Agilent 1260 2

The stability was assessed by analyzing PVR sample solution five times within 24 hours. Variation was evaluated by the RSD. The robustness studies were carried out by analyzing the reference solution with the developed method with small changes in method parameters as follows: flow rate (1.0 ± 0.1 mL/min) and column temperature (35 ± 3°C). The developed method was also tested on 3 different Agilent Poroshell 120 EC-C18 columns (batch number: B15046, B18386, and B19476) and 2 different instruments (Agilent 1260 I and Agilent 1260 II). With chlorogenic acid as the reference compound, the RRT values and the content of neochlorogenic acid and cryptochlorogenic acid in PVR samples were calculated for evaluation. Furthermore, the resolution of the 3 target peaks in PVR sample solutions was also used to evaluate the robustness.

HPLC-C18 fractionation was carried out according to the conditions described in [35 (link)]. Briefly: The fractionation of extracts of dinoflagellates was carried out on an Agilent 1100 G1312A LC system coupled to an Agilent 1260 II automatic fraction collector with an Agilent 1260 II UV detector (Agilent Technologies, Waldbronn, Germany). A Kinetex® LC-C18 column (4.6 × 250 mm, 5 µm, 100 Å, Phenomenex) was used for the fractionation. Water with 5 mM of ammonium formate and 0.1% of formic acid (A) or methanol (B) constituted each mobile phase. The mobile phase gradient started from 60% B to 100% B, taking 85 min at a flow rate of 1 mL/min. The injection volume was 100 µL. A total of 49 fractions were collected. The solvent was removed by evaporation under N₂ at 50 °C and reconstituted in 1 mL of MeOH LC-MS.
The initial methanolic extract of the G. australes (IRTA-SMN-17-271) was fractionated under the above-described conditions. A portion (1 mL) of the initial methanol extract, containing the equivalent of 609,011 cells, was evaporated to dryness under N₂ at 50 °C and reconstituted in 100 µL of methanol and filtered through 0.22 µm (Syringe Driver filter Unit, Millex®-CV 0.22 um, 13 mm, Millipore, Billerica, MA, USA) before its injection into the HPLC system.

The separation and quantification of the four purines and uric acid were performed using an Agilent 1260II ultra-HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a G7104C quaternary pump, G7129C autosampler, G7116A column heater, and variable wavelength detector (VWD). An Agilent TC-C18 (2) (4.6 mm × 250 mm, 5.0 µm) was used as the analytical column, and the injection volume was 10 µL. In order to establish a suitable HPLC detection method, the following parameters: mobile phase (7 × 10⁻³ M KH₂PO₄, 0.02 M KH₂PO₄, and methanol-water solutions); flow rate (0.8, 1.0 and 1.2 mL/min); column temperature (25 °C, 28 °C, 30 °C, and 35 °C) and absorption wavelength have been optimized. Data were collected and processed using OpenLab CDS Data Analysis software (product version 2.5; Agilent Technologies, Santa Clara, CA, USA).