Method development and Validation
Tomato and digested tomato
100 mg of lyophilized and homogenized tomato/digested tomato + internal standard mixture + 2ml of pure methanol, vortex, shake, sonicate and centrifuge.
Collect the supernatant and repeat again all the steps after adding 2ml of pure methanol. Dry the collected supernatant completely, reconstitute in 0.4 ml of ultrapure water/methanol = 9/1 + 0.3 ppm rosmarinic acid (external standard), vortex and filter using 0.2 um RC filter.
Analyse using LC-Qtrap
Method validation parameters: linearity (calibration curve in solvents), precision (intra and inter method and instrumental repeatability), extraction recovery, matrix effect, limit of quantification and detection
Based on the present concentration of selected compounds we perform method validation on concentrated, 100 and 1000 times diluted matrix
Fecal fermentation extracts
Add internal standard mixture, centrifuge and filter the fecal sample using RC 0.2 um membrane filters
Inject directly to LC-Qtrap
Based on the concentration of selected compounds we perform method validation on concentrated and 100 times diluted matrix