Development and optimization of a LC-MS/MS compatible method for quantification of losartan, hydrochlorothiazide and their impurities using AQbD approach and measurement uncertainty evaluation

Abstract

The concept of Analytical Quality by Design (AQbD) comes as a more robust, economical, and scientifically based alternative for analytical development, to the detriment of OFAT (one factor at a time). This new understanding applicable to analytical development is recommended since regulatory flexibility can be achieved and ensure more reliable results throughout the life of the product. This new approach was applied to develop an analytical procedure indicative of stability for a pharmaceutical product of association of Losartan Potassium and Hydrochlorothiazide, considered a potential first line for the treatment of hypertension. The first stage of the analytical development consisted of defining analytical target profile (ATP), follow by a bibliographic survey of the physicochemical properties of the molecules in question to define an initial method. After defining the initial analytical conditions, statistical tools for design of experiments (DoE) were used for the screening and optimization steps. In the screening stage, the Plackett-Burman design was chosen, using 11 factors and 2 levels, through which it was possible to evaluate numerous variables and determine their significance in relation to the responses. Next, optimization was carried out with the experimental design of a central composite with 4 factors and 5 levels, which allowed modeling a complex response surface and evaluating the phenomena of interactions between the factors. Thus, the optimized analytical conditions were defined, considering a 0.3% formic acid gradient as eluent A and a mixture of acetonitrile and methanol (80:20) as eluent B, X-Bridge C18 chromatographic column (150 mm × 4 .6 mm × 3.5 μm), column temperature of 40°C, flow rate of 1.3 mL/min, injection volume of 10 μL. Through this methodology, it was possible to identify an unknown degradation product of Hydrochlorothiazide, formed by the reaction with lactose (excipient present in the drug formulation), proving that the method can be applicable both to DAD detectors and to spectrometry and mass detectors. It was also proven through the forced degradation study that the method is indicative of stability, in addition to being validated and robust for its purpose.

Keywords: Analytical development; High performance liquid chromatography; Mass spectrometry; Quality by design (AQbD); Stability study.