Dgu 20a5

High-performance liquid chromatography (HPLC) fingerprinting of ME and EA was conducted to detect the presence of patuletin. The purity of patuletin was also determined as described earlier (Li et al., 2010). Briefly, both samples and standards including patuletin, patulitrin, and methyl protocatechuate were prepared in the mobile phase. An HPLC-Prominence System Controller CBM-20A (Shimadzu) equipped with a degasser (DGU-20A5), quaternary pump (LC-20AT), autosampler (SIL-20A), and diode array detector (SPD-M20A) and software LC Solution were used for analysis.

The quantification of clonidine hydrochloride was performed using an HPLC system (Shimadzu Prominence UFLC) comprising a binary pump (LC-20AD), a solvent degasser (DGU-20A5), a multiple wavelength photodiode array detector (SPD-M20A), a refrigerated autosampler (SIL020AC HT) and a column oven (CTO-20AC). Integration and peak purity were calculated with the help of the HPLC software (LabSolutions 5.54 sp5, Shimadzu Corporation).
The separation was performed on a Kinetex XB- C18 chromatographic column (3.0 x 100 mm, 5 μm, Phenomenex, Torrance, CA, USA). The column temperature was kept at 40°C and the UV detector wavelength was set at 210 nm. The flow rate was 1 mL/min and the injection volume was 10 μL. The composition of mobile phase A was a mixture of 923.0 mL of purified water, 77.0 mL of acetonitrile and 0.5 mL of trifluoroacetic acid, while mobile phase B was acetonitrile. Between 0 and 4 min, mobile phase A was 100%. Then mobile phase A proportion was reduced to 30% in 0.5 min and remained the same for 1 min. Finally mobile phase A was set back to its initial conditions in 0.5 min, for re-equilibration until 15 min.

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.

The method was developed and validated on a Shimadzu LC-20AT HPLC system (Shimadzu, Kyoto, Japan) consisting of a pump (model LC-20AD), a degasser (model DGU-20A5), a PDA detector (model SPD-M20A), an autosampler (model SIL-20ACHT), and a control module (model CBM-20Alite). This system used LC solution software version 1.25 SP4 for data processing and evaluation. Analytical column was a Luna C18 column (250 × 4.6 mm, 5 μm) of Phenomenex (Torrance, CA, USA).