Sampe preparation for GC-MS analysis of phytohormones
Collect fresh samples and keep on ice or store at -70 oC.
Grind (as little as 0.2 g) better about 1-2 g of tissue in liquid N2.
Add about half of the expected amount as internal standard (50 - 500 ng,
D3-ABA, 13C-IAA, D2-JA,
14C-JA) and radioactive tracer (ca. 10,000 cpm of
3H-IAA, 3H-JA and 14C-SA).
Extract samples in 10 mL isopropanol overnight at 4C.
Collect 2500 g supernatant (5 min at 4C) and collect three additional
extractions with 2 mL of 100% isopropanol each in a 25 mL flask.
Reduce the combined extract to aqueous phase in a rotary evaporator and
diluted to ca. 20 mL with water and 0.5 mL of 0.1M (imidazole) buffer pH 7.0.
Prime NH2 Solid-Phase-Extraction columns with methanol, 5% acetic
water, and apply the extract. Wash with hexane and methanol, and elute with
5% acetic acid in 50% methanol.
Dry sample (rotary evaporator) and take up in 3x50 µL 36% MeOH.
Apply 100 µl of sample to a 50 mm, C18 reversed-phase column and gradient-elute
with 1% acetic acid in 36% MeOH (1 mL/min, 19 min), raise MeOH to 50%
within 6 min, and keep at 50% MeOH for further 10 min.
Under these conditions, the retention time for IAA, SA, ABA and JA are 10, 14,
21 and 30 min, respectively.
Combine the two 1-mL fractions with the highest radioactivity of each compound,
dry in vacuo, and methylate with 0.5 mL ethereal diazomethane.
Dry and take up in 50 µl of ethyl acetate.
For SA, use 100 µl of ethyl acetate and use half of that amount to determine the
radioactivity to determine yield.
Inject 1 µL samples of the methylated samples in a GC (HP- 5973) equipped
with a 15 m x 0.25 mm, J&W DB-1071 column and helium as carrier gas. All
samples are injected (250C).
The temperature profile for ABA and IAA starts at 160C and is ramped after 1
min at 20C×min-1 to 280C.
JA analysis begins at 120C and after 1 min raised at 20C×min-1.
SA analysis starts at 60C with a ramp of 20C×min-1 after 1 min at to
(and to keep the column clean at 40C× min-1 to 280C).
Under these conditions, the retention times are 5.25, 4.0, 4.5, and 4.3 min for
ABA, IAA, JA, and SA, respectively. The quantification is based on the
abundance of representative fragments for ABA (m/z 190/193), IAA (m/z
130/136), JA (m/z 151/153) and 120 for SA. SA was quantified based on the
amount of 14C in the sample.