A liquid chromatography–electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation

Cyclotides are plant-produced, bioactive, cyclic mini-proteins with interesting pharmaceutical and agricultural applications. A reverse phase liquid chromatography electrospray ionization mass spectrometry (RP-LC–ESI-MS) method for analysis of cyclotides in plant materials with a minimum of sample pre-treatment is presented. Three exemplary cyclotides (kalata B1, kalata B2 and cycloviolacin O2) were used as reference substances for the method development. Linearity (r2 > 0.99) was achieved in the concentration range 0.05–10 mg/L and the limit of detection was 1.7–4.0 μg/L. The present study is the first to demonstrate that cyclotides dissolved in water sorb to glass vials, but the addition of 15% of acetonitrile or 40 mg/L of bovine serum albumin is sufficient to keep the cyclotides in solution. Cyclotides were extracted from candied violets, violet tea, and the plants Oldenlandia affinis and Viola odorata using 70% methanol containing 0.1% formic acid (v/v). The plant content was determined to be 23.5–14,200 μg/g (dry weight). The highest content of cyclotide was found in wild Danish V. odorata, and it is the highest content of cyclotide in a plant reported hitherto. Candied violets contained 0.00–8.66 μg/g (dry weight), while no cyclotides were detected in commercial violet tea.

► An RP-LC–ESI-MS method for cyclotide analysis in plants was developed.
► We demonstrated that cyclotides dissolved in water sorb to glass vials.
► Adding 15% of acetonitrile or 40  mg/L of BSA kept the cyclotides in solution.
► We determined the plant content to be 23.5–14,200 μg/g (dry weight).
► We reported the highest cyclotide plant content determined hitherto.