Luna 5 m c18 2 100

The specific rotation values and IR spectra were measured using a JASCO P-2000 digital polarimeter and a THERMO Scientific Nicolet iS5 FT-IR spectrophotometer, respectively. ESIMS and HRESIMS were recorded using a BRUKER 7 Tesla solariX FTMS system. NMR spectra were obtained from a JEOL Resonance ECZ 400S or an ECZ 600R NMR spectrometer, with the residual signals of CHCl₃ (δ_H 7.26 ppm) and CDCl₃ (δ_C 77.0 ppm) used as the internal standards for ¹H and ¹³C NMR, respectively. Coupling constants (J) are provided in Hz. Column chromatography was carried out with a silica gel (230~400 mesh, MERCK) column. Thin-layer chromatography was performed on plates precoated with silica gel 60 F₂₅₄ (0.25-mm-thick, MERCK); the plates then sprayed with 10% (v/v) H₂SO₄ in methanol, followed by heating to visualize the spots. A normal-phase (NP) HPLC was performed using a system comprised of a HITACHI 5110 pump, a RHEODYNE 7725i injection port and a NP column (YMC pack SIL, 5 μm, 12 nm, 250 × 20 mm, YMC group). Reverse-phase (RP) HPLC was performed using a system comprised of a HITACHI L-2130 pump, a HITACHI L-2455 photodiode array detector, a RHEODYNE 7725i injection port and a RP column (Luna 5 µm C18(2) 100 Å, 250 × 21.2 mm, Phenomenex).

Nucleotide extraction was performed at 5 and 6 dpf, as we previously described [35] (link), but now using a Precellys 24 homogenizer (Bertin Technologies) at 6800 rpm (3 ×20 s, CK28 beads). ATP, ADP and AMP were quantified using HPLC (reversed-phase column; 250 ×4.6 mm Luna 5 µm C18(2) 100 Å; Phenomenex) with a diode-array detector (Agilent 1100 series), measuring 260 nm absorbance. Energy charge was calculated with the formula: ([ATP]+0.5[ADP]) ÷ ([ATP]+[ADP]+[AMP]) [49] (link).

A semi-preparative LC system (Octave 10 Semba Bioscience, Madison, USA) was used to separate the FM2 mixture into 80 fractions in a total run time of 80 min. The chromatographic conditions used are reported in the paper by Citti et al.^{4 (link)}. A Luna C₁₈ with a fully porous silica stationary phase (Luna 5 µm C18(2) 100 Å, 250 × 10 mm) (Phenomenex, Bologna, Italy) was the column employed and a mixture of acetronitrile:0.1% aqueous formic acid 70:30 (v/v) was used as mobile phase at a constant flow rate of 5 mL/min. The fractions containing CBDHA (retention time 13.0 min) was isolated as reported in our previous work^{2 (link)}. The fractions containing CBDHA (13.0 min) were analyzed by UHPLC-HESI-Orbitrap and dried on the rotavapor at 70 °C. The residue was placed in an oven at 120 °C for 2 h to achieve decarboxylation. An amount of about 0.3 mg of CBDH was obtained.