Robust analysis of underivatized free amino acids in soil by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry
Introduction
Amino compounds (amino acids and amino sugars) play an important role in soil N cycling. These compounds account for 30–80% of total soil N and constitute the majority of soil organic N [1], [2], [3], [4], [5]. Amino acids are the key component of soil Dissolved Organic Nitrogen (DON) and they are closely related with the Soil Organic Carbon (SOC) pool in terms of the dynamics and stabilization of dissolved organic carbon [6], [7]. Amino acids not only represent a crucial N source for microorganism but also can be directly utilized by some plants [8], [9]. Additionally, these compounds can serve as biomarker to indicate their sources (e.g. microbial vs. plant residues) [5].
Amino acids are zwitterionic compounds, i.e. they can exist as an overall neutral form, a weak acid or a weak base. Due to the lack of specific chromophores for ultraviolet (UV) or fluorescence detection for most of amino acids [10], derivatization is widely used to improve the separation and the sensitivity of detection for analysing amino acids in biological and environmental samples [11], [12], [13], [14], [15], [16]. Overall these techniques can guarantee good separation, sensitive detection and high reproducibility but they have some drawbacks, such as being time-consuming and displaying instable derivatives, insufficient reproducibility of derivative yield, lack of analyte specificity and side effects of reagents [10], [17]. Therefore, recently, the techniques for direct analysis of underivatized amino acids have gained more attention and several methodologies have been developed. Casella and Contursi [18] measured 12 amino acids in milk using ion chromatography (IC) coupled to amperometric detection. High performance anion exchange chromatography combined with pulsed amperometric detection (HPAEC-PAD) was successfully applied to analyse soil amino acids [3], [4], [19]. Desiderio et al. [20] demonstrated that capillary electrophoresis (CE) linked to mass spectrometry (MS) detection enabled analysis of amino acids without previous derivatization. Ion-pairing liquid chromatography (LC) coupled to MS detection is another alternative approach to analyse underivatized amino acids [21], [22], [23], [24]. However, there are still serious drawbacks connected to all described techniques, including low throughput, reagents-induced retention time shift and contamination of analytical system [10], [23].
Most recently, hydrophilic interaction liquid chromatography (HILIC) has attracted considerable attention because of the many advantages over conventional normal phase LC and reverse phase LC [25]. HILIC is capable of separating a broad range of polar compounds, including amino acids, peptides, carbohydrates, polar drugs, metabolites and biologically important compounds in proteomics, glycomics and clinical analysis [25], [26], [27], [28]. Guo et al. [27] have demonstrated that HILIC is a reliable technique to analyse amino acids in food. The successful application of HILIC in detecting medicine and cellular metabolites was displayed as well [29], [30]. As part of these efforts, various HILIC columns have been tested for separation of amino acids, such as silica, amide, bridged ethyl hybrid (BEH) amide and zwitterion [26], [27], [31]. While successfully applied in the before-mentioned matrices, the applicability of HILIC has yet to be tested for the analysis of amino acids from soils, which form a complex and difficult matrix because of the plethora of binding sites for various compounds including amino acids. In the mineral soil compartment, many interfering compounds can be co-extracted from soils together with the targeted amino acids [32], [33], [34], and the free amino acid concentrations in soil solutions are generally low [35]. Therefore, in contrast to the application in matrices such as food, purification and concentration of soil extracts are most likely necessary prior to the analysis of soil amino acids. Solid phase extraction (SPE) has become a widely-used technique to clean up and concentrate extracted amino acids in complex samples through isolating analytes of interest from a wide variety of matrices, such as biological matrices [21], [36], [37] and sediment [38]. However, only a few studies have reported the performance of the SPE technique in purifying and concentrating soil extracts prior to the measurement of free amino acids. Dell’mour et al. [26] reported that the overall recoveries of 12 free amino acids in soil extracts through SPE procedure ranged between 13% and 70%.
Therefore, the aim of the present study was to develop and test a novel, robust and fast procedure to extract and analyse amino acids from soils through combining HILIC-HPLC–MS/MS and SPE technique. For this purpose, ten soils representing a broad range of compositions (pH, texture class, organic C content, etc.) were augmented with 20 amino acids that are common in soils. These were subsequently used to develop and test the new procedure.
Section snippets
Chemicals
Acetonitrile (ACN, LC/MS grade), methanol (MeOH, ULC/MS grade) and formic acid (FA, ULC/MS grade) were purchased from Biosolve B.V. (Valkenswaard, The Netherlands). Analytical grade ammonium formate (NH4HCO2) and ammonium acetate (NH4OAc) were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Analytical grade hydrochloric acid (37%), ammonium hydroxide (25 wt%) and acetic acid (AcOH) were purchased from Merck KGaA (Darmstadt, Germany). Analytical grade sodium azide (NaN3) was
Optimization of MS/MS condition
The retention time, transitions and optimal MS conditions for all the targeted amino acids and IS are listed in Table 4. Positive ionization mode was selected in MRM detection in line with the study of Guo et al. [27]. For all amino acids except Gly-D5, the protonated molecular ion [M + H]+ was selected as the precursor ion (Table 4). The precursor mass of Gly-D5 was based on the [M-D3 + H3 + H+]+ ion because its acidic deuterium atoms are exchanged with H upon aqueous dissolution [49]. The qualifier
Conclusion
For the first time a reliable method for quantifying 20 common amino acids in 10 different soils spanning a wide range of matrix composition (texture, pH, organic C content etc.) was developed and validated. The method that consists of a combination of extraction and pre-concentration with SPE and subsequent analysis with HILIC-HPLC–MS/MS yielded good separation and accurate determination of the 20 amino acids without derivatization for all soils and all amino acids, with the exception of Arg
Acknowledgements
We wish to thank the technicians of the Earth Surface Science (ESS) group (Leo Hoitinga, Joke Westerveld, Peter Serne, Leen de Lange and Jorien Schoorl) for the laboratory assistance and partial financial support from the China Scholarship Council and the Deutsche Forschergemeinschaft DFG (FOR1806, “The Forgotten Part of Carbon Cycling: Organic Matter Storage and Turnover in Subsoils (SUBSOM)”.
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