LC Method For Simultaneous Determination Research Articles

Inside Back Cover: Development of a validated liquid chromatography–diode array detection method for simultaneous determination of olanzapine and fluoxetine in their combined formulation: Application to greenness assessment

DOI: 10.1002/sscp.202200013 The cover picture shows the development of a greener LC method for simultaneous determination of olanzapine and fluoxetine in a fixeddose combination. This current AES approach assessed method greenness corresponding to reagents and instruments used. Afterward, risk assessment and DoE based robust method was developed and validated to match the method intent for routine application purpose.

AQbD-Oriented Development of a New LC Method for Simultaneous Determination of Telmisartan and Its Impurities

Analytical quality by design (AQbD)-oriented liquid chromatographic method development for determination of telmisartan and its impurities A, C, and 1 is determination is presented. Step-by-step process was conducted in order to define reliable design space. At the beginning, critical process parameters with the highest influence on method performance were defined: acetonitrile content in the first (ACN 1) and second (ACN 2) gradient step and time (t 2) the second gradient step. These factors were varied according to Box–Behnken plan of experiments and their influence on retention times of impurities A and C, S value between telmisartan and impurity 1 and peak capacity were followed. In this way, the relationship between the critical process parameters and critical quality attributes was established. The obtained mathematical models and Monte Carlo simulations were used to identify the design space. Fractional factorial design was applied for experimental robustness testing, and the method was validated to verify the adequacy of selected optimal conditions. Finally, all validation parameters were tested, and adequacy of the method was confirmed. Applicability as a routine method was confirmed by analysis of commercially available tablets.

A validated stability-indicating LC method for simultaneous determination of quinapril and hydrochlorothiazide in pharmaceutical samples

Abstract A stability-indicating liquid chromatographic method was developed and validated for simultaneous determination of quinapril and hydrochlorothiazide in drug substances and dosage forms. Chromatographic separation of quinapril, hydrochlorothiazide and its degradation products was achieved on a RP-18 column, using acetonitrile and phosphate buffer (pH 4.6) as mobile phase in a gradient mode and detection at 216 nm. Stress testing was performed under hydrolytic, oxidative, thermal and photolytic conditions. The degradation products were well resolved from main peaks, proving the stability-indicating power of the method. The assay was linear for quinapril and hydrochlorothiazide concentrations of 40–200 µg mL−1 and 25–125 µg mL−1, respectively. The developed method was selective, accurate and precise for quinapril and hydrochlorothiazide determination. This method was used to quantify both drugs in combined commercial tablets. The results showed that the proposed method was found to be suitable for quantitative determination and the stability study of quinapril and hydrochlorothiazide in pharmaceutical samples.

Development and validation of a stability-indicating LC method for simultaneous determination of related compounds of guaifenesin, terbutaline sulfate and ambroxol HCl in cough syrup formulation

A new liquid chromatographic method has been developed and validated for the determination of terbutaline sulfate (TLS), guaifenesin (GFN) and ambroxol HCl (AML), for its potential impurities in drug substances and drug products. Efficient chromatographic separation was achieved on X-Terra RP-18 column with a simple mobile phase combination containing a gradient mixture of solvents A and B at a flow rate of 1.0mLmin−1 and quantitation was carried out using ultraviolet detection at 222nm with column temperature of 35°C. The resolution between TLS, GFN and AML, its associated impurities was found to be greater than 1.5. Regression analysis shows an r value (correlation coefficient) greater than 0.998. This method was capable to detect all the process impurities of TLS, GFN and AML, at a level below 0.015% with respect to a test concentration of 0.125, 5.0 and 1.5mgmL−1, respectively. The % RSD for the inter-day and intra-day precisions for all the impurities of TLS, GFN and AML were found to be less than 3.0. The method has shown good, consistent recoveries. The drugs were subjected to stress conditions of acid, base, water hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by international conference on harmonization (ICH).

An Ultra-Sensitive LC Method for Simultaneous Determination of Rosuvastatin, Alprazolam and Diclofenac Sodium in API, Pharmaceutical Formulations and Human Serum by Programming the Detector

