Quantification of Violacein and Indolmycin

Extraction of violacein and indolmycin for quantitative studies was tested using propan-2-ol, methanol, ethanol, and acetonitrile. Acetonitrile created a two-phase system of variable size due to the media salts and could not be used. Methanol and ethanol had to be added at 5 and 2 times the sample volume, respectively, to extract the violacein quantitatively. This resulted in a lower concentration of the analytes in the mixture and also a need to inject more of the mixture to maintain a reasonable UHPLC (Ultra High-Performance Liquid Chromatography) peak shape for di-demethylindolmycin. Overall, propan-2-ol provided the most accurate extraction, so 5 ml of bacterial culture (fresh or thawed from -20°C storage) were prepared for the quantification of violacein, indolmycin, and indolmycin precursors by the addition of an equal volume of propan-2-ol.

The mixture was inverted until homogeneous and placed in an ultrasonication bath for 30 min. Tubes were centrifuged (4,000 × g, 15 min) to pellet the biomass (colorless) from the extracted violacein, indolmycin and related compounds in solution. Seven hundred and fifty microlitres of supernatant was transferred to an autosampler vial. UHPLC-UV/Vis analysis was performed on a Dionex RSLC Ultimate 3000 system (Sunnyvale, CA, USA) using a 150 mm × 2 mm i.d., 2.6 μm Kinetex C₁₈ column (Phenomenex, Torrance, CA, USA), running at 800 μl min^-1 and 60°C using a binary linear solvent system of water (A) and acetonitrile (B) (both buffered with 50 μl l^-1 trifluoroacetic acid; TFA). The gradient program was: t = 0, 15% B; t = 0.5 min 25% B; t = 6 min 65% B; and t = 7 100% B, keeping this for 1 min, then reverting to 15% in 1 min. A sample volume of 1.5 μl was injected.

Violacein (RT 2.76 min) was detected recording absorption at 578 ± 2 nm, and indolmycin (1.98 min), di-demethylindolmycin (RT 1.21 min), and the two mono-demethylindolmycin (1.60 and 1.64 min, integrated as one peak) by absorption at 219 ± 2 nm.

Quantification by external standard calibration using six different concentrations of violacein and indolmycin for the calibration (40, 20, 10, 5, 2, and 1 μM) resulted in a linear calibration curve with R² of 0.9999 and 0.9997, respectively. Extraction efficiency was tested by extracting various batches of samples three consecutive times, showing an extraction efficiency of >97%. The di- and mono-demethylindolmycins were quantified using the indolmycin calibration curve, since these compounds have the same chromophore and thus molar extinction coefficient. Detection limits were 1 μM for violacein, and 10, 5, and 1 μM for di-demethylindolmycin, mono-demethylindolmycin, and indolmycin, respectively.