Towards safer food supplements: importance and challenges of multi-analyte measurement

Abstract

Plants such as Aloe, Rheum, Rhamnus and Cassia senna, are often used in food supplements to improve bowel function. The downside of these preparations is the presence of hydroxyanthracene derivatives (HADs), naturally occurring plant substances. Although HADs are considered important biologically active components with their recognized laxative effect, recently, the European Commission prohibited a number of HADs due to toxicological concerns. In this view, validated analytical methods for the determination of multiple HADs in food are in high demand for regulatory analysis and dietary intake studies. Currently, the available methods target only two to five substances. Having diverse HADs in the method is pivotal to having more complete data on the levels and type of HADs, which was also emphasized by a previous report on large variability in total HAD content in food supplements from the Belgian market. However, data on individual HADs were lacking. Consequently, the estimation of the exposure to HADs from supplements was performed only for a limited number of substances due to the lack of occurrence data for the others. The objective of this study was to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of 16 individual HADs in food supplements. The method included sennosides, (gluco)frangulins, aloins, aloe-emodin, emodin, danthron, rhein, physcion and chrysophanol. The main challenges of this method were the diverse physicochemical properties of the targeted compounds, jeopardizing simultaneous analysis and the isomeric nature of some HADs, potentially hampering individual quantification. The latter issue became apparent for aloins and sennosides that produced the same abundant precursor and product ions in the MS, making them indistinguishable in Multiple Reaction Monitoring mode. However, using the ACQUITY UPLC BEH C18 column and carefully optimizing the gradient of the mobile phase, the LC peaks of these isomers were baseline-resolved, ensuring the desired quantification scope. Another bottleneck of the method, sample preparation, was tackled by testing several organic solvents (methanol, acetonitrile, ethanol, acetone) and/or their combinations for the extraction. The addition of water to the extraction mixture improved the results for sennosides due to their more polar character, however, significantly reducing recovery of less polar HADs (e.g., physcion). The proportion of water was optimized, and the final procedure was a compromise for analyzing all target HADs with an easy-to-apply protocol and good sensitivity. The validation revealed good performance characteristics of the method, with the exception of physcion for which recovery correction was required for quantification. The importance of putting in place methods encompassing multiple HADs was demonstrated by the results of a market study on food supplements. Analysis of commercial products by the developed method revealed that about 60% of the samples significantly exceeded the threshold proposed by the European Commission. Interestingly, two products contained 13 out of 16 target HADs simultaneously. This study provided a sensitive method for delivering accurate and reproducible results. This method is a critical tool in collecting occurrence data for a wider range of HADs in plant-based products and facilitating a more accurate dietary exposure assessment.