Nexera system

Anidulafungin (0.5 to 0 μg/ml), caspofungin (0.1 to 20 μg/ml), and fluconazole (0.2 to 20 μg/ml) were measured in plasma by separate validated UHPLC-MS/MS methods on a Shimadzu Nexera system coupled to a Shimadzu 8030+ triple quadrupole mass spectrometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). The stationary phase was a Kinetex C8 (50 × 2.10 mm, 1.7 μm) UHPLC column (Phenomenex, Torrance, CA, USA). The mobile phase was a gradient of acetonitrile and 0.1% formic acid (anidulafungin and caspofungin) or acetonitrile and 0.1% formic acid with 10 mM ammonium formate (fluconazole). Ionization was by positive-mode electrospray, with analytes detected at the following MRMs: 1140.4 → 343.10 (anidulafungin), 1,093.50 → 1,033.50 (caspofungin), 306.7 → 238.2 (fluconazole), 470.10 → 160.20 (dicloxacillin), and 350.0 → 281.1 (voriconazole). Plasma (100 μl) was spiked with internal standard (dicloxacillin for anidulafungin/caspofungin, and voriconazole for fluconazole) and treated with acetonitrile to precipitate proteins before instrumental analysis.
For anidulafungin analysis, the supernatant was washed with dichloromethane to remove lipid-soluble components. Sample analysis met batch acceptance criteria. The methods were validated for linearity, LLOQ, matrix effects, precision and accuracy, and stock stability by using the FDA criteria for bioanalysis [17 ].

CA42 EPS (50 μg) was completely hydrolyzed using 2 M trifluoroacetic acid (TFA) at 100°C for 3 h. The obtained monosaccharides were labeled with 4-aminobenzoic acid ethyl ester (ABEE) and analyzed using high-performance liquid chromatography (HPLC; Nexera System; Shimadzu, Kyoto, Japan), as described previously (Urai et al., 2006 (link)). To determine the absolute configuration of CA42 EPS, the TFA hydrolysate of EPS was converted into acetylated (−)-2-butyl glycoside and analyzed using gas–liquid chromatography (GLC) (Leontein et al., 1978 (link); Gerwig et al., 1979 (link)).

LC-MS/MS based metabolomics was performed as previously described (Kirkwood et al., 2012 (link)). In short, high-pressure liquid chromatography (HPLC) was performed on a Shimadzu Nexera system (Shimadzu, Columbia, MD, United States) with a phenyl-3 stationary phase column (Inertsil Phenyl-3, 4.6 × 150 mm, GL Sciences, Torrance, CA, United States) coupled to a quadrupole time-of-flight mass spectrometer (Sciex TripleTOF 5600) operated in information-dependent MS/MS acquisition mode in both positive and negative ion mode. The flow rate was 0.4 ml/min, the injection volume was 10 μL, and the mobile phases consisted of water (A) and methanol (B), both with 0.1% formic acid. The elution gradient was as follows: 0 min, 5% B; 1 min, 5% B; 11 min, 30% B; 23 min, 100% B; 35 min, 100% B; 37 min, 5% B; and 47 min, 5% B. Samples were randomized, and a pooled QC sample was analyzed every 5 samples. Auto-calibration was performed after every two samples.

Metabolomics analysis was performed using a previously established method^{22 (link)}. Briefly, the metabolomics analysis, ultra-high performance liquid chromatography (Shimadzu Nexera system; Shimadzu; Columbia, MD) coupled to a high resolution hybrid quadrupole-time-of-fight (TOF) mass spectrometer (MS) (TripleTOF 5600; Sciex; Framingham, MA) was utilized. Every sample underwent analysis via two different methods using either reverse phase and HILIC columns in both the negative and positive ion modes^{21 (link)}. High performance liquid chromatography (Agilent 1100 Series) coupled to LTQ-XL (Thermo Fisher) was used to analyze plasma levels of phosphatidylcholine and lysophosphatidylcholine^{67 (link)}. Peak area of each metabolite, measured within the linear range of the mass detector, was normalized to plasma volume for each sample and presented as mean ± SEM.

Ultra-pressure liquid chromatography was performed as previously reported on a Shimadzu Nexera system (Shimadzu, Columbia, MD) coupled with a quadrupole time-of-flight mass spectrometer (AB SCIEX, Triple TOF 5600) operated in information dependent MS/MS acquisition mode⁶³ . Data was imported into PeakView software for relative quantification and identification. Sphingolipids, CHOL and fatty acids species were confirmed by high resolution MS, MS/MS fragmentation, and isotopic distribution, and then compared using the PeakView database⁶³ . Sphingolipids, TAG and CHOL were identified in positive ion mode as [M+H]+ except ceramide as [M+H-H2O]+, and fatty acids in negative ion mode as [M−H]-, respectively.

Betacyanins were identified by LC (Nexera system; Shimadzu, Kyoto, Japan)–triple quadrupole mass spectrometry (LCMS-8060; Shimadzu). Samples (5 μL) were injected into an InertCore C18 column (150 mm × 2.1 mm I.D., 2.4 μm particle size; GL Sciences) at a flow rate of 400 μL/min. A gradient was produced by changing the mixing ratio of the two eluents: A, 0.1% (v/v) formic acid; and B, acetonitrile containing 0.1% (v/v) formic acid. The gradient was started with 5% B with a 4-min hold; this was then increased to 30% B for 30 min and then increased immediately to 100% B with a 3-min hold, following which the mobile phase was immediately adjusted to its initial composition and held for 4 min to re-equilibrate the column. The column temperature was set at 40°C. The autosampler (kept at 4°C) was equipped with a black door to prevent the samples from being exposed to light. Data acquisition for the estimation of betacyanins was performed at λ = 538 nm with a UV-vis high-performance liquid chromatography (HPLC) detector coupled with positive ion electrospray ionization LC–MS analysis (electrospray voltage, 4.0 kV; capillary temperature, 300°C; sheath gas, N₂, 10 L/min).

Mass spectrometry based metabolomic profiling was performed as previously described [15] (link). Briefly, liquid chromatography (LC) was performed on a Shimadzu Nexera system and metabolites separated on an Inertsil phenyl-3 stationary phase (GL Sciences, 5 uM, 4.6 × 150 mm). Mass spectrometry was performed on an AB SCIEX Triple TOF 5600 quadrupole-time-of-flight mass spectrometer. MS/MS spectra were gathered on the fly by information dependent acquisition. Most metabolites were identified by mass, isotope distribution, MS/MS fragmentation, and when standards were available, retention time. In the absence of chemical standards, MS/MS spectra were compared to those in the METLIN online database.
To account for analytical and sample preparation variation, samples were normalized to total ion count. Central energy metabolites (metabolites of the tricarboxylic acid cycle, pentose phosphate pathway, and glycolysis and amino acids) were targeted post-data acquisition and in addition, untargeted statistical analysis (Student's t-test p-value plotted against fold-change) revealed large (>10-fold) changes in cyclic AMP and cyclic GMP, and subsequently, related metabolites (purines and pyrimidines) were targeted post-data acquisition.