For further comparison, M. extorquens AM1 pCM160 was cultivated in shake flasks in the presence of kanamycin on minimal medium with natural labeled methanol containing the default cobalt concentration and on minimal medium with 99% 13C methanol containing a 10 times increased cobalt concentration.
Isopropylidene-protected vicinal diol 4 3b was treated with TFA in a THF/H2O mixture to furnish diol 5, which, after solvent evaporation, was immediately taken up in methanol containing a catalytic amount of HCl to furnish the corresponding methyl ester 6 in 77 % yield.
To 100 μl of sample (plasma, salivary gland tissue homogenates or calibrator), 100 µl of phosphoric acid 4% and 1 ml of methanol containing hexestrol as an internal standard were added.
The LC mobile phases (delivered at 0.28 mL/min) included water containing 0.5 mM HCOOH for solvent A and methanol containing 0.5 mM HCOOH for solvent B. A specially designed LC binary gradient elution was performed and the gradient program was as fallows: 0 1 min at 0% B; from 0% B to 80% B in 0.01 min; from 80% B to 100% B in 2.5 min; back to 0% B in 0.01 min; maintained 2.5 min.
The two mobile phases were water (UPLC MS grade, Sigma) with 0.1% formic acid (Buffer A), and methanol containing 0.1% formic acid, (Buffer B).
The mobile phase consisted of 100% water containing 0.5% (w/w) trifluoroacetic acid (solvent A), 100% methanol containing 0.5% (w/w) trifluoroacetic acid (solvent B), and 100% acetonitrile containing 0.5% (w/w) trifluoroacetic acid (solvent C).
Using this port, the liquid solvent (methanol) containing standard aflatoxins or an extracted sample was directly introduced into the injection port.
Next, the samples were reconstituted with acidified methanol containing synthetic cerebrin as an internal calibrant.
The optimized analytical method for ultra fast high performance liquid chromatography (UFLC) was a gradient elution using water containing 0.04‰ formic acid (A) and methanol containing 0.04‰ formic acid (B), at a flow rate of 0.3 mL/min.
Resveratrol and ferulic acid derived products were eluted with an isocratic mobile phase of water containing 0.1% trifluoroacetic acid (A) and methanol containing 0.1% trifluoroacetic acid (B) in a ratio of 73:27 (A B, v/v) with a flow rate of 1.0 mL/min.
