Acquity upc2 system

Optical rotations were measured using an Autopol VI polarimeter. IR spectra were obtained from Perkin-Elmer 577 spectrometers. Ultraviolet absorption spectra were recorded in MeCN (25 μg/mL) on a UV-2401 PC spectrophotometer. ECD spectra were recorded in MeCN (50 μg/mL) on a Chirascan-plus spectrometer (Applied Photophysics Ltd., Surrey, United Kingdom). NMR spectra were measured in DMSO-d₆ on Bruker AV-400 and Bruker AV-600 spectrometers and calibrated by the solvent peak used. Mass spectrometry was performed on a SYNAPT G2-Si HDMS (Waters Corp., Manchester, United Kingdom) with an electrospray ion source (Waters, Milford, MA) connected to a lock-mass apparatus, which performed real-time calibration correction. Column chromatography was performed with CHP20P MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Japan) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). A Waters 2535 Series machine equipped with a X-bridge C₁₈ column (4.6 × 250 mm, 5 μm) was used for HPLC analysis, and a preparative X-bridge Prep C₁₈ OBD column (19 × 250 mm, 5 μm) was used for sample preparation. Enantioseparations were performed by a Waters Acquity UPC² system (Waters, Milford, MA, United States) with a sample manager, binary solvent manager, compensation solvent pump and column manager on a Daicel Chiralpak IG column (5 μm, 250 × 4.6 mm).

All experiments were performed on a Waters Acquity UPC
² system (Waters) equipped with a binary solvent manager delivery pump; a sample manager autosampler, which included a 10‐µL loop for partial loop injection; a column oven; and a two‐step (active and passive) backpressure regulator (ABPR). 2‐Propanol and methanol/water 50:50, v/v, were used as the weak and strong solvents, respectively, with volumes of 600 and 200 µL, respectively. The chromatographic system was combined with a Waters PDA detector set at 214 nm for UV detection in a compensated mode (compensation reference of 350–410 nm).

We used a validated bioanalytical method using supercritical fluid chromatography coupled with tandem mass spectrometry to quantify MBQ-167 in tumors and tissues, as previously described [26 (link)]. The analysis was performed on an Acquity UPC² system (Waters Corp., Milford, MA, USA) coupled to a triple quadrupole tandem mass spectrometer (MS/MS). An Acquity UPC² BEH (3.0 × 100 mm², 1.7 μm) column was used for separation purposes.

The extracted β-carotene was characterized quantitatively using supercritical fluid chromatography based ultra-performance conversance chromatography (Acquity UPC² system, Waters Technologies, USA) equipped with a reverse-phase analytical polymeric High Strength Silica C18 (HSS C18 SB), 3 × 100 mm with particle size 1.8 µm, as reported by Runco et al.³⁷ . Empower³ software was used to operate the system during the quantitative analysis of samples.

Total anthocyanins were extracted following a previously described method⁴⁸ with some modifications. One gram of pericarp sample was ground into fine powder in liquid nitrogen and incubated in 3 ml of 1% HCl in methanol (v/v) overnight at room temperature. An additional 3 ml of chloroform was then added and mixed thoroughly to remove fat-soluble pigments. After centrifugation at 12,000 × g for 2 min, the supernatants were collected and filtered through a 0.45-μm filter. The content of anthocyanins was determined spectrophotometrically at 535 nm and expressed as A₅₃₅ g⁻¹ fresh weight. Average values were calculated from three independent replicates.
Carotenoids were extracted as described previously⁴⁹ . The carotenoid content was measured using a Waters ACQUITY UPC2 System (Milford, MA, USA) equipped with a C18 column, according to the manufacturer’s recommendations. The individual carotenoids (phytoene, lycopene, and β-carotene) were identified according to the retention time and quantified using standard curves.

The conditions of UPC²-Q TOF MS were set according to a previously reported laboratory procedure [26 (link),27 (link),28 (link),29 (link)]. Waters Acquity UPC² system (Milford, MA, USA) equipped with an Acquity UPC² BEH HSS C18 column (150 mm × 3.0 mm i.d.; 1.7 µm) was utilized for the separation of TAGs. All the other instrument conditions were similar as previous reported method. A Waters 1525 single pump was used as the compensated pump, and pumped 0.3 mL/min of 0.1% ammonium formate in methanol into the MS source. The TAG compositions of the berry seed oils were analyzed and semi-quantified with a Waters Xevo-G2 Q-TOF MS system as previously described. The ion mode, capillary voltage, cone voltage, temperatures of source and desolvation, as well as other parameters were similar as our previous publications. The collision energy was 6 eV in MS1, and information of fragment ions was collected in the MS2 mode and the collision energy was 35 eV, with the scan time set at 0.2/s.

SFC analysis was performed using an Acquity UPC2 system (Waters, Milford, MA) with a binary solvent pump, a column oven, an autosampler, PDA detector and backpressure regulator (BPR). Methanol was used as the needle wash solvent. Data acquisition and control of UPC2 were carried our using the Waters Empower 3 software. The SFC final analysis was performed at 25 °C on ACQUITY UPC BEH column (100 × 3 mm, 1.7 μm, Waters, Milford, MA). The elution gradient (eluent A: CO₂/B: MeOH) involved: 0-6 min, 95–65% A, 6–8 min, 65–65% A, 8–10 min, 65–90% A. The backpressure was set to 13.7 MPa, the flow rate was 1.5 mL/min, and the injection volume was 4 µL. The PDA detection wavelength was set at 254 nm.