Waters acquity uplc h class

Frondoside A, the major saponin compound of sea cucumber extract, was quantified using UPLC-ESI-MS. The filtered samples (2 µL) were injected into the UPLC system (Waters Acquity UPLC H-class, Waters Limited, Milford, MA, USA) equipped with a phenyl-hexyl X-Bridge column (4.6 mm × 100 mm, 5 µm) and a tandem quadrupole mass detector (Waters Quattro Micro, Waters Limited, Milford, MA, USA). The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). The flow rate of 0.7 mL/min was set in a linear gradient mobile phase of solvent B applied at time t (min) as follows (t, B%): (0, 10%), (7, 30%), (16, 50%), (17, 50%), (18, 80%), (25, 80%), (27, 10%), and (35, 10%). The MS conditions were ESI in negative ion mode (ESI-) with a capillary voltage of 3000 V, nebulizer gas (N2) temperature of 375 °C, source temperature of 150 °C, and cone temperature of 60 °C. Frondoside A was detected (retention time of 21.9 min) using the single ion mode of m/z = 1312.

A second UHPLC system (Waters Acquity UPLC H-Class, Waters Corporation) equipped with an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm, Waters Corporation) was used for the analysis of anthocyanins. Used eluents were 0.1% formic acid (A) and acetonitrile (B), sample injection volume was 3 µl, solvent flow rate 0.3 ml min⁻¹, and the gradient as follows: 0.0–1.1 min, 5–10% B in A (linear gradient); 1.1–5.7 min, 10–60% B in A (linear gradient); 5.7–9.0 min, 60–90% B in A (linear gradient); 9.0–11.0 min, 90–100% B in A (linear gradient); column wash and stabilization. Quantification of anthocyanins was done by UV detection at 520 nm. Cyanidin-3-glucoside was used as an external standard and the calibration curve was determined at a concentration range of 0.59–63.83 µg ml⁻¹.
The identification on anthocyanins was confirmed on a Waters Q-Tof Premier mass spectrometer coupled to a Waters Acquity UPLC using the same column and gradient as for anthocyanin quantification. Electrospray ionization was used in positive mode and ions were detected between m/z 100–1200. The conditions were set at as follows: capillary voltage 3.0 kV, sample cone voltage 45 V, ion source temperature 120 °C, desolvation gas (N₂) flow and temperature 800 l h⁻¹ and 270 °C, and collision gas (Ar) flow 0.60 l h⁻¹. Reserpine was used as a lock spray compound.