Аспекти розробки екобезпечної методики визначення азилсартану медоксомілу методом високоефективної рідинної хроматографії

A novel, simple, rapid, and highly selective stability indicating RP-HPLC method was developed and validated for simultaneous estimation of azilsartan medoxomil (AZL) and amlodipine besylate HCl (AMLO) in tablet dosage form having strength of 20 mg and 2.5 mg, respectively. The effective chromatographic separation was achieved on a Phenomenex luna ODS C18 (15 cm X 4.6 mm internal diameter, 3.5 μm Particle size) with a mobile phase composed of phosphate buffer (pH-2.5) adjusted with ortho phosphoric acid : acetonitrile in the ratio of 60:40 v/v. The mobile phase was pumped using an isocratic HPLC system at a flow rate of 0.7 mL/min with injection volume 20μl and quantification of the analytes was done at detection wavelength 254 nm. The retention times were found to be 5.918 min and 14.901 min for AMLO and AZL, respectively. The proposed HPLC method was validated with respect to linearity, ranges, precision, accuracy, specificity, robustness, LOD, and LOQ as per ICH Q2 (R1) guideline. Calibration plots were linear over the concentration range of 75-125 µg/mL and 600-1000 µg/mL with correlation coefficients 0.9966 and 0.9948 for AMLO and AZL, respectively. Forced degradation studies were performed using hydrolysis, oxidation, photolytic, and thermal degradation conditions with good resolution between the degradants and analytes. Degradation products resulting from the stress studies did not interfere with the detection of AMLO and AZL, thus the proposed method is sensitive and stability-indicating. The validated HPLC method was successfully applied to the analysis of AMLO and AZL in tablet dosage form.

Aims: To develop methods with complete validation according to ICH guidelines and to be applied for the determination of both drugs in laboratory prepared mixtures and in synthetic tablets.
 Study Design: Ultra high performance liquid chromatography (UHPLC), High performance thin layer chromatography (HPTLC) and visible spectrophotometric methods are developed for determination of amlodipine besilate and azilsartan medoxomil in laboratory-prepared mixtures and in synthetic tablets.
 Methodology: Two techniques have been developed for the simultaneous determination of amlodipine besilate and azilsartan medoxomil in pure form and synthetic tablets. The first was UHPLC in which separation was achieved on a C18 column using 0.1% o-phosphoric acid - acetonitrile - methanol (60:10:30, by volume) as mobile phase with detection at 243nm. The second was HPTLC where separation was performed on silica gel 60 F254 plates using chloroform- tolune-methanol-glacial acetic acid (7: 1.5: 1.5: 0.5 by volume) as a developing system and UV detection at 243nm. In addition, visible- spectrophotometric method was developed for determination of amlodipine besilate in presence of azilsartan medoxomil through formation of yellowish orange colored product after reaction of amlodipine besilate with anisaldehde in acid medium with λmax at 443 nm.
 Results: UHPLC method was linear over the concentration ranges of 2-20 μg/ mL and 4-40 μg/ mL while HPTLC method was linear over the concentration ranges of 0.2 -4.0 μg/ spot and 0.5-8.0 μg/ spot for amlodipine besilate and azilsartan medoxomil, respectively. The visible spectrophotometric method was found to be valid over the concentration range of 10–80 μg/mL for amlodipine besilate.
 Conclusion: The proposed three techniques are rapid, accurate and precise, thus can be effectively applied for the routine estimation of both drugs in bulk and in their combined formulations.

Expert system for the selection of high-performance liquid chromatographic methods in pharmaceutical analysis : Validation of the rules for the selection of the mobile phase

Compound danshen dripping pills affect the pharmacokinetics of azisartan by regulating the expression of cytochrome P450 2B1, 2C6, and 2C11 in rats

Application of green methodology to pharmaceutical analysis using eco-friendly ethanol-water mobile phases

