L pal3 autosampler

GC–TOF–MS analysis was performed using an Agilent 7890A GC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an L-PAL3 autosampler and Pegasus^® HT TOF-MS system (LECO Corp., St. Joseph, MI, USA). Metabolites were separated using an RTX-5MS column (30 m length × 0.25 mm inner diameter × 0.25 μm particle size, Restek Corp., St. Joseph, MI, USA) with a constant flow of helium (1.5 mL) as the carrier gas. For analysis, all dried samples were oximated with 50 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) for 90 min at 30 °C and silylated with 50 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide for 30 min at 37 °C. The derivatized samples (1 μL) were injected into the GC column in splitless mode. The analytical program for sample analysis was adopted from our previous study [22 (link)]. Moreover, metabolite analysis was performed in a random manner to reduce bias and systematic errors.

GC–MS measurements were performed on an Agilent 7890 gas chromatograph (Agilent USA) with an L-PAL3 autosampler (Leco USA) coupled with a Pegasus BT TOF (time of flight) mass spectrometer (Leco, USA). Separation was performed on HP-5 MS column (30 m × 0,25 mm, 0,25 µm, Agilent, USA). Injection 1 μl splitless, injector temperature 250 °C. Oven program was as follows: 50 °C (1 min.) to 300 °C (10 °C/min.) 5 min. The carrier gas was helium, constant flow 1 mL/min. Transfer line temperature was 250 °C. Mass spectrometer parameters was as follows: source temperature 250 °C, mass range 40–650 m/z and acquisition rate 20 spectra/s.

For gas chromatography–time-of-flight mass spectrometry (GC-TOF-MS) analysis, the reconstituted samples (50 μL) were dried using a speed vacuum concentrator. The dried samples were derivatized using the following protocol. First, 25 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) was added to the dried samples and placed on a thermomixer for 90 min at 30 °C to protect the ketone and aldehyde groups. Then, 25 μL of N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA), being used as a silylating agent, was incubated for 30 min at 37 °C. One microliter of each derivatized samples was injected into an Agilent 7890A GC system (Santa Clara, CA, USA) equipped with an L-PAL3 autosampler and Pegasus^® HT TOF-MS system (LECO Corp., St. Joseph, MI, USA). Metabolites were separated using an RTX-5MS column (30 m length × 0.25 mm inner diameter × 0.25 μm particle size; Restek Corp., St. Joseph, MI, USA) with a constant flow of helium (1.5 mL/min) as the carrier gas. The analytical parameters and program were adopted from our previous study [13 (link)]. All samples were run in a randomized manner to reduce systematic errors and bias.