Agilent 1200 series high performance liquid chromatography

Sample analysis was performed on the basis of a previously validated approach using liquid chromatography–tandem mass spectrometry.20, 21 Samples were analyzed using an Agilent 1200 series high‐performance liquid chromatography (HPLC) system (Agilent Technologies, Palo Alto, CA) interfaced with an ABSciex 5500 hybrid triple quadrupole/linear ion trap mass spectrometer (Concord, ON, Canada) equipped with an electrospray ionization source operating in the positive/negative switch mode.
Briefly, 50 μL serum sample was combined with 400 μL cooled methanol, incubated for protein precipitation overnight, and dried in a SpeedVac concentrator centrifuge (Thermo Fisher Scientific/Savant, Waltham, MA). Reconstitution was performed in 20 μL of water/methanol (80:20) and subjected to a modified targeted metabolomics analysis with relative quantification (165 metabolites in the current assay).

¹H and ¹³C NMR spectra were recorded in CDCl₃ on an AVANCE 600 spectrometer (Bruker, Rheinstetten, Germany) at 400 and 100 MHz, respectively, using tetramethylsilane as an internal standard, and chemical shifts are given in δ (ppm). Distortionless enhancement by polarization transfer spectra was acquired using the Bruker software. UV spectra were obtained in methanol or acetone on a UVICON 933/934 spectrophotometer (Kontron, Milan, Italy), and mass spectra on a JMS-DX 303 spectrometer (Jeol, Tokyo, Japan). Silica gel 60 (0.063–0.2 mm) (Merck, Darmstadt, Germany) was used for column chromatography. Merck precoated silica gel plates (Kieselgel 60 F₂₅₄) were used for analytical thin-layer chromatography (TLC). An Agilent 1200 series high-performance liquid chromatography (HPLC) (Agilent, Santa Clara, CA) was used for isolation of active principles.

The analysis was conducted as described previously with an Agilent 1200 series high-performance liquid chromatography (Agilent Technologies, Santa Clara, CA, USA) coupled to a 6530 Q-TOF mass spectrometer (Agilent Technologies) [42 (link)]. The autosampler was set at 4°C for all procedures. In positive ion mode, ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 μm; Waters) was maintained at 40°C, and metabolite separation was conducted by binary gradient elution with a flow rate of 0.4 mL/min. Mobile phase A was water with 0.1% formic acid; mobile phase B was ACN with 0.1% formic acid. The gradient was 0 min, 2% B; 1 min, 2% B; 3 min, 20% B; 8 min, 90% B; 14 min, 90% B; 14.5 min, 2% B; 18 min, 2% B. Essential mass spectrometer parameters are given in S3 Table in S2 File. In negative ion mode, the ZIC-HILIC column (100 × 2.1 mm, 3.5 μm; Merck, Darmstadt, Germany) was maintained at 35°C, and metabolite separation was conducted by binary gradient elution with a flow rate of 0.5 mL/min. Mobile phase A was ACN/water (5:95, v/v) with 10 mM ammonium acetate; mobile phase B was ACN/water (95:5, v/v) with 10 mM ammonium acetate. The gradient was 0 min, 99% B; 1 min, 99% B; 15 min, 50% B; 17 min, 50% B; 17.1 min, 99% B; and 22 min, 99% B. The mass spectrometer was operated using equivalent conditions to positive ion mode.