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well as labeled 13C6 UA, as an internal standard. For the preparation of the internal standard solution 13C6 UA was dissolved in acetonitrile/dimethylsulfoxide (1/1, v/v) to a concentration of 1.00 mg/mL and subsequently diluted to 100,000 ng/mL. Further dilution to a concentration of 2500 ng/mL was done in methanol. In case signal inter- ference compromised the quanti fi cation of the analyte and/ or the internal standard, the sample was repeated (if suf fi - cient sample volume was available). The concentration of the repeated analysis was reported in case of the absence of the interfering signal. Otherwise, no value was reported for that sample. For quality control in general, 10% of the samples were reanalyzed. Repeat samples were chosen randomly by the analyzing laboratory from the samples belonging to the different study interventions. To measure the levels of UA glucuronide in both plasma and in human whole blood collected in DBS validated methods were developed for each of these matrixes. Analysis and quan- ti fi cation of UA glucuronide was performed for both of these matrixes using a synthesized reference sample con- sisting of and equal mix of the two isoforms of UA glu- curonide as the reference item, as well as labeled 13C6 UA glucuronide as an internal standard. The measurement of UA sulfate in plasma was performed using a validated method, using a characterized reference sample of synthe- sized UA sulfate as the reference item, as well as labeled 13C6 UA sulfate as an internal standard. The limit of quanti fi cation in plasma was 5.00 pg/ml for parent UA and 5.00 ng/ml for both the UA glucuronide and UA sulfate its metabolites. An analytical method to determine UA glu- curonide in human whole blood collected by DBS using liquid chromatography coupled to mass spectrometry was also developed and validated. The limit of quanti fi cation for UA glucuronide in human DBS samples (6-mm spots) range was 5.00 – 5000 ng/mL for the analysis of human DBS samples. The quanti fi cation of UA glucuronide was per- formed by column separation with reversed phase liquid chromatography followed by detection with triple-stage quadrupole MS/MS in the selected reaction monitoring mode. The concentration of the analytes was calculated using the internal standardization method. The acquisition and processing of data was performed using LCquan ver- sion 2.5.6 and Xcalibur version 2.0.7 (Thermo Fisher Sci- enti fi c, San Jose, CA, USA). Quanti fi cation of ellagic acid, punicalagins, and UA in commercial grade food products and dietary supplements Validated HPLC analytical methods were used to detect EA and punicalagin. For determination of the parent UA levels in commercial grade food products and dietary supple- ments, a validated HPLC analytical method was developed
centrifuged at 1500 g for 10 min at 4 °C. Within 30 min of centrifugation the top layer of human plasma was trans- ferred into a prelabelled polypropylene tube containing approximately 1500 μ L of plasma. Tubes from each time point were capped immediately and the plasma was frozen at − 80 °C for storage. DBS were also collected on blood collection cards (Whatman Filter Paper 903 spots card). To collect the sample, a fi ngerpick with a lancet was performed and three to four blood spots (each containing approx. 20 – 40 mL of whole blood) were spotted on each DBS card. The cards were dried and stored in sealable biohazard foil bags containing desiccant at room temperature until analy- sis. Each participant was also supplied with a stool sample collection kit (OMNIGene OMR-200, DNA Genotek, Kanata, ON, Canada) at their screening visit to collect a sample at home prior to study visit V2. Upon return to the study site, the stool samples were stored at − 80°C.
UA bioavailability measurements
A total of 600 human plasma samples were analyzed from the study (i.e., six samples per subjects with three time points per intervention: T0, T6 hours, and T24 hours) over different batches. Each batch included eight calibration samples, at least two blank samples, one double blank plasma sample, as well as three different quality control samples in duplicate. The inter-batch precision was between 3.1 and 12.3%, whereas the inter-batch accuracy was in the range from 96.6 to 99.3% of nominal concentration. Mea- surements were performed according to the FDA Guidance for Industry and EMA guidelines on bioanalytical method validation as previously described [10]. The quanti fi cation of UA and its metabolites, i.e., UA glucuronide and UA sulfate in plasma, was performed by column separation with reverse phase chromatography using an Agilent 1200 HPLC system (Agilent Technologies, Waldbronn, Germany) fol- lowed by detection with a TSQ Vantage triple-stage quad- ropole MS/MS (ThermoFisher Scienti fi c, San Jose, CA, USA) in the selected reaction mode. The plasma samples were diluted with 200 µL water containing 0.1% formic acid, and thereafter forti fi ed with internal standard. A solid- phase extraction with a Bond-Elut focus plate (Agilent Technologies, Waldbronn, Germany) using water as wash- ing solvent and MeOH as elution solvent was used for sample cleanup, and after elution the extraction solvent was evaporated. Subsequently, the sample was reconstituted in 200 µL of a water/methanol mixture (1/1, v/v) and 50 µL was injected into the HPLC MS/MS system. Separation was achieved using a C18 reverse phase column (YMC Co, Ltd., Kyoto, Japan). For the measurements of parent UA in plasma, a validated method was developed. Analysis and quanti fi cation were performed using a characterized refer- ence sample of synthesized UA, as the reference item, as
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