Lichrocart

Left ventricular specimen were homogenized on ice with 0.4 mmol/L perchloric acid and precipitated with KOH. The samples were centrifuged and supernatants were injected twice onto a pre-equilibrated RP18 column (LiChroCART, Merck, Darmstadt, Germany) as previously published (Salameh et al., 2015 (link)). For ATP detection a HPLC-apparatus from Knauer (Berlin, Germany) and an UV-detector (PDA Detector 2800, Knauer, Berlin, Germany) were used. Peaks were measured at 259 nm. Standard curves were generated with 4 concentrations of ATP (25-12.5-6.25-3.125μg/ml) and measured together with the ventricular samples.

For separation, a LiChroCart (Merck, Darmstadt, Germany) RP-C₁₈ column (125 mm × 3.0 mm, 3 µm) connected to a guard column (LiChroCart RP-C₁₈ 4.0 mm × 4.0 mm, 5 µm) was used with a mobile phase consisting of an aqueous formic acid solution (0.1%; A) and methanol (B). The flow rate was 0.3 mL/min and started with a mixture of 90% A and 10% B, raised to 60% B and 40% A during 75 min, turned back to 90% A in 5 min and kept for 15 min for reconditioning before the next injection. A total of 20 µL was injected into the column kept at room temperature.
Mass spectrometer conditions (only for system 2): negative mode, capillary temperature, 275 °C; source voltage, 4.5 kV; capillary voltage, −5.0 V; sheath gas (N₂) flow at 80 arbitrary units and auxiliary gas (N₂) flow rate at 10 arbitrary units. During the chromatographic run, mass spectra of the eluate were recorded from m/z 100 to m/z 1000 and tandem mass spectrometry experiments were carried out. For quantification the SIM mode was used.

HPLC analysis was carried out on a Thermo Scientific Dionex UltiMate 3000 (Dionex Corporation, Sunnyvale, CA, USA) equipped with a pump LPG-3400SD, column oven TCC-3000SD, detector DAD-3000 and autosampler WPS-3000TSL. Chromatographic separations were carried out using column RP-18 LiChroCART, 125 mm × 3 mm, 5 µm (Merck, Darmstadt, Germany) at a temperature of 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was 1.0 mL/min with the following gradient elution: starting at 7% mobile phase B and holding for 0.5 min, reaching 50% in 4 min and 95% in 4.5 min and holding for 1 min. From 5.5 min, the gradient returned to 7% mobile phase B and stopped after 7 min in this concentration. The injection volume of the sample was 10 µL, and detection was performed at 280 nm wavelength. The commercial TC of various concentrations was used to prepare the calibration curve (y = 0.2625x − 0.0771, R² = 0.998).

Analyses were carried out using an Agilent HP1100 HPLC system equipped with an RP-C18 column, LiChroCART (250 × 4 mm, 5 μm;Merck KGaA, 64271, Darmstadt, Germany) connected to a Finnigan LTQ (Thermo Electron Corp, Dreieich, Germany) operated in the APCI mode (vaporizer temperature: 500 °C, capillary temperature 300 °C). Standard compounds for identification were either purchased (Sigma-Aldrich (St. Louis, MO, USA) or synthesised. Isoxazolin-5-one glucoside and its esters were synthesised according to previously described protocols (Becker et al.2013 (link), 2015 (link)).
Samples were analyzed by injection (5 μl) and by the application of a gradient elution. The following protocol was used: 100 % solvent A (H₂O + 0.1 % HCOOH) and 0 % solvent B (MeCN + 0.1 % HCOOH), linear gradient to 60 % solvent B in 35 min. Extract samples of whole larvae were analyzed by injecting a 5 μl sample and using an isocratic elution with 35 % solvent B (v/v) in H₂O +0.1 % HCOOH. For identification and quantification, the formic acid adducts [M+HCOOH-H]⁻ were used (m/z 292 for 2-(β-D-glucopyranosyl)-3-isoxazolin-5-one (5), m/z 393 for 2-[6′-(3″-nitropropanoyl)-β-D-glucopyranosyl]-3-isoxazolin-5-one (6), m/z 331for salicin (3), and m/z 377 for 8-hydroxygeraniol-8-O-β-D-glucoside (1).

Left ventricular specimen were homogenized at 4°C with 0.4 mmol/L perchloric acid and precipitated with KOH. Thereafter, the samples were centrifuged for 10 min and 20 μl of the supernatant was injected twice onto a pre-equilibrated RP18 column (LiChroCART, Merck, Darmstadt, Germany) as previously published (Salameh et al., 2015a (link)). For ATP, ADP AMP, and adenosine detection a HPLC-apparatus from Knauer (Berlin, Germany) and an UV-detector (PDA Detector 2800, Knauer, Berlin, Germany) were used. Peaks were measured at 259 nm, standard curves were generated with 4 concentrations of ATP, ADP, AMP, and adenosine respectively (25–12.5–6.25–3.125 μg/ml) and measured together with the ventricular samples.