Studije forsirane degradacije amlodipin-besilata i bisoprolol-fumarata primjenom tečne hromatografije hidrofilnih interakcija

Currently, in pharmaceutical analysis, great importance is given to forced degradation studies, which can greatly help to predict the shelf life of the drug, but also for identification of possible degradation products. These studies enable investigation of stability indicating method, then it is used to test the active substances intrinsic/inner molecular stability, as well as defining active substances impurity profiles. In this work forced degradation studies of amlodipine besylate and bisoprolol fumarate either individually and in mixtures, was performed, where the method of hydrophilic interaction liquid chromatography was used, and any possible changes in the concentration of samples were followed. The results showed that the test compounds are sensitive to the tested stress agents, especially amlodipine besylate, and that both of these compounds showed increased stability in the mixture in comparison to individual analysis.U savremenim farmaceutskim analizama danas se veliki značaj pridaje studijama forsirane degradacije, koje mogu u velikoj mjeri pomoći u predviđanju roka upotrebe lijeka, ali i u identifikaciji mogućih proizvoda degradacije. Ove studije omogućavaju ispitivanje specifičnosti stability indicating metode, zatim koriste se za ispitivanje intrinzičke/unutrašnje stabilnosti molekule aktivnih supstanci, kao i za definisanje profila nečistoća aktivnih supstanci. U ovom radu vršena je studija forsirane degradacije amlodipin-besilata i bisoprolol-fumarata pojedinačno i u smješi, gdje se kao metoda koristila tečna hromatografija hidrofilnih interakcija, kojom su se pratile promjene koncentracije ispitivanih uzoraka. Rezultati ispitivanja pokazali su da su ispitivana jedinjenja osjetljiva na većinu ispitivanih stres agenasa, naročito amlodipinbesilat, kao i da su oba ova jedinjenja pokazala veću stabilnost u smješi, nego kad su pojedinačno analizirana

Design of experiments and Derringer's desirability function in optimisation and validation of RP-HPLC method for the analysis of enrofloxacin and its impurities

Using the Design of Experiments methodology (Response-Surface Methodology and Derringer's Desirability Function), a simple, fast and robust RP-HPLC method was developed for the analysis of enrofloxacin (EFC), its impurity A (fluoroquinolonic acid, FQ) and impurity B (ciprofloxacin, CPX). Gradient elution of samples was performed on a Zorbax Eclipse XDB C18 column (150 × 4.6 mm, 3.5 μm) with a mobile phase consisting of 32 mM phosphate buffer pH 3.5-methanol (0 min-19.6% methanol; 15.5 min-19.6% methanol; 29.5 min-80% methanol; 30 min-19.6% methanol; 35 min-19.6% methanol), delivered at a flow rate of 1.5 mL min-1, wavelength of detection 278 nm (for EFX and CFX) and 265 nm for FQ. A good linear response was achieved in the range 15-35 μg mL-1 (EFX) and LOQ-150% for impurities (CFX and FQ). Other validation parameters were also tested: precision, accuracy, sensitivity and robustness. The developed method was shown to be simple, practical and suitable for the analysis of EFC and its impurities (CPX, FQ) in veterinary drugs

Testiranje robusnosti metode tečne hromatografije za određivanje itrakonazola i njegovih nečistoća primenom frakcionog faktorskog dizajna

Test of robustness is a part of the method validation and it is carried out in the end of the method development or in the beginning of the method validation. The purpose of robustness testing is to avoid problems in interlaboratory studies and to define parameters with the greatest impact on the method. The chosen factors are investigated within the range that slightly exceeds the expected variations when the method is transferred from one instrument/laboratory to another. The application of experimental design is recommended to perform robustness testing and two most often utilized - Plackett-Burman and fractional factorial design - are described. Furthermore, the manners of evaluation of factor significance, the methods for calculation of the range of insignificance for significant factors and the procedure for determination of system suitability test parameters are described. Such an approach of robustness testing was applied to the high performance liquid chromatographic method for determination of itraconazole and its impurities B and F. The robustness is tested by using fractional factorial design and 4 factors in 11 experiments were analyzed. After the application and the analysis of all the suggested steps for robustness, the impact of factors on the system responses was evaluated, insignificance ranges were defined and system suitability test limits for suggested method were determined.Test robusnosti metode predstavlja deo validacije metode, a izvodi se na kraju razvoja metode ili na početku validacije. Testiranje robusnosti je uvedeno da bi se izbegli problemi u međulaboratorijskim ispitivanjima i da bi se definisali faktori koji imaju najveći uticaj na metodu. Izabrani faktori ispituju se u intervalu koji blago prevazilazi varijacije koje se očekuju kada se metoda prenosi sa jednog instrumenta/laboratorije na drugi. Za testiranje robusnosti korisno je primeniti eksperimentalni dizajn. U ovom radu opisana su dva najčešće korišćena, Plakett-Burman-ov i frakcioni faktorski dizajn. Takođe, opisani su i načini procene značajnosti faktora, zatim načini izračunavanja intervala neznačajnosti za značajne faktore, kao i postupak određivanja parametara za procenu pogodnosti sistema. Ovakav pristup procene robusnosti metode primenjen je na metodu tečne hromatografije za određivanje itrakonazola i njegovih nečistoća B i F. Robusnost je testirana primenom frakcionog faktorskog dizajna, a analizirana su četiri faktora kroz 11 eksperimenata. Sprovodeći i analizirajući sve predložene korake za robusnost procenjen je uticaj faktora na faktore rezolucije kao odgovore sistema, definisani su intervali neznačajnosti i određeni limiti za proveru pogodnosti sistema predložene metode

