Nexera ultra high performance liquid chromatograph

The analytical system used was a Shimadzu LCMS-8040 triple-quadrupole mass spectrometer; LC-MS/MS (Shimadzu, Kyoto, Japan) coupled to a Nexera ultra high performance liquid chromatograph (Shimadzu, Kyoto, Japan) and data was analyzed using Shimadzu LabSolutions software (Version 5.72). The LC-MS/MS was operated in DUIS mode (ESI/APCI) using multiple reaction monitoring (MRM). The LC-MS/MS conditions consisted of a desolvation line temperature of 250 °C and heating block temperature of 400 °C. Nebulizing gas flow was 2 L/min and drying gas was 15 L/min.
Tissue and plasma samples were analyzed for creatine-¹³C. The analytical column used was a Primesep 200 (3 μm, 2.1 × 100 mm) (SIELC Technologies, Wheeling, IL, USA) mixed function cation exchange column. The mobile phase consisted of a pH gradient with mobile phases A (0.05% aqueous formic acid) and B (1% formic acid in acetonitrile). A linear gradient was applied from 0% to 85% B over 4 min, held at 85% B for 2 min followed by a step down to 0% B and held for 4 min to recondition and equilibrate the column prior to the next injection. The total flow rate of the system was 0.4 mL/min and the column oven was set at 40 °C. The following transitions were monitored in positive MRM mode: m/z 133.1 > 90.1 (collision energy (CE) of 15 eV) for creatine-¹³C and 135.1 > 93.1 (CE of 15 eV) for creatine-d₃.

The LC/MS/MS system was comprised of a Shimadzu Nexera ultra-high-performance liquid chromatograph and a Shimadzu LCMS-8040 triple quadrupole mass spectrometer equipped with an ESI ion source (Shimadzu Co., Kyoto, Japan). The conditions for LC/MS/MS analysis were: column, InertSustain (2.1 × 150 mm; particle size, 3 μm; GL Sciences Inc., Tokyo, Japan); column temperature, 40°C; mobile phase, 20 mM ammonium acetate in water (A) or methanol (B); flow rate, 0.35 mL min⁻¹; gradient curve, 75% B at 0 min, 99% B at 22 min, 99% B at 39 min, 75% B at 39.1 min, and 75% B at 45 min; injection volume, 2 μL; mass analysis mode, both positive and negative ion mode with a polarity switching time of 15 ms; electrospray voltage, 4.5 kV for positive- and −3.5 kV for negative-ion mode; nebulizer gas flow, 3.0 L min⁻¹; drying gas flow, 15.0 L min⁻¹; desolvation temperature, 250°C; heat block temperature, 400°C; and detector voltage, 1.62 kV. The MRM mode with a dwell time of 5 ms per channel was used. Other optimized MRM parameters for each lipid and its related metabolite are shown in Table 1. One pooled QC sample was repeatedly analyzed at each fifth sample injection in this study.