As 4150

RP-HPLC analysis was performed on a Jasco Extrema (JASCO, Inc., 28600 Mary’s court, Easton, Maryland 21601, USA) connected with an in built autosampler (AS-4150), quaternary pump (PU-4180) with an on-line degasser, column oven (CO-4061), UV/Vis detector (UV-4075) and an interface box LC-NetII/ADC. Chromatographic data was collected and analysed using ChromNAV Ver.2 software. A second HPLC system (Agilent 1100 series) equipped with variable wavelength detector (G1314A VWD), autosampler (G1313A) and isocratic pump system (G1310A). This system automated with a Chemstation (Agilent technologies) software and which was used for intermediate precision (repeatability) testing and data collection. Chromatographic separations were achieved by using SOLAS 100 Å C18 150 mm × 4.6 mm 5 µm column (Glantreo limited, Ireland). Different HPLC columns such as SOLAS ODS C18 150 mm × 4.6 mm 5 µm, SOLAS ODS C18 150 mm × 4.6 mm 3 µm, SOLAS BDS C18 150 mm × 4.6 mm 5 µm, SOLAS BDS C18 150 mm × 4.6 mm 3 µm and EIROSHELL C18 150 mm × 4.6 mm 2.6 µm (Glantreo limited) were used for this method development purpose. Each Chemical and test samples were accurately weighed using an analytical balance (Adventurer Pro AS214, Ohaus corporation, Pine Brook, NJ USA).

Quantification of trimesic acid (H₃BTC), H₄TazBz, AzA and Nic was performed using HPLC using a reversed phase Jasco LC-4000 series system, equipped with a PDA detector MD-4015 and a multisampler AS-4150 controlled by ChromNav software (Jasco Inc., Easton, MD, USA). A Purple ODS reverse-phase column (5 µm, 4.6 × 150 mm² Análisis Vínicos) was employed. Note here that AzA was analyzed after its derivatization (der-AzA), as previously described [42 (link)]. The mobile phase consisted of a 50:50 solution (v/v) of buffer (0.04 M, pH = 2.5) and methanol (MeOH) for H₃BTC and H₄TazBz; 20:80 solution (v/v) acetonitrile:buffer (0.5 mM, pH = 4) for der-AzA; and 5:95 solution (v/v) acetonitrile:buffer pH = 4 for Nic. The injection volume was set at 30 µL with a flow rate of 1 mL min⁻¹ and the column temperature fixed at 25 °C (H₃BTC and H₄TazBz) and 40 °C (der-AzA and Nic). The standard calibration curves showed a good correlation coefficient ≥0.99. The chromatogram of standard solutions showed a retention time (rt) of 3.51 min (identified as H₃BTC, λmax at 225 nm, Figure S1), 21.36 min (identified as H₄TazBz, λmax at 205 nm, Figure S2), 9.12 min (identified as der-AzA, λmax at 255 nm, Figure S3), and 5.56 min (identified as Nic, λmax at 213 nm, Figure S4).

All the analyses were performed on an integrated LC-4000 Series JASCO RHPLC system (JASCO, Tokyo, Japan) equipped with a quaternary pump (model No. PU-4180, Tokyo, Japan), a vacuum degasser, an autosampler (model No. AS-4150, Tokyo, Japan), a column oven (model No. CO-4062, Tokyo, Japan), and a fluorescence detector (model No. FP-4020, Tokyo, Japan) operating at the excitation and emission wavelengths of 378 nm and 492 nm, respectively. The detector signal was amplified tenfold. System control and data acquisition processes were performed using the ChromNAV2 software. Spectra Manager ver. 2 was used to analyze the spectra.
The samples (5 μL) were injected onto a 150 × 4.6 mm, 3.6 μm particle size Aeris™ WIDEPORE XB-C18 column (Phenomenex, CA, USA) using an autosampler. The mobile phase consisted of 0.1% TFA in water (A) and 0.1% TFA in ACN (B). All the analyses were performed at room temperature and the flow rate of the mobile phase was 1 mL min⁻¹. Chromatographic separation of HSA and low-molecular-weight thiols was achieved in 35 min with gradient elution as follows: 0–5 min, 5% B; 5–11 min, 5–8% B; 11–18 min, 8–15% B; 18–20 min, 15–30% B; 20–25 min, 30–40% B; 25–26 min, 40–50% B; 26–30 min, 50–70% B; 30–33 min, 70–5% B; 33–35 min, 5% B. Peaks were identified by comparing retention times and fluorescence spectra with corresponding data from the authentic standard.

Polyphenol identification was performed on a Jasco LC Net II, equipped with the AS-4150 autosampler, the PU-4180 pump and the MD-4010 PDA detector. The method and instrumental conditions were previously optimised (A1-A5) by the authors Lončarić et al. [27 (link)]. Quantification has been performed by external standard calibration and the calibration range for each phenolic standard was 0.1–10 µg/g. The concentration of individuals polyphenols was expressed as µg/g of dry weight (dw).

Quantification of trimesic acid (H₃BTC) was performed using HPLC using a reversed phase Jasco LC-4000 series system, equipped with a PDA detector MD-4015 and a multisampler AS-4150 controlled by ChromNav software v.2 (Jasco Inc., Easton, MD, USA). A Purple ODS reverse-phase column (5 µm, 4.6 × 150 mm² Análisis Vínicos, Ciudad Real, Spain) was employed. The mobile phase consisted of a 50:50 solution (v/v) of buffer (0.04 M, pH = 2.5) and methanol (MeOH). The injection volume was set at 30 µL with a flow rate of 1 mL·min⁻¹ and the column temperature fixed at 25 °C. The standards used for the calibration curve consisted of trimesic acid solutions in MilliQ water solution with a concentration range from 9.65 to 0.01 µg·mL⁻¹ (correlation coefficient > 0.99). The chromatogram of the standard solution showed a retention time (rt) of 2.70 min (λ_max at 225 nm).

Experiments were performed on an integrated LC-4000 Series JASCO RHPLC system (JASCO, Tokyo, Japan) equipped with a quaternary pump (model No. PU-4180, Tokyo, Japan), a vacuum degasser, an autosampler (model No. AS-4150, Tokyo, Japan), a column oven (model No. CO-4062, Tokyo, Japan), and a fluorescence detector (model No. FP-4020, Tokyo, Japan). The detector operated at the excitation and emission wavelengths of 264 nm and 338 nm, respectively. System control and data acquisition processes were performed using the ChromNAV2 software. Spectra Manager ver. 2 was used to analyze the spectra.

The total and free amino acids contents were analyzed by RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) using an integrated system from Jasco (Jasco, Japan) equipped with two high-pressure pumps (PU-980), an automatic injector (AS-4150), a fluorescence detector (FP-2020 Plus), and a UV/Vis absorption detector (MD-2015 Plus). The samples (extracts of total and free amino acids) were derivatized online with OPA/3-MPA and FMOC as described by Machado et al. [25 (link)]. Amino acids were identified based on the retention time of the corresponding standards. The quantification of each amino acid was based on the response of the fluorescence signal of each standard, converted into units of concentration through calibration curves obtained for each compound, using the internal standard method. The determination of the total and free amino acid contents was performed in triplicate.