Targetlynx application manager

Tissue samples collected at each necropsy (one femur sample per animal and samples of white fat, brown fat, and cerebrum) were analyzed for the presence of four metabolites of AGIQ (quercetin, quercetin 3-O-glucuronide, kaempferol, and isorhamnetin [3-o-methylquercetin]) using a validated UPLC-MS/MS method at the David H. Murdock Research Institute (DHMRI, Kannapolis, NC). The femur and white fat samples were analyzed on a Sciex Exion LC-X500R QTOF system (SCIEX, Framingham, MA), whereas the brown fat and cerebrum samples were analyzed on a Waters Acquity UPLC-Triple Quadrupole system (Waters Corp., Milford, MA) due to instability issues with these samples on the Sciex platform. Both systems were operated in ESI negative mode using mobile phases of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The raw data generated by the Waters UPLC-MS system were processed using the TargetLynx application manager (Waters Corp., Milford, MA), whereas the raw data generated by the Sciex UPLC-MS system were processed using Sciex Analytics.

The raw LC−MS/MS data files were processed with TargetLynx Application Manager (Waters Corp., Milford, MA) to extract peak area and retention time of each metabolite. The raw GC−TOFMS data files were processed with Chroma TOF software (Leco Corp., St Joseph, MI) to extract peak signal and retention time for each metabolite. The detected metabolites were annotated with our internal standard library using an automated mass spectral data processing (AMSDP) software package [51 (link)]. IPA was applied to visualize the overall difference between FANCC-expressing high and low cells along with SIMCA-P 12.0.1 (Umetrics, Umeå, Sweden). Nonparametric statistical analysis (i.e., the Mann−Whitney U test) was used for searching the significantly different metabolites between the groups with a critical p-value of 0.05 and 0.01.

A 500-μL aliquot of isopropynal/hexane (4:1) with 2% phosphate (2 M) and 10 μL of one internal standard (5 μg/mL of nonadecylic acid-d37) was added to 10 mg of caecal content sample, homogenized for 5 min, and centrifuged at 13,200 g at 4 °C for 15 min. A total of 400 μL supernatant was transferred into a 1.5-mL tube. An aliquot of 800-μL hexane and 300 μL of water were then added to the tube, and the mixture was vortexed for 2 min and centrifuged for 10 min at 12,000 g. An aliquot of 800 μL upper layer was transferred to a new tube and dried under vacuum. The residue was reconstituted with 80 μL of methanol and subjected to analysis.
Free fatty acids were analyzed by UPLC/QTOFMS. The elution solvents were water (A) and acetonitrile/isopropyl (v/v = 80/20, B) with a flow rate of 400 μL/min. The initial gradient was 70% B and kept for 2 min, increased to 75% B in 3 min, increased to 80% in 5 min, increased to 90 in 3 min, increased to 99% in 3 min, and kept at 99% for 5 min before switching back to initial condition. The MS was operated at a positive electrospray ionization mode. One standard calibration solution with 65 free fatty acid standards at 10 different concentration levels was analyzed every five sample injections. The peak annotation and quantitation was performed by TargetLynx application manager (Waters Corp., Milford, MA, USA).

Samples were prepared according to the provided instructions (Waters AccQTag derivatization kit, Manchester, UK) and were applied to ultra-performance liquid chromatography (UPLC) for separation (ACQUITY UPLC system, Waters, Manchester, UK). A mass spectrometer (Xevo TQ-XS, Waters) was used for monitoring. The respective amino acid levels were analyzed by Waters MassLynx 4.2 software and quantified by Waters TargetLynx application manager (Waters, Manchester, UK).

Ileal bile acids were quantified by ultraperformance liquid chromatography triple-quadrupole mass spectrometry (LC-TQMS) assays as described previously [32 (link),33 (link),34 (link)]. Briefly, each 100 µL of ileal juice was lyophilized to dry powder in a BA-free matrix using a freeze dryer. The residues were reconstituted in 1:1 (v/v) mobile phase B (acetonitrile/methanol = 95:5, v/v) and mobile phase A (water with formic acid) and centrifuged at 13,500× g and 4 °C for 20 min. The supernatant was then transferred to a 96-well plate for LC-TQMS analysis. A UPLC-TQMS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) was used to quantify BAs in the human samples. The raw data were processed using the TargetLynx application manager (Waters Corp., Milford, MA, USA) to obtain calibration equations and the measured concentration of each BA in the individual samples. The intra- and inter-batch CVs are less than 10% and the recovery rate is higher than 95–110% for all BAs in quality control samples in the current study. To ensure the comparability between groups, lab personnel were blinded to disease status of biospecimens.

Raw data from UPLC—MS/MS was processed using the TargetLynx application manager (Waters Corp., Milford, MA, USA) to obtain calibration equations and quantitative concentrations of each metabolite in the samples. Raw data from GC-TOFMS analysis were exported to the ChromaTOF software (v4.50, Leco Co., CA, USA) for baseline correction, smoothing, noise reduction, deconvolution, library searching, and area calculation. For the GC−TOFMS generated data, identification was processed by comparing the mass fragments and the retention time with our in-house library or the mass fragments with NIST 05 Standard mass spectral databases in NIST MS search 2.0 (NIST, Gaithersburg, MD, USA) software using a similarity of more than 70%. The detected metabolites from GC-TOFMS were annotated and combined using automated mass spectral data processing software [9 (link)]. Samples or compounds with significant loss of data (10% of data was missing) were excluded from further analysis. These quantification protocols using authentic standards resulted in quantitative profiles for the following four classes of metabolites: Bile acids (BA, 42 metabolites), phospholipids (lipids, 109 metabolites), and other small molecules including free fatty acids (FFA) (128 metabolites total).