Extraction and quantification of geranate,
3-methyl-crotonate and 3-methyl-3-butanoate in aqueous phase
One hundred microliters of cell culture were collected and centrifuged
at 10,000 g for 10 min. Four hundred microliters of methanol was mixed
with the obtained supernatant and vortexed for 1 h. The mixture was then
centrifuged at 10,000 g for 10 min to remove the precipitated enzymes
and the supernatant was then analysed. An Ultra Performance Liquid
Chromatography (UPLC, Waters ACQUITY) linked with a Time-of-flight Mass
Spectrometry (TOFMS, Bruker micrOTOF II) was used to quantify geranate,
3-methyl-crotonate (3-MC) and 3-methyl-3-butanoate (3-MB). The column
used was Poroshell 120 EC-C18 column (2.1 × 50 mm, 2.7 μm, Agilent
Technologies). Two mobile phases were used: A) 0.1 % trifluoroacetic
acid in water; B) acetonitrile. The gradient was as follows: 0–2 min,
95% A; 2-2.5 min, 95%-90% A; 2.5-3.5 min, 90% A, 3.5-5.5 min,
90%-60% A, 5.5-9 min, 60% A; 9-10 min, 60%-95% A; 10-11 min, 95%
A. The flow rate was 0.25 mL/min. Electrospray ionization was used and
the mass spectrometry was operated to scan 50–800 m/z in positive mode
with 2,500 V end plate voltage and 3,800 V capillary voltage. Nebulizer
gas was provided in 1 bar, drying gas temperature was 9 mL/min, and dry
gas temperature was 200 °C. Sample injection volume was 10 μL.