Cto 10a

Mass spectra were obtained using the Shimadzu Prominence system comprising a Shimadzu CBM-20A system controller, a Shimadzu LC-20AD binary HPLC pump, a Shimadzu CTO-10AS column oven, a Shimadzu SIL-20ACHT cooling autosampler, and a Shimadzu SPD-20MA diode array detector (Shimadzu, Tokyo, Japan). The ESI-MS parameters were as follows: positive and negative mode; ESI interface voltage 4.5 kV; detector voltage 1.15 kV; nebulizing gas flow 1.5 mL·min⁻¹; drying gas flow 15 mL·min⁻¹; heat block temperature 200 °C; temperature of desolvation line pipe 250 °C; SCAN mode 300–700 m/z. The mobile phase (acetonitrile) flow rate was 0.3 mL min⁻¹; chromatograms were analyzed using LabSolutions software ver. 5.75 SP2 (Shimadzu, Kyoto, Japan).

RP-HPLC-DAD setup consisted of a degassing unit DGU-20A, a liquid chromatograph LC-20AT, a column oven CTO-10AS with a C18 5 µm column (250 × 4.6 mm I. D.), a refractive index detector RID-20A, and a photodiode array detector SPD-M20A, all of which were manufactured by Shimadzu, Japan. The mobile phase was an isocratic flow of 20% methanol and water with 0.1% TFA at a flow rate of 1 mL/min and an injection volume of 20 µL. Prior to injection, samples were dissolved in 20% MeOH containing 0.1N NaOH to allow for increased solubility of lignin. The liquid extracts from the various extraction processes of the different bark species were used. As reference substances DHBA (dihydroxybenzoic acid), HBA (hydroxybenzoic acid), trans-CA (trans-cinnamic acid), SA (syringic acid), CA (coumaric acid), FA (ferulic acid), V (vanillin) and T (taxifolin) were applied.

Vitamin C was estimated for tubers obtained from field experiments. For each potato cultivar and each repetition, three tubers were collected. Potato tubers were peeled and then rubbed on a plastic grater. About 6 g of sample was weighed into a 50 mL test tube. It was supplemented with an extraction solution (metaphosphoric acid, acetic acid, pH 2) at a weight of 25 g, which prevented the breakdown of vitamin C. The sample was shaken on a Thermomixer compact (Eppendorf AG, Hamburg, Germany) for 15 min at 4 °C and after this was spun for 10 min at 4 °C and 604× g in Centrifuge 5417R (Eppendorf AG, Hamburg, Germany). After centrifugation, the clear solution was filtered through a 0.40 µm PTFE syringe filter. The injection volume was 2 µL. The mobile phase was water: acetonitrile in relative 2:98, flow 1.5 mL min⁻¹. Analyses were performed using Column Luna 3u HILIC 150 × 4.6 mm (Phenomenex, Torrance, CA, USA). Vitamin C concentrations were evaluated using ultra-high efficiency HPLC liquid chromatography kit (LC-20AD, DGU-20A 3, SIL-20AC HT, CTO-10AS) with a UV detector (SPD-20A) from Shimadzu (Kyoto, Japan).

All analytical HPLC analyses were performed with the Shimadzu Prominence System (Shimadzu, Kyoto, Japan) consisting of a DGU-20A mobile phase degasser, two LC-20AD solvent delivery units, a SIL-20AC cooling auto sampler, a CTO-10AS column oven and SPD-M20A diode array detector. Chromatographic data were collected and processed using Shimadzu Solution software at a rate of 40 Hz and detector time constant of 0.025 s. The Chromolith Performance RP-18e monolithic column (100 × 3 mm i.d., Merck, Darmstadt, Germany) coupled with a guard column (5 × 4.6 mm; Merck, Darmstadt, Germany) was used. Mobile phase acetonitrile/water/formic acid (80/20/0.1, v/v/v, phase A) and acetonitrile/water/formic acid (5/95/0.1, v/v/v, phase B) were employed in the analyses; gradient: 0–10 min 7%–80% A; 10–12 min 80% A, 12–14 min 80%–7% A. The flow rate was 1.2 mL/min at 25 °C. The photodiode array (PDA) data were acquired in the 200–450 nm range, and 360 nm signals were extracted.

An aliquot was taken from each serum bottle using a sterile syringe every day and then filtered through a 0.45 μm membrane filter for measuring nitrate and nitrite concentrations using an ion chromatography (Metrohm Compact IC-861, Switzerland). The eluent was prepared as a mixture of 3.2 mM Na₂CO₃ and 1.0 mM NaHCO₃ and was degassed before use. A 20.0 mM H₂SO₄ was used as a regeneration solution. The instrument was calibrated on a daily basis using appropriate standard solutions. For antibiotics analysis, an aliquot was periodically sampled from each serum bottle. The sampled solution was also filtered through a 0.45 μm membrane filter and then transferred to amber colored vials. A high performance liquid chromatographer (HPLC; SCL-10A, Shimadzu, Japan) equipped with an autosampler (SIL-10AD; Shimadzu, Japan) and a UV-VIS detector (SPD-10A; Shimadzu, Japan) was used. A reverse-phase Sunfire C18 column (4.6 mm × 250 mm; Waters, USA) was employed in a column oven (CTO-10AS; Shimadzu, Japan). The mobile phase A was a mixture of 99.9% deionized water and 0.1% formic acid, while mobile phase B comprised 99.9% acetonitrile and 0.1% formic acid. The eluent was pumped as 70% mobile phase A and 30% mobile phase B in a binary gradient mode at the rate of 0.32 mL/min. A 10 μL aliquot of the sampled solution was injected into the column at 25°C and the absorbance was measured at 254 nm.

The LC-MS-system (Shimadzu, Kyoto, Japan) consisted of a CBM-20A controller unit, pumps (LC-20AD), a SIL-20A_HT auto sampler, a CTO-10AS column oven, a valve unit FCV-20AH₂ and an MS-8030. The mobile phase was 0.2% HCOOH. MeOH was added in the following gradient: 0–3.0 min 1% MeOH, 3.1–6.0 min 15% MeOH, 6.1–10.0 min 1% MeOH. The flow rate was set to 1 mL/min. A C18 column (Kinetex 4.6 × 100 mm 2.6 µm 100 Å) was kept at 50 °C. Samples (10 µL) were auto-injected.
For MS detection, electrospray ionization (ESI) was applied as the ion source, while the combination of a triple-quadrupole and an electron multiplier were used as analyzer and detector. Detection times were set from 3.0 min to 10.0 min.
PTCA and TTCA were measured using the positive selected ion monitoring mode in the third quadrupole (Q3-SIM), while PDCA and TDCA were measured using the negative Q3-SIM mode. PDCA and PTCA were identified both by reference substances and mass-to-charge ratio (m/z) (PDCA 154.0; PTCA 200.0). TDCA and TTCA were identified by mass-to-charge ratio (TDCA 172.0; TTCA 218.0) only. The latter were not commercially available. The area under the curve (AUC) obtained by MS analysis was normalized to the total count of all events in the range of 0.5 µm to 50 µm (see Section 4.6). The AUC per count (AUC/count) was used to compare relative amounts of specific degradation products.