A Vanquish UHPLC paired with a Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific) was used to perform LC-MS/MS analysis of IBC and the internal standard, isobutyryl L-carnitine-d3 (Cayman Chemical, Ann Arbor, MI). Chromatographic separation of analytes was achieved on an Accucore aQ column (150 mm × 2.1 mm, dp = 2.6 μm) with a C18 AQUASIL guard cartridge (2.1 mm × 10 mm, dp = 3 μm). The temperature of the column and autosampler was retained at 40 and 4°C, respectively. The mobile phase contains solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile-methanol, 50:50 v:v). The gradient elution was 5.0 min at a flow rate of 0.4 ml/min, and conditions were as follows: 0–0.5 min, 0% B; 0.5–2.3 min, 30% B; 2.3–3.8 min, 30–95% B; 3.8–4.2 min, 95% B; 4.2–5.0 min, 0% B. The extracted samples (5 μl) were injected for analysis, and following parameters were established for the mass spectrometer: 40 Arb, 12 Arb, 3.3 Arb, 350, and 375°C for sheath gas, aux gas, sweep gas, ion transfer tube, and vaporizer temperature, respectively. The ion source was managed by heated ESI in positive ion mode with ion spray voltage at 3,500 V. Argon was used as a collision gas at a pressure of 1.5 mTorr. Precursor molecular ions and product ions were recorded for confirmation and detection of IBC (232.144 > 85.083) and the internal standard (236.056 > 85.056). Assay validation studies demonstrated that the within-day precision and between-day precision ranged from 0 to 6.16%, and the accuracy ranged from 92.8 to 105%. The lower limit of quantification for IBC was 0.1 ng/ml.

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