Determination of Perillic Acid in Bioconversion Supernatants by Gas Chromatography

Abstract

Perillic acid can be obtained from microbial oxidation of the exocyclic methyl group of limonene. Due to the pharmacological potential of such a metabolite, the biotransformation processes leading to its synthesis have been approached in recent studies. A robust analytical method is needed to assess the performance of such studies. An analytical method was developed and validated to determine perillic acid in the supernatants of a yeast-induced bioconversion of limonene, involving gas chromatography (GC) and an acid-induced precipitation during the sample preparation. GC analysis was performed using a column with polyethylene glycol as stationary phase (HP-Innowax) which resulted in higher loads and better peak shape. The sample preparation involved the supernatant initial filtration and precipitation with 0.6 M HCl followed by centrifugation and dissolution in ethyl acetate. GC analysis conditions were oven from 50°C to 250°C at 20°C·min-1, and then held 5 min (total runtime 15 min). Injector was set at 280°C, and detector at 300°C. Helium was the carrier gas at 1 ml·min-1. Injections of 1.0 μl were at the split ratio 25:1. The method was validated: Linearity with R2 of 0.9992, Accuracy of 98.3% in the range 190 - 950 μg·ml-1; Limit of detection of 10.4 μg·ml-1; Repeatability of 2.1% RSD. Thus, a complete methodology to determine perillic acid in a bioconversion supernatant was developed and validated. This overall approach may be useful for bioconversions of monoterpenes by other microorganisms that metabolize limonene.