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3 protocols using nh4hco2

1

Optimized nLC-ESI-MS/MS Lipid Profiling

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To determine the nLC–ESI–MS/MS run condition, 28 lipid standards from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) were utilized: 12:0-LPC, 16:0-LPC, 18:1-LPC, 12:0/12:0-PC, 13:0/13:0-PC, 16:0/16:0-PC, 18:1/18:0-PC, 20:0/20:0-PC, 14:0-LPE, 18:0-LPE, 12:0/12:0-PE, 14:0/14:0-PE, 18:0/22:6-PE, 12:0-LPG, 15:0/15:0-PG, 18:0-LPS, 18:0/18:0- PS, 16:0/18:2-PI, 18:0-LPA, 12:0/12:0-PA, (18:1)4-CL, d18:0/12:0-SM, d18:1/24:0-SM, d18:1/12:0-MHC, d18:1/16:0-DHC, d18:1/12:0-Cer, 16:0/18:1-DAG, and 16:0/16:0/18:1-TAG. All solvents used in this study were of HPLC grade. H2O, CH3CN, CH3OH, isopropanol, and methyl-tert-butyl ether were obtained from Avantor Performance Materials (Center Valley, PA, USA), and CHCl3 was purchased from Sigma-Aldrich (St. Louis, MO, USA). NH4HCO2 and NH4OH (Sigma-Aldrich) were used as ionization modifiers and added to LC mobile phase solutions. All PL standards were diluted with a binary solvent mixture (9:1, v/v, CH3OH:H2O). Silica capillary tubes used for capillary columns, and all connections were purchased from Polymicro Technology, LLC (Phoenix, AZ, USA), with all tubes having inner diameters of 20, 50, 75, or 100 μm (outer diameter of 360 μm for all).
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2

Antifungal AmB Characterization Protocol

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AmB analytical standard (87.8%), Methanol (MeOH) High Performance Liquid Chromatography (HPLC) grade, formic acid, Sabouraud Dextrose (SBD) Broth, NaOH, MgCl2, CaCl2, NHCO3 and NH4HCO2 were obtained from Sigma Aldrich (Germany); AmB active pharmaceutical ingredient (API) powder (85%) from Cayman Chemical (USA); Bovine Serum Albumin Protease Free Powder Fraction V (BSA), dimethyl sulfoxide (DMSO), dextrose, Na2HPO4, NaH2PO4, NaCl and KCl were obtained from Fisher Scientific (USA); Sabouraud dextrose (SBD) agar was obtained from Oxoid (UK), 25 mL sterile universal culture tubes were obtained from Sterilin Thermo Scientific (UK); 10 μL plastic loops from Microspec (UK); GF/D (pore size 2.7 μm, 25 mm diameter) and GF/F (pore size 0.7 μm, 25 mm diameter) filters were obtained from Whatman (UK) and regenerated cellulose (RC) filters 0.45 µm 13 mm diameter from Cronus (UK).
The yeast strain used in the microbiology experiments was Candida albicans SC5314 [18] .
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3

Analytical Standard Characterization Protocol

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AmB analytical standard (87.8%), methanol (MeOH) high performance liquid chromatography (HPLC) grade, formic acid mass spectrometry grade, NaOH, MgCl2, CaCl2, hexadecyltrimethylammonium bromide (CTAB), cholesterol (CH), NHCO3 and NH4HCO2 were obtained from Sigma Aldrich (Germany); ethylenediaminetetracetic acid anhydrous (EDTA) from Sigma Aldrich (USA); AmB API powder (85%) from Cayman Chemical (USA); BSA protease free powder fraction V, dimethyl sulfoxide (DMSO), dextrose, sodium dodecyl sulphate (SLS), Na2HPO4, NaH2PO4, KH2PO4, NaCl and KCl from Fisher Scientific (USA); Tween 80 from Amresco (USA); phosphatidylcholine from egg from Lipoid GmbH (Germany); sodium taurocholate from Prodotti Chimici e Alimentaria (Italy); GF/D (pore size 2.7 μm, 25 mm diameter) and GF/F (pore size 0.7 μm, 25 mm diameter) filters from Whatman (UK) and regenerated cellulose (RC) filters 0.45 µm 13 mm diameter from Cronus (UK).
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