Acquity uplc equipment

Analytical and semipreparative HPLC analyses were conducted using a Waters Alliance (Waters Corporation, Milford, MA, USA) chromatographic system with a SunFire C18 column (10 µm, 10 × 250 mm, Waters). For UPLC analysis an Acquity UPLC equipment with a BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters) was used. Optical rotations were measured using a Jasco P-2000 polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were obtained with an Agilent 1100 DAD (Agilent Technologies, Santa Clara, CA, USA). IR spectra were recorded on a JASCO FT/IR-4100 spectrometer (JASCO Corporation, Tokyo, Japan) equipped with a PIKE MIRacle^TM single reflection ATR accessory (PIKE Technologies Inc., Madison, WI, USA). NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for ¹H and ¹³C NMR, respectively) equipped with a 1.7 mm TCI MicroCryoProbe^TM (Bruker Biospin, Falländen, Switzerland), using the signal of the residual solvent as internal reference (δ_H 3.31 and δ_C 49.0 ppm for CD₃OD). HRESIMS spectra were acquired using a Bruker maXis QTOF spectrometer (Bruker Daltonik GmbH, Bremen, Germany).

Semipreparative HPLC was performed with an Alliance chromatographic system (Waters Corporation, Mildford, MA, USA) and an Atlantis C18 column (10 μm, 10 × 150 mm, Waters). For UPLC analysis an Acquity UPLC equipment (Waters) with a BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters) was used. Optical rotations were determined with a JASCO P-2000 polarimeter (JASCO Corporation, Tokyo, Japan). IR spectrum was measured with a JASCO Fourier transform infrared (FT/IR)-4100 spectrometer (JASCO Corporation) equipped with a PIKE MIRacle^TM single reflection ATR accessory. NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for ¹H and ¹³C NMR, respectively) equipped with a 1.7 mm TCI MicroCryoProbe^TM (Bruker Biospin, Fällanden, Switzerland), using the signal of the residual solvent as internal reference (δ_H 2.50 and δ_C 39.5 ppm for DMSO-d₆). ESI-TOF MS spectra were acquired with a Bruker maXis QTOF spectrometer (Bruker Daltonik GmbH, Bremen, Germany).

The identification and quantification of carotenoids, by comparison to authentic standards (when available) was performed on Acquity UPLC equipment (Waters corp., MA, USA), and the following conditions were used: column: ACQUITY UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 um) (Waters, Milford, MA, USA), column temperature: 34°C, and eluents were A: water + 0.1% formic acid and B: acetonitrile + 0.1% formic acid. The flow rate was set at 0.3 ml min⁻¹ and the linear gradient elution was: START: 25% A: 75% B; 20 min: 0% A: 100% B; 28 min: 0% A: 100% B; the sample temperature was kept constant at 20°C and the injection volume was 2 μl. Injections have been repeated three times. The detector was an Acquity TUV Detector (Waters, Milford, MA, USA), and the wavelength was set at 445 nm. Data were processed with Empower 3 workstations. Limit of detection (LOD) and limit of quantification (LOQ) were calculated according to Ermer et al. (20 (link)). Chromatographic separation was followed by a mass spectrometry (LCQ Fleet Thermofisher, MA, USA) analysis, according to a previous study (21 (link)). A positive electrospray mode was used for the ionization of molecules with a capillary voltage of 50 V and at a capillary temperature of 275°C. The heater temperature was set at 40°C, the gas flow rate was 20 (arb), and the spray voltage was 4.0 kV. The monitored mass range was m/z 100–900.