise, rapid, and reproducible RP-HPLC method is developed for the simultaneous estimation of brimonidine tartrate and timolol maleate present in ophthalmic dosage forms.
 Method: Gradient RP-HPLC is carried out at 35℃ on column C18 (25 cm X 4.6 mm, 5 µm) with mobile phase a ACN and buffer (25 ml ACN & 475 ml buffer) and mobile phase B water and ACN (37.5 ml water & 212.5 ml ACN) at a flow rate of 1.2ml/min. detection is carried out using DAD detector at 248 nm. Parameter such as linearity, accuracy, precision, specificity, LOD, LOQ and robustness are studied as per ICH guidelines Q2 (R1). 
 Results: The linearity range of Brimonidine tartrate and Timolol maleate are 10-50 µg/ml and 4-20 µg/ml. The accuracy for both Brimonidine Tartrate and Timolol Maleate are in range of 100-101%. The RSD for Brimonidine Tartrate and Timolol Maleate are less than 2 for precision, specificity, robustness respectively. 
 Conclusion: This RP-HPLC method can be highly suitable for analysis of brimonidine tartrate and timolol maleate without interference in combined dosage form.

RP-HPLC Method Development for Simultaneous Estimation of Oral Iron Chelator Deferiprone and its related Impurity

Purpose: This study aims to develop and validate a RP-HPLC method for simultaneous estimation of deferiprone and its synthesized impurity. Method: Structure of impurity was confirmed by spectral analysis while their purity was confirmed by HPLC analysis. The chromatographic separation was achieved on Agilent Zorbax Bonus-RP (250 x 4.6mm, 5µ) using mobile phase of 20 ACN-80 0.1% TFA. Detection was carried out at 280 nm using PDA detector with flow rate of 1 ml/min and temperature of 30℃. Result: The method was validated as per ICH guidelines. The percent recovery was found to be within limit and %RSD for deferiprone and the impurity was found to be 0.09 and 0.48. The standard curve was linear over the concentration range of 60-140µg/ml and shows correlation coefficient (r2) of 1. The LOD and LOQ values for deferiprone were found to be 0.21µg/ml and 0.62µg/ml respectively. The method was found to be robust even by change in flow rate of ±0.2ml/min. Conclusion: Validation studies demonstrated that the proposed HPLC method is specific, reliable, rapid and reproducible. The developed method can be successfully employed for routine analysis for deferiprone in pharmaceutical dosage form.

Validated HPTLC Method Development for Simultaneous Estimation of Spironolactone and Hydrochlorothiazide in Formulation

This study describes a new TLC method for simultaneous quantification of Spironolactone and Hydrochlorothiazide in bulk and tablet dosage forms that is simple, precise and accurate. The drugs were separated using aluminium precoated plate with silica gel 60 F-254 as the stationary phase using Toluene: Ethyl acetate: Methanol 6: 4: 1 (v/v/v) as the mobile phase. At 231 nm, the separated zones were densitometrically evaluated. The two drugs were satisfactorily resolved with RF values of 0.78 ± 0.02 and 0.33 ± 0.02 for Spironolactone and Hydrochlorothiazide. The accuracy and reliability of the method were assessed by evaluation of linearity (120-270 ng/spot for Spironolactone and 100-350 ng/spot for Hydrochlorothiazide), precision (intra-day RSD 0.98-1.80 % and inter-day RSD 0.76-1.92 % for Spironolactone, and intra-day RSD 0.84-1.85 % and inter-day RSD 0.31-1.68 % for Hydrochlorothiazide), accuracy (100.78 ± 0.49 % for Spironolactone and 98.54 ± 1.80 % for Hydrochlorothiazide, by ICH guidelines. These methods have been applied to formulation and no interference of excipients is seen which proves that the method is specific.

Smart Spectrophotometric Method Development for Simultaneous Estimation of Antidiabetic Drugs in Formulations

Smart Spectrophotometric Method Development for Simultaneous Estimation of Antidiabetic Drugs in Formulations

Design of experiment-based LC-MS/MS method development for simultaneous estimation of nateglinide and metformin hydrochloride in rat plasma.

