Abstract

Elucidation of regulatory mechanisms of plant secondary metabolism requires quantitation of metabolic fluxes. A method of high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) has been developed for the trace analysis of eight components (rosmarinic acid, lithospermic acid B, L-phenylalanine, t-cinnamic acid,4-coumaric acid, L-tyrosine, 4-hydroxyphenylpyruvic acid and homogentisic acid) involved in lithospermic acid B biosynthesis pathway in Salvia miltiorrhiza hairy root cultures. The separation was performed by a ZORBAX SB-C₁₈ column (3.5 µm, 2.1 x 150 mm, I.D. Agilent Corporation, MA, USA) and a C₁₈ guard column (5 µm, 4.0 x 2.0 mm, Agilent Corporation, MA, USA) with an isocratic mobile phase consisting of acetonitrile-water (55:45, v/v) at a flow rate of 0.3 ml/min. The components were detected by an Agilent G6410A triple quadrupole LC/MS system equipped with a MassHunter interface. The components were detected using electrospray ionization (ESI) in negative-ion mode and quantified by multiple-reaction monitoring (MRM) mode using the transition mass of m/z 359→161, 717→519, 164→147, 147→103, 163→119, 180→119, 179→107 and 167→123 for rosmarinic acid, lithospermic acid B, L-phenylalanine, t-cinnamic acid, 4-coumaric acid, L-tyrosine, 4-hydroxyphenylpyruvic acid and homogentisic acid, respectively. The recovery of the method was in the range of 95.4 to 105.5%, and all the components showed good linearity (r＞0.995) in a relatively wide concentration range. This method has been successfully applied to the determination of the eight components in different elicitor (Methyl jasmonate and silver ions) treated S. miltiorrhiza hairy root cultures, for describing elicitor-based regulatory effect to lithospermic acid B metabolic fluxes.

Key words: Salvia miltiorrhiza, biosynthesis pathway, liquid chromatography–tandem mass spectrometry, phenolic acids.