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

Abstract

Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [15N], has been frequently used. In plants, it is usually applied in form of [15N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [15N]-nitrate/[14N]-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 [15N]-nitrate to [14N]-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 [15N]-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 [14N]-nitrate as the sole nitrogen source.

Quantification of Arbutin in Cosmetics, Drugs and Food Supplements by Hydrophilic-Interaction Chromatography

“…For initial experiments (optimisation of the HPLC conditions), an LC-10 HPLC system (Shimadzu, Nakagyo-ku, Japan) configured as described previously [ 39 ] was used. The column oven was operated at 25 °C and the UV detector was set to 284 nm.…”

The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation

“…Polyamines were quantified by HPLC after derivatization with dansyl chloride (details are also given in the Supplementary Information ). Nitrate was quantified by ion-pair chromatography as described previously ( Schramm et al, 2019a ). For analysis of minerals homogenized frozen plant material was weighed into a quartz crucible and incinerated at 500°C for 5 h. The ash was dissolved in 1 ml 1 M nitric acid and water was added to a final volume of 10 ml.…”

“…For analysis of minerals homogenized frozen plant material was weighed into a quartz crucible and incinerated at 500°C for 5 h. The ash was dissolved in 1 ml 1 M nitric acid and water was added to a final volume of 10 ml. For analysis of potassium, the solution was diluted 1:20 with distilled water and analyzed by cation exchange chromatography as described previously ( Schramm et al, 2019a ). The phosphate content was analyzed by a modified molybdate method as described in the Supplementary Information .…”