Mestrenova 9

Identification of the CPC purified compounds was performed by ESI-MS with an Advion compact mass (Advion, Ithaca, NY, USA) and NMR. The ESI-MS spectra conditions were as follows: positive ion mode; mass range, m/z 100–1200; capillary temperature, 200 °C; capillary voltage, 150 V; source voltage offset, 30; source voltage span, 10; source gas temperature, 150 °C; and ESI voltage, 3500 V. ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) were measured on a Bruker model digital Avance III 400 NMR in CDCl₃. The NMR spectra were processed by the MestReNova 9.0 software (Mestrelab Research, Santiago de Compostela, Spain).

For the sample preparation of all types of edible oils, only the addition of deuterated solvent was required as a single step. 600 μL of CDCl₃ (containing 1% TMS, Tetramethylsilane) and 50 μL of oil were placed in a 5 mm NMR tube and mixed thoroughly during 10 s. The spectra were acquired using a Bruker DPX 300 MHz NMR spectrometer, equipped with a BACS-60 robotic autosampler (which allows for fully automated analysis of up to 60 samples at a time) and a 5 mm Z-gradient QNP probe. For the acquisition, 32 K complex points were recorded, the spectral width was set to 12 ppm, the frequency offset was set to 5 ppm, the recycle delay was set to 4 s, the acquisition time was 4.56 s, the excitation pulse was a 90° hard pulse, the number of scans was set to 8 and the number of dummy scans was equal to 2. The total experimental time was 1 min and 26 s. A temperature of 30 °C was chosen for the experiments. The data were acquired automatically using the software ICON-NMR (Bruker BioSpin, Rheinstetten, Germany).
The resulting spectra were processed manually and automatically with the software MestreNova 9.0.1 (Mestrelab Research SL, Santiago de Compostela, Spain) and Topspin 2.1 (Bruker Biospin, Rheinstetten, Germany). No window functions were applied. The chemical shift scale was referenced using the signal of the TMS (0 ppm).

MestReNova 9.0 (Mestrelab Research, S.L., Santiago de Compostela, Spain) was used to process all the acquired raw data, including phase correction, baseline adjustment, and integration, which were conducted manually, as previously described [18 (link),23 (link)]. The average values from six times replicated processing were performed for all the phase correction and integration procedures to minimize analyst error [24 (link)]. The chemical shifts were referenced to the IS resonance (δ 5.86, s), and the obtained peak area was used for the quantitation. The calculation of the analytes was accorded to our previous work [18 (link)] using the formula below: $${\mathrm{W}}_{\mathrm{s}}={\mathrm{W}}_{\mathrm{r}}\times \frac{{\mathrm{A}}_{\mathrm{s}}}{{\mathrm{A}}_{\mathrm{r}}}\times \frac{{\mathrm{E}}_{\mathrm{s}}}{{\mathrm{E}}_{\mathrm{r}}}$$
where W_s stands for the weight of IS, A_s and A_r denote the peak area of analyte and ISTD, respectively, while E_s and E_r represent the ratios of molecular weight to nuclei number for analyte and IS, respectively.