HPLC glucosinolate analysis

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Method synopsis

Extraction and quantification of desulfated glucosinolates from plant leaves using high-performance liquid chromatography with diode array and charged aerosol detection. Follows established desulfation protocols for GSL analysis.

Synthesized from method descriptions across 6 papers using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Novel host unmasks heritable variation in plant preference within an insect population (2022), Evolution. Implementations in other papers (listed below) may differ.

1. Methanol extract preparation
   Fresh host plants collected from field. Leaves removed from stems, weighed in small packets, transferred to liquid nitrogen to make methanol extracts. Modified extraction from Agerbirk and Olsen (2011): leaves frozen, lightly crushed, boiled in 70% MeOH for several minutes before filtering. Excess MeOH evaporated, filtrate left to evaporate for 24 h. Small amount of 70% MeOH added to achieve equal concentrations (10 g fresh weight/L).
   Quantities: 24 samples of C. cordifolia, 19 of T. arvense collected. Final concentration 10 g fresh weight/L in both extractsDuration: 24 h evaporation period, several minutes boiling timeConditions: Extracts stored in dark, cool fridge to prevent light-sensitive glucosinolate degrading
   Equipment: liquid nitrogen, 70% MeOH, filtering equipment, dark cool storage
2. Glucosinolate extraction and quantification
   Sixth leaf from apical meristem of 15 plants each species collected into screw-cap microcentrifuge tubes containing 70% MeOH. Leaf samples kept in cool, dark incubation for 8 months. Glucosinolates leached into surrounding MeOH, desulfated and quantified following Prasad et al. (2012) and Keith and Mitchell-Olds (2017) protocols.
   Quantities: 15 plants each of C. cordifolia and T. arvense, sixth leaf from each plant collectedDuration: 8 months cool, dark incubation periodConditions: Cool, dark storage conditions to preserve glucosinolates
   Equipment: screw-cap microcentrifuge tubes, 70% MeOH, HPLC equipment for quantification

Typical Equipment

• screw-cap microcentrifuge tubes
• 70% MeOH
• high-performance liquid chromatography (HPLC)
• diode array detector (DAD)
• charged aerosol detector (CAD)

Output Measurements

• glucosinolate concentrations
• GSL structural identification

Papers Using This Protocol (6)

Species Studied With This Protocol