Pda eλ detector

The analytes were separated and quantified using a Waters Acquity TM Ultra Performance LC system (Waters Corp., Madrid, Spain) equipped with an autosampler and a PDA eλ Detector (Waters). The analytical column was an ACQUITY UPLC^®BEH C18 column (1.7 µm, 2.1 × 100 mm) that was maintained at 30 °C. The separation was performed under a gradient elution mode using a mobile phase consisting of water (solvent A) and acetonitrile (solvent B). The elution began by using 20% of solvent B and linearly increased to using 60% within 8 min. The flow rate was maintained at 0.5 mL/min and the injection volume was 5 μL. The detection was performed at 290 nm.

L-Glu and L-Gln contents were determined by ultra-performance liquid chromatography (UPLC) with an AccQ-Tag Ultra system (Waters, Milford, MA, USA) as previously reported [16 (link)]. The brain tissues were homogenized in T-PER (tissue protein extraction reagent) (Pierce, Rockford, IL, USA) and the supernatant was collected after centrifugation at 12,000 rpm for 30 min. Protein concentration in the supernatant was determined using the Bicinchoninic Acid (BCA) reagent (Pierce, Rockford, IL, USA). The sample was ultra-filtered using a SmarPor Syringe Filter (25 mm, 0.2 µm, Woongki Ltd., Seoul, Korea). Once the filtrate was diluted to a suitable concentration, fluorescence derivatization was performed according to the AccQ-Tag manufacturer’s instruction. A 20-µL sample solution, 60 µL of AccQ-fluor borate buffer, and 20 µL of AccQ-fluor reagent were mixed together, and the mixture was incubated for 5 min at 55 °C. Derivatized amino acids were separated on an AccQ-Tag Ultra column (2.1 × 100 mm, Waters) by gradient elution (AccQ-Tag Ultra eluent A and B) at 30 °C. The derivatized amino acids were detected by a PDA eλ detector (Waters) [17 (link),18 (link)]. The chromatography data were analyzed using Empower software (Waters). To determine amino acid concentrations, a standard solution containing known concentrations of amino acids were analyzed with samples in every series.

The chromatographic separation was performed using a Waters ACQUITY UPLC system with a PDA e-λ detector (Manchester, UK) and a Waters ACQUITY UPLC HSS T3 Column (100 Å, 1.8 μm, 2.1 mm × 50 mm). The binary mobile phase consisted of 0.2% acetate acid in water (A) and acetonitrile (B). The flow rate was controlled precisely by a binary solvent pump at 0.4 mL/min. Linear gradients from 10%B/90%A to 100%B/0%A over 20 min were used (the linear gradient sequence was 0–8 min: 10% B; 8–10 min: 40–80% B; 10–16 min: 80–100% B; 16–20 min: 100% B). The injection volume of the sample was 2 μL (partial loop with Needle overfill) and the absorbance was recorded at 210 and 254 nm.

Fractions from the organic extract were diluted in methanol suitable for LC-MS at 1.0 mg/mL, filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane, and analyzed in a Waters ACQUITY class-H UPLC^TM system using an Acquity UPLC^® BEH (1.7 μm) C18, reverse phase LC column, 2.1 × 100 mm, a Waters PDA eλ Detector, and a XEVO^® TQD spectrometer (Waters Corporation, Milford, MA, USA) supplied with an electrospray ionization (ESI) source. The gradient for the chromatographic separation was carried using a step UPLC run of acetonitrile and water (H₂O), starting at 10:90% AcCN-H₂O to 100% AcCN in 10 min and returning to initial condition of 10:90% AcCN in 2 min at a flow rate of 0.2 mL/min throughout the run. The ESI-MS/MS parameters were set as follows: capillary voltage 3.5 kV; cone voltage 20 V; source temperature 150 °C; desolvation temperature 450 °C; desolvation gas flow 600 L/h. Mass spectrometry data were acquired in positive ion mode with a detection range of 175–2000 m/z using the MS Survey Scan method for MS/MS fragmentation. MassLynx^TM software (version 4.2) was used for data acquisition and processing.

The extraction of phenolic acids from mushroom samples and UPLC determination was according to Gąsecka et al. [40 (link)]. The samples were mixed with 80% methanol and were shaken for 8 h at room temperature. They were then centrifuged and evaporated to dryness at 40 °C. For further analysis, the extracts were redissolved in 1 mL of 80% methanol and filtered. An ACQUITY UPLC H-Class System and PDA eλ Detector (Waters Corporation, Milford, MA, USA) were used for phenolic acid identification. The separation of the acids was conducted on an Acquity UPLC BEH C18 column (2.1 mm×150 mm, 1.7 µm, Waters) thermostated at 35 °C. Gradient elution was performed with water and acetonitrile (both containing 0.1% formic acid, pH = 2) at a 0.4 mL/min flow rate.