Multipurpose sampler

Pegasus GCxGC-TOFMS (Leco Corporation Joseph, MI, USA) that uses an Agilent 7890A GC (Atlanta, GA) coupled to a time-of-flight mass spectrometer (TOFMS) (Leco Corporation, St Joseph, MI, USA) equipped with a Gerstel Multipurpose sampler, was used for chromatographic analyses of the derivatized samples. One µL of serum extract was randomly injected at a split ratio of 1:50 and the carrier gas used was helium at a flow rate of 1 ml min⁻¹. For the entire run, the temperature of the injectors was kept constant at 270°C. A Restek Rxi-5Sil MS capillary column (29.145 m × 0.25 μm d.f.) was used as the primary column. The primary oven was programmed to an initial temperature of 70°C for 2 min to obtain a compound separation. Subsequently, this was followed by a 4°C per minute increase to a final temperature of 300°C where it was maintained for 2 min. The second separation of compounds was achieved using a Restek Rxi-17 (1.400 m, 0.25 µm i.d., 0.25 μm d.f.) column. The secondary column used was set to the same temperature parameters as that of the primary column. The filament bias was EI at 70 eV while the detector voltage was at 1,600 V. Subsequently, the mass spectra were collected at an acquisition rate of 200 spectra per second with a source temperature of 220°C and a solvent delay of 400 s from 50 to 800 m/z (Du Preez and Loots 2013 (link)).

For global metabolomics, urine (60 µL) was aliquoted and treated with 5 µL (160 mg/mL) urease for 1 h at 37 °C with gentle agitation and serum (30 µL) was aliquoted without pretreatment. Quality control (QC) samples were prepared by aliquoting an equal volume from each sample. Samples were deproteinated with 1 mL 100% cold methanol with internal standards (final concentration 20 µg/mL), incubated on ice for 10 min, and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was evaporated under N₂ to ~100 µL, transferred to a GC vial with a 250 µL glass insert, and evaporated to dryness in a speedvac without heat. Samples were derivatized inline with a Gerstel (Linthicum, MD, USA) multipurpose sampler for 1 h at 60 °C with rigorous shaking with MOX (10 µL) and an additional 1 h at 60 °C with MSTFA/1% TMCS (90 µL). For TCA intermediate analysis, urine (40 µL) was prepared as above, however, samples were deproteinated with 1 mL cold methanol (internal standard citric acid-d₄ at a final concentration of 2 µg/mL) without urease pretreatment and batch derivatized per day.

Metabolite derivatization was performed by using a multi-purpose sampler (GERSTEL, Germany). Dried samples were dissolved in 15 μl pyridine, containing 20 mg/ml methoxyamine hydrochloride, at 40°C for 60 min under shaking. After adding 15 μl N-methyl-N-trimethylsilyl-triflouroacetamide (MSTFA) samples were incubated at 40°C for 30 min under continuous shaking.
GC-MS analysis was performed by using an Agilent 7890A GC coupled to an Agilent 5975C inert XL MSD (Agilent Technologies, Germany). A sample volume of 1 μl was injected into a Split/Splitless inlet operating in splitless mode at 270°C. The gas chromatograph was equipped with a 30 m DB-35MS capillary column + 5 m DuraGuard capillary in front of the analytical column (Agilent J&W GC Column).

SPME fiber (Supelco, Bellefonte, Penn.) was installed on a MultiPurpose sampler (Gerstel, GER) and combined with 7890B-7000D triple quadrupole gas chromatography mass spectrometry (Agilent Technologies, Palo Alto, CA, USA) to detect VOCs that ZBP and ZBL. Weigh three samples from each batch for experiment. weigh 0.01g for 20 mL headspace vials for each batch of samples. Incubation temperature was 75 °C, incubation time was 5 min, extraction time was 40 min, vial penetration was 21.00 mm, injection penetration was 54.00 mm desorption time was 5 min, desorption temperature was 250 °C.

The Coccidioides species culture filtrates and medium blanks were allowed to thaw at 4°C overnight, and 2 ml was then transferred and sealed in sterilized 10-ml GC headspace vials with polytetrafluoroethylene (PTFE)-silicone septum screw caps. All samples were stored for up to 12 days at 4°C until analyzed. Samples were randomized for analysis. Volatile metabolite sampling was performed by solid-phase microextraction (SPME) using a Gerstel multipurpose sampler directed by Maestro software. Sample extraction and injection parameters are provided in Table S3 in the supplemental material (autosampler method). Volatile metabolite analysis was performed by two-dimensional gas chromatography–time of flight mass spectrometry (GC×GC-TOFMS) using Leco (St. Joseph, MI) Pegasus 4D and Agilent 7890 GC instruments. Chromatographic, mass spectrometric, and peak detection parameters are provided in Table S3 (GC×GC method and mass spectrometry method). An external alkane standard mixture (C₈ to C₂₀; Sigma-Aldrich, St. Louis, MO) was sampled multiple times for calculating retention indices (RIs). The injection, chromatographic, and mass spectrometric methods for analyzing the alkane standards were the same as the methods for the samples.