Here, we report a rapid and efficient liquid chromatographic method with UV detection for the simultaneous determination of rosuvastatin, alprazolam and diclofenac sodium in API, pharmaceutical formulations and human serum employing a reversed phase Bondapak C18 (25 cm, 0.46 cm, 10 μm) column at room temperature using methanol: water (80:20 v/v) and pH adjusted to 2.9 with 85% o-phosphoric acid. Separation was achieved within a time span of 6 min. The detector response was monitored at 240 nm and the flow rate was set at 1.0 mL.min-1. Various LC parameters were optimized and developed method was validated as per ICH (2006) guidelines. Linear calibration range was determined to be 0.02-0.64, 0.125-4.0 and 0.05-1.6 μg.mL-1 and detection and quantitation limits were found to be 4.0, 17.0, 7.0 and 12.0, 52.0, 22.0 ng.mL-1 for rosuvastatin, alprazolam and diclofenic sodium respectively. Method was linear for all analytes with correlation coefficients >0.998. The intra-day and inter-day accuracy and precision of the assay were in acceptable range of 98.21-101.91% recovery and 0.20-2.07% RSD. The sensitivity of the method was enhanced when the chromatograms were analyzed by programming the detector at individual λmax of 244, 222 and 284 nm for rosuvastatin, alprazolam and diclofenac sodium respectively. High peak intensity was observed on injecting same sample concentration after the detector was programmed. Linearity was obtained even at lower concentrations of 0.02-0.64, 0.05-1.60 and 0.02-0.64 μg.mL-1. LOD and LOQ values shifted down to 3.0, 6.0, 1.0 and 9.0, 19.0, 3.0 ng.mL-1 respectively. Recovery was found to be in the range of 98.32-101.87% and inter-day and intra-day precision was less than 2.10%. The proposed method was found to be robust and successfully employed for the determination of studied drugs in commercial formulations and human serum without interference of excipients or endogenous components of serum.

A Validated Micellar Lc Method For Simultaneous Determination of Furosemide, Metoprolol And Verapamil in Human Plasma

An accurate and precise micellar LC method coupled with UV and fluorimetric detectors was developed and validated for the simultaneous analysis of furosemide, metoprolol and verapamil in human plasma. The total analysis time was 25 min (12 min for sample preparation and 13 min for drug separation). All drugs possessed linear behavior (r > 0.999 for calibration curves) in their therapeutic concentrations. The mean drug recoveries were 101.9, 100.1 and 100.2% for furosemide, metoprolol and verapamil, respectively. The accuracies (relative error %) were less than 15% for all drugs. Intra- and inter-day precisions (RSD%) were less than 15% and the stability data were acceptable according to the US FDA guideline for bioanalytical method validation.

A Validated Stability-Indicating LC Method for Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Samples

A stability-indicating liquid chromatographic method was developed and validated for the simultaneous determination of enalapril maleate and hydrochlorothiazide in drug substances and dosage forms. The method was developed using a RP-18 column with mobile phase containing methanol - tetrahydrofuran - phosphate buffer (pH 2.2; 0.01M) (32:5:63, v/v/v) at a flow rate of 1.0 mL min-1 and UV detection at 210 nm. Enalapril and hydrochlorothiazide were subjected to hydrolytic, oxidative, thermal and photolytic stress conditions. The degradation products were well resolved from main peaks, proving the stability-indicating power of the method. The assay was linear for enalapril and hydrochlorothiazide concentrations of 40-140 μg/mL and 100-350 μg/mL respectively. The developed method was selective, accurate and precise for enalapril and hydrochlorothiazide determination. This method was successfully applied for determination of enalapril and hydrochlorothiazide in combined commercial tablets. The proposed method was found to be suitable for quantitative determination and the stability study of enalapril and hydrochlorothiazide in pharmaceutical preparations. Keywords: Enalapril, Hydrochlorothiazide, Pharmaceutical preparations, Stability-indicating, Stress testing, Validation, LC Assay, Limits of Detection, Limit of Quantification, Thermal Degradation

A Validated LC Method for Simultaneous Determination of Three Major Components in Entada phaseoloides

A high performance liquid chromatography method was developed for the simultaneous determination of three major active constituents in Entada phaseoloides, namely phaseoloidin (1), entadamide A (2), entadamide A-β-d-glucopyranoside (3), respectively. The samples were separated on an Aglient Eclipse XDB-C18 column with gradient elution of acetonitrile and 0.3% phosphoric acid (v/v) at a flow rate of 1.0 mL min−1 and detected at 280 nm. The three target compounds were completely separated within 10 min. All calibration curves showed good linearity (r > 0.9999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and RSD values were less than 1.04%. The recoveries were between 97.15 and 101.95%. The method was successfully applied to the analysis of three compounds in 22 commercial samples of E. phaseoloides. The results indicated that the developed LC assay was readily utilized as a quality control method for E. phaseoloides.

A Validated LC Method for Simultaneous Determination of Phenolic, Coumarin and Phthalide Compounds in the Ethanolic Extract of Angelica tenuissima

In this study, an LC method for the simultaneous determination of six bioactive compounds from Angelica tenuissima, namely chlorogenic acid, ferulic acid, Z-ligustilide, nodakenin, decursin and decursinol angelate was developed and validated. Chromatographic analysis was carried out on a C18 column with a mobile phase consisting of 0.1% formic acid, methanol and acetonitrile at a flow rate of 0.8 mL min−1 and the effluent from the column was monitored by UV detector at 325 nm. The excellent linear behavior was observed over the investigated concentration range for reported compounds. The intra- and inter-day precision over the concentration range of compounds were lower than 1.7% (as relative standard deviation), and accuracy was between 97.2 and 106.0%. These results showed that the developed method is accurate, reproducible, and consequently applicable for the quantitation of bioactive components from the ethanolic extract of Angelica tenuissima Nakai.

