TITLE:
Determination of Perillic Acid in Bioconversion Supernatants by Gas Chromatography
AUTHORS:
Eliane G. Carvalho, Antonio C. Siani, Igor C. Cardoso, Virginia Garcia Correia, Maria A. Ferrara, Elba P. S. Bon, Marcelo R. Romero Tappin
KEYWORDS:
Limonene, Perillic Acid, Sample Preparation, Gas Chromatography
JOURNAL NAME:
American Journal of Analytical Chemistry,
Vol.8 No.10,
October
26,
2017
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.