Sampliq c18

Due to the high quantity of tyrosine in I. umbellifera, and tyrosine's low solubility, a special extraction protocol was developed. Twenty‐five milligrams of dried leaf sample were homogenized as above and extracted in 2 mL of acidified 10% MeOH (90% water adjusted to pH = 3 with acetic acid/10% MeOH; v/v) for 20 min at 80°C. Samples were centrifuged at 13,250 × g and ambient temperature for 5 min. The resulting supernatant was retained, and the extraction was repeated. The combined supernatants were separated on preweighed Agilent SampliQ C18 solid‐phase extraction columns (500 mg ODS). The supernatant was added to the prepared column and washed with an additional 2 mL 10% MeOH (pH 3). The 10% MeOH wash contained tyrosine and was dried under vacuum (0.8 torr) and redissolved in 20 mL of 10% MeOH. Samples were then separated on a Hitachi LaChrom Elite with an Omnisphere C18 250 × 2.0 mm column isocratically using 10% MeOH/90% HOH with 0.1% formic acid. Tyrosine was detected at 275 nm and quantified based on peak area and external calibration curves. The SampliQ columns were dried and reweighed. As I. umbellifera does not contain saponins (Coley et al. 2005), the difference in final minus initial weights was considered to be the mass of the phenolic fraction trapped on the column.