Diterpene resin acid extraction and analysis
Diterpenes were extracted from lyophilized ground tissue in 1 ml of
methanol, as adapted from Mullin et al . (2021). Samples were
vortexed for 30 sec, then sonicated for 10 min, and left for 24 h in the
dark at room temperature, and then vortexed for 30 sec and centrifuged
(18,213 rcf) for 15 min. Extracts were transferred into 2 mL glass vials
and stored at −40 °C. Diterpenes were analyzed in an UHPLC, fitted with
a reverse-phase InfinityLab Poroshell 120 EC-C18 column (2.1 x 150 mm
1.9µm, Agilent Tech.) and a diode array detector (UV/Vis, 1290 DAD,
Agilent Tech.). We performed a gradient analysis with a binary solvent
system, a 1.7% v/v glacial acetic acid in distilled deionized water
(channel A) and 100% pure methanol (channel B) flowing at 0.3 mL
min-1 for 17 min with solvent gradients. A 5 µl
injection volume was used. The system began at 75% B for 1 min, then
increasing to 85% B over 9 min, held for 2 min, then decreased to 75%
B over 14-17 min, and held for 3 min.
Diterpenes were quantified using analyte absorbance at wavelengths of
240, 268, and 282 nm (Kersten et al. 2006) and applying standard
curves. The curves were generated from dilutions prepared from
analytical standards of dehydroabietic acid (CP: >99%),
sandaracopiramic acid (CP: >90%), levopiramic acid (CP:
>95%), neoabietic acid (CP: >99%), palustric
acid (CP: >92%), and abietic acid (CP:
>75%). All chemicals were obtained from CanSynth Chem.
Corporation (Toronto, ON, CAN), except for abietic acid from Sigma
Aldrich. Diterpene concentrations were reported as
µg mg-1 of dried
weight.