All analytical standards used in this study were >97% purity and purchased from Sigma Aldrich (St. Louis, MO), or AccuStandard (New Haven, CT). Isotopically labelled internal standards were purchased from Cambridge Isotope Laboratories (Andover, MA) or Toronto Research Chemicals (North York, ON) and were >98% purity. Acetonitrile (ACN; LCMS grade) and methanol (MeOH; LCMS grade) were acquired from Honeywell – Burdick & Jackson (Muskegon, MI). Tert-butyl methyl ether (MTBE, HPLC grade) was obtained from ACROS organics (Morris Plains, NJ). Phosphoric acid (85% w/w%) solution (99.99% trace metals basis), formic acid (ACS grade), and ammonium fluoride (HPLC grade) were purchased from Sigma Aldrich (St. Louis, MO). Ethylenediaminetetraacetic acid (EDTA), hydrochloric acid, ammonium hydroxide and sodium phosphate were purchased from Fisher Scientific (Hampton, NH). Double-deionized (DDI, 18.2MOhm*cm) low TOC (<50ppb) water was produced by a Milli-Q® Integral 5 Water Purification System. Solid Phase Extraction cartridges were purchased from Waters (Oasis HLB, 60 um particle size, 500 mg, 6 cc, Milford, MA) and Agilent Technologies (Bond Elut Plexa (45 um particle size, 500 mg, 6 cc, Santa Clara, CA), Bond Elut ENV (45 um particle size, 500 mg, 6 cc, Santa Clara, CA)). PTFE syringe filters were purchased from Agilent Technologies (Captiva, 15mm diameter, 0.2um pore size).
Bond elut plexa
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
Bond Elut Plexa is a solid-phase extraction (SPE) sorbent developed by Agilent Technologies. It is designed for the efficient extraction and purification of a wide range of analytes from various sample matrices. The core function of Bond Elut Plexa is to facilitate the selective separation and concentration of target compounds prior to instrumental analysis.
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Lab products found in correlation
Qtrap 5500 mass spectrometer, by AB Sciex (2 mentions)
Sodium phosphate, by Thermo Fisher Scientific (1 mentions)
Ammonium hydroxide, by Thermo Fisher Scientific (1 mentions)
Acs grade formic acid, by Merck Group (1 mentions)
Oasis hlb, by Waters Corporation (1 mentions)
Bond elut env, by Agilent Technologies (1 mentions)
Captiva, by Agilent Technologies (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Vacutainer, by BD (1 mentions)
Reagent grade formic acid, by Merck Group (1 mentions)
Multiquant, by AB Sciex (1 mentions)
Millex gp filter unit, by Merck Group (1 mentions)
0.22 m sterile filter, by Merck Group (1 mentions)
0.2 μm membrane, by Merck Group (1 mentions)
Ce standard bare fused silica capillary, by Agilent Technologies (1 mentions)
Synergi reverse phase c18 column, by Phenomenex (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Multiquant 2, by AB Sciex (1 mentions)
Easy spray ion source, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano liquid chromatography system, by Thermo Fisher Scientific (1 mentions)
12 protocols using bond elut plexa
1
Analytical Standards Purification Protocol
2
Quantification of Venlafaxine and Metabolite
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Working standards of venlafaxine hydrochloride and O-desmethylvenlafaxine of 99.83% and 99.82% purity respectively were purchased from Vivan Life Sciences (India). Deuterium labeled venlafaxine-D6 hydrochloride (VEN-D6) of 99.46 atom% D-isotropic enrichment and 99.68% purity and Rac-O-desmethylvenlafaxine-D6 succinate hydrate (ODV-D6) of 99.92 atom% d -isotropic enrichment and 99.02% purity was also procured from Vivan Life Sciences (India) and used as an internal standard (IS). Reagent grade ammonium formate and HPLC grade acetonitrile and methanol were obtained from Honeywell Specialty Chemicals (GmbH). Reagent grade orthophosphoric acid and liquor ammonia were acquired from Thermo Fisher Scientific (India). Reagent grade formic acid was obtained from Sigma-Aldrich Chemicals (India). All aqueous solutions and buffers were prepared using ultrapure Milli-Q water. SPE cartridges (Bond Elut PLEXA, 30 mg/1 cc) were obtained from Agilent (USA). Sodium heparin-containing blood collection tubes, BD VACUTAINER, were purchased from Becton Dickinson (USA). Efexor XR 37.5 mg was purchased from Pfizer Ireland Pharmaceuticals (Ireland).
