Planta Med 2013; 79 - PH2
DOI: 10.1055/s-0033-1352079

Improving method reliability through selective removal of glycerolipid and chlorophyll interferences

S Bijttebier 1, E D'Hondt 2, S Apers 3, N Hermans 3, S Voorspoels 2
  • 11: Flemish Institute for Technological Research (VITO), Industrial Innovation, Boeretang 200, 2400 Mol, Belgium. 2: University of Antwerp, Natural Products & Food – Research and Analysis (NatuRA), Universiteitsplein 1, 2610 Antwerp, Belgium
  • 2Flemish Institute for Technological Research (VITO), Industrial Innovation, Boeretang 200, 2400 Mol, Belgium
  • 3University of Antwerp, Natural Products & Food – Research and Analysis (NatuRA), Universiteitsplein 1, 2610 Antwerp, Belgium

Exact quantitative determination of carotenoids and other secondary plant metabolites is essential in the estimation of potential valorisation pathways. Erroneous quantification can cause major differences in the end result of an economical calculation and can make the difference between potential profit or loss.

A generic Accelerated Solvent Extraction (ASE) procedure was optimized for the complete extraction of carotenoids from organic biological material. As the extraction procedure is generic, other apolar compounds like glycerides and chlorophylls are also extracted, which can induce ionization suppression of co-eluting carotenoids in mass spectrometric detection or can disturb ultra violet detection. Extra purification steps are therefore necessary to minimize matrix influence of triglycerides and chlorophyll derivatives on detection.

Saponification is generally used to hydrolyse glycerolipids and chlorophylls. However, in nature carotenoids are regularly esterified with fatty acids and saponification also hydrolyses these esterified carotenoids. The natural composition of carotenoids present in the sample is therefore lost with implications on future valorisation steps.

Lipase enzymes were evaluated to selectively remove glycerolipids like di- and triglycerides from the sample matrix, leaving esterified carotenoids untouched. Lipases are enzymes that are naturally present in animals for the digestion of glycerolipids. The optimized lipase clean-up method resulted in hydrolysis of more than 96% of triglycerides present in the samples.

For the selective removal of chlorophylls, preparative open bed column chromatography, selective removal of chlorophylls using dioxane and a new selective 'Chlorofiltr™' (UCT) adsorbent were investigated.

The optimized extraction method was coupled to the clean-up steps, resulting in an effective generic sample preparation flowchart for the qualitative and quantitative screening of apolar metabolites in organic biological samples.