Method development with high-throughput enhanced matrix removal followed by UHPLC-QqQ-MS/MS for analysis of grape polyphenol metabolites in human urine.

Grape and grape derived products contain many bioactive phenolics which have a variety of impacts on health. Following oral ingestion, the phenolic compounds and their metabolites may be detectable in human urine. However, developing a reliable method for the analysis of phenolic compounds in urine is challenging. In this work, we developed and validated a new high-throughput, sensitive and reproducible analytical method for the simultaneous analysis of 31 grape phenolic compounds and metabolites using Oasis PRiME HLB cleanup for sample preparation combined with ultra-performance liquid chromatography with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Using this new method, the accuracy achieved was 69.3 % ∼ 134.9 % (except for six compounds), and the recovery achieved was 52.4 % ∼ 134.7 % (except for two very polar compounds). For each of the 31 target analytes, the value of intra-day precision was less than 14.3 %. The value of inter-day precision was slightly higher than intra-day precision, with a range of 0.7 % ∼ 19.1 %. We report for the first time on the effect of gender and BMI on the accuracy and recovery of human urine samples, and results from analysis of variance (ANOVA), and principal component analysis (PCA) indicated there was no difference in the value of accuracy and recovery between different gender or BMI (>30) using our purposed cleanup and UHPLC-QqQ-MS/MS method. Overall, this newly developed method could serve as a powerful tool for analyzing grape phenolic compounds and metabolites in human urine samples.

Assessment of Digestion, Absorption, and Metabolism of Nanoencapsulated Phytochemicals Using In Vitro and In Vivo Models: A Perspective Paper.

Nanoencapsulation delivery systems have been used to enhance the absorption and bioefficacy of phytochemicals. With modified physical and chemical properties, nanoencapsulated phytochemicals differ from their free forms in digestion, absorption, and metabolism. These pharmacokinetic processes can be assessed using a combination of various in vitro/in vivo models and analytical strategies, but each approach has its limitations. The correlation between current models and physiological conditions and their feasibility for nanoencapsulation systems require further validation. More detailed studies are still needed to clarify how nanoencapsulation affects the phytochemical and host interaction. Future investigations must take extra caution in model selection and result interpretation.

Method development and validation for analysis of phenolic compounds in fatty complex matrices using enhanced matrix removal (EMR) lipid cleanup and UHPLC-QqQ-MS/MS.

Reliable analysis of phenolic compounds in fatty matrices is a challenging task. In this work, a robust analytical method was developed and validated for 55 phenolic compounds employing QuEChERS (quick, efficient, cheap, easy, rugged and safe) and Enhanced Matrix Removal (EMR)-lipid cleanup in 96-well plates for sample preparation, coupled with ultra-high performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). Seven high-fat matrices of pork brain, belly and liver; horse serum, beef, salmon and avocado were explored for method validation and led to promising stepwise recoveries of extraction, clean-up, drying-reconstitution of most analytes ranging from 75% to 113%, and with an accuracy of 78%∼117%, except for six catechin-analogues. The matrix removal efficiency of EMR was determined using UHPLC-quadruple time of flight (QTOF)-MS, and results indicated that 56%∼77% of co-extractives were removed. This method would be readily extended to wide range of applications demanding high-throughput and sensitive analysis of phenolic compounds in fatty samples.

Changes in polyphenol serum levels and cognitive performance after dietary supplementation with Concord grape juice in veterans with Gulf War Illness.

