Lc 1200

The contents of each phenolic acid in free and bound phenolic extracts from the raw and cooked triticale samples were evaluated via three repetitions using the HPLC method described by Irakli et al. [22 (link)] with minor adjustments. A 20 μL aliquot of sample solution was fractionated using an HPLC system (LC1200, Agilent Technologies, Santa Clara, CA, USA) with a diode array detector. An Inspire C18 column (5 µm, 4.6 mm × 250 mm; Dikma tech, Lake Forest, CA, USA) was used for fractionation at 30 °C. The mobile phase, with 1 mL/min of the flow rate, consisted of solvent A (methanol) and solvent B (acetic acid/water, 1:99, v/v). A 58 min linear gradient was performed as follows: 0–13 min, 90–80% B; 13–15 min, 80–75% B; 15–28 min, 75–65% B; 28–43 min, 65–35% B; 43–52 min, 35–0% B; 52–58 min, 0% B. The detected wavelengths were 260 nm for IS, PRCA, p-OHBA, VA, and t-CA, 270 nm for GA and SRA, and 320 nm for CA, p-CA, FA, and SA. The spectra were recorded from 190 to 400 nm. Phenolic acids in the free and bound phenolic extracts were recognized by comparing their UV spectra and relative retention times with those of the authentic compounds, and the concentration of each compound was calculated based on the peak area ratio of analytes to IS at 10 μg/mL using an internal standard method. The phenolic acid content was expressed as micrograms per gram of dry sample (μg/g).

GPC was performed using an Agilent LC 1200 setup equipped with an isocratic pump, a PL-gel 5 µm Mixed-C column PL 1110–6500, and a refractometric detector. The system, operating at 40 °C with an eluent (THF) flow rate of 1 mL/min, was calibrated against narrow polystyrene standards (ranging from 162 to 6,035,000 g mol⁻¹).

Silica gel (Kieselgel 60, Merck 0.063–0.200 mm) and Sephadex LH-20 were used in column chromatography (CC). Technical solvents used in extraction were previously distilled and dried according to standard procedures. The extraction and purification of 1,2,3,4, and 5 were carried out essentially as described^33–36 (link). The purity of all compounds was estimated by using a HPLC-PDA (≥ 98%) (Agilent LC-1200). Tau 4R (four microtubule binding domain) recombinant protein was expressed in E coli. The expressed protein was purified by HPLC using a ProPac IMAC-10 Column. Aggregation and fluorescence assays were performed in a Biotek H1 monochromator-based multi-mode microplate reader. Samples were prepared on highly ordered pirolytic graphite (HOPG) as substrate, and atomic force images were obtained using a Nanoscope III equipment in tapping mode. Raman spectra and maps were obtained through a Confocal Raman Microscopy Alpha 300 (WITec GmbH) .

To determine the contents of drug loading (LC) and entrapment efficiency (EE) of the FA-DM1-NPs, a predetermined amount of nanoparticles were dissolved in 1 mL methylene dichloride under vigorous vortexing. The solution was transferred to 5 mL of mobile phase consisting of acetonitrile and deionized water (50:50, v/v). A nitrogen stream was introduced to evaporate the methylene dichloride for approximately 20 min. And then a clear solution was obtained for high-performance liquid chromatography (HPLC) analysis (LC 1200, Agilent Technologies, Santa Clara, CA, USA). A reverse-phase C₁₈ column (250 mm × 4.6 mm, 5 μm, C₁₈, Agilent Technologies, Santa Clara, CA, USA) was used at 25°C. The flow rate of mobile phase was 1 mL/min. The column effluent was detected using a UV detector at λ_max of 227 nm. The measurement was performed in triplicate. Drug loading and encapsulation efficiency of the drug-loaded nanoparticles were calculated according to the following equations, respectively.
$\begin{array}{c}\mathrm{L}\mathrm{C}\left(\%\right)=\left(\mathrm{\text{Weight}}\mathrm{\text{of}}\mathrm{D}\mathrm{M}1\mathrm{\text{in}}\mathrm{\text{the}}\mathrm{\text{nanoparticles}}\right)/\hfill \\ \left(\mathrm{\text{Weight}}\mathrm{\text{of}}\mathrm{\text{the}}\mathrm{\text{nanoparticles}}\right)\times 100\%\hfill \end{array}$
$\begin{array}{c}\mathrm{E}\mathrm{E}\left(\%\right)=\left(\mathrm{\text{Weight}}\mathrm{\text{of}}\mathrm{D}\mathrm{M}1\mathrm{\text{in}}\mathrm{\text{the}}\mathrm{\text{nanoparticles}}\right)/\hfill \\ \left(\mathrm{\text{Weight}}\mathrm{\text{of}}\mathrm{\text{the}}\mathrm{\text{feeding}}\mathrm{D}\mathrm{M}1\right)\times 100\%\hfill \end{array}$
Briefly, 10 mg of FA-DM1-NPs were introduced into Eppendorf tubes and dissolved in 1 mL acetonitrile and diluted by 0.1 M citric acid. Meanwhile, the amount of DM1 in the solution was determined by HPLC.

Pools of peptide samples of control tissue (C), as well as the MS tissues from gray and white matter were prepared (300 μg each) and separated using Hydrophilic Interaction Liquid Chromatography (HILIC) (Agilent LC1200 equipped with a column Polyamin II 250 × 3.0 mm 120 Å, 5 μm). The applied gradient was formed of the two solvents: A: 75% ACN, 8 mM KH₂PO₄ and B: 5% ACN, 100 mM KH₂PO₄ (pH4.0) for 60 min. Fractions of 1 ml were collected in 27 tubes (detailed protocol as Figure S2). To reduce the number of the samples to be analyzed on the mass spectrometer the fractions were pooled from two tubes. Before injection, samples were purified on “Finisterre SPE” columns (Wicom International, Heppenheim, Germany). Samples of 1 ml were vacuum dried and dissolved in an appropriate buffer, 3%ACN/0.1%TFA. In total 11 fractions for each tissue sample (control and MS of gray and white matter) were prepared. After another vacuum drying, peptides were dissolved in 3%ACN/0.1%FA buffer. Concentrations were measured with Nanodrop instrument and adjusted to 0.25 μg/μl. Reference peptides (iRT) were added (iRT, Biognosys, Schlieren, Switzerland) to each sample.