Kinetic 5 c18 100a

Ground leaf tissue was weighed and collected into 200 µl methanol containing 40 µM caffeine or 10 µM esculin as an internal standard and incubated at 60 °C for 2 hours. After a 30 minute centrifugation step at 5000 g, an aliquot of the supernatant (25 µl) was mixed with an equal volume of water and analyzed on a Thermo-Finnigan instrument equipped with a Deca XP ion trap detector or a Shimadzu IT-TOF. In both systems the column used was a Phenomenex Kinetic 5 µ C18 100A (100 x 2.10 mm x 5 µm) and the binary solvent system consisted of acetonitrile (ACN) and 0.1% formic acid in water. The elution program was as follows: 1 minute isocratic at 12% ACN, 3.5 minute gradient up to 25% ACN, 2.5 minute gradient up to 50% ACN, 1 minute gradient up to 100% ACN, 6 minute isocratic at 100% ACN, 1 minute gradient down to 12% ACN, and 2.5 minute isocratic at 12% ACN. Peak areas were calculated using the ICIS algorithm in Finnigan’s Xcalibur software or LCMS solutions and normalized by leaf mass (fresh weight) and the peak area of the internal standard.