AIMS: We investigated whether the consumption of Concord grape juice (CGJ) was associated with increased bioavailability of serum metabolites and their potential impact on cognitive performance in Veterans with Gulf War Illness (GWI). MAIN METHODS: Twenty-six veterans were selected from a cohort of 36 enrolled in a 24-week randomized, double-blind, Phase I/IIA clinical trial exploring whether the consumption of Concord grape juice (CGJ) was tolerable and safe in Veterans with GWI and improved cognitive function and fatigue. These 26 veterans were selected based on their completion of the entire 24-week protocol and documented adherence to the study beverage ≥80%. Differences in serum metabolite levels between CGJ and placebo at midpoint and endpoint were evaluated using two-way repeated measures ANOVA with post hoc Sidak's multiple comparison test. Bivariate correlations to assess for possible relationships between change in serum metabolite levels and change in cognitive function as measured by the Halstead Category Test-Russell Revised Version (RCAT) were also conducted. KEY FINDINGS: Seventy-six metabolites were identified and quantified in this study, with three (cyanidin-glucuronide, me-cyanidin-glucuronide, and me-malvidin-glucuronide) found to be significantly higher (p < 0.05) in the CGJ group compared to placebo at 24 weeks. Significant associations between changes in cognitive function and changes in serum levels of epicatechin-sulphate (r = 0.48, p = 0.01) and petunidin-glucuronide (r = 0.53, p < 0.01) from baseline to 24 weeks were also observed. SIGNIFICANCE: Our data suggest that dietary supplementation with CGJ is associated with increased bioavailability of specific phenolic metabolites, some of which may be correlated with cognitive performance.

Development and validation of a micro-QuEChERS method with high-throughput enhanced matrix removal followed with UHPLC-QqQ-MS/MS for analysis of raspberry ketone-related phenolic compounds in adipose tissues.

Raspberry ketone (RK) is a major flavor compound in red raspberries, and it has been marketed as a popular weight-loss dietary supplement with high potential in accumulating in fatty tissues. However, challenges in extracting and characterizing RK and its associated phenolic compounds in fatty tissues persist due to the complex matrix effect. In this work, we reported a high-throughput sample preparation method for RK and 25 related phenolic compounds in white adipose tissues using an improved micro-scale QuEChERS (quick, efficient, cheap, easy, rugged and safe) approach with enhanced matrix removal (EMR)-lipid cleanup in 96-well plates, followed by UHPLC-QqQ-MS/MS analysis. The absolute recovery was 73-105% at the extraction step, and achieved 71-96% at the EMR cleanup step. The EMR cleanup removed around 66% of total lipids in the acetonitrile extract as profiled by UHPLC-QTOF-MS/MS. The innovative introduction of a reversed-phase C18 sorbent into the extract significantly improved the analytes' recovery during SpeedVac drying. The final accuracy achieved 80-120% for most analytes. Overall, this newly developed and validated method could serve as a powerful tool for analyzing RK and related phenolic compounds in fatty tissues.

Improving the bioaccessibility and bioavailability of carnosic acid using a lecithin-based nanoemulsion: complementary in vitro and in vivo studies.

Carnosic acid (CA) represents one of the most effective antioxidants that can be applied for the prevention of degenerative and chronic diseases. However, the intrinsic hydrophobic nature of CA results in low solubility and poor dissolution in the gastrointestinal (GI) tract, which limits its applications in a variety of functional food systems. In order to address these issues, we encapsulated CA in a lecithin-based nanoemulsion (CA-NE) to improve its bioaccessibility and bioavailability which are evaluated using in vitro and in vivo digestion models. The CA-NE demonstrated a loading capacity of 2.6-3.0%, an average particle size of 165 nm, a ζ-potential value of -57.2 mV, and good stability during 4-weeks of storage at 4, 25, and 37 °C. The in vitro static pH-stat lipolysis model and dynamic TNO gastrointestinal (TIM-1) model demonstrated a 12.6 and 5.6 fold increase in the total bioaccessibility of CA encapsulated in nanoemulsion, respectively, as opposed to CA in suspension form. Moreover, the in vivo pharmacokinetics study on a rat model (Male Sprague Dawley) confirmed that the bioavailability of CA in nanoemulsion showed a 2.2 fold increase, as compared to that of CA in suspension form. In conclusion, the bioaccessibility and bioavailability of CA were remarkably improved by encapsulation of CA in a lecithin-based nanoemulsion. Moreover, the combined in vitro and in vivo study could serve as a useful approach for the comprehensive evaluation of oral lipid-based delivery systems.