Chemometrically assissted optimization and validation of RP-HPLC method for the analysis of itraconazole and its impurities

This paper presents the chemometrically assisted optimization and validation of the RP-HPLC method intended for the quantitative analysis of itraconazole and its impurities in pharmaceutical dosage forms. To reach the desired chromatographic resolution with a limited number of experiments in a minimum amount of time, Box-Behnken design was used to simultaneously optimize some important chromatographic parameters, such as the acetonitrile content in the mobile phase, pH of the aqueous phase and the column temperature. Separation between itraconazole and impurity F was identified as critical and selected as a response during the optimization. The set optimal mobile phase composition was set as acetonitrile/water pH 2.5 adjusted with o-phosphoric acid (50:50, V/V). Separations were performed on the Zorbax Eclipse XDB-C18 4.6 × 150 mm, 5 m particle size column with the flow rate 1 mL min–1, column temperature set at 30 °C and UV detection at 256 nm. The established method was then subjected to method validation and the required validation parameters were tested. For the robustness evaluation, fractional factorial 24–1 design was utilized and factors that might significantly affect the system performance were defined. As other validation parameters were also found to be suitable, the possibility to apply the proposed method for the determination of itraconazole, its impurities B and F in any laboratory under different circumstances has been proven

Quality by Design oriented development of hydrophilic interaction liquid chromatography method for the analysis of amitriptyline and its impurities

This paper presents integration of Quality by Design concept in the development of hydrophilic interactions liquid chromatographic methods for analysis of amitriptyline and its impurities (A, B, C, and F). This is the first time that HILIC method for amitriptyline and its impurities is developed. Using QbD concept, it is possible to design a robust method and incorporate quality directly into its development. QbD concept in combination of Design of Experiments methodology (DoE) enables creation of well-defined design space. In this study, for method optimization a Box-Behnken design was used to test the effect of acetonitrile content, buffer concentration and pH of water phase on critical system responses such as retention factor of impurity A, resolution between impurity B and impurity C, amitriptyline peak asymmetry factor and retention time of last eluted impurity F. The defined mathematical models and Monte Carlo simulations were used to identify the design space. For robustness testing, fractional factorial design was applied. Optimal chromatographic conditions were the analytical column ZORBAX NH2 (250 mm x 4.6 mm, 5 mu m particle size); mobile phase consisted of acetonitrile-water phase (60 mM ammonium acetate, pH adjusted to 4.5 with glacial acetic acid) (92.5:7.5 v/v); column temperature 30 degrees C, mobile phase flow rate 1 mL min(-1), wavelength of detection 254 nm. Finally, method was fully validated and applicability of the method in tablet analysis was confirmed

Study of forced degradation of amlodipine besylate and bisoprolol fumarate by application of hydrophilic interaction liquid chromatography

Currently, in pharmaceutical analysis, great importance is given to forced degradation studies, which can greatly help to predict the shelf life of the drug, but also for identification of possible degradation products. These studies enable investigation of stability indicating method, then it is used to test the active substances intrinsic/inner molecular stability, as well as defining active substances impurity profiles. In this work forced degradation studies of amlodipine besylate and bisoprolol fumarate either individually and in mixtures, was performed, where the method of hydrophilic interaction liquid chromatography was used, and any possible changes in the concentration of samples were followed. The results showed that the test compounds are sensitive to the tested stress agents, especially amlodipine besylate, and that both of these compounds showed increased stability in the mixture in comparison to individual analysis

Investigation of the retention mechanisms of amlodipine besylate, bisoprolol fumarate, and their impurities on three different HILIC columns

