Highly sensitive analysis of polyphenols and their metabolites in human blood cells using dispersive SPE extraction and LC-MS/MS

Abstract

Blood cells, particularly erythrocytes, present a significant compartment for distribution of drugs and endogenous compounds and have been suggested to be factored in pharmacokinetic and pharmacodynamic evaluations. We previously detected the binding of polyphenols to red blood cells and found indications for a facilitated uptake of the bioactive procyanidin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) into human erythrocytes. The purpose of the present investigation was to develop an effective, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify low concentrations of polyphenols in human blood cells. Various sample preparation methods including classic sample clean-up techniques and variations of the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach were compared regarding compound recovery, matrix effects and overall process efficiency. The QuEChERS technique which involves a liquid-liquid extraction and clean-up by dispersive solid-phase extraction yielded best results. The method was fully validated for the six analytes: (+)-catechin, ferulic acid, M1, taxifolin, caffeic acid and δ-3-methoxy-4-hydroxy-phenyl- γ-valerolactone (M2) in human blood cells with an optimised QuEChERS sample preparation and prior enzymatic hydrolysis of analyte conjugates. The lower limits of quantification for the analytes ranged from 0.12 ng/mL for M1, M2 and taxifolin to 48.40 ng/mL for caffeic acid. The application of the method to a blood cell sample of a volunteer ingesting 100 mg/day of the standardised pine bark extract Pycnogenol(®) over the course of 3 weeks revealed measurable steady-state concentrations of catechin, M1, taxifolin, ferulic acid and M2. To our knowledge, this is the first report of using the QuEChERS approach for detection and quantification of plant-derived compounds in human blood cells. The method can be applied in pharmacokinetic studies to determine the distribution of polyphenols and their metabolites in human whole blood, blood cells or erythrocytes. This might contribute in gaining deeper insights into the in vivo distribution of polyphenols and their metabolites.

Profiling a gut microbiota-generated catechin metabolite's fate in human blood cells using a metabolomic approach

“…For M1 metabolite detection blood samples were subjected to two different sample preparation techniques, a simple protein precipitation with methanol and a dispersive solid-phase extraction method known as QuEchERs [27]. In an earlier study, the latter method was optimized for sensitive detection of M1 [11]. In contrast, in the present study, both methods gave comparable results regarding detection of M1 metabolites, though the protein precipitation allowed the discovery of more metabolites than the QuEchERs approach.…”

“…Retention time (t R ) for M1 was 7.10 and 4.40 min for M1-GSH. The identity and purity of M1-GSH in the fraction at t R of 4.40 min was determined by LC-MS/MS according to [11] and NMR before UPLC-ESI-qTOF-MS analysis.…”

“…Blood cell samples were extracted either by protein precipitation with methanol (see Section 2.4) or were subjected to sample preparation using the QuEChERS approach as described previously [11]. Briefly, 2.0 mL of human blood cells were diluted with PBS buffer to 10 mL and 275 L of 4% o-phosphoric acid were added (pH 5.0).…”

“…This has been previously shown to produce detectable plasma and also blood cell concentrations of M1 in vivo [5,11]. For M1 metabolite detection blood samples were subjected to two different sample preparation techniques, a simple protein precipitation with methanol and a dispersive solid-phase extraction method known as QuEchERs [27].…”

“…facilitated uptake [9,10]. Recently, M1 was also quantified in human blood cells in vivo after intake of Pycnogenol ® by a human volunteer [11]. To our knowledge, the absolute stereochemistry of this bacteria-generated compound has not been clarified yet, although it can be assumed that only a single enantiomeric form is generated by the gut bacteria [10].…”