Trifluoracetic acid

Analytical and chromatographic grade reagents were used for this study: acetonitrile, neochlorogenic acid, cryptochlorogenic acid, quercetin 3-O-(6″-O-malonyl)-β-d-glucoside (quercetin malonylglucoside in text), isorhamnetin 3-O-rutinoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, cyanidin 3-O-arabinoside, β-carotene, ascorbic acid, malic acid, fructose, glucose, sorbitol, sucrose, xylose, calcium carbonate, BHT, hexane, potassium persulfate, 2,2-azinobis (ethyl-2,3-dihydrobenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), Trolox were purchased from Sigma–Aldrich GmbH (Steinheim, Germany); 99.8% trifluoracetic acid, chlorogenic acid, hyperoside, isoquercitrin, rutin, astragalin were purchased from Carl Roth GmbH (Karlsruhe, Germany); 96.3% ethanol was purchased from Stumbras SC (Kaunas, Lithuania). Purified deionized water (18.2 mΩ/cm) was produced using the Millipore (Burlington, MA., USA) water purification system.

All reagents used for the study were of analytical and chromatographic grade. Acetonitrile, methanol, (+)-catechin and (−)-epicatechin, -hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-azinobis (ethyl-2,3-dihydrobenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulphate were purchased Fluka, epigallocatechin, epigallocatechin gallate, gallic acid, quercetin, delphinidin, cyanidin, 1-butanol, Sephadex LH-20, hydrochloric acid, acetone, ferric ammonium sulphate, phosphoric acid, 85 wt %, sodium acetate trihydrate, iron(III) chloride hexahydrate, and 2,4,6-tripyridyl-s-triazine (TPTZ) from Sigma-Aldrich (Steinheim, Germany), 99.8% trifluoracetic acid from Carl Roth (Karlsruhe, Germany), 96% ethanol from Vilniaus degtine AB (Vilnius, Lithuania). The purified deionized water (18.2 mΩ/cm) was produced using the Millipore water purification system. The Pelargonium sidoides root extract (PSRE) was purchased from UNILAB, LP (Rockville, MD, USA) (extraction medium 50% methanol).

From each TMA, a section of 4 μm was adhered to an indium-tin-oxide (ITO) slide (Bruker Daltonics, Bremen, Germany). Sample preparation has previously been described in detail [15 (link),16 (link)]. Briefly, sample slides were heated to 80 °C prior to dewaxing with xylene (Carl Roth GmbH, Karlsruhe, Germany), and subsequent rehydration with increasingly concentrated ethanol washes (Carl Roth GmbH, Karlsruhe, Germany). Afterward, the samples were subjected to heat-induced antigen retrieval in MilliQ water at 95 °C for 20 min. A trypsin (Promega, Mannheim, Germany) solution was prepared in 40 mM ammonium bicarbonate (Sigma-Aldrich Chemie GmbH, Munich, Germany) to a final concentration of 0.1 µg/µL. The enzyme solution was sprayed with an automatic sprayer (TM Sprayer, HTX Technologies, Chapel Hill, NC, USA) in 16 cycles with a fixed spraying flow of 150 μL/min. On-tissue digestion was carried out for 2 h at a controlled temperature of 50 °C. Following digestion, four cycles of matrix solution (10 mg/mL of alpha-cyano-4-hydroxycinnamic acid matrix (Sigma-Aldrich Chemie GmbH, Munich, Germany) in 70% acetonitrile aqueous solution with 1% trifluoracetic acid (Carl Roth GmbH, Karlsruhe, Germany)) were deposited with a defined flow of 120 μL/min and a temperature of 75 °C.