Simultaneous analysis of 21 bioactive compounds in biorefinery oil: Multivariate optimization of a method based on liquid chromatography, atmospheric pressure chemical ionization and tandem mass spectrometry

A HPLC method coupled to atmospheric pressure chemical ionization (APCI) and tandem MS was optimized for the analysis of tocotrienols, tocopherols, retinol, squalene, phytosterols and carotenoids in lipid matrices. The aim of the research was to obtain a specific, rapid and robust method for bioactive compounds in by-products from the agri-food industry, in order to discover new high added-value products. The separation of 21 analytes, by non-aqueous reverse phase (NARP) liquid chromatography, was optimized by using design of experiments and response surface methodology. The best compromise among peak resolution and rapidity consisted in a run of 25 min. The problem of coelution of some substances was overcome thanks to the mass detection in multiple reaction monitoring mode, which also allowed maximization of sensitivity. The instrumental method was characterized by calibration curves with R-2 of 0.989-0.999, limits of detection in the range 0.6-27 mu g L-1, relative standard deviation always <20% and excellent specificity (4 identification points for each compound). The method was applied, with minimal sample pre-treatment, to vegetable oil mixtures and processed products from a first-generation biodiesel plant. Negligible matrix effect and accuracy of 80-90% were observed for most analytes, thanks to the use of APCI and dilution factors ranging from 500 to 7500 (volume to mass ratio). The most concentrated species were phytosterols, squalene and tocopherols, which showed levels of 10-30 mg g(-1) in some samples, indicating the possibility of exploiting these by-products for the recovery of highvalue chemicals.

Automated summary

An optimized HPLC-APCI-MS method was developed for analyzing bioactive compounds in lipid matrices, achieving a balance between peak resolution and speed within a 25-minute run. The use of multiple reaction monitoring mode helped overcome coelution issues and improve sensitivity in the analysis.