Elsevier

Journal of Chromatography A

Volume 1449, 3 June 2016, Pages 78-88
Journal of Chromatography A

Robust analysis of underivatized free amino acids in soil by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry

https://doi.org/10.1016/j.chroma.2016.04.071Get rights and content

Highlights

  • A method for the reliable analysis of 20 soil free amino acids was developed.

  • The solid phase extraction technique had high and stable overall recoveries.

  • The application of this method was validated by testing a broad range of soils.

Abstract

Amino acids are an important and highly dynamic fraction of organic N in soils and their determination in soil without derivatization is challenging due to the difficulties in separation and detection of trace amounts of these polar analytes. In the present work, we developed an analytical method to quantify 20 free amino acids in aqueous soil extracts without derivatization. The method employed hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) technique combined with a cation exchange solid phase extraction (SPE). Four stable isotope labelled amino acids were used as internal standards to improve the method performance. Good separation of 20 underivatized amino acids was achieved within 12 min. The limit of detection (LODs) and limit of quantification (LOQs) were in the range of 13–384 ng g−1 and 43–1267 ng g−1 (dry soil basis), respectively. The results showed that overall recoveries with high precision were obtained for the extracted free amino acids from ten different soils. The overall recoveries of 18 amino acids were similar for the ten soils used, which differed substantially in organic C content and in other properties as soil texture and pH. For most of the amino acids, the average recoveries from soil extracts were between 74% and 117%, with the exception of Met (31%), Pro (52%) and Arg (68%). Variability was within acceptable limits (relative standard deviations were between 4% and 13%), with the exception of Met (relative standard deviation = 90%) and Arg (relative standard deviation = 53%). Thus the proposed method with high throughout and high analyte specificity shows great promise for consistent analysis of free amino acids extracted from soils and offers new horizons for the analysis of amino acids in terrestrial and aquatic ecosystem.

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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