Using theoretical models, retention mechanisms of amlodipine besilate and its impurities D, E, and F were tested, as well as bisoprolol fumarate and its impurities A, C, K, and L. Appropriate theoretical retention models for tested compounds were used, which enabled the determination of retention mechanisms responsible for the separation of tested compounds on three different HILIC column: silica, amino, and diol. In order to describe the effect of ratio of aqueous and organic portion of the mobile phase on the retention behavior of tested compounds, as well as the consideration of shares of partition and adsorption processes in HILIC retention mechanism in all three columns, partitioning and adsorption retention theoretical models were calculated. For this purpose, the water content was varied in the range from 4% to 29%, corresponding to the HILIC region of mobile phase composition. During the experiment, values of other factors of mobile phase were maintained at a constant level. Regression coefficients of partition and adsorption retention models and the corresponding determination coefficients were determined (R-2). Partitioning models for all tested compounds on amino column had higher R-2 values, while adsorption models for all compounds tested on silica and diol column had higher R-2 value. [GRAPHICS]

Monitoring of bisoprolol fumarate stability under different stress conditions

Stability studies of drugs by stress study is a very important process which is done by treating the study drug with different stress agents, with the aim to define the critical factors affecting the stability of the drug, to accurately define the storage conditions of the drug, as well as to identify the resulting degradation products. In this paper, stress studies of bisoprolol fumarate were performed, in order to examine what are the stress agents that affect its stability. For the analysis previously optimized and validated HILIC method was used It was demonstrated that oxidative stress agent has the largest effect on the degradation of bisoprolol fumarate, and then the acid and base stress agent successively. Water, as a neutral medium, and light had no significant effect on the stability of bisoprolol fumarate. During degradation under acid conditions impurity A was formed and it was confirmed with UPLC/MS/MS method. In order to more clearly define the processes of degradation, kinetic studies of degradation of bisoprolol fumarate have been carried out, in order to determine the order of the reaction rate of degradation and degradation half-time, which provided clearer definition of the mechanism of degradation

STRESS DEGRADATION OF LISINOPRIL DIHYDRATE IN DIFFERENT AQUEOUS MEDIA

Lisinopril is an antihypertensive drug from the ACE inhibitor group. It is often used in practice and, in oral administration is characterized by very low percentage of resorption (~25%). Therefore, it is important to ensure the stability of this drug, since any degradation leads to a decrease in the percentage of resorption and an increased risk of sub-dosage of patients. In accordance with modern trends of pharmaceutical analysis, the chemical stability of lisinopril under the influence of various aqueous media was tested: distilled water, 0.1M HCl, 0.1M and 0.01M NaOH and 3% hydrogen peroxide solution. In order to clearly define the effect of elevated temperature on the stability of this drug, tests were carried out at 25 °C and 50 °C. High-pressure liquid chromatography was used as the method for stability testing. The obtained results clearly define that lisinopril is the most sensitive to hydrolysis in the base environment, where at 25 °C, in the first minute, it degrades ~100%. In this paper, kinetic parameters (degradation constant rate, half-life of degradation, order kinetics,  and activation energy) were also determined, which enabled clear definition of the stability and identification of degradation products of lisinopril under the influence of various stress agents. In the degradation of lisinopril in neutral and acidic medium, impurity D is identified as the main degradation product

Design of Experiments in Optimization and Validation of a Hydrophilic Interaction Liquid Chromatography Method for Determination of Amlodipine Besylate and Bisoprolol Fumarate

In this paper chemometrically-assisted optimization and validation of hydrophilic interaction liquid chromatographic (HILIC) method for the quantitative analysis of amlodipine besylate (AB) and bisoprolol fumarate (BF) in pharmaceuticals are presented. In this study, the development of HILIC method for AB and BF separation was performed by a systematic approach which included Design of Experiments (DoE) methodology. First, in method optimization, a central composite design was applied. Influence of the acetonitrile content in the mobile phase, pH of the aqueous phase, and concentration of ammonium acetate on selected chromatographic responses was investigated. Optimal chromatographic conditions in terms of adequate separation and minimal analysis duration were set by graphical optimization overlapping the contour plots as: acetonitrile-water phase (10mM ammonium acetate, pH 4.0 adjusted with glacial acetic acid) (92:8, v/v), column Luna HILIC 200 A, 150mmx4.6mm, 5 mu m particle size, column temperature 30 degrees C, flow rate 1mL min(-1), and UV detection at 230nm. After this step, method robustness was tested using a 2(5-1) fractional factorial design. Statistical and graphical evaluation of results confirmed robustness of the method and then the method was fully validated. Finally, the proposed method was successfully applied for AB and BF determination in pharmaceuticals