Objectives: A selective, precise and accurate RP-HPLC stability indicating assay method has been developed for the simultaneous estimation of Azilsartan medoxomil and Cilnidipine in tablet dosage form. Materials and Methods: The efficient chromatographic separation of drug was achieved by using C18 (150mm×4.6mm, Agilent 5μm) Column at ambient temperature. Mobile phase contains triethylamine buffer (pH 3.5 adjusted with ortho-phosphoric acid) and acetonitrile (40:60 V/V). Flow rate of mobile phase 1.0 ml/min using isocratic mode .Wavelength selected at 249 nm by using photo diode array detector. Results: The retention time of Azilsartan medoxomil peak 1, Azilsartan medoxomil peak 2 and Cilnidipine were noticed to be 2.16 min, 3.90 min and 9.52 min respectively. The linearity range for Azilsartan medoxomil and Cilnidipine were found to be 50 -150 μg/ml and 12.5-37.5 μg/ml and percent recoveries were noticed to be 99.27±0.58 and 98.65±0.49 respectively. Various stress testing conditions such applied to the drug ingredients and drug formulation. The degradants and drugs efficiently separated by using enhanced chromatographic conditions. The developed method was validated as per recommendation parameters of International council on harmonization guideline Q2(R1). Conclusion: The validation parameters stated that the drug substances were efficiently separated from its degradants and developed method can be routinely applied for the simultaneous estimation of Azilsartan medoxomil and Cilnidipine in tablet formulation in a laboratory.

A combination of Azilsartan Medoxomil and Cilnidipine is prescribed in the treatment of Hypertension. An accurate and precise high‐performance liquid chromatographic method has been developed for the estimation of Azilsartan Medoxomil and Cilnidipine in a combined tablet dosage form. Symmetry C18 (250 × 4.6 mm, 5 μm) column was used as the stationary phase, and methanol:water (85:15 v/v; pH 3.5) was used as the mobile phase. The method was linear in the concentration range of 4–20 μg/ml for Azilsartan Medoxomil and 1–5 μg/ml for Cilnidipine with a correlation coefficient ( r 2 ) 0.995 for Azilsartan Medoxomil and 0.995 for Cilnidipine. The proposed method was validated with respect to linearity, accuracy, precision, and robustness as per International Council on Harmonization Q2 (R1) guideline. A forced degradation study was performed to find out the intrinsic stability of the molecules. The degradation products were well resolved from the drug peak and did not interfere with the analysis. The method was successfully applied for the analysis of Azilsartan Medoxomil and Cilnidipine in combined tablet formulation.

A stability-indicating reversed-phase high-performance liquid chromatography method using a photodiode array detector has been developed for simultaneous estimation of chlorthalidone (CLR) and azilsartan medoxomil (AZL) in combined solid dosage form. The method was developed based on statistical design of experiments (DoE) followed by optimization using the response surface methodology. Separation was achieved on a double end-capped C18 column (150 mm × 4 mm, 5 µm). The effects of % acetonitrile (v/v) and buffer salt concentrations on the retention time of the two drugs and on their resolution were investigated and optimized. A robust design space was created by the overlay contour plot method. The optimum chromatographic condition within the design space was found to be isocratic mobile phase consisting of 10 mM Tris(hydroxymethyl)aminomethane buffer (pH 7.7) and acetonitrile at ratio of 60:40 (v/v) with flow rate of 1 mL min−1 for 7 min. The retention times of CLR and AZL were found to be 2.6 and 4.9 min, respectively. The method was validated according to International Conference on Harmonization (ICH) and Food and Drug Administration (FDA) guidelines, and various validation parameters were determined. Forced degradation studies were also carried out in acid, base, oxidation, and reduction media with a view to establishing the specificity and stability-indicating property. The practical applicability of the method was confirmed by determining CLR and AZL in combined dosage form. This reliable and validated stability-indicating method for simultaneous estimation of CLR and AZL is available for routine analysis in the pharmaceutical industry as well as research laboratories.

The recently approved angiotensin II receptor blocker, azilsartan medoxomil (AZL), was determined spectrophotometrically and spectrofluorimetrically in its combination with chlorthalidone (CLT) in their combined dosage form. The UV-spectrophotometric technique depends on simultaneous measurement of the first derivative spectra for AZL and CLT at 286 and 257 nm, respectively, in methanol. The spectrofluorimetric technique depends on measurement of the fourth derivative of the synchronous spectra intensities of AZL in presence of CLT at 298 nm in methanol. The effects of different solvents on spectrophotometric and spectrofluorimetric responses were studied. For, the spectrofluorimetric study, the effect of pH and micelle-assisted fluorescence enhancement were also studied. Linearity, accuracy, and precision were found to be satisfactory over the concentration ranges of 8–50 μg mL−1 and 2–20 μg mL−1 for AZL and CLT, respectively, in the spectrophotometric method as well as 0.01–0.08 μg mL−1 for AZL in the spectrofluorimetric method. The methods were successfully applied for the determination of the studied drugs in their co-formulated tablets. The developed methods are inexpensive and simple for the quality control and routine analysis of the cited drugs in bulk and in pharmaceuticals.

