Estimation of confidence intervals for quantitation of coeluted peaks in liquid chromatography-Photodiode array detection through a combination of multivariate curve resolution-alternating least-square and Bayesian inference techniques

There is a dramatic increase in drug candidates that exhibit complex structures and do not comply with Lipinski's rule of five. One of the most critical and complex technical challenges in the quality con-trol of such drug candidates is the control of analogous substances contained in active pharmaceutical ingredients and related formulations. Although the development of ultrahigh-performance liquid chro-matography and high-performance columns has improved efficiency per unit time, the difficulty of peak separation to quantify impurities with similar structures and physicochemical properties continues to rise, and so does the probability of failure to achieve the necessary separation. Coeluting peaks observed in the case of high-performance liquid chromatography (HPLC) with photodiode array detection can be separated using the multivariate curve resolution-alternating least-square (MCR-ALS) method exploiting differences in analyte UV spectra. However, relatively large quantitation errors have been observed for coeluting analogous substances, and the reliability of the corresponding quantitative data requires im-provement. Herein, Bayesian inference is applied to separation by the MCR-ALS method to develop an algorithm assigning a confidence interval to the quantitative data of each analogous substance. The use-fulness and limitations of this approach are tested using two analogs of telmisartan as models. For this test, a simulated two-component HPLC-UV dataset with an intensity ratio (relative to the main peak) of 0.1-1.0 and a resolution of 0.5-1.0 is used. The developed algorithm allows the prediction confidence interval, including the true value, to be assigned to the peak area in almost all cases, even when the intensity ratio, resolution, and signal-to-noise ratio are changed. Finally, the developed algorithm is also evaluated on a real HPLC-UV dataset to confirm that reasonable prediction confidence intervals includ-ing true values are assigned to peak areas. In addition to allowing the separation and quantitation of substances such as impurities challenging to separate by HPLC in a scientifically valid manner, which is impossible for conventional HPLC-UV detection, our method can assign confidence intervals to quanti-tative data. Therefore, the adopted approach is expected to resolve the issues associated with assessing impurities in the quality control of pharmaceuticals.& COPY; 2023 Elsevier B.V. All rights reserved.