High-precision quadruple isotope dilution method for simultaneous determination of nitrite and nitrate in seawater by GCMS after derivatization with triethyloxonium tetrafluoroborate

Highlights

• High-precision determination of nitrite and nitrate in seawater.

• Use of quadruple isotope dilution.

• Aqueous Et₃O⁺[BF₄]⁻ derivatization chemistry for GCMS analysis of nitrite and nitrate.

Abstract

Quadruple isotope dilution mass spectrometry (ID⁴MS) has been applied for simultaneous determination of nitrite and nitrate in seawater. ID⁴MS allows high-precision measurements and entails the use of isotopic internal standards (¹⁸O-nitrite and ¹⁵N-nitrate). We include a tutorial on ID⁴MS outlining optimal experimental design which generates results with low uncertainties and obviates the need for direct (separate) evaluation of the procedural blank. Nitrite and nitrate detection was achieved using a headspace GCMS procedure based on single-step aqueous derivatization with triethyloxonium tetrafluoroborate at room temperature. In this paper the sample preparation was revised and fundamental aspects of this chemistry are presented. The proposed method has detection limits in the low parts-per-billion for both analytes, is reliable, precise, and has been validated using a seawater certified reference material (MOOS-2). Simplicity of the experimental design, low detection limits, and the use of quadruple isotope dilution makes the present method superior to the state-of-the-art for determination of nitrite and nitrate, and an ideal candidate for reference measurements of these analytes in seawater.

Introduction

The precise and accurate determination of nitrite, nitrate and other nutrients in seawater is a mature topic in marine biology and the importance of this subject was recently highlighted by the Intergovernmental Panel on Climate Change (IPCC): “changes in nutrient concentrations can provide information on changes in the physical and biological processes that affect the carbon cycle and could potentially be used as indicators for large-scale changes in marine biology” [1].

In spite of the need for high-quality data for nutrients in seawater, most of the classical analytical chemistry methods for nitrite and nitrate are still based on spectrophotometric and electrochemical detection [2], [3]. These approaches cannot deliver specificity and sensitivity and can be significantly affected by the seawater matrix. Furthermore, direct colorimetric assays are available for nitrite, but not for nitrate, which is routinely determined as nitrite after heterogeneous-phase reduction [4]. Analytical chemistry approaches for nitrite and nitrate in seawater are, therefore, limited when high-precision is demanded, and mass spectrometry should be preferred.

Mass spectrometry detection can address specificity and sensitivity and it allows isotope dilution calibration [5]. Isotope dilution is widely recognized as one of the most accurate approach of modern quantitative analysis [6], [7]. Since its introduction, however, isotope dilution has encountered many evolutions, including the recent formulation of the concept of quadruple isotope dilution (ID⁴MS) [8]. ID⁴MS achieves quantitation of the analyte by determination of the isotopic composition of four blends which are made by mixing the sample and the primary standard solution of the analyte with the isotopic internal standard. When an optimal experimental design (exact-matching) is adopted, ID⁴MS provides results with high metrological quality [8]. In this paper ID⁴MS has been applied for the first time to quantitate nitrite and nitrate in seawater. To facilitate the use of this new method, we also include a tutorial explanation of the ID⁴MS along with an Excel spreadsheet (Fig. 1).

The application of ID⁴MS for the speciation of nitrite and nitrate depends on the availability of mass spectrometric methodologies that are able to detect the analytes while avoiding any artifacts which may affect the results [5]. Despite the use of ESIMS [9], [10] and thermal ionization mass spectrometry [11] for such measurements, GCMS-based methods are more attractive due to the wide availability of the GCMS [12]. However, neither nitrite nor nitrate are volatile, therefore they cannot be subjected to gas chromatography without derivatization. In this vein, the nitration of an aromatic compound was first proposed for the conversion of nitrate into nitroarenes [13]. This approach requires a strongly acidic reaction medium which can cause the unwanted conversion of nitrite to nitrate [14], [15]. A modern alternative to nitration is alkylation with pentafluorobenzyl bromide (F₅BzBr). Alkylation with F₅BzBr was proposed for nitrite [16], and later extended to both nitrite and nitrate [17], [18]. Despite the specificity and sensitivity of this method, sample preparation requires a non-aqueous reaction medium (acetone), elevated reaction temperature (50 °C for 1 h), and a toluene extraction after the evaporation of the reaction medium. To overcome the disadvantages of the F₅BzBr alkylation, we recently proposed an alternative carbon-based derivatization chemistry for nitrate and nitrite [19] based on the use of triethyloxonium tetrafluoroborate, Et₃O⁺[BF₄]⁻ to convert nitrite and nitrate to ethyl nitrite and ethyl nitrate, respectively. Triethyloxonium tetrafluoroborate [20], [21], [22] is a water soluble reagent that is able to perform ethylation in aqueous media and at room temperature. Both EtONO and EtONO₂ are volatile and can be sampled in the headspace, allowing their separation from the sample matrix before the GCMS analysis. This simple state-of-the-art GCMS method in combination with quadruple exact-matching isotope dilution calibration provide a robust approach for the speciation of nitrite and nitrate in seawater, producing high-precision metrological results.