Lc ms vials

The acylcarnitine internal standard mix was made up of the following four deuterated acylcarnitines, each at a concentration of 200 μM: d3-acetylcarnitine, d3-butyrylcarnitine, d3-octanoylcarnitine, and d3-decanoylcarnitine. Ten μL of the acylcarnitine internal standard mix was spiked into 190 μL of human urine. Following the previous protocol, urine was dried down under nitrogen (N₂) at 60°C and resuspended in 200 μL of methanol. Samples were vortexed and centrifuged at 14,000 rpm for 5 min. at room temperature. Urinary acylcarnitines were selectively isolated by solid-phase extraction (SPE) using SEP-PAK VAC 6 cc silica gel cartridges (Waters, Milford, MA, USA) following a literature procedure. This cartridge type is useful for acylcarnitines because it adsorbs analytes of even weak hydrophobicity from aqueous solution. Acylcarnitines were eluted off the SPE cartridges with 1.5 mL methanol:water:acetic acid (50:45:5, v/v/v). Extracts were then dried down under N₂ and resuspended in 1 mL of methanol:water:formic acid (70:30:0.1, v/v/v). Samples were vortexed and centrifuged at 14,000 rpm for 5 min. at room temperature and ultimately transferred to LC-MS vials (Agilent, Palo Alto, CA, USA). Flow injection analysis (FIA) was utilized for sample introduction into the ESI-DMS-MS/MS platform.

The diluted contrast agent samples were placed in separate vials (2-mL clear glass liquid chromatography-mass spectrometry [LC-MS] vials [#5183–2067], Agilent Technologies, Santa Clara, CA, USA). Each vial was placed at the isocenter of a 1.5-T Unity MR-Linac (Elekta, Crawly, UK) and irradiated with 7 MV photons to doses of approximately 2 Gy, 8 Gy, 15 Gy, or 30 Gy; two non-irradiated vials were used as baselines for comparison (controls).

Firstly, 5.26 μL of 120 μM deuterated acetylcarnitine internal standard solubilized in water (100%) was spiked into 100 μL of NHP urine (final concentration of IS = 6 μM), dried down under nitrogen (N₂) at 60°C, resuspended in 105 μL of methanol, vortexed and centrifuged at 14,000 rpm for 5 minutes at room temperature. Acetylcarnitine was then extracted by solid-phase extraction using SEP-PAK VAC 6cc silica gel cartridges (Waters, Milford, MA, USA). Acetylcarnitine was eluted with 1.5 mL methanol:water:acetic acid (50:45:5, v/v/v)³⁸ . The extract was then dried down under N₂, resuspended in 526.3 μL of methanol:water:formic acid (70:30:0.1, v/v/v), vortexed, centrifuged at 14,000 rpm for 5 minutes and transferred to LC-MS vials (Agilent, Palo Alto, CA, USA). Flow injection analysis (FIA) enabled sample introduction into the mass spectrometer for DMS-MS/MS analysis (Figure 1).