Kromasil

Chromatographically pure eremomycin amide and (adamantyl-2) eremomycin amide were provided by the Gause Institute of New Antibiotics (Moscow, Russia). Chloreremomycin was isolated from the cultural liquid provided by JSC «Biohimik» (Saransk, Russia) and purified using preparative reversed-phase HPLC.
5 µm silica (Kromasil, Akzo Nobel, Sweden) with a specific surface area of 313 m²/g and pore diameter of 11 nm was used as a matrix for CSPs synthesis. Pure 3-glycidoxypropyltriethoxysilane (Sigma-Aldrich, Milwaukee, WI, USA) was used to activate silica with epoxy-functional groups.
An amino acid standard kit was purchased from Sigma-Aldrich (USA). Chromatographically pure methanol and acetonitriles were obtained from Panreac (Spain). Glacial acetic acid (pure for chemical analysis, Vekton, Moscow, Russia), ultrapure phosphotic acid, pure 25% ammonium hydroxide (both from Khimmed, Moscow, Russia), triethylamine (pure, Sigma-Aldrich, CIIIA), ultrapure perchloric acid (Reakhim, Moscow, Russia), and pure for analysis sodium dihydrophosphate (Sigma-Aldrich, CIIIA) were used as buffers and additives to eluents. Deionized water was purified by the Werner system (Leverkusen, Germany).

The content of chlorogenic acid from the permeation studies was evaluated by HPLC-DAD. The separation was performed in a reverse phase column (C-18, 250 mm × 4.6 mm × 5.0 µm; Kromasil, Akzo Nobel, Nashville, TN, USA) in an HPLC-equipped with: ultimate 3000 pump LPG; auto sampler WPS-3000 TSL; columns compartment TCC-3000 SD and diode array detector (DAD) (Thermo Fisher Scientific, São Paulo, Brazil). Water-formic acid 0.1% v/v (A) and acetonitrile-formic acid 0.1% v/v (B) were used as mobile phases in the gradient mode: (i) 0–20 min, 10% B; (ii) 21–26 min, 100% B; (iii) 27–37 min, 10% B. The flow rate used was 1.0 mL/min and the injected volume of 70 µL. The detection was performed at 325 nm [29 ,31 (link)].

Milk fat hydrolysis was monitored by determination of lipid classes following a procedure adapted from Tan and Brzuskiewicz [43 (link)] in a HPLC (Pump LC-20at, Degasser DGU-20A5 and communicator module CBM-20A; Shimadzu^®, Japan) (Figure S1). Samples were dissolved in an injection solution, composed of acetonitrile:isopropanol:hexane (2:2:1, v/v/v) and a reversed-phase HPLC column (C18, 5 µm, 250 mm × 4.6 mm, Kromasil^®; AkzoNobel^®, Sweden) was used. The lipid analytes were eluted with a mobile phase gradient of acetonitrile (A) and isopropanol (B), at 1.0 mL/min, from 0 to 69% B from 0 to 60 min, followed by re-equilibration until 0% B, from 60 to 76 min (Table S1). The eluate was monitored with an evaporative light scattering detector (ELSD-LT II; Shimadzu^®, Japan), using N₂ as nebulizer gas at 0.65 mL/min, and drift tube temperature at 40 °C. Lipid classes of TAGs-DAGs and FFAs-MAGs were identified comparing with commercial standards and quantified by internal normalization. Results were expressed in g/100 g of total lipids.