Enantiomeric Resolution of Pidotimod and Its Isomers in Pidotimod Oral Solutions Using Chiral RP-HPLC with Quadrupole Dalton Analyzer Detection

This study aimed to develop and validate a high-performance liquid chromatography system combined with a single-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 °C 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; S,S ) and 0.5 to 3.5 μg·mL −1 ( R,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 evaluations suggested that the sorbent-analyte interactions of amylose tris(3,5-dimethylphenycarbamate) can be used to successfully interpret and even predict the chromatographic separation results. The validated method showed high sensitivity and can potentially be used to analyze the isomer impurities of raw materials for quality control purposes.