Prodigiosin purification

Ethanol extract of strain N4-5, prepared as described above, was concentrated to dryness in a Rotovap and dissolved in 3 mL acetone. Prodigiosin was purified from this extract by flash chromatography via elution through a silica (Sigma silica, Cat. No. 236722, 60 Å, 200–245 mesh) prep column with hexane: acetone: ammonium hydroxide (85:15:1; v:v:v) as the elution solvent. Orange fractions containing prodigiosin were collected, combined, dried under N₂, and resuspended in methanol. Purity of the prodigiosin fractions was determined with the LC/MS–MS with an ESI source and X-bridge C-18 reverse phase column (150 mm × 2.1 mm i.d., 5 μm). For this, the diluted aliquots of the prodigiosin fractions were introduced into the C-18 LC column and eluted using gradient separation where solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. The LC operation was the same as described above except the solvent gradient program was terminated after the final gradient reached 100% solvent B when it was brought and held at initial conditions for 8 min. Mass scans of fractions from 100 to 1200 m/z were conducted to verify the purity of the prodigiosin preparation (98% pure). Fractions containing only a single peak at 22.6 min, indicative of pure prodigiosin, were combined.

Quantitation of prodigiosin was performed using multiple reaction monitoring (MRM) mode where the collision induced dissociation (CID) transition of parent to daughter ion was 324 m/z to 252 m/z. This transition was monitored over the time window as prodigiosin was observed to elute under the above conditions, e.g. 22.6 min. The prodigiosin standard was used to create a calibration curve and determine the concentrations of prodigiosin in the ethanol extracts and the purified prodigiosin preparation.