Liquid chromatograph

The estimation of the three metabolites were performed on a Waters liquid chromatograph equipped with a Waters 600 controller, a Waters Delta 600 solvent delivery system, a Rheodyne 7125 sample injector fitted with a 20 µL loop, and a Waters 2996 Photodiode Array Detector, with Waters Empowered 2.154 software. A Supelco 516 C₁₈ (4.6 mm×25 cm) reverse phase analytical column equipped with a Waters µBondapak C₁₈ 10 µm precolumn was used for estimation. The wavelength scan range of the PDA was set to 190–350 nm and the presence of withanosides-V, withaferin-A and withanolide-A was detected at 227 nm. A. The isocratic mobile phase consisted of 60% acetonitrile containing 0.1% acetic acid (solvent A) and 40% water containing 0.1% acetic acid (solvent-B) at a flow rate of 1.0 mL min⁻¹. The metabolites were estimated in comparison to the external standards and the results were presented as µg mg⁻¹ of dry weight of leaf tissue.

The extraction and measurement of procyanidins by the phloroglucinol test were performed according to the method by Lachowicz et al. [7 (link)]. Phloroglucinolysis was analyzed using a liquid chromatograph (Waters, Milford, MA, USA) consisting of a diode array, scanning fluorescence detectors, and a column manager. Separation was carried out using a Cadenza CD C18 column (75 mm × 4.6 mm, 3 μm) kept at 15 °C. For phloroglucinolysis investigation, the following solvent system (mobile phase A (25 mL of acetic acid and 975 mL of water) and mobile phase B (acetonitrile)) was applied. Fluorescence was recorded at emission wavelength 360 nm and excitation wavelength 278 nm. The calibration curves and quantification were evaluated using standards: (−)-epicatechin, (+)-catechin, (−)-epicatechin-phloroglucinol, and (+)-catechins-phloroglucinol. The degree of polymerization was analyzed by evaluating the molar ratio of all the flavan-3-ol units. All measurements were noted three times and expressed as mg/100 g d.w.

Crocin solutions were analyzed by an isocratic reversed-phase HPLC using a Waters Liquid Chromatograph fitted with a model 740 integrator, a 5 µm, 25 × 0.46 cm Ultrasphere ODS column (Beckman, CA, USA) or Ultremex ODS column (Phenomenex, Torrance, CA, USA) and a 4.6 × 0.45 cm precolumn. The eluent was a mixture of water and 0.1 M di-n-octylamine acetate in MeOH (1:2 vol/vol) at a flow rate of 1 ml min⁻¹.
Before injection, samples (40 mL) from the aqueous solutions were diluted with 0.1 M di-n-octylamine acetate in MeOH (80 mL); samples from the MeOH solutions were mixed with one equal volume of H₂O and one of 0.1 M di-n-octylamine acetate in MeOH. This precaution avoided undesirable peaks in the chromatograms due to solvent interferences. The mixtures were centrifuged and 50 mL were injected into the column.
Peaks were monitored at 440 nm (absorption maximum) and identified by reference to standards. Cis isomer peak areas were corrected for the difference in the extinction coefficient at 440 nm in the HPLC mobile phase with respect to all-trans (a factor of 1.36).

Samples were analyzed through a targeted hydrophilic-interaction ultraperformance liquid chromatography–tandem mass spectrometry (HILIC-UPLC-MS/MS) method, as previously described [52 (link)], on an ACQUITY liquid chromatograph in tandem with a Xevo TQD MS System (Waters, Milford, MA, USA), equipped with the MassLynx software, using an ACQUITY HILIC, BEH amide column (2.1 mm × 150 mm, 1.7 μm) at 40 °C. A gradient elution of solvent A (acetonitrile-water 95:5 (v/v)) versus solvent B (acetonitrile-water 30:70 (v/v), 0.01% ammonium formate) was used at a flow rate of 0.5 mL/min, and the sample volume injected was 5 μL. A polarity switching mode (both positive and negative) of electrospray ionization was applied. The capillary voltage was +3.5 KV, while the voltage of the cone and the collision energy were adjusted for each analyte as described [52 (link)], where chromatographic and mass spectrometric characteristics of each identified metabolite are reported. Block and desolvation temperatures were 150 and 350 °C, respectively. The total analysis time was 40 min. A mix of standards of the targeted metabolites at three concentration levels was used as a quality control sample and was periodically injected to confirm the stability of the analytical system. Urine samples were analyzed spectrophotometrically for lactate as described [21 (link)].

Carotenoids were determined in a saponified sample of paprika extract using the internal standard by HPLC analysis. This methodology was described in the previous paper [51 (link)]. After saponification, carotenoids were analyzed using a Waters Liquid chromatograph equipped with DAD 2996 detector. Separation of carotenoids was carried out using 4.6 × 150 mm chromatographic column YMC^TM Carotenoid with granulation of 3 μm additionally equipped with a protective column YMC^TM Carotenoid S-3. The mobile phase was a mixture of methanol, acetonitrile, and water (75:10:15, v:v:v) in gradient elution with dichloromethane. The flow rate was 1 mL/min and the column temperature was set at 25 °C. The injection volume was 20 μL.
Capsanthin of 96% purity, capsorubin of 98% purity, β-carotene of 99% purity, β-cryptoxanthin of 97% purity, violaxanthin of 95% purity, and zeaxanthin of 97% purity standards purchased from CaroteNature GmbH (Münsingen, Switzerland) were used for identification and quantification based on a calibration curve. β-Apo-8′-carotenal of ≥96% purity obtained from Sigma-Aldrich (Buchs, Switzerland) was used as internal standards. The qualitative analysis was based on a comparison of the retention time of peak with an appropriate carotenoid standard.

Meldonium (purity > 99.9%) was purchased from Yuanye Pharmaceutical Co. (Shanghai, China). L-carnitine (purity > 99.9%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A stable isotope labeled internal standard compound, acetonitrile and methanol, were obtained from Merck Chemical Co., (Darmstadt, Germany). Formic acid and ammonium acetate were purchased from Thermo Scientific (Waltham, United States). All the other chemicals were of analytical grade or better.
The liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consisted of a liquid chromatograph (Waters Technologies, Massachusetts, United States) and coupled with an AB SCIEX 6500 triple quadrupole mass spectrometer (Sciex Corp., Framingham, United States). A FLYDWC50-1A animal simulated hypobaric hypoxia chamber was purchased from Guizhou Fenglei Oxygen Chamber Co., Ltd. (Anshun, China). An FLPI-2 laser doppler blood flow monitor (Moor Instruments, Axminster, UK), a high-content imaging analysis microscope (Molecular Devices, California, United States), an ABL90 Flex blood gas analyzer (Radiometer Medical, Copenhagen, Denmark), and an IX51 light microscope (Olympus, Japan) were also applied in this study.