Enantiomeric resolution of pidotimod and its isomers in pidotimod oral solutions by using HPLC QDa

Abstract

Abstract This study aimed at developing and validating a high-performance liquid chromatography (HPLC) system combined with a quadrupole Dalton mass detector for the separation and determination of pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl)carbonyl]-thiazolidine-4-carboxylic acid) and its three isomers. The separations were achieved using a Lux Amylose-1 column operated at 40℃ using acetonitrile/0.1% trifluoroacetate (TFA) and isopropanol/0.1% TFA (90:10, v/v). The enantiorecognition mechanism was also elucidated using the ORCA 5 program. Four isomers were successfully separated with a resolution (Rs) > 1.5, with the method showing satisfactory linearity in the ranges of 0.2 to 3.5 µg·ml− 1(S,R; R,S) and 0.7 to 3.5 µg·ml− 1(S,S; R,R), along with good specificity, accuracy, and precision. Further, three batches of pidotimod oral solutions were evaluated and the S,S-isomer in each batch was detected at about 0.05%. Moreover, computational studies on the sorbent-analyte interactions of amylose tris(3,5-dimethylphenycarbamate) suggested that they can be used to successfully interpret or even predict the chromatographic separation results. The validated method showed high sensitivity, and can potentially be used to analyze the isomer impurities of the raw material for quality control purposes.