This research aims to develop and validate a bioanalytical method for simultaneous estimation of an antidiabetic combination using LC-MS/MS in rat plasma. Nateglinide and metformin hydrochloride are commonly used combination for clinical management of Type 2 diabetes. Hence, simultaneous determination in plasma is essential for the rapid analysis of samples from the pharmacokinetic studies. Statistical optimization was carried out for liquid chromatography (LC) parameters and mass spectroscopic (MS) parameters by design of experiment (DoE) (Design Expert Version 11, Stat Ease Inc., USA) approach. A 33 full factorial design was used for optimization of LC parameters; %methanol, %formic acid, and flow rate were selected as independent variables, whereas peak area and tailing factor were considered as dependent variables for both drugs. Box-Behnken design was used to optimize MS parameters including drying gas flow rate, nebulizing gas flow rate, DL temperature, heat block temperature, and positive voltage as independent factors, and responses selected were [M + H]+ intensity of nateglinide and metformin hydrochloride. The [M + H]+ intensity of the optimized method for nateglinide and metformin hydrochloride were 2,462,838 and 11,873,826, respectively. The model was found significant for optimizing LC and MS parameters with p < 0.05 for both nateglinide and metformin hydrochloride. The optimized method was validated as per the ICH-M10 guideline, which was accurate, precise, and selective. The method was cost-effective and capable of quantitating concentrations in picogram levels for nateglinide and metformin hydrochloride simultaneously.

RP-HPLC method development for simultaneous estimation of empagliflozin, pioglitazone, and metformin in bulk and tablet dosage forms

RP-HPLC method development for simultaneous estimation of empagliflozin, pioglitazone, and metformin in bulk and tablet dosage forms

A Stability Indicating RP-HPLC Method Development for Simultaneous Estimation of Alogliptin, Pioglitazone, and Metformin in Finished Pharmaceutical Formulations

A Stability Indicating RP-HPLC Method Development for Simultaneous Estimation of Alogliptin, Pioglitazone, and Metformin in Finished Pharmaceutical Formulations

Gradient RP-HPLC Method development for simultaneous estimation of Dextromethorphan hydrobromide, Phenylephrine hydrochloride, and Triprolidine hydrochloride in Liquid Dosage Form

A novel, sensitive, and specific gradient RP-HPLC method with UV detection is proposed for the simultaneous quantification of multicomponent liquid dosage form. The separation of the three drugs was achieved using a C18 column (250 mm×4.0 mm, 5 μ) as a stationary phase. No preliminary extraction procedure is required for liquid formulations and a very simple extraction procedure is required for tablets and creams. A mixture of two mobile phases was used with gradient time programing at a flow rate of 1 ml/min. Detection was performed by using PDA detector at 270 nm. Phenylephrine HCl (RT=7.705 min), triprolidine HCl (RT=26.510 min) and dextromethorphan HBr (RT=29.166 min) were separated with good resolution in a single chromatographic run of 50 min. Each drug obeys Beer-Lambert's law in the linearity range of 0.080–0.12 mg/ml for dextromethorphan HBr with a correlation coefficient of 0.998, 0.040–0.060 mg/ml for phenylephrine HCl with a correlation coefficient of 0.996, and 0.010–0.015 mg/ml for triprolidine HCl with a correlation coefficient of 0.998. The recoveries for dextromethorphan HBr and phenylephrine HCl were found to be 99.35% and 99.53%, respectively and for triprolidine HCl was 98.88%. Validation results show satisfactory linearity, precision, accuracy and specificity.