Objective: A quick simultaneous separation and quality control assay of two antihypertensive representatives, Azilsartan (AZIL) and Cilnidipine (CLIN) in bulk and tablet formulation was developed and validated using a Reverse phase (RP) HPLC method within a run time of 10 min. Methods: All chromatographic separations of AZIL and CILN were operated on a “Supelco C18 column (250 × 4.6 mm, 5 μ)”, using a mobile phase of Na2SO4 (0.1 M, pH 4.0): methanol at 60:40 (v: v) ratio and the samples were analyzed at 239 nm. Stability assessments of AZIL and CILN were carried out as per the ICH Q1A (R2) regulation. The methodology for determining AZIL and CILN in bulk and formulations tablets was verified by adhering to International Conference on Harmonization (ICH) recommendations. Results: Retention times of AZIL and CILN samples were 4.023 and 5.732 min, respectively, indicating a quick elution time. Over the tested range of 20–60 µg/ml for AZIL and 5–15 µg/ml for CILN determination, calibration curves have displayed linearity and satisfactory results. LOD of AZIL and CILN are 0.083 µg/ml and 0.056 µg/ml, respectively. The approach suggested herein has satisfactory precision (RSD: 0.1013% for AZIL and 0.4944% for CILN) and accuracy (recovery: 99.20 to 100.34 % for AZIL and 100.17 to 101.59 % for CILN). Furthermore, the approach has also been shown to be effective in detecting degradants of AZIL and CILN and resolving them with high resolution. Conclusion: This approach is shown to be acceptable for the accurate quality control assay of two antihypertensive representatives, AZIL and CLIN in both bulk and tablet formulation.

Simultaneous determination of azilsartan and chlorthalidone in rat and human plasma by liquid chromatography-electrospray tandem mass spectrometry.

A combination of Azilsartan Medoxomil and Cilnidipine is prescribed in the treatment of Hypertension. The present work represents an accurate and precise high-performance thin layer chromatographic method for the estimation of Azilsartan Medoxomil (AZL) and Cilnidipine (CLN) in combined tablet dosage form. Pre-coated silica gel- G60 F254 aluminum sheet (100 × 100 mm, 0.2 mm layer thickness) were used as stationary phase and Ethyl Acetate: Toluene: Glacial Acetic Acid (5: 4.9: 0.1 %v/v/v) in the mixture was used as mobile phase. The method was linear in the concentration range of 200 - 2000 ng/band for AZL and 50 -500 ng/band for CLN with a correlation coefficient (r2) of 0.996 for AZL and 0.999 for CLN. The proposed method was validated with respect to linearity, accuracy, precision, and robustness as per ICH Q2 (R1) guideline. A forced degradation study was performed to assess the stability indicating the nature of the method. All the degradant peaks were well resolved from the peak of the drug without any interference. The method was successfully applied for the analysis of AZL and CLN in combined tablet formulation.

A sensitive and robust method for determination and quantification of potential genotoxic impurities in sartans has been developed. These impurities need to be controlled at trace levels during quantification in drug substances and drug products for safe consumption. Recent regulatory requirements also suggested the need to have highly sensitive analytical method for trace level quantification of nitrosamine impurities. In this paper, we have described a simple, rapid and sensitive liquid chromatography-mass spectrometry method for six potential genotoxic nitrosamine impurities: N-Nitroso dimethyl amine (NDMA), N-Nitroso diethyl amine (NDEA), N-Nitroso Ethyl Iso propylamine (NIPEA), N-Nitroso-Nmethyl-4-aminobutyric acid (NMBA) N-Nitroso diisopropylamino (NDIPA) and N-Nitroso dibutyl amine (NDBA) in Azilsartan (AZL), Valsartan (VAL), Telmisartan (TEL), Olmesartan (OLM), Losartan (LOS) and Irbesartan (IRB) with a limit of quantification of less than 0.003ppm. Chromatographic separation is achieved using Poroshell HPH- C18, 150 × 4.6mm, 2.7μm column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at a flow rate of 0.5mL/min using gradient mode of elution at a total run time of 20min. Six nitrosamine impurities are ionized and quantified in positive mode of atmospheric pressure chemical ionization using multiple reaction monitoring. As per ICH guidelines, method validation is performed and evaluated the limit of quantification and detection and found to give good S/N ratios with good linearity range of 0.003-0.045ppm with regression coefficient > 0.999 for all the six nitrosamine impurities. Method recoveries are also established using three-step sample preparation and are found to be satisfactory within 80-120%. The single method can be used routinely applied for the detection of nitrosamines in AZL, VAL, TEL, OLM, LOS and IRB.

Strategic approach for method selection in high-performance liquid chromatography

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis : II. Optimization for selectivity in normal-phase systems