Ultimate 3000

The sample, control and synthetic indicaxanthin were each dissolved in aqueous MeCN solution (60%). These solutions were filtered and injected (1 μl) into a UHPLC system (Ultimate 3000, Thermo Fisher) equipped with a reverse-phase column (Kinetex^® EVO C18; 50 × 2.1 mm, 1.7 μm, Phenomenex), a DAD (Ultimate 3000) and a mass detector (TSQ Quantum Ultra, Thermo Fischer). The eluent was composed of MeCN (0.1% formic acid) and H₂O (0.1% formic acid), the flow rate was 0.4 μl/min, and the temperature of the column oven was 40°C. The compound was detected in positive SRM mode targeting the typical fragmentations of indicaxanthin of 309 Da to 217 Da (23 eV) and 309 Da to 263 Da (17 eV).

Standard stock solutions of ten different substances—quinic acid, gallic acid, chlorogenic acid, caffeic acid, catechin, quercetin, kaempferol, rutin, apingenin, and apingenin glycoside—were prepared in 50% acetonitrile (2 mg/mL). All solutions were filtered prior to analysis through a 0.45 μm syringe filter and injected three times into the HPLC. The phenolic compounds were identified by analyzing the same extracts with no added standard compounds and with added standard compounds. LC analysis was performed on a DIONEX UltiMate 3000 UHPLC+ focused system (Thermo Fisher Scientific, Waltham, MA, USA), which consists of an UltiMate 3000 pump, UltiMate 3000 autosampler, UltiMate 3000 column compartment, UltiMate 3000 variable wavelength detector, and Chromeleon software. As a stationary phase, an Ascentis Express 90 Å AQ-C18 column (15 cm × 3.0 mm, 2.7 μm, Supelco, Darmstadt, Germany) was used. The mobile phase was composed of 0.1% aqueous trichloroacetic acid (v/v) (A) and acetonitrile (B) with the following gradient elution: 0 min—95% A, 10 min—75% A, 25 min—20% A, and 30 min—95% A. Before each sample analysis, a 4 min equilibration with 5% B was performed. The flow rate was set at 0.425 mL/min, the column temperature was 40 °C, the injection volume was 1 μL, and the time of analysis was 30 min.

LC-MS/MS analysis was using either a capillary HPLC (Ultimate 3000, Thermo) in series with an ion trap MS (Esquire HCT Ultra, Bruker)^{35 (link)} or an Ultimate 3000 nanoLC in series with a Q-Exactive Plus Orbitrap MS (Thermo)^{45 (link)}. Search settings for the ion trap data were: Enzyme = trypsin, MS tolerance = 1.5 Da, MS/MS tolerance = 0.8 Da, missed cleavages = 1, fixed modifications = carbamidomethyl (Cys), optional modifications = oxidation (M). For the orbitrap data, the MS tolerance was set at 10 ppm and the MS/MS tolerance at 0.2 Da. Proteins were considered identified when at least two peptides were identified that were significant (p < 0.05). Peptides identified with a Mascot score of <15 were excluded from the identified peptide count. The quantitative analysis of whole cell lysates was conducted as above on an Ultimate 3000 nanoLC in series with an Orbitrap Fusion Lumos Tribrid MS (Thermo) with a 90 min run time^{46 (link)}. Three replicate injections of both the ATCC 33277 and W50 whole cell lysates  were analyzed.

The amount of MAP-1 or MAP-2 in the samples was analyzed using a 15-cm-long, 4.6 mm-inner diameter Inertsil ODS-4V column (GL Science, Tokyo, Japan), a UltiMate 3000 pump (Thermo Fisher Scientific), a UltiMate 3000 autosampler, and a UltiMate 3000 UV detector. The mobile phase was set at a flow rate of 1 mL/min, and the UV detector set at a wavelength of 254 nm.

The analysis was conducted using an HPLC system (Thermo Scientific, UltiMate 3000, Waltham, Mass, USA) equipped with a C18 (Acclaim^TM RSLC 120, Thermo Scientific, UltiMate 3000, Waltham, Mass, USA) analytical column (2.2 µm, 120 Å, 2.1 × 100 mm). The mobile phase consisted of acetonitrile and 0.1% TFA at a ratio of 80:20 (v/v). The system was run at a flow rate of 0.3 mL/min, with the column temperature at 30 °C. The detection was performed at the wavelength of 215 nm, and the injection volume was 1 µL.
The samples were prepared as follows: deionized water was added to the prepared FA-PC, followed by constant shaking under 100 rpm and 25 °C for 24 h. Subsequently, the resultant liquid was centrifuged at 12 krcf and 4 °C for 5 min to separate the undissolved drug. Then, the supernatant was collected and diluted with the mobile phase, and a 10 µL aliquot of the solution was injected into the HPLC system. In addition, FA powder was also dissolved in 1 mL of mobile phase, and a 10 µL aliquot of the solution was injected into the HPLC system.

Fatty acids and Ppc-1 in sera, tissues, and cells were analyzed by C18 reverse-phase liquid chromatography coupled to high resolution MS as reported [42 (link),43 (link)]. Briefly, lipids and organic compounds were extracted by the method of Bligh and Dyer [44 (link)]. The free fatty acid compositions were analyzed on high performance liquid chromatography (HPLC)-MS/MS system A (HPLC, UltiMate 3000; MS, TSQ Vantage, Thermo Scientific) controlled by XCALIBUR (version 2.2, Thermo Scientific). To assess relative concentrations, the peak areas obtained from the MRM chromatogram for each fatty acid were normalized to the peak area of C17:0 margaric acid added as an internal standard. For the detection of Ppc-1, the extracts were analyzed on HPLC-MS/MS system B (HPLC, UltiMate 3000; MS, Orbitrap, Thermo Scientific) controlled by XCALIBUR. The m/z list of Ppc-1 was obtained from Metworks (version 1.3, Rococo Co., Ltd, Osaka, Japan) software for the prediction of m/z of drugs and their derivatives. The peak values of Ppc-1 were calculated from mass spectra obtained by positive acquisition polarity in a mass spectrometer.

The plasma MDA concentration was determined using the high-performance liquid chromatography (HPLC) system Ultimate 3000 (Dionex, USA), after prior derivatization of MDA with 2, 4-dinitrophenylhydrazine as described previously [25 ]. The selenium concentration in whole blood was analyzed using hydride generation atomic absorption spectrometry – HG AAS (SOLAAR, Thermo Scientific, USA). The samples for Se determination were prepared by mineralization with HNO₃ and H₂O₂ using the microwave digestion system ETHOS TOUCH CONTROL (Milestone, Italy) followed by evaporation. Whole blood GPx activity was measured with a RANSEL kit (Randox Laboratories Ltd., UK) using a UV method based on that of Paglia and Valentine [26 (link)]. Serum TAS was also measured using standardized kits supplied by Randox Laboratories Ltd. For determination of GPx and TAS an automatic Konelab 20XT biochemical analyzer (Thermo Fisher Scientific, Finland) was used. Vitamin A and E and beta carotene concentrations in serum were determined using the HPLC system Ultimate 3000 (Dionex, USA). Blood samples for vitamins determination were prepared by extraction (hexane) followed by evaporation and dissolution in mobile phase (methanol).