Accela 1250 hplc system

The AsA concentration was measured according to a method used in previous studies [46 (link),47 ], with minor modifications. Approximately 1.0 g frozen samples were ground to a fine powder in liquid nitrogen and extracted with 5 mL 0.1% metaphosphoric acid solution. The supernatants and AsA standards (GWL8-54KE, Beijing, China) were neutralized with 1 M NaOH, and after centrifugation, the final pH of all samples was adjusted between 5 and 6. Then, 5 mM of DL-dithiothreitol (DTT) was added to the neutralized supernatants for a 30 min pre-treatment at room temperature. The supernatants were filtered using a 0.22 μm filter, and 10.0 μL of each filtered supernatant was injected into an Accela 1250 HPLC system (Thermo Fisher Scientific, Boston, MA, USA) equipped with a monomeric C18 column (WONDASIL C18, COLUMNS 4.6 × 150 mm, GL Sciences Inc., Shanghai, China) with a mobile phase of 0.1% metaphosphoric acid and acetonitrile (98/2, v/v) and a flow rate of 0.5 mL/min. Finally, standard curves of AsA were generated as a reference for quantifying AsA concentrations in the measured samples.

LC-MS/MS and GC-MS/MS methods are routinely implemented in our laboratory for accurate quantitation of intracellular metabolites in cell extracts from various organisms. Quantitation is based on selected reaction monitoring (SRM) and the relative response to the respective fully ¹³C-labeled metabolites added to each sample via the U¹³C-labeled cell extract to account for errors introduced during sample-pretreatment, the analytical process as well as differences in ionization efficiency. Both methods have been validated, the corresponding analytical figures of merit are available elsewhere.^{42 (link),43} For the purpose of this study, yeast cell extracts were analyzed on both platforms to provide reference values for comparison with RP-PGC-TOFMS.
RPLC-MS/MS was performed employing a silica-based Atlantis T3 C18 column (2.1 × 150 mm, 3 μm particle size, Waters) on a Thermo Accela 1250 HPLC system coupled to a Thermo TSQ Vantage triple quadrupole mass spectrometer. A detailed method description is available elsewhere.^{42 (link)}Quantification by GC-MS/MS was based on the on-time two-step derivatization (ethoxymation and trimethylsilylation) of intracellular metabolites using a Gerstel MPS2 dual rail Multi-Purpose Sampler followed by GC-MS/MS analysis using a Thermo TSQ Quantum XLS Ultra triple quadrupole GC-MS system. A detailed method description is available elsewhere.⁴³

Lipid concentrations were determined using an Accela1250 HPLC system coupled with an ESI–MS Orbitrap Exactive (ThermoFisher Scientific) as described by de Kok et al. (33 ). In short, samples were prepared by applying 150 µl distilled 1-butanol, mixing by vortex and separating by centrifugation (16.000 RCF, 2 min), and this process was repeated once more. The lipid layer containing 1-butanol was collected in glass vials and dried under a nitrogen gas stream. Lipid films were subsequently dissolved in methanol and injected on a Waters ACQUITY Premier CSH C18 (1.7 µm, 2.1 × 150 mm) column. Lipid separation was achieved by applying a changing gradient of eluent A: MQ:MeCN (40:60) containing 5 mM ammonium formate and eluent B: MQ:MeCN:1-BuOH (0.5:10:90) also containing 5 mM ammonium formate. Mass spectrometry specifications and settings have been described previously (34 (link)). Thermo Scientific XCalibur processing software was used to analyze spectral data by applying the Genesis algorithm-based automated peak area detection and integration. Total ion counts for each extracted lipid were normalized for the internal standard (10:0 PG).

To investigate the major product’s mass, a CYP3A4 reaction mixture containing 0.40 µM of P450, 500 µM of phloretin, and NGS was used in 0.25 mL of a potassium phosphate buffer (100 mM, pH 7.4). After these reaction mixtures were incubated at 37 °C for 30 min, injection samples were prepared as described above. Analyses of the mass values of phloretin and its products were performed using a TSQ Quantum™ Access MAX Triple Quadrupole Mass Spectrometer on an Accela 1250 HPLC system (ThermoFisher Scientific, Waltham, MA, USA). The samples were separated on a ZORBAX SB-C18 column (250 mm × 4.6 mm i.d. 5 µm; Agilent, Santa Clara, CA, USA) at a flow rate of 1 mL/min. The mobile phases were (A) 0.1% (v/v) formic acid in water and (B) acetonitrile. The isocratic flow of the mobile phase was (A) 60% and (B) 40% on HPLC. The injection volume was 10 µL. The mass spectra were recorded via electrospray ionization in positive mode to characterize phloretin metabolites. The collision energy and scan rate were 10 V and 0.5 spectra/s, respectively.