Translating bumblebee dialect…
Reconstituted extracted alkaloid fraction with measured volume of methanol containing 1.5 μg/mL caffeine (internal standard), transferred ~1 mL to 1.5 mL GC autosampler vials for GC/FID or GC/MS analysis.
GC/FID analysis
Analyzed samples using Shimadzu GC-2010 gas chromatograph equipped with Shimadzu AOC-20i autosampler and J&W DB-5 column (30 m × 0.32 mm, 0.25 μm film thickness) with flame ionization detector. Injected samples (1.0 μL) splitless at 250°C using helium carrier gas at constant flow rate of 2.0 mL/min. Column oven temperature program: 100°C for 1 min, increased to 200°C at 50°C/min, then to 260°C at 5°C/min, then to 320°C at 50°C/min, held at 320°C for 8.8 min for total run time of 25 min.
Quantities: 1.0 μL injection volume, 2.0 mL/min helium flow rate, 25 min total run timeDuration: 25 min per sampleConditions: Splitless injection at 250°C, temperature program from 100°C to 320°C
Equipment: Shimadzu GC-2010, Shimadzu AOC-20i autosampler, J&W DB-5 column, FID detector
GC/MS analysis
Performed GC/MS analysis using Finnigan MAT GCQ (Finnigan MAT, San Jose, CA, USA) equipped with split/splitless injector and DB-5MS (30 m × 0.25 mm; J&W Scientific) column. Injection port temperature 250°C, oven heated in splitless mode. Split vent flow rate 50 mL/min and purged after 0.80 min. Oven temperature 100°C for 1 min, 100-200°C at 40°C/min, 200-275°C at 5°C/min, then held at 275°C for 1.5 min. Used electron impact ionization (EI) at 70 eV with ion source temperature of 200°C.
Quantities: 50 mL/min split vent flow rate, 0.80 min purge time, 1.5 min final hold time, 70 eV ionization energyDuration: Variable run time based on temperature programConditions: Splitless injection at 250°C, ion source at 200°C, EI at 70 eV
Equipment: Finnigan MAT GCQ, DB-5MS column, split/splitless injector