Agilent 1100 series hplc instrument

Chromatographic analysis of the secondary metabolites was carried out on an Agilent series 1100 HPLC instrument (Agilent Technologies, Waldronn, Germany) with a DAD, on a reverse-phase Zorbax SB C-18 (Agilent, Newport, Delaware, USA) analytical column (250 × 4.6 mm, 5 µm particle size) thermostatted at 25 °C. Prior to HPLC analysis, the extracts were filtered through nylon syringe filters (Captiva syringe filters, 0.45 µm, 13 mm, Agilent Technologies). The mobile phase consisted of solvent A (1%, v/v solution of orthophosphoric acid in water) and solvent B (acetonitrile, J.T. Baker, Deventer, the Netherlands), using gradient elution previously published by Popović et al. [20 (link)]. Briefly, samples of belowground and aboveground plant parts were separated as follows: 98–90% A 0–5 min, 90–85% A 5–17 min, 85% A 17–20 min, 85–70% A 20–30 min, 70–0% A 30–39 min, and 0% A 39–42 min. The flow rate was 1 mL/min. The injection volume of samples was 5 µL; the detection wavelengths were set at 260 and 320 nm. The quantification of secondary metabolites was done using the external standard method by preparing calibration standards ranging from 0.01 to 0.5 mg/mL and recording the calibration curves at 260 nm for secoiridoids and flavonoids, and at 320 nm for xanthone mangiferin. The results are presented as milligrams per gram of dry weight (dw).

To detect the possible post-induction changes in the levels of individual ganoderic acids in the fruit body, the methanol extract was used to perform the assay on an Agilent series 1100 HPLC instrument (Agilent, USA) equipped with a quaternary pump, a diode-array detector (DAD), an autosampler, and a column compartment. The sample (10 μL) was separated on a Zorbax SB-C₁₈ column (5 μm, 4.6 × 250 mm; Agilent). The mobile phase consisted of acetonitrile (CH₃CN) and water containing 0.1% (v/v) CH₃COOH, with a gradient from 30% to 32% CH₃CN over the first 40 min, then up to 40% in 20 min, and held at 40% CH₃CN for another 5 min. The flow rate was 1.0 mL/min, and column temperature was set at 35°C. The DAD detector was monitored at 252 nm. The mixture of ganoderic acids A, C, C2, E, F, and lucidenic acid A in methanol was used as the reference standard.

An agilent series 1100 HPLC instrument (Agilent, France) equipped with a quaternary pump, a diode array detector and an autosampler was used for analyses. Sample extracts (5 mg/mL) were separated on a C18 column. The mobile phase was a binary solvent system composed of formic acid in water (0.5/99.5 v/v) labelled solution A and formic acid in acetonitrile (0.5/99.5 v/v) named solution B. The linear solvent started from 95 % A − 5 % B, up to 60 % A – 40 % B within 60 min, at 0.8 mL∙min-1. 325 and 280 nm were used for this analysis. The levels of polyphenols were expressed in micrograms/milligrams (µg/mg).