Zorbax rrhd sb c18 column

The assay was performed on LC-Q/TOF-MS using Agilent 1290 Infinity LC coupled with an electrospray ionization (ESI) source and Agilent 6530Q-TOF mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The LC separation was carried out on a ZORBAX RRHD SB-C₁₈ column (2.1 × 100 mm, 1.8 μm, Agilent) with the column temperature maintained at 40°C. The mobile phases consisted of ultrapure water (A) and acetonitrile (B) both containing 0.1% (v/v) formic acid; the gradient program was shown in Table 1. The injected volume of sample was 4 μL and temperature of autosampler was 4°C.
Nitrogen was used as both nebuliser gas and cone gas. Detection mode of flight tube was type V. The mass spectrometric data was collected in both positive and negative modes with the following parameters: capillary voltage 4 kV (positive mode) and 3.5 kV (negative mode), sampling cone voltage 35 kV (positive mode) and 50 kV (negative mode), source temperature 100°C, desolvation temperature 350°C (positive mode) and 300°C (negative mode), cone gas flow rate 50 L/h, desolvation gas flow rate 600 L/h (positive mode) and 700 L/h (negative mode), extraction cone voltage 4 V, and full scan mode scanning from m/z 50–1000 with a scan time of 0.03 s and an interscan time of 0.02 s. Leucine enkephalin was used as the lock mass ([M+H]⁺ = 556.2771 in the positive mode and [M−H]⁻ = 554.2615 in the negative mode).

The HPLC analyses were carried out using an Agilent 1290 Infinity LC system coupled with photodiode array detector. Separation was performed at 25°C on a ZORBAX RRHD SB-C18 column (1.8 μm, 2.1 mm × 150 mm; Agilent Technologies, Singapore). Two solvents were used for the mobile phase: 0.1% formic acid in distilled water (v/v; solvent A) and 0.1% formic acid in acetonitrile (v/v; solvent B). The following optimized gradient elution (expressed in % B) was used: 0–2 min, 5% B; 2–6 min, 5–14% B; 6–38 min, 14–40% B; 38–40 min; 40–90% B; 40–42 min, 90–5% B; 40–45 min, 5% B. Aliquots (20 μl) of phenolic extracts from each time point were injected into the column. The flow rate was 0.3 ml/min, and detection was performed at 280 nm. Phenolic acids were identified by comparing their retention times and UV visibility with the standards under same analysis conditions. Quantitation was based on linear calibration curves of phenolic acid standards prepared using concentrations ranging from 0.78125 to 100 mg/L. All measurements were performed in triplicate and all the samples were injected in duplicate. The final concentrations of phenolic acids were expressed as μg/g.