Derivatization of Proline for the Enantiomeric Separation and Estimation of D-Proline in L-Proline by NP-HPLC

Abstract

The core object of this exertion is to progress and validate a modest, competent and explicit method for the exodus of D and L isomers of proline in a racemic mixture and limit the content of D-Proline in commercial L-Proline. This has been technologically advanced in a chiral method using normal phase HPLC on CHIRALPAK-IA (250X4.6 mm) 5 µ column. This progress advanced with polar mobile phase ethanol encompassing modifier/additive TFA in 0.1% concentration. Due to the chromophore’s deficiency in proline, proline’s derivatization has been done using fluorescent reagent NBD-Cl. After derivatization, proline has made UV detectable at 465 nm. Within run time of 20 minutes, D and L isomers of proline were eluted at 6.72 and 9.22 minutes, respectively. The technologically advanced method was validated as per ICH guidelines. Linearity regression coefficients for both D and L-Proline are obtained as 0.999. Retrieval for D-Proline was obtained at 93 to 95% range for 4 levels. LoD and LoQ for both D-and L-Proline were detected as 0.6 and 2 ppm, respectively. Hence this method is newer, modest, particular and explicit chiral method.