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Samples (1 µL of 100 times-diluted samples in methanol) were injected manually in splitless mode through the injector.
The extracts dissolved in methanol were injected in split mode (10 1 ratio).
Samples (2 μL in methanol) were injected into the ESI source directly and after separation by an Ultimate 3000 (DIONEX) high-performance liquid chromatography system equipped with a 136/CF column and an auxiliary UV detector (λ = 256 nm); the flow rate was 0.15 mL min-1.
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Instead, a small amount of water and anti-freeze (eg, methanol) is injected into the cylinder just as the liquid nitrogen is drawn in, causing it to boil and expand rapidly thereby forcing the piston down inside the the cylinder.
Air is supplied through the feed side and methanol is injected in the CO2 (sweep gas) stream through the permeate side.
Initially pure methanol was injected in both ESI modes to detect the presence of any background contamination due to the solvent or instrument (Additional file 3: Figure S1 and Additional file 4: Figure S2).
Prodigiosin dissolved in methanol was injected into the LC MS and MS was performed using positive ion electrospray ionization with the following settings: capillary voltage 3.4 V, cone voltage 30 V at a source temperature 100 °C.
An equal volume of berberine in methanol was injected as standard marker.
1 μL of extract diluted with methanol was injected into GC/MS and the compounds were identified based on the molecular structure, molecular mass, and calculated fragment ratio of resolved spectra with that of mass spectra available from the library.
To assess the chemical composition of CRAE with conditions as literature reports [ 5, 6] with some modifications, 10 μL of CRAE (0.5 mg/mL in methanol) was injected and eluted through RP-C18 column (250 mm x 4.0 mm, 5 μm, Alltech, USA) with mobile phase as acetonitrile-25 mM potassium dihydrogen phosphate (25:75).
Dry material was resuspended in 0.5 mL of methanol and 10 µL of methanol solutions were injected into LC/MS for initial analysis (Agilent 1200 series HPLC/6130 mass spectrometer, Phenomenex C18(2) 4.6 mm×10 100, 10–100% aqueous CH3CN with 0.1% formic acid over 20 min).
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