Agilent 6546 lc q tof

Aliquots (40 µL for (positive mode) and 120 µL for (negative mode)) of thawed plasma (NIST SRM 1950 Metabolites in Frozen Plasma, Sigma, St. Louis USA) were each extracted using a modified Folch extraction procedure [36 (link)] and reconstituted in 100 µL of a methanol/chloroform mixture (9:1, v/v). LC separation was performed on an Agilent 1290 Infinity II LC System, with a 19 min gradient time on a reverse phase C18 column (Agilent InfinityLab Poroshell 120 EC-C18, 3.0 × 100 mm, 2.7 µm). Mobile phase consisted of 10 mM ammonium acetate and 0.2 mM ammonium fluoride in 9:1 water/methanol, while mobile phase B consisted of 10 mM ammonium acetate and 0.2 mM ammonium fluoride in 2:3:5 acetonitrile/methanol/isopropanol. Negative and positive polarity data was acquired on the Agilent 6546 LC/Q-TOF using iterative MS/MS acquisition mode on 6 injections of extracted plasma for each polarity [37 ]. Detailed experimental methods for chromatography and mass spectrometry can be found in Supplemental Table S1 and Table S2, respectively, and in the Agilent application note 5994-0775en [37 ]. Two methods were used, a high-load and a low-load method, to determine the effect of high injection volumes/concentration on the number of annotations using the Agilent 6546 LC/Q-TOF.

The metabolites formed during biodegradation of RB4 were extracted from the cell free supernatant by using resin XAD-16 absorption then eluted by methanol and concentrated using a rotary evaporator for further analysis. The RB4 dye and its degraded products were evaluated by LC-HRMS chromatography. The degraded metabolite dyes were analyzed using LC-MS (Agilent 6546 LC/Q-TOF).
HPLC analysis was carried out with a ZORBAX C18 column (50 mm × 2.1 mm Agilent Technologies Inc., CA, United States). The binary mobile phase used in the experiment was composed of (A) water (0.1% FA) and (B) methanol gradient with the following elution program: 0–0.5 min, 90% A; 0.5–10 min, 90-0% A; 10–13 min, 0% A; 13–16 min, 0-90% A; 13–20 min, 90% A. The MS interphase is equipped with electron spray ionization (ESI) in both positive and negative ionization modes. The samples were eluted at a flow rate of 0.3 mL min^–1 and monitored at 280 nm. For MS analysis, N2 was used as the nebulizing gas (35 psig), heated sheath gas (350°C), and drying gas (10 L min^–1). The method of HPLC and MS analysis was based on previous research (Navada et al., 2018 (link); Alam et al., 2020 (link)).