engleski

HPLC-DAD-MS stability-indicating method for proposed fixed-dose combination of azathioprine and folic acid

With the rise of fixed-dose combinations (FDC) in the pharmaceutical industry, there is an increasing need for analytical support for new formulations. The chemical compatibility and physicochemical characteristics of active pharmaceutical ingredients (API) need to be investigated, as well as analytical methods for quality control developed. To improve patient compliance to therapy of inflammatory bowel disease, a FDC of immunosuppressive azathioprine (AZA) and nutrient folic acid (FA) is proposed. The aim of this work was to ascertain whether AZA and FA are compatible in a FDC by means of forced degradation study and degradant characterization, as well as to develop a method for determination of FA, AZA and their impurities. Measurements were made on an Agilent 1100 system coupled with LC/MSD Trap VL mass spectrometer (Agilent, USA). Zorbax SB- C8 column (Agilent, USA) 150 × 4.6 mm, particle size 5 µm, thermostated at 35 °C, was used as stationary phase, while the mobile phase consisted of 0.1% HCOOH (v/v) in ultrapure water and acetonitrile (mobile phase components A and B, respectively), using a gradient program. Analyte detection and quantification was carried out at wavelengths of 285, 320 and 368 nm, while MS was used to identify the degradants. Standards (FA, AZA and 4 of their impurities) and sample (mixture of FA and AZA tablet dosage forms) were dissolved in 30 % DMF (v/v) and sonicated for 15 min ; the sample was additionally centrifuged for 10 min. Forced 1 / 2 degradation studies were conducted on each API individually, their mixture and sample. Acid and base hydrolyses were carried out using 0.1 M HCl and NaOH at 60 °C for 6 h, respectively. Photolytic degradation was carried out for 3 days for solids and 30 min for solutions and suspensions. Thermal degradation on solids was carried out at 70 °C for 5 days, while oxidative degradation was carried out using 3 % H2O2 at 60 °C for 30 min. Method validation was carried out according to ICH guidelines. The developed method was able to successfully resolve all of the analytes, as well as observed degradation products, in 27 min. No significant increase in degradation was observed in FA and AZA mixtures as opposed to each individual API. The method was validated and showed good linearity (R2 ≥ 0.9987), precision (RSD ≤ 5.19 %) and accuracy (ranging from 94.79 to 110.49 %). Finally, it was applied to the sample, successfully determining FA, AZA and the impurities above LOQ (FA impurity A and AZA impurity G). In this work, a HPLC-DAD-MS stability- indicating method for determination of FA, AZA and their impurities was developed and successfully applied to the sample. Seeing that no significant difference in degradation of each drug individually and their mixture was observed, it can be presumed that they are chemically compatible and further studies shall be conducted. This work has been supported in part by the Croatian Science Foundation under the project number [UIP-2017- 05-3949].

azathioprine ; folic acid ; stability-indicating

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