Selective microemulsion liquid chromatography analysis of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera by using a monolithic silica column

• A method is proposed for the assay of LE300 and its N-methyl metabolite by MELC.
• Higher resolution, higher efficiency and faster separations are achieved in MELC.
• Application to pharmacokinetic study demonstrates the method is fit-for-purpose.
• Low LOD, good recoveries and interferences-free endogenous serum were obtained.

A highly selective, sensitive, and rapid microemulsion liquid chromatography (MELC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and pindolol (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis by using a monolithic column was performed by directly injecting the sample after appropriate dilution with the microemulsion mobile phase. The chromatographic behaviour of these compounds was studied to demonstrate their chromatographic efficiency, retention, and peak symmetry. The MELC method was validated for its specificity, linearity, accuracy, precision, robustness and stability. An experimental design was used during validation to evaluate method robustness. The calibration curves in serum showed excellent linearity (r = 0.997) over concentrations ranging from 10 to 400 ng mL−1 for LE300 and 15 to 500 ng mL−1 for its N-methyl metabolite. The mean relative standard deviation (RSD) of the results of inter- and intra-day precision and accuracy of LE300 and its N-methyl metabolite were ≤5%. The overall recoveries of LE300 and its N-methyl metabolite from mouse sera were in the range 97.9–101.5% with %RSD ranging from 0.98% to 3.63%, which were in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study.