Enantiomeric and Diastereomeric Resolutions of Chiral Pesticides Using UltraPerformance Convergence Chromatography (UPC2) with UV Detection

The development of analytical methods for the separation of chiral compounds is important in many areas of research because it is well known that enantiomers can react differently in a chiral environment. Biochemical reactions can be stereo or enantioselective - and while one enantiomer may deliver the desired effect (referred to as the eutomer) to the target species, the other enantiomer may be less effective to the target or completely ineffective. It is estimated that 30% of pesticides on the market today have optical isomers and there are reports that 40% of the pesticides used in China are chiral. The study of enantioselectivity is important to the agricultural chemicals manufacturing industry since the knowledge of the efficacy of each individual enantiomer could facilitate a significant reduction in the total amount of pesticide applied. In order to improve our knowledge of enantioselectivity, analytical methods that provide reliable and reproducible separations in a rapid time frame are needed. Supercritical fluid chromatography (SFC) has become known as an effective chiral separations technique possessing many advantages over conventional high performance liquid chromatography (HPLC). The properties of a supercritical fluid allow high efficiency separations with shorter analysis times to be achieved. The structural complexity of new pesticides is increasing, which means that there is a greater likelihood that multiple chiral centers may be present in a molecule and high efficiency techniques are needed to perform successful separations.

In this application note, we present the enantiomeric and/or diastereomeric separations of three pesticides: metalaxyl-M, S-metolachlor (acetanilide class of pesticides), and difenoconazole (a triazole fungicide). Metalaxyl has one chiral center, while metolachlor and difenoconazole have two chiral centers. The structures are shown in Figures 1 to 3.

Separations were performed using Waters^® ACQUITY^® UltraPerformance Convergence Chromatography™ System (UPC^2®). Convergence chromatography is a complimentary separation technique to liquid chromatography, providing orthogonal selectivity and using supercritical CO₂ as the primary mobile phase. Empower^® 3 Software was used for chromatographic data processing.

ACQUITY UPC² allows high-efficiency separations that can significantly increase the sample throughput when compared with traditional normal-phase separations. The time taken to develop a method from the column and co-solvent screening step to the final optimized method is decreased. The methods described here use supercritical CO₂ as the primary mobile phase and predominantly 2-propanol as the organic modifier. The need to use large volumes of potentially hazardous solvents is reduced as is the cost associated with solvent waste disposal. The %RSD’s obtained were comparable to those obtained by UPLC^®/UV methods.

The study of enantioselective toxicity and environmental fate has previously been a challenge due to the difficulty in resolving chiral compounds. The benefit of having faster analytical methods to resolve chiral compounds means that critical information pertaining to their stereoselective behavior can be obtained more rapidly.