Development and Validation of an LC Method for Simultaneous Determination of Amiloride Hydrochloride and Hydrochlorothiazide in Human Urine

An HPLC method with photometric detection has been developed for determination of a binary mixture of amiloride hydrochloride and hydrochlorothiazide in human urine using chlorthalidone as the internal standard. Reversed-phase chromatography was performed at room temperature on a cyanopropyl column with the mobile phase consisting of a 10 mM KH2PO4 solution (pH 4.5)-methanol (70 + 30, v/v) at a flow rate of 1 ml/min. The detector was set at 214 nm. The total analysis time was 10 min. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability, and re-injection reproducibility. The procedure shows good accuracy, repeatability, and selectivity. Moreover, the method was applied directly to urine that had not undergone prior treatment. The intra- and interday coefficients of variation for all compounds were below 4%, and the method was highly accurate, with a relative error for all compounds that was below 8%. No interference from endogenous compounds in urine samples was found. The proposed method, which is rapid, simple, and does not require any separation steps, has been successfully applied to the assay of human urine containing amiloride hydrochloride and hydrochlorothiazide.

A Validated RP–LC Method for Simultaneous Determination of Losartan Potassium and Amlodipine Besilate in Pharmaceutical Preparations

A simple RP–LC method for simultaneous quantification of losartan and amlodipine and separation of their degradation products has been developed. For this purpose we tested appropriated mobile phase pH range, flow rate, temperature and different columns. The method was validated with an ODS column. A gradient of acetonitrile and phosphate pH 3.0 buffer was utilized as mobile phase. The linearity was determined at 50–150% level. Individual recoveries at 70–130% level ranged from 98.8 to 100.5% for losartan and 96.4–101.2% for amlodipine. The robustness was also evaluated. Although losartan has much higher quantities than amlodipine in commercial tablets, this method allowed simultaneous quantification for both drugs.

Development and Validation of LC Method for Simultaneous Determination of Two Binary Mixtures Containing Indapamide

A new sensitive, simple, rapid, and precise RP LC method with hydrochlorothiazide as internal standard has been developed for resolving two binary mixtures, perindopril with indapamide and captopril with indapamide, in pharmaceutical formulations. The drugs were separated at room temperature on a 250 mm × 4.6 mm, 5-μm particle, cyanopropyl column with 10 mm KH2PO4, pH 6.0-methanol 55:45 (v/v) as mobile phase at a flow rate of 1 mL min−1. Detection was at 210 nm. Factors affecting the separation process were studied and optimized. The linearity, accuracy, and precision of the method were good, and the method was successfully applied to the determination of the two binary combinations in synthetic mixtures and commercial pharmaceutical products.

Development and Validation of an LC Method for Simultaneous Determination of Ascorbic Acid and Three Phenolic Acids in Sustained Release Tablets at Single Wavelength

A simple, rapid and sensitive column liquid chromatographic method was developed and validated to measure simultaneously the amount of ascorbic acid and phenolic acids at single wavelength (240 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new sustained release tablet formulation and its subsequent stability studies. A combined isocratic and linear gradient reversed-phase LC method was carried out at 240 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.042 and 0.150 mg mL−1 for ascorbic acid, 0.084–0.250 mg mL−1 for chlorogenic acid, 0.053–0.360 mg mL−1 for caffeic acid, and 0.016–0.250 mg mL−1 for ferulic acid (r > 0.99 for all analytes) were established. The recovery of the active ingredients from the samples was at the range of 92.3–102.9%. Intra- and inter-day precisions were less than 2.5%. The limits of detection and quantification were 8 and 24 μg mL−1 for ascorbic acid, 18 and 54 μg mL−1 for chlorogenic acid, 37 and 112 μg mL−1 for caffeic acid, and 11 and 34 μg mL−1 for ferulic acid. The determination of the four active ingredients was not interfered by the excipients of the products. Samples were stable in the release mediums (37 °C) at least for 12 h.

A rapid and selective LC method for simultaneous determination of cyclosporin a and its major metabolite (AM1) in human serum at room temperature

A simple and highly selective isocratic reverse-phase high performance liquid chromatography (RP-LC) method at room temperature is developed in order to determination of Cyclosporine A (CyA) and its major metabolite (AM1) in serum samples of kidney transplanted patients. The method uses a phenyl column stationary phase, acetonitrile–water–methanol 47:50:3 as mobile phase and 215 nm detector wavelength, at room temperature. The solid phase extraction procedure using cyano disposable extraction column was carried out to separtate the CyA and AM1 with recovery 99±6 and 98±10, respectively. A linear correlation was found at the range of 40–1000 ng ml −1 for CyA and 25–500 ng ml −1 for AM1. The average intra and inter-day variations were 5.03 and 7.89% for CyA, 5.92 and 8.12% for AM1, respectively. The detection limit of 20 ng ml −1 was found for CyA and 12.5 ng ml −1 for AM1. Also, the clinical application of the method using serum concentration against time profile from kidney transplantated patients is reported.