Accela uplc

100 µl culture medium samples were extracted with 300 µl ice-cold methanol containing the internal standards (¹³C₆ L-arginine and D4 L-citrulline). After centrifugation, methanolic extracts were evaporated to dryness. Then, dried extracts were reconstituted in 85% acidified acetonitrile and injected into the UPLC-MS/MS system. Analytes were resolved using an Accela UPLC (Thermo Scientific, UK) equipped with 1.7 µm HILIC Kinetex 2.1 × 50 mm UPLC column (Phenomenex, UK) and a mobile phase gradient of buffer A (water + 0.1% formic acid) and buffer B (acetonitrile + 0.1 formic acid) at a flow rate of 250 µl/min. Eluting compounds of interest were detected using a TSQ Vantage mass spectrometry system (Thermo Scientific, UK) in positive-ion mode. The optimum transitional daughter ions mass of each analyst was as follows: argininosuccinate m/z 291.1 → 70.2, arginine m/z 175.1 → 70.2, citrulline m/z 176.1 → 70.2, ¹³C₆ arginine m/z 181.0 → 74.2, and D4 citrulline m/z 180.2 → 74.2. Plasma argininosuccinate was measured using untargeted metabolomics assay as described previously [26 (link)].

The abundance of PA, DAG, triglyceride, and CL in mouse hearts was determined as described with modification (22 (link)). The LC-MS analysis was performed either with a Shimadzu 10A HPLC system and a Shimadzu SIL-20AC HT auto-sampler coupled to a Thermo Scientific TSQ Quantum Ultra triple quadrupole (TQ) mass spectrometer operated in selected reaction monitoring mode or with a Thermo Fisher Scientific Vantage TSQ mass spectrometer with Thermo Accela UPLC operated by Xcalibur software using selected ion monitoring mode. PA-(14:0)₂, DAG-(15:0)₂, triglyceride-(17:0)₃, and CL-(14:0-14:0)₂ were used as internal standards. Quantification of lipids was based on the ratio of the peak area of the analyte to the internal standard. For example, the ratio of CL-(18:2/18:2)₂ and CL-(14:0-14:0)₂ is used for measurement of CL-(18:2/18:2)₂.

To characterize the metabolic profile of [³H]-MMAE, the bile samples were analyzed across time points that covered the majority of the radioactivity (up to 6 h with 75% of the total radioactivity injected) excreted. Due to the low radioactivity recovered in the urine, the urine samples were analyzed for up to 8 h. Both the bile and urine samples were investigated using Accela UPLC coupled with a Linear Trap Quadrupole (LTQ)-Orbitrap Velos system (Thermo Scientific, San Jose, CA, USA) and an online β-RAM 5C radiodetector (Lab Logics, Tampa, FL, USA) for radioprofiling.
Chromatographic separation was performed on a Luna C18 column (150 × 4.6 mm, 3 μm particle size, Phenomenex, Torrance, CA, USA) with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a constant flow rate of 1 mL/min. The gradient was as follows: initial holding at 5% B for 2 min, increased to 15% B at 4 min, 42% B at 44 min, 75% B at 49 min, 95% B at 50 min, holding at 95% B until 55 min, decreasing to 5% B at 55.1 min, and then column re-equilibration until 60 min. The flow was split 3:1 post-column for radiomeasurements and mass spectrometry, respectively. As the majority of the metabolite was the intact [³H]-MMAE, only the intact [³H]-MMAE was quantified.