Development of a MS compatible HPLC-HILIC method for the analysis of allantoin and glycolic acid in snail slime and related dosage forms: Focus on the enantioseparation of allantoin.

Glycolic acid and allantoin are known to be responsible, at least in part, for the main activities of snail slime, such as moisturizing, skin regeneration, antioxidant, soothing and anti-inflammatory. Accordingly, a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of allantoin and glycolic acid in snail slime samples was developed by a conventional UV-Vis detector and the diol-type Luna® HILIC column. An optimized eluent made up of acetonitrile/water/methanol/formic acid (90:5:5:0.1 v/v/v/v) allowed the separation of the two investigated compounds from each other and from lactic acid and hydantoin used as internal standards. Being allantoin a chiral compound, an enantioselective HPLC protocol was also developed. The chiral analysis of allantoin was performed with the Lux® 3 μm i-Amilose-3 chiral stationary phase using the same mobile phase as for the achiral analysis. The chiral method was efficiently transferred to a HPLC system coupled to triple-quadrupole mass spectrometry (MS/MS). HPLC-MS/MS analysis revealed the racemic nature of allantoin in the pure snail slime. The same profile was also observed in a prototype hydrogel prepared with snail slime. Moreover, the same method allowed to measure the amount of glycolic acid in the two investigated samples (pure snail slime and gel) and ascertain a valuable extraction recovery. The enantiomeric elution order (S)<(R) with the amylose-based phase was established coupling the results of experimental electronic circular dichroism analysis to time-dependent density functional theory simulations.