Ms 500 mass spectrometer

The phenolic composition was investigated by LC/DAD-ESI-MS following the procedure described by Pinto et al. [13 (link)], using an Agilent 1260 liquid chromatography (LC) equipment attached to a diode array detector (DAD), a electrospray ion (ESI) source operating in negative ion mode and a Varian 500-MS mass spectrometer (MS). The column used was an Agilent Eclipse C18 3 × 100 mm (3.5 µm) and the elution was ternary. The mobile phases were water with 1% formic acid (A), acetonitrile (B), methanol (C), and water with 0.1% formic acid (D) applied in the following gradients: 0–12 min, 5% A, 5% B and 90% D; 12–18 min, 5% A, 15% B, 20% C and 60% D; 18–20 min, 5% A, 70% B, 20% C and 5% D; and then, 5% A, 5% B and 90% D. The flow rate and the injection volume were 400 µL/min and 10 µL, respectively. Turbo data-depending scanning (TDDS) was used to attain the fragmentation patterns of the eluted compounds. The ultraviolet (UV) spectrum was also confirmed for the compounds identified in comparison with the respective standards and literature. For quantification purposes, ellagic acid, protocatechuic acid, and pyrogallol were used as standards. The results are presented as µg of each phenolic compound per gram on DW.

Separation of polyphenols was achieved on an Agilent Eclipse XDB C-18 (3.0 × 150 mm) 3.5 μm eluting with acetonitrile (A) and H₂O 0.1% formic acid (B). A gradient program was used as follows: 0 → 15th min: A:B (5:95) → A:B (15:85) 15 → 35th min: A:B (85:15) → A:B (100:0) 48 → 53th min: A:B(100:0) → A:B (5:95). After the chromatography column a “T” junction split the flow to DAD and Varian MS 500 mass spectrometer with ESI as ion source. Flow rate was set at 500 µL/min. For qualitative purposes, MSⁿ spectra was used for identification of the compound, with the use of reference when available. For quantification, DAD detector was set at 280, 330 and 350 nm. Compounds were identified on the basis of MS data and UV spectra and classified in four classes namely proanthocyanidins, dihydrochalcones, hydroxycinnamic acids and flavonols. For quantification of these classes of compounds the following reference compounds were used namely catechin, phloridzin, chlorogeinc acid and rutin. Standard solutions were prepared for the calibration curves in the concentration range of 5–100 µg/mL: catechin at 280 nm, y = 20.525x + 3.2962 (r² = 0.999); phloridzin at 280 nm, y = 87.029x – 1.832 (r² = 0.9998); chlorogenic acid at 330 nm, y = 47.359x + 439.99 (r² = 0.9951); rutin at 350 nm, y = 27.788x + 330.7 (r² = 0.9981).