Formic acid

MRM analyses were performed on a 6500 QTRAP mass spectrometer (SCIEX) equipped with an electrospray source, a 1290 Infinity UPLC system (Agilent Technologies, Santa Clara, CA), and a XBridge Peptide BEH300 C18 (3.5 μm, 2.1 mm × 150 mm) column (Waters, Milford, MA). Liquid chromatography was carried out at a flow rate of 400 µL/min, and the sample injection volume was 30 µL. The column was maintained at a temperature of 60 °C. Mobile phase A consisted of 0.1% formic acid (Sigma Aldrich) in water (Thermo Fisher Scientific), and mobile phase B consisted of 0.1% formic acid in acetonitrile (Thermo Fisher Scientific). The gradient with respect to %B was as follows: 0–1.5 min, 5%; 1.5–2 min, 5–15%; 2–5 min, 15%; 5–7.1 min, 15–20%; 7.1–8.1 min, 20–80%; 8.1–9.0 min, 80%; and 9.0–9.1 min, 80–5%. 9.1–16 min, 5%. The instrument parameters for 6500 QTRAP mass spectrometer were as follows: Ion spray voltage of 5500 V, curtain gas of 20 psi, collision gas set to “medium”, interface heater temperature of 400 °C, nebulizer gas (GS1) of 80 psi and ion source gas (GS2) of 80 psi, and unit resolution for both Q1 and Q3 quadrupoles as previously described in Ravipaty et al. 2017^{19 (link)}.

Liquid chromatography analysis was performed using a DIONEX Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). From each sample, 10 µL was injected onto a Synergi 4 µm Hydro-RP 80 Å, 250 × 3.0 mm column (Phenomenex, Le Pecq, France). The mobile phases were composed of 0.1% formic acid (Thermo Fisher Scientific) in water (A) and 0.1% formic acid in acetonitrile (B). The gradient was set as follows with a flow rate of 0.9 mL/min: 0% phase B from 0 to 5 min, 0–95% B from 5 to 21 min, holding at 95% B until 21.5 min, 95–0% B from 21.5 to 22 min, holding at 0% B until 25 min for column equilibration. Mass spectrometry analysis was carried out on a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) with a heated electrospray ionization source, HESI II, operating in both positive and negative mode. High-resolution accurate-mass full-scan MS and the top 5 MS2 spectra were collected in a data-dependent fashion at a resolving power of 70,000 and 35,000 at m/z 400, respectively. This standard procedure has been described in more detail in the cited publications [20 (link),21 (link),22 (link),23 (link),24 (link),25 (link)]. The analyses were performed separately on each of the two groups: the first group consisted of the 51 tumors of the training set and the second of the 49 tumors of the validation set.

Plasma extraction of anthocyanins and their metabolites was based on the method described recently [40 (link)]. Briefly, each aliquot (1 mL of plasma acidified with 30 µL of 50% aqueous formic acid (Lgc Promochem, Wesel, Germany)) was loaded onto an Oasis-HLB (1 mL/30 mg) SPE cartridge (Waters, Inc., Eschborn, Germany), preconditioned with 1 mL of methanol (Thermo Fischer Scientific, Langenselbold, Germany) and 1% formic acid, followed by 1 mL of acidified water (1% formic acid). The cartridge was then washed with 1 mL of acidified water, after which anthocyanins and their metabolites were eluted with 1 mL of acidified methanol. Afterwards, eluates were dried under N₂ for approximately 3 h [40 (link)]. For cell migration studies with HT-29 and Caco-2 cells, dried anthocyanins isolated from plasma as well as their metabolites (PAM) were resolved in the same volume of culture media (1 mL; pH 7.2) as the original plasma volume (1 mL).

High-performance liquid chromatography (HPLC) grade methanol and chloroform and LC–MS grade acetonitrile with 0.1% formic acid (v/v) and water with 0.1% formic acid (v/v) were purchased from Thermo Fisher Scientific (Waltham, USA). 1,4-dioxane, boron trifluoride diethyl etherate, ethanethiol, pyridine, and tricin were purchased from Sigma-Aldrich (St. Louis, USA). 4,4′-Ethylidenebisphenol was purchased from TCI America (Portland, USA). BSTFA + 1% trimethylchlorosilane (TMCS) silylation reagent was purchased from Thermo Fisher Scientific (Waltham, USA).