3
Oxylipins Quantification by LC-MS/MS
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Samples were lyophilized and resuspended in 1 ml 10% methanol containing deuterated internal standards (Standards are listed in Suppl. Table 1 ) followed by an extraction using solid reverse phase extraction columns (Bond Elut Plexa, Agilent). Fatty acid derivatives were eluted into 1.0 ml of methanol, lyophilized and resuspended in 100 µl of water/acetonitrile/formic acid (70:30:0.02, v/v/v; solvent A) and analysed by LC-MS/MS on an Agilent 1290 separation system. Samples were separated on a Synergi Hydro reverse-phase C18 column (2.1 × 250 mm; Phenomenex) using a gradient as follows: flow rate 0.3 µl/min, 1 min (0% solvent B: acetonitrile/isopropyl alcohol, 50:50, v/v;), 3 min (25% solvent B), 11 min (45% solvent B), 13 min (60% solvent B), 18 min (75% solvent B), 18.5 min (90% solvent B), 20 min (90% solvent B), 21 min (0% solvent B). The separation system was coupled to an electrospray interface of a QTrap 5500 mass spectrometer (AB Sciex). Compounds were detected in scheduled multiple reaction monitoring mode. For quantification a 12-point calibration curve for each analyte was used. Data analysis was performed using Analyst (v1.6.1) and MultiQuant (v2.1.1) (AB Sciex, Switzerland). Oxylipins measured are detailed in Suppl. Tables 2 –3 .
4
Yogurt Protein Sample Preparation
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Sample preparation was according to Kunda et al. (2012) (link) with the addition of a size separation step to select proteins smaller than 3 kDa. Briefly, 0.2 mL yogurt sample was centrifuged at 12000 x g for 30 min in Amicon® Ultra-0.5 centrifugal filter device (3 kDa), and the filtrate (<3 kDa) collected. The resulting filtrate was diluted with 2.5 ml of reduction buffer (6 M urea, 5 mM sodium citrate, 5 mM DTT, pH 8.0) and incubated for 1 h at room temperature, followed by centrifugation at 3200 x g for 30 min. Any resulting fat layer was removed by pipetting and the clear solutions were filtered through 0.22 μm PES (Millex GP filter Unit, Millipore) before solid-phase extraction (SPE) with Bond Elut Plexa (Agilent, UK) polymeric SPE cartridges (60 mg of sorbent). The SPE cartridges were conditioned with 2 mL of methanol followed by 2 mL of water. Sample (2 mL) was applied to the SPE column, rinsed with two 1 ml washes (water), and the retained compounds were eluted with 1 ml of 80:20 (v/v) methanol: water and 0.1% (v/v) of formic acid. Flow rate was maintained at approximately 1 ml/min during SPE using Cerex System 48 pressure processor. Eluate was evaporated to dryness and final residue was reconstituted with 98% water, 2% acetonitrile, 0.1% formic acid before LC-MS experiments.
5
Extraction and Purification of Blueberry Compounds
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Goldtraube blueberries, kindly provided by Mirtilusa SA (Sever do Vouga, Portugal), were stored at −20 °C until processing, and extracted as described elsewhere [20 (link)]. Briefly, ethanolic extracts were produced and purified using solid phase extraction columns (Bond Elut Plexa, Agilent Technologies, Santa Clara, CA, USA). The resulting extract powder was then dissolved in deionized water (2000 µg mL−1) and sterilized using a 0.22 µm sterile filter (Millipore, Burlington, MA, USA). Henceforth, whenever extract is mentioned, it refers to the solution obtained in this step.
6
Quantification of Ferulic Acid and Vanillin
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The ferulic acid and vanillin in the samples obtained after colon simulation were analyzed using a previously published method [34 ]. The analysis was done with an Agilent CE instrument equipped with a diode array detector (DAD) and CE standard bare fused silica capillary (Agilent Technologies, Waldbronn, Germany) having an internal diameter of 50 μm and an effective length of 72 cm. As a migration electrolyte (BGE), 45 mM tetraborate buffer was used with 0.9 mM sodium dodecyl sulphate (SDS), adjusted to a pH of 9.35 with 1 M HCl. The method required the use of 30 kV voltage, constant temperature of 300 °C, and direct UV absorption at 280 nm.
