Greenness, Blueness, and Whiteness Assessment of Novel Chromatographic Methods for Separation and Simultaneous Analysis of Zuclopenthixol and Its Major Potentially Toxic Impurity: A Comparative Study

Zuclopenthixol was reported to treat schizophrenia, psychoses, and acute bipolar mania. 2‐Chlorothioxanthon is reported to be zuclopenthixol official Impurity C (IMP C), and it is considered to be zuclopenthixol synthetic precursor. IMP C was reported to have toxicity on mRNA and protein expression. Environmentally validated HPTLC and HUPLC were developed for the simultaneous analysis of zuclopenthixol and IMP C in the analytical standard form and in pharmaceutical form, according to ICH guidelines. Regarding the HPTLC method, zuclopenthixol and its toxic impurity were separated using a mobile phase consisting of dichloromethane:ethyl acetate:triethylamine (6:4.5:0.4, v/v), and the scanning wavelength was 257 nm. For the HUPLC method, the simultaneous separation was completed within 2 min using Waters, CORTECS C 8 (4.6 mm × 150, 2.7 μm) as a stationary phase. The applied mobile phase was an isocratic mixture of methanol:water (80:20, v/v), pH was 3.25 ± 0.02 using orthophosphoric acid, pumping at 3 mL/min, and the detection wavelength was 257 nm. To evaluate the greenness value of the presented methods, the Eco‐Scale Assessment method, Analytical Greenness Metric, and Green Analytical Procedure Index were applied. The practicality of the analytical methods was evaluated using the Blue Applicability Grade Index. Additionally, the analytical whiteness was determined via the RGB algorithm.