Quality by Design Based Method Development for Simultaneous Estimation of Montelukast Sodium and Theophylline in Tablet Dosage Form

Background: Analytical Quality by design (AQbD) is one of the upcoming regulatory requirements in the field of pharmaceutical analysis for the development of reliable and robust analytical methods. Montelukast sodium and theophylline available in tablet dosage forms are used widely for the treatment of airway disorders. Liquid chromatographic methods for estimation of the drugs are reported but those chromatographic methods developed by the AQbD approach are not available. Methods: A systematic design of experiments has been employed to develop a liquid chromatographic method for simultaneous estimation of montelukast sodium and theophylline. Critical process parameters are defined and optimization of methanol content in mobile phase, the concentration of trifluoroacetic acid and flow rate was employed within the framework of the full factorial design. The statistical analysis of the experimental data for critical quality attributes namely tailing factor of both the drugs and resolution was performed. Results: A multi-dimensional design space for the critical process parameters was obtained. The optimal chromatographic separation of the two API was achieved using C18 (250mm×4.6mm, 5µm) column as stationary phase and mobile phase composed of acetonitrile, trifluoroacetic acid (TFA) (0.2%v/v in water) and methanol in the ratio of 25:15:60 (v/v/v) at flow rate of 1.0 ml min-1. The influence of the CPP was evaluated by various data tables for example, analysis of variance and lack of fit. Interpretation of various plots obtained from the experimental data such as predicted vs. actual, predicted vs. run, pareto chart, perturbation plot and contour plot provided the information on the manner in which each factor affects the critical quality attributes. Conclusion: A design space generated by design of experiments is an evidence for the robustness of the method. The assay results show the applicability of the method for analysis of commercially available tablets.

Application of quality by design approach in RP-HPLC method development for simultaneous estimation of saxagliptin and dapagliflozin in tablet dosage form

A simple, sensitive, precise, accurate and robust high performance liquid chromatographic method has been developed for simultaneous estimation of saxagliptin (SAXA) and dapagliflozin (DAPA) in pharmaceutical formulation. Design of experiments (DoE) was applied for multivariate optimization of the experimental conditions of RP-HPLC method. Risk assessment was performed to identify the critical method parameters. Three independent factors; mobile phase composition, flow rate and column temperature were used to design mathematical models. Central composite design (CCD) was used to study the response surface methodology and to study in depth the effects of these independent factors. Desirability function was used to simultaneously optimize the retention time and resolution of SAXA and DAPA. The optimized and predicted data from contour diagram consisted of acetonitrile and ortho phosphoric acid (0.1%) in the ratio of 50:50 respectively, at a flow rate of 0.98 ml/min and column temperature 31.4 °C. Using these optimum conditions baseline separation of both drugs with good resolution and run time of less than 6 min were achieved. The optimized assay conditions were validated according to ICH guidelines. Hence, the results clearly showed that Quality by design approach could be successfully applied to optimize RP-HPLC method for simultaneous estimation of SAXA and DAPA.

HPTLC Method Development for Simultaneous Estimation of Lidocaine and Prilocaine in Topical Formulation.

HPTLC Method Development for Simultaneous Estimation of Lidocaine and Prilocaine in Topical Formulation.

Formulation and Evaluation of Floating Pulsatile Drug Delivery System of Ibuprofen and Ranitidine Combination

Since rheumatoid arthritis patients experience severe pain, inflammation, and joint stiffness in the early morning hours a pulsatile drug delivery system of a suitable anti-inflammatory drug that is administered at bedtime but release the drug in the early morning would be a promising system. The objective of this work was to develop a pulsatile release tablet containing a combination of ibuprofen and ranitidine HCl from which ibuprofen gets released after a lag time of 6-7 hours. The methodology involves; analytical method development for simultaneous estimation of combination drugs, development of pulsatile release tablet and an in vivo study in rats. Lag time was controlled by coating the rapid release core tablet with different grades and concentrations of HPMC polymer. A floating gastroretentive layer was applied on top to prevent metabolism of ranitidine at the last part of the intestine. Six different formulations were prepared with three different concentrations of HPMCK4M and HPMCK100M. In vitro studies showed that the lag time increased with an increase in both concentration and viscosity of polymers. The formulation where the core tablet was coated with 100 mg of HPMCK100M had an optimum lag time of 6.3 hrs. In vivo study evaluated the ulcer protection of the formulation in four animal groups; first group remained as control, the second group received ibuprofen at a dose of 180 mg/kg, third and fourth groups received a combination of both the drugs but ranitidine in different doses. Based on this research, it can be concluded that pulsatile release of ibuprofen tablets can be successfully formulated by using HPMC polymers. The usage of ranitidine along with ibuprofen reduces the ulcerogenecity of the later.

