Set-up of a liquid chromatography-tandem mass spectrometric method for the quantitation of (poly)phenolic metabolites in human urine with on-line solid phase extraction

(Poly)phenols (PPs) are plant secondary metabolites found in a wide variety of foods and derived products. After food intake, PPs are absorbed and metabolized by host gut microbial enzymes (Absorption-Distribution- Metabolism-Excretion, ADME), originating a complex mixture of derived metabolites that may additively or synergistically account for the reported beneficial health effects of phenolic-rich diets. Analytical chemistry can effectively increase the knowledge about the dietary exposure of consumers to PP metabolites by the development of analytical platforms for their quantitation in human biofluids, to evaluate the bioavailable and excreted concentration levels. The aim of our research is to develop and validate an on-line SPE-LCMS/ MS method for the quantitative analysis in human urine of 34 structurally heterogeneous PP metabolites, selected accordingly to literature findings on the berry intake biomarkers. First, the chromatographic behaviour of the selected analytes was investigated, focusing on the analytical challenging separation of isobaric metabolites (e.g., glucuronidated urolithins and quercetins), by testing different stationary and mobile phases. To develop a “wide-scope” method feasible to the targeted and quantitative analysis of PP metabolites in urine postprandial samples from a berry-based intervention study, the on-line SPE step was performed using a mixed-mode sorbent (i.e., Isolute ENV+), combining hydrophobic and polar interactions, capable to enrich a broad range of PP metabolites with different physicochemical properties. Urine samples were collected from volunteers after the intake of 25 g of bilberry (Vaccinium myrtillus) or blueberry (Vaccinium corymbosum) supplement mixed with 500 mL of drinking water. Instrumental analysis was performed on a Shimadzu (Kyoto, Japan) chromatographic system coupled with a 5500 QTrap mass spectrometer (Sciex, Framingham, MA, USA), equipped with a Turbo V® interface with an ESI probe. On-line SPE and chromatographic analysis was performed using a two position six-port switching valve connected to a Nexera X2 LC-30AD quaternary pump (for desorption and chromatographic separation), and one isocratic pump LC-20AD XR (dedicated to the on-line SPE cartridge loading and washing). The optimization of the on-line SPE extraction process on Isolute ENV+ sorbent phase was carried out by analysing three different conditions: (i) continuous desorption from the cartridge at room temperature, (ii) methanol assisted desorption followed by post-SPE dilution with water, and (iii) thermal-assisted desorption. Three stationary phases were tested to reach the best separation conditions, i.e., Kinetex (i) octadecyl, (ii) biphenyl, and (ii) phenyl-hexyl. The optimization of the chromatographic separation was carried out focusing on critical isobaric groups. Once the optimal conditions for the recovery of the 34 analytes were found, the on-line SPE-LC-MS/MS method was finally applied to the analysis of a pooled urine sample from the aforementioned study. The metabolites most present in the analysed samples were hydroxybenzoic acids and related sulfonate conjugates, contributing for the 62% of the total metabolic component, and among them, 2,5-Hydroxybenzoic acid exhibited the highest concentration value. Such result is not unexpected, since hydroxybenzoic acids are metabolic end-products of some major PP occurring in bilberries, i.e., anthocyanins and flavonols. Hydroxycinnamic acid sulfonates and urolithins with their conjugates showed comparable concentration values in the investigated sample, each group accounting for the 8.3-8.6% of the total metabolic contribution. Other hydroxycinnamic acids exhibited a relevant contribution to the metabolome, such as glucuronidated conjugates (3.6%), free acids and their derivatives (4.7%), i.e., Chlorogenic acid and 5-O-Feruloylquinic acid. Similar contributions were highlighted for hippuric acids (1.3%) and quercetin conjugates (1.0%), whereas the less present metabolites category was the one of resveratrol and their conjugates (0.2%), since such metabolites are mainly associated to wine and/or tea consumption, and its contribution may derive from pre- or post-intervention food sources. Finally, Abscisic acid and 3-Hydroxyphenilacetic acid were included in the “others” category, accounting for the 10.5% of the metabolic contribution. The relevant impact of 3-Hydroxyphenilacetic acid derives from the metabolic pathway of the major components of the supplements, i.e., anthocyanidins, ending with the formation of protocatechuic and phenylacetic acids. Keeping in mind the critical aspects of both extraction and separation steps a Quality by Design workflow will be built, encompassing a screening procedure and an RSM optimization. The selected factors will be: Desorption Temperature, Exposure Time of the SPE cartridge to the chromatographic gradient, Loading solution pH, Wash Organic Percentage, Loading Flow, Wash Volumes and two different sorbent beds ISOLuteENV+ and Oasis HLB.