Q exactive hybrid quadrupole orbitrap high resolution mass spectrometer

A Shimadzu Prominence UFLC system consisting of two LC-30AD binary pumps, a SIL-30AC auto-sampler, a DGU-20A5 degasser, and a CTO-30A column oven (Shimadzu Corporation, Kyoto, Japan) was used throughout. Chromatographic separation was carried out on a BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters Co., Milford, MA, USA). The mobile phase consisted of water containing 0.1% (v/v) acetic acid (A) and acetonitrile containing 0.1% (v/v) acetic acid (B). The gradient program was: 0–20 min at 15–55% B; 20–45 min at 55–95% B; 45–53 min at 95% B; 53–54 min at 95–15% B; and 54–60 min at 15% B. The flow rate was set at 0.3 mL/min and the LC column temperature was maintained at 40 °C. The sample injection volume was 10 µL.
Mass spectrometric analysis was performed with a Q-Exactive hybrid quadrupole orbitrap high resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using a heated electrospray ionization source in the positive ionization mode. The operating parameters were as follows: spray voltage, 3.5 kV; sheath gas pressure, 35 arbitrary units (arb); auxiliary gas pressure, 10 (arb); capillary temperature, 320 °C; auxiliary gas heater temperature, 350 °C; scan modes, full MS (resolution 60,000) and scan range, m/z 200–1200. All data were acquired using the Xcalibur 2.0 software (Thermo Fisher Scientific Inc., Waltham, MA, USA).

The metabolites were extracted from each sample as previously described [105 (link)] using 10 µL of 2× diluted Metabolomics IS Mix and 200 µL of 8:1:1 Acetonitrile/Methanol/Acetone, and incubated for 30 min at 4 °C. The samples were centrifuged at 20,000× g at 4 °C for 10 min, and 190 µL of the supernatant was dried under nitrogen gas at 30 °C. All samples were stored at −80 °C. Samples were reconstituted to 25 µL in 0.1% formic acid, and analyzed on a Q Exactive™ Hybrid Quadrupole-Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The metabolomics injection standards mix is constituted by Leucine-13C6 (4 µg/mL), Creatine-D3 H2O (methyl-D3) (4 µg/mL), L-Leucine-D10 (4 µg/mL), L-Tryptophan-2,3,3-D3 (40 µg/mL), L-Tyrosine Ring-13C6 (4 µg/mL), L-Phenylalanine Ring-13C6 (4 µg/mL), N-BOC-L-tert-Leucine (4 µg/mL), N-BOC-L-Aspartic Acid (4 µg/mL), Propionic Acid 13C3 (8 µg/uL), Succinic Acid-2,2,3,3-d4 (8 µg/mL), Salicylic Acid D6 (4 ug/mL), Caffeine-d3 (1-methyl-d3) (4 µg/mL) and was reconstituted in 0.1% formic acid. A Red Cross Plasma positive control and an extraction blank negative control were extracted as Quality Controls (QC). A Neat QC was made with 1:1:3 Metabolomics IS Mix/Amino Acids Mix/0.1% formic acid.

Untargeted metabolomics data were performed as described (17 (link)). Metabolites were extracted from plasma samples using acetonitrile (2:1, vol/vol). Stable isotopes caffeine-¹³C3, tyrosine-¹⁵N, and progesterone-d⁹ were used as internal standards. LC-MS/MS analysis was performed with an HPLC-UV, 1220 Infinity (Agilent Technologies, Santa Clara, CA, USA) coupled with Q Exactive hybrid Quadrupole-Orbitrap high-resolution mass spectrometer (Thermo Fisher, Waltham, MA, USA). Reverse phase C18 chromatography was performed with Zorbax Eclipse Plus C18 column (4.6 × 150 mm², 3.5 µm Agilent) and positive electrospray ionization.