Determination of the [(15)N]-Nitrate/[(14)N]-Nitrate Ratio in Plant Feeding Studies by GC–MS

Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [(15)N], has been frequently used. In plants, it is usually applied in form of [(15)N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [(15)N]-nitrate/[(14)N]-nitrate ratio in leaves are useful for the planning and evaluation of feeding and pulse–chase experiments. Here we describe a simple and sensitive method for determining the [(15)N]-nitrate to [(14)N]-nitrate ratio in leaves. Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were sufficient for analysis, allowing a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was analyzed by gas chromatography–mass spectrometry with electron ionization. Separation of the derivatized samples required only 6 min. The method shows excellent repeatability with intraday and interday standard deviations of less than 0.9 mol%. Using the method, we show that [(15)N]-nitrate declines in leaves of hydroponically grown Crassocephalum crepidioides, an African orphan crop, with a biological half-life of 4.5 days after transfer to medium containing [(14)N]-nitrate as the sole nitrogen source.