Sample preparation: First, 15 mL of each sample was purified through solid phase extraction with a C18 cartridge (Bond Elut Plexa, Agilent, Waldbronn, Germany). The cartridge was preconditioned with methanol (10 mL) and washed with water (5 mL), after which the sample was applied. After the sample passed through, the cartridge was washed with 5 mL water and finally with 5 mL methanol (solvent for the extraction of the polyphenols). The methanolic effluent thus collected was concentrated to 1 mL, filtered through 0.2 μm membranes (Millipore, Bedford, MA, USA) and degassed before injection. Standard addition was further employed, to evaluate the extraction efficiency.
Sample preparation: First, 15 mL of each sample was purified through solid phase extraction with a C18 cartridge (Bond Elut Plexa, Agilent, Waldbronn, Germany). The cartridge was preconditioned with methanol (10 mL) and washed with water (5 mL), after which the sample was applied. After the sample passed through, the cartridge was washed with 5 mL water and finally with 5 mL methanol (solvent for the extraction of the polyphenols). The methanolic effluent thus collected was concentrated to 1 mL, filtered through 0.2 μm membranes (Millipore, Bedford, MA, USA) and degassed before injection. Standard addition was further employed, to evaluate the extraction efficiency.
7
Trace Contaminants Analysis of Sludge
8
Quantification of 6k-PGF1α and PGE2 via LC-MS/MS
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6k-PGF1α and PGE2 in CM of ascTAM, ascTU and CAF were quantified as described previously [27 (link)] with slight modifications. Samples (1 mL) were spiked with 100 µL internal standard (PGE2-d4 and 6k-PGF1α-d4, each 9.8 ng/mL) in methanol and extracted using solid reverse phase extraction columns (Bond Elut Plexa, Agilent, Santa Clara, CA, USA). After elution and lyophilization, samples were resuspended in water/acetonitrile (70:30) with 0.02% formic acid (solvent A). Analysis was performed by LC-MS/MS on an Agilent 1290 device coupled to a QTrap 5500 mass spectrometer (AB Sciex, Darmstadt, Germany). Samples were separated at a flow rate pf 0.3 mL/min on a Synergi reverse-phase C18 column (2.1 × 250 mm; Phenomenex, Aschaffenburg, Germnay) using the following gradient: 1 min (0% solvent B: acetonitrile/isopropyl alcohol, 50:50, v/v), 3 min (25% B), 11 min (45% B), 13 min (60% B), 18 min (75% B), 18.5 min (90% B), 20 min (90% B), 21 min (0% B), 26 min (0% B). 6k-PGF1α and PGE2 were detected in scheduled multiple reaction monitoring mode (transitions: PGE2 351 → 271, PGE2-d4 355 → 275, 6k-PGF1α 369 → 163, 6k-PGF1α-d4 373 → 167). For quantification, a 11-point calibration curve was used (0.06–60 ng/mL). Data analysis was performed using Analyst 1.7.2 and MultiQuant 2.1.1 (AB Sciex, Darmstadt, Germany).
9
Enrichment and Quantification of Co and As
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The SFI sample was enriched with SPE (Bond Elut Plexa, 6 mL/200 mg, Agilent). After washing with water, the methanol eluent was collected and evaporated to dry. The residue was dissolved in 5.0 mL water with a final concentration 0.6 g/mL Co or As.
The other samples, including Co_E, and As_E, were prepared with the same procedure, and the concentration was adjusted to a final concentration 0.6 g/mL Co or As.
The other samples, including Co_E, and As_E, were prepared with the same procedure, and the concentration was adjusted to a final concentration 0.6 g/mL Co or As.
10
Anthocyanin-rich Blueberry Extract Isolation
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The extracts were produced using an ethanolic solid–liquid extraction and purified using solid phase extraction columns (Bond Elut Plexa, Agilent Technologies, Santa Clara, CA, USA) as described elsewhere [11 (link)]. The resulting powder, henceforth referred to as the extract, contained 637 mg g−1 of anthocyanins with all 15 different anthocyanins found in blueberries (malvidin, cyanidin, delphinidin, petunidin and peonidin arabinosides, glucosides and galactosides) being present in the extract.
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