A gas chromatography–mass spectrometry method for the determination of delta-aminolevulinic acid in plant leaves

Abstract

Delta-aminolevulinic (δ-ALA) acid is an important intermediate for tetrapyrroles biosynthesis and it has recently received great attention in plant physiology and human toxicology. However, the colorimetric method which is the most common method for determination of δ-ALA is time consuming and is not specific. In this study, a method for determination of δ-ALA in plant tissues was developed based on the trimethylsilyl (TMS) derivative of the pyrrole formed from the reaction of δ-ALA with ethyl acetoacetate via Knorr condensation. The δ-ALA in the HCl extract was reacted with ethyl acetoacetate to form a pyrrole. Then, the pyrrole compound was extracted using ethyl acetate and the solvent was evaporated to dryness. The dried sample was derivatized to its TMS ester and analyzed using GC–MS. The concentration of δ-ALA in citrus leaves incubated with levulinic acid was also determined by the conventional colorimetric method. The linear range was 10–200ppm in the full scan mode and 0.1–20ppm in the selected ion monitoring (SIM). The limit of detection was 6ppm in the full scan and 0.05ppm in SIM mode, representing a four-fold increase in sensitivity compared to the colorimetric method. The GC–MS method developed in this study is simple, accurate, sensitive, and could also be used to measure δ-ALA in other biological samples.