Simultaneous Estimation of Paracetamol, Ambroxol Hydrochloride, Levocetirizine Dihydrochloride, and Phenylephrine Hydrochloride in Combined Tablet Formulation by First-Order Derivative Spectrophotometry

Paracetamol, ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride are used in combination for the treatment of chronic sinusitis, rhinitis, fever, nasal discharge, sore throat, and wheezing. The present work deals with method development for simultaneous estimation of paracetamol, ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride in tablet formulation by first-order derivative spectrosphotometry. For determination of sampling wavelength, 10 μg/mL of each of paracetamol, ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride was scanned in 200–400 nm ranges and sampling wavelengths were found to be 305.5 nm for paracetamol, 321 nm for ambroxol hydrochloride, 244 nm for levocetirizine dihydrochloride, and 280 nm for phenylephrine hydrochloride in first-order derivative spectrophotometry. In this method, linearity was observed in the ranges of 20–140 μg/mL for paracetamol and 10–70 μg/mL for ambroxol hydrochloride, levocetirizine dihydrochloride, and phenylephrine hydrochloride. The % recovery was within the range between 98 and 102%, and % relative standard deviation for precision and accuracy of the method was found to be less than 2%. The method is validated as per International Conference on Harmonization Guidelines. The method can be successfully applied for the simultaneous analysis of these drugs in pharmaceutical dosage forms.

Spectrophotometric Simultaneous Determination of Salbutamol Sulfate and Ketotifen Fumarate in Combined Tablet Dosage Form by First-Order Derivative Spectroscopy Method

Salbutamol sulfate and ketotifen fumarate are used in combination for the treatment of asthma. The present work deals with method development for simultaneous estimation of salbutamol sulfate and ketotifen fumarate in two-component tablet formulation by first-order derivative spectroscopy. For determination of sampling wavelength, 10 μg/mL of each of salbutamol and ketotifen was scanned in 200–400 nm ranges and sampling wavelengths were found to be 257 nm for salbutamol and 278 nm for ketotifen in first-order derivative spectroscopy. In this method, linearity was observed in the ranges of 5–45 μg/mL for salbutamol and 5–35 μg/mL for ketotifen. The % recovery was within the range between 98 and 102%, and % relative standard deviation for precision and accuracy of the method was found to be less than 2%. The method is validated as per international conference on harmonization guidelines. The method can be successfully applied for the simultaneous analysis of both drugs in pharmaceutical dosage forms.

Stability Indicating Method Development for Simultaneous Estimation of Ezetimibe and Atorvastatin in Pharmaceutical Formulations by RP-HPLC

Simple, specific, economical and precise high performance liquid chromatographic method for the simultaneous determination of ezetimibe and Atorvastatin in API (active pharmaceutical ingredient) and formulation has been developed and validated. Chromatography was carried out at 30°C on a prepacked Zorbax SB C18 (5 mm, 250×4.6 mm) column with the 0.02 M Potassium dihydrogen phosphate: Acetonitrile: Methanol (10:40:50, v/v/v) was used as the mobile phase. The UV detection was carried at 236 nm. The results obtained showed good agreement with the declared contents. Ezetimibe and Atorvastatin separated in less than 10 min with good resolution and minimal tailing and without interference of excipients. The retention times of ezetimibe and Atorvastatin were 5.7 min and 9.1 min, respectively. The method was linear in the range of 5–50 μg/ml for Ezetimibe concentration with a correlation co-efficient 0.9992 and in the range 5–60 μg/ml for Atorvastatin concentrations having correlation co-efficient 0.9994 and the recovery was 99-102%. The method was validated according to ICH guidelines and the acceptance criteria for accuracy, precision, linearity, specificity and system suitability were met in all cases. The proposed method can be used for quantitative determination of Ezetimibe and Atorvastatin combination from API and formulations.