The Analysis of Pyrrolizidine Alkaloids in Jacobaea vulgaris; a Comparison of Extraction and Detection Methods

Introduction - Pyrrolizidine alkaloids (PAs) serve an important function in plant defence. Objective - To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC-NPD) and liquid chromatography tandem mass spectrometry (LC-MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. Methodology - Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC-NPD, reduction is required to transform PA N-oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. Results - The lowest PA concentration measured with GC-NPD was approximately 0.03 mg/g and with LC-MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC-NPD. With the LC-MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC-NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC-MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC-NPD analysis with sulfuric acid extraction and PA N-oxide reduction with zinc and purification. Conclusions - In terms of sensitivity and discrimination, formic acid extraction in combination with LC-MS/MS detection is the method of choice for analysing PAs (both free and N-oxides forms) in plant material. Copyright (C) 2009 John Wiley & Sons, Ltd.