Ctc pal

Amino acids, organic acids, and sucrose were analyzed using a GC-MS system. Amino acids and organic acids were derivatized by methoximation, followed by tert-butyldimethylsilylation. Sucrose was derivatized by methoximation, followed by trimethylsilylation. Samples were analyzed by an Agilent 7890 GC system (Agilent, Santa Clara, CA, USA) coupled to an Agilent 5975C inert XL Mass Selective Detector (Agilent, Santa Clara, CA, USA) with an autosampler (CTC PAL; Agilent, Santa Clara, CA, USA). Metabolites were separated by an Agilent VF5ms GC column, 30 m × 0.25 mm × 0.25 m with 10 m guard column (Part number: CP9013; Agilent, Santa Clara, CA, USA). For amino acids and organic acids, 1 μL of the derivatized sample was injected into 10 split mode with helium carrier gas with a flow rate of 1.2 mL min⁻¹. The oven temperature gradient was: 100°C (4 min hold), increased by 5°C/min to 200°C, then by 10°C/min to 320°C, and held at 320°C for 10 min. Electron ionization (EI) is at 70 eV and the mass scan range was 100–600 amu. The ionization source temperature was set at 150°C and the transfer line temperature at 300°C. The fragment ions used for the isotopologue analysis are described in the Supplemental Table S2.

Mass spectrometry for anion exchange LC-MS/MS and GC-EI-MS were carried out using the methods described in ref. 12 (link) and detailed in Dataset S5. Reverse-phase LC-MS/MS and GC-chemical ionization (CI)-MS had the following changes: Samples for reverse-phase liquid chromatography-tandem mass spectrometry were analyzed by an ACQUITY UPLC pump system (Waters) coupled with Waters XEVO TQ-S ultra-performance liquid chromatography tandem mass spectrometry (Waters) by the method described in ref. 12 (link). Samples for gas chromatography-electron ionization-mass spectrometry were analyzed by an Agilent 7890B GC system (Agilent) coupled to an Agilent 7010B triple quadrupole gas chromatography-electron ionization-mass spectrometer with an autosampler (CTC PAL) (Agilent). An Agilent VF5ms GC column, 30 m × 0.25 mm × 0.25 m with 10-m guard column was used. One microliter of the derivatized sample was injected with helium carrier gas at a flow rate of 1.2 mL⋅min⁻¹. The oven temperature gradient was: 40 °C (1-min hold), increased at 40 °C/min to 150 °C, then a 10 °/min to 250 °C, then a 40 °C/min to 320 °C, and finally held at 320 °C for 4.5 min. CI was used, and the mass scan range was 150 amu to 650 amu with step size 0.1 amu. The ionization source temperature was set at 300 °C, and the transfer line temperature was 300 °C.

The levels of co-trimoxazole in plasma were determined using liquid chromatography–tandem mass spectrometry (LC-MS). Sulphamethoxazole and trimethoprim were individually weighed and dissolved in dimethyl sulfoxide (DMSO) to give 50 mg/mL (sulfamethoxazole) and 5 mg/mL (trimethoprim) stock solutions. Dextrometorphan (DXT) was dissolved in DMSO to give a 1-mg/mL stock solution and diluted to 1 μg/mL with acetonitrile containing 2.5% DMSO to give the working internal standard (IS) solution. Marmoset plasma standard (50 μL) or sample was mixed with 150 μL of the IS solution and centrifuged, and the supernatant decanted into a 250-μL HPLC polypropylene vial. The supernatants were reduced in volume using a Genevac centrifugal evaporator for 90 min at 40°C. Twenty microliters of 0.1% formic acid were added and mixed by vortex before being injected onto the LC-MS system (Agilent 1100, CTC PAL, Sciex 3000). Calibration curves were produced over the range 50 to 50,000 μg/mL for sulfamethoxazole and 5 to 1,000 ng/mL for trimethoprim.