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2 protocols using d2 ga15

1

Comprehensive Phytohormone Profiling Protocol

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All reagents were obtained from LabBox (Vilassar de Dalt, Spain). All phytohormones, unlabeled (GA1, GA3, GA4, GA7, GA8, GA12, GA15, GA19, GA20, GA29, GA44, GA51, GA53, ABA, JA, IAA) and deuterium-labeled (d2-GA1, d2-GA3, d2-GA4, d2-GA7, d2-GA8, d2-GA12, d2-GA15, d2-GA19, d2-GA20, d2-GA29, d2-GA44, d2-GA51, d2-GA53, d6-ABA, d6-JA, d5-IAA) standards, were purchased from OlChemIm (Olomouc, Czechia). SPE columns and OASIS® HLB 1cc and OASIS® PRIME HLB 1cc were purchased from Waters (Milford, MA, United States). Fixed insert vials and pre-slit PTFE screw cap were purchased from Teknokroma (Sant Cugat del Vallès, Spain). The HPLC column Kinetex® 2.6 μm XB-C18 100 Å (302.1 mm) was purchased from Phenomenex (Torrance, CA, United States), and the HPLC column Mediterranea Sea 18 column (100.2 cm, 2.2 μm) was purchased from Teknokroma (Sant Cugat del Vallès, Spain).
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2

Quantifying Wheat Grain Gibberellins

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Wheat samples
(100 mg taken from 10 flash-frozen, pooled, and magnalised grains)
were extracted in triplicate overnight in 1.0 mL of acidified methanol
pH 4.0 [80/20, methanol/5.0 mM formic acid-containing butylated hydroxytoluene
(3–5 crystals)]. As internal tracers, d2-GA1, d2-GA4, d2-GA8,
d2-GA9, d2-GA15, d2-GA19, d2-GA20, and d2-GA29 (20 pmol each, Olchemim)
were added. After purification on an RP-C18 cartridge (500 mg), samples
were derivatized with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (1.0 mg/sample, pH 4.0, 60 min,
37 °C under continuous shaking, Eppendorf thermomixer). Next,
these derivatized samples were analyzed using a UPLC-MS/MS equipped
with an electrospray interface in positive mode (ACQUITY, TQD, Waters).
Samples (6.0 μL) were injected on an ACQUITY BEH C18 column
(2.1 × 50 mm; 1.7 mm, Waters) using a column temperature of 30
°C and eluted at 450 μL/min with the following gradient
of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile
(solvent B): 0–0.8 min isocratic 92% A, 8% B; 0.8–5
min linear gradient to 60%A, 40%B; 5–5.5 min linear gradient
to 10% A, 90% B.
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