Isolation of underivatized amino acids by ion-pair high performance liquid chromatography for precise measurement of nitrogen isotopic composition of amino acids: Development of comprehensive LC×GC/C/IRMS method

Nitrogen isotopic composition of amino acids has been widely applied to biochemical, ecological, archeological, and biogeochemical studies in an attempt to trace nitrogen source and transformation processes. For accurate isotope analysis of individual amino acids, we validated a preparative method involving the isolation of underivatized amino acids by ion-pair chromatographic separation and confirmed the consistency of nitrogen isotope composition. Ion-pair reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS) and gas chromatography/combustion coupled with isotope ratio mass spectrometry (GC/C/IRMS) were conducted for the purpose of separation of underivatized amino acids and nitrogen isotopic analysis, respectively. Firstly, we confirmed the resolution of proteinogenic and non-proteinogenic amino acids by the preparative ion-pair LC separation. Diagnostic product ions determined by mass spectrometry can support the rapid identification of individual amino acids in screening analyses. Secondly, we observed no dependency on nitrogen isotopic composition for the injection amount of underivatized amino acids and even for different chemical formula including neutral, acidic, sulfur-containing, heterocyclic, and aromatic species. The recovery during the LC was 91.7 ± 4.3% (n = 3). The present method and strategy of LC coupled with GC/C/IRMS (i.e., comprehensive LC × GC/C/IRMS) are useful for the high precision determination of the nitrogen isotopic composition of amino acids, in conjunction with an appropriate pre-treatment of cation-exchange chromatographic procedures. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

Focusing on nitrogen isotopic composition, although Tripp et al. reported ion-pair separation of amino acids for isotopic measurements and an isotopically-dispersed validation by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS), resulting the nitrogen isotopic difference (i.e., 15 N, the difference between ␦ 15 N before and ␦ 15 N after LC separation) ranged from −9.0‰ (vs. Air; valine) to +9.6‰ (vs. Air; proline) (mean, −0.1 ± 4.6‰, n = 13; [13]). Subsequently, Broek et al. also investigated an ion-pair LC procedure using the SiELC Primesep A column with elemental analyzer/isotope ratio mass spectrometry (EA/IRMS) for amino acids (nitrogen isotopic difference, 15 1. (a) The concept of comprehensive LC × GC/C/IRMS (liquid chromatography × gas chromatography/combustion/isotope ratio mass spectrometry) for precise measurement of nitrogen isotopic composition of amino acids. (b) Experimental workflow for high-precision CSIA for ␦ 15 N values of amino acids investigated in chemical, biological, and geological samples, with or without (w/o) a matrix effect. The asterisk (*) represents this study. Fractionation and purification of amino acids by cation-exchange chromatography were previously validated using appropriate resins (e.g., AG50W-X8 200-400 mesh [21]; Dowex AG50W-X8 200 mesh (e.g., [39,40])). (c) Precision and accuracy of working standard for GC/C/IRMS by the reference mixtures of 9 amino acids (alanine, glycine, leucine, isoleucine, aspartic acid, methionine, glutamic acid, phenylalanine, and hydroxyproline: each amino acid in 1 sigma range) with known ␦ 15 N values for EA/IRMS (elementary analyzer combined with an isotope ratio mass spectrometry). The summary data is shown in Supplementary Information. Abbreviations: Gly, glycine; Ser, serine; Ala, alanine; Asp, aspartic acid; Pro, proline; Glu, glutamic acid; Val, valine; Leu, leucine. (b) Protein and other amino acids. Small amounts of Asn and Gln were coinjected with Asp and Glu, respectively. Norleu was used as an internal standard for GC/C/IRMS analysis. Abbreviations: Hyp, hydroxyproline; Asn, asparagine; Gln, glutamine; Norleu, norleucine. Asn and Gln will convert to Asp and Glu, respectively, after hydrolysis. (c) Comparison of responses determined by other online detectors for corona CAD and photodiode array detector (DAD) for a UV absorbance of 260 nm. The summary data are also shown in Table 1 and Supplementary Information for other non-protein amino acids.

Table 1
Summary of the ion-pair reversed-phase LC for underivatized amino acids showing elution order, chemical formula, molecular weight, parent ion, and fragment (m/z) by electrospray ionization mass spectrometry. In the right-hand column, 'P' and 'NP' represent protein amino acid and non-protein amino acid, respectively. #1, hydroxyproline is one of the important amino acids in collagen protein. #2, #3, Asparagine (Asn) and glutamine (Gln) will convert to aspartic acid (Asp) and glutamic acid (Glu), respectively, after hydrolysis. #4, The chromatographic co-elution of leucine and isoleucine may occur on this ion-pair LC separation. However, if the eluent and gradient program was modified with same ion-pair reagent, leucine and isoleucine were separated as shown in Supplementary Information. Please see other non-protein type amino acids shown in Fig. 3  chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) [14]. Here, we report the development and validation of a method for underivatized amino acid separation by the ion-pair LC procedure. Then, we focused on the verification of nitrogen isotopic composition by separation, injection volume, the ion pairing interaction, and overall elution procedures using a reversed-phase column. We established a preparative isolation method for the high-precision measurement of the nitrogen isotopic composition of proteinogenic (protein type) and non-proteinogenic (non-protein type) amino acids.

Separation and detection for underivatized amino acids
For the separation of underivatized amino acids, we used an ion-pair liquid chromatograph (Agilent Technologies Inc., 1100 series; Tokyo, Japan) coupled with either an electrospray ionization mass spectrometer (Agilent Technologies Inc., 1100 series; Tokyo, Japan) or a corona charged aerosol detector (Corona CAD; Dionex K.K./Thermo Fisher Scientific Inc.; Kanagawa, Japan) and a Table 2 Recovery of representative amino acids between before ion-pair LC and after LC. The recovery average during the LC was 91.7 ± 4.3% (n = 3) for the injection of 20 nmol by the GC/NPD multiple run analysis. The initial abundance of each amino acid (without ion-pair LC) was defined as 100%, and the yield of the derivatization reaction (N-pivaloyl iso-propyl esters) was also assumed as 100% in both.
The mobile phase consisted of two solvents. Solvent A was 20 mM NFPA in distilled water, while solvent B was acetonitrile.   Table 1 and Supplementary Information for other non-protein amino acids.  Table 1. For corona CAD condition, the nitrogen gas pressure was constantly 35 ± 0.1 psi unit with the corona voltage (<3400 V).
After a preparative collection of underivatized amino acids by the ion-pair LC and subsequent dry-up by nitrogen flow, we conducted the derivatization procedure for N-pivaloyl isopropyl esters of amino acids [15][16][17][18] (cf. the relationship between GC stationary columns and nitrogen isotopic composition of amino acids; [15]). As it is important to keep combustion efficiency of the CuO/NiO system on GC/C/IRMS [19,20], we carefully eliminate the ion-pair reagents (i.e., fluorinated compounds) by liquid/liquid extraction (i.e., water/organic phase separation) in the derivatization process prior to GC/C/IRMS analysis [15]. The recovery of representative amino acids between before ion-pair LC and after LC was determined by gas chromatography (GC) using a 6890N GC instrument connected to the flame ionization detector (FID) and nitrogen phosphorus detector (NPD) (Agilent Technologies Inc., Tokyo, Japan). The separation was performed by VF-35ms capillary column (30 m × 0.52 mm; film thickness, 0.50 m; Agilent Technologies Inc., Tokyo, Japan).

GC/C/IRMS analysis for N-pivaloyl iso-propyl esters of amino acids
The nitrogen isotopic composition of the individual amino acids (derivatized as N-pivaloyl iso-propyl esters) was determined using GC/C/IRMS (Thermo Finnigan Delta Plus XP; Thermo Fisher Scientific Inc., Kanagawa, Japan) combined with an Agilent 6890 N GC system (Agilent Technologies Inc., Tokyo, Japan). For the GC separation, we used an Ultra-2 capillary column (25 m × 0.32 mm i.d., 0.52 m film thickness; stationary phase, 5% phenyl 95% methyl polysiloxane; Agilent Technologies Inc.; Tokyo, Japan) [15,18]. The GC oven temperature was programmed as follows: heating from 40 to 110 • C at a rate of 15 • C min −1 after 3 min at the initial temperature, heating from 110 to 150 • C at a rate of 3 • C min −1 , 150 • to 220 • C at a rate of 6 • C min −1 , and then holding isothermally at 220 • C for 17.3 min. The combustion furnace was performed in a micro-volume ceramic tube with CuO, NiO, and Pt wires at 950 • C. The reduction furnace was performed in a micro-volume ceramic tube with a