As 4050

An integrated HPLC system (Jasco, Tokyo, Japan) was applied, which is built up from an autosampler (AS-4050), a binary pump (PU-4180), and a fluorescence detector (FP-920). Chromatographic data were evaluated employing ChromNAV2 software (Jasco, Tokyo, Japan). OTA was quantified as it has been recently reported [55 (link)]. Briefly, samples (20 μL) were driven through a SecurityGuard precolumn (C18, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, USA) linked to a Kinetex EVO-C18 (150 × 4.6 mm, 5 μm; Phenomenex) analytical column with 1.0 mL/min flow rate, at room temperature. Isocratic elution was performed, where sodium borate buffer (10 mM, pH 10.0) and ACN (87:13 v/v%) were applied in the mobile phase. OTA was detected at 446 nm (λ_ex = 383 nm). Samples were measured in triplicates.
The linearity of the method was determined between 10 nmol/L and 1.0 μmol/L (4.0–403.8 μg/L) concentrations (R² = 0.9996). Limit of detection (2 nmol/L or 0.8 μg/L) and limit of quantification (6 nmol/L or 2.4 μg/L) values were defined as the lowest concentrations when the signal-to-noise ratios reached 3 and 10, respectively. The intraday repeatability was evaluated based on the intraday coefficient of variation (0.55%; n = 7).

Total anthocyanins: The total anthocyanins were determined by the pH differential method according Lee et al. (2005) [46 (link)] by spectrophotometry (spectrophotometer V-700, Jasco, Tokyo, Japan). All analyses were carried out in triplicate.
Anthocyanin profile by HPLC-DAD: Anthocyanin analysis was done in a chromatographic system HPLC consisting of a Jasco interface LC-NetII/ADC with a diode array detector MD-4010 and an autosampler AS-4050 (Jasco, Tokyo, Japan) with a C18 column (Kromasil-100, 3.5 mm i.d × 150 mm). The samples (20 μL) were then injected. The mobile phases, A (0.2% v/v TFA in water), B (0.2% v/v TFA in acetonitrile), and C (0.2% v/v TFA in methanol), were used under the following conditions: initial, 8% B and C maintained for 5 min; an 11 min linear change to 12% B and 7% C; a 12 min linear change to 11.5% B and C; a 20 min linear change to 13.5% B and C; a 25 min linear change to 20% B and C; a 30 min linear change to 50% B and C.

We applied an integrated HPLC system (Jasco, Tokyo, Japan) built up from an autosampler (AS-4050), a binary pump (PU-4180), and a fluorescence detector (FP-920). Chromatographic data were evaluated employing ChromNAV2 software (Jasco, Tokyo, Japan). WAR concentrations were quantified using the previously reported method [35 (link),50 (link)]. Briefly, samples (20 μL) were driven through a guard column (SecurityGuard C18, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, USA) linked to a Nova-Pak C18 analytical column (150 × 3.9 mm, 4 μm; Waters, Milford, MA, USA). The isocratic elution was performed at room temperature with 1.0 mL/min flow rate applying sodium phosphate buffer (20 mM, pH 7.0), methanol, and acetonitrile (70:25:5 v/v%) as the mobile phase. WAR was detected at 390 nm (λ_ex = 310 nm).

The method was described in detail in a recent publication [24 (link)]. Briefly, the threefold volume of ACN was added to a 50 μL aliquot of plasma samples. After vortexing and centrifugation (10 min, 14,000× g, 4 °C), the supernatants were diluted with water then directly injected into the HPLC. Samples were analyzed using an HPLC system (Jasco, Tokyo, Japan), which included an autosampler (AS-4050), a binary pump (PU-4180), and a fluorescence detector (FP-920); the chromatograms were evaluated employing ChromNAV software. Samples (20 μL) were driven through a Security Guard™ (C₁₈, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, USA) guard column linked to a Gemini NX-C₁₈ (150 × 4.6 mm, 3 μm; Phenomenex, Torrance, CA, USA) analytical column with a 1.0 mL/min flow rate, at room temperature. The eluent contained ACN and sodium phosphate buffer (10 mM, pH 7.0) (30:70 v/v% in eluent A, and 55:45 v/v% in eluent B). We applied 100% eluent A from 0 to 4 min, then the eluent was linearly changed to 100% eluent B from 4.0 to 4.5 min, after which 100% eluent B was used from 4.5 to 14.9 min. SZV 1287 and its metabolites (oxaprozin, L 2799, L 2805, and L 2811) were detected applying 320 and 368 nm excitation and emission wavelengths, respectively. Solutions applied for the calibration curves were freshly prepared and diluted every day. The calibration curves showed good linearity (R² > 0.99).

HPLC analysis was conducted on a JASCO^® equipment with a reverse-phase C-18 column (LiChroCART^® 250-4, LiChrosorb^®, RP-18, 250 mm × 4 mm, 5 µm), a DAD (MD-4010, Jasco^®, Oklahoma City, OK, USA), an autosampler (AS-4050, Jasco^®, Oklahoma City, OK, USA), and a pump (PU-4180, Jasco^®, Oklahoma City, OK, USA). ChromNav^® 2.0 was the software employed for data acquisition and analysis, which was set at room temperature with an injection volume of 10 µL. Detection was performed at 254 and 368 nm. Different mobile phase mixtures were evaluated in an isocratic elution. Previously purified water was filtered with a 0.22 µm nylon filter, and all solvents were degassed by ultrasound. A ratio of 55:40:5 of water/acetonitrile/methanol, acidified with 1.5% acetic acid, was used as the mobile phase. The flow rate was as follows: 1.000 (0–3.5 min), 1.300 (3.5–8 min), and 1.000 (8 min until analysis finale) mL/min. The chromatographic run was set for 10 min.

3-Ga used for Apo-transferrin
coated NPs was prepared using a previously reported procedure.^{14 (link),33 (link)} In short, commercially available chemicals were purchased from Sigma-Aldrich,
Merck, and Chem Intel and used as received unless otherwise stated.
Apo-transferrin is from Sigma-Aldrich (St. Louis, MO, US). Analytical
reagent (AR)-grade solvents were used for the reactions and column
chromatography. Pyrrole was subjected to filtration using a column
packed with neutral aluminum oxide before use, while the remaining
reagents were employed without further purification. Unless otherwise
stated, synthesis was performed in ambient conditions. Produced 3-Ga for in vivo studies was recrystallized
in DCM/hexanes 1:1 and washed with cold hexanes for maximal purity.
HPLC analysis was performed on a combination of a JASCO organizer,
a diode array detector MD-4010, an autosampler AS-4050, and an RHPLC
pump PU4180. Silica gel (230–400 mesh) used for column chromatography
was obtained from E. Merck Ltd. Either flash or preparative thin-layer
chromatography was performed to purify the compounds. A purity of
>97% for 3-Ga was determined with HPLC and UV detection
at their Soret band region; a summary of the HPLC result and the HPLC
condition of 3-Ga is shown in the Supporting Information
(Figure S12–S14).