Determining¹⁵N enrichment of dissolved organic nitrogen in environmental waters by gas chromatography/negative-ion chemical ionization mass spectrometry

Abstract

To minimize the required sample volume and simplify the overall procedure for determining 15N enrichment of dissolved organic nitrogen (DON) in environmental waters, we employed derivatization of NO3− with pentafluorobenzyl bromide and gas chromatography/negative‐ion chemical ionization mass spectrometry (GC‐NICI‐MS). The pretreatment processes to remove inorganic nitrogen were optimized to ensure recovery of labile DON molecules, and the derivatization procedure for NO2− and NO3− was optimized for environmental samples. The required sample volume is 10 mL or less and the detection limit is about 0.015 µmol 15N L−1, or 0.3 atomic percent excess of 15N for water samples containing 5 µmol DON L−1. This method can also be applied to 15N enrichment analysis of particulate organic nitrogen. We applied the developed method to measure DON production rate by the hermatypic coral Porites cylindrica during incubation with 15N‐labeled NO3−.

A streamlined method to quantify the fates of 15N in seawater samples amended with 15N‐labeled organic nitrogen

“…This result is consistent with a previous study that UV oxidation efficiency may decrease at higher DON concentration (Bronk et al 2000). The calculated oxidation efficiency was lower than for other methods (e.g.,~95% in Miyajima et al 2005), but comparable to reported recovery rates for the UV oxidation method (Bronk et al 2000). Based on the oxidation efficiency vs. N concentration relationship, optimized results can be obtained if total N in solution is in the range of~0.5-40 μmol L −1 .…”

“…The proposed method is more timesaving than conventional methods for determining 15 N in different forms (e.g., Axler and Reuter 1987;Slawyk and Raimbault 1995;Hasegawa et al 2000). Conventional methods, and even newly developed methods (Miyajima et al 2005;Isobe et al 2011), require complex preparation processes, such as the alkaline isolation of NH 4 + as NH 3 and subsequent trapping of NH 3 to ammonium salt, which is not only the rate-limiting step (Miyajima et al 2005;Isobe et al 2011), but problematic in terms of NH 3 carryover to other N pools (Slawyk and Raimbault 1995). Also they are subject to potential interference from labile organic N such as amino sugars, amino acids, and amines during the process (Miyajima et al 2005).…”

“…This technique is robust and reliable, but very laborintensive (Bronk 2002). It requires a number of complex preparation processes prior to mass spectrometry analysis and often requires sample sizes >100 mL (Miyajima et al 2005;Isobe et al 2011) for sufficient analytical sensitivity in 15 N-spiked studies, due to the high background concentration of N 2 gas.…”

“…Alternative methods for 15 N analysis are needed to simplify sample preparation and handle samples with limited volume. One method for 15 N analysis involved derivatizing NO 3 − with pentafluorobenzyl bromide (PFB-Br) and analyzing the nitric acid ester of PFB-alcohol (PFB-ONO 2 ) using gas chromatography mass spectrometry (GC-MS; Miyajima et al 2005). Concentration of each 15 N form can be determined by transforming it to 15 NO 3 − before analysis.…”

“…Alkaline removal of NH 4 + is time consuming (takes 7 to 14 d to complete; e.g., Slawyk and Raimbault 1995;Miyajima et al 2005;Isobe et al 2011), and may be affected by sample salinity (Miyajima et al 2005). Isolation of NH 4 + at elevated temperature under alkaline conditions may also lead to degradation or hydrolysis of labile organic nitrogen such as peptides and amines (Miyajima et al 2005), which can be a major issue when labile organic nitrogen compounds, for example, glutamate (Ogawa et al 2001), leucine (Heissenberger and Herndl 1994), or other amino acids, are used as the incubation substrate during the controlled bioassay experiment. Furthermore, the incomplete removal and subsequent carryover of inorganic NH 4 + into other N pools cause concern (Slawyk and Raimbault 1995).…”