Abstract:

A quantitative method based on high performance liquid chromatography-tandem mass spectrometry
(HPLC-MS) has been established for simultaneous analysis of nine phytohormones in Porphyra haitanensis. The optimized
sample preparation procedure involved extraction with methonal-H2O-formic acid (15:4:1, V/V) and re-dissolution in
methonal-H2O-acetic acid (90:10:0.05, V/V) containing 10 mmol/L ammonium acetate. The chromatography was performed
using a Thermo Hypersil Gold C18 column (100 mm × 2.1 mm, 3 μm) as the stationary phase, and the mobile phase consisted
of aqueous solution containing 10 mmol/L ammonium acetate-methanol. Nine phytohormones (indole-3-acetic acid, N6-(2-
isopentyl) adenosine, N6-(2-isopentenyl)adenosine, trans-zeatin riboside, zeatin, abscisic acid, salicylic acid, gibberellin A3,
and brassinolide) were quantified using selective reaction mode. As results, good linear relationships between concentrations
and the corresponding peak areas of nine standards were achieved with correlation coefficients (R2) higher than 0.991. The
recoveries of eight standards except gibberellin A3 were higher than 71%. The limits of detection (LODs) of phytohormones
by this method were all lower than 3 μg/L and the limits of quantification (LOQs) were in the range from 0.45 to 5 μg/L.
Using the established method, the concentrations of nine phytohormones in different varieties of Porphyra haitanensis and at
different cultivation periods were successfully investigated and correlated with its morphology.

Key words: Porphyra haitanensis, phytohormones, high performance liquid chromatography coupled with triple quadrupole-tandem mass spectrometry (HPLC-QqQ-MS/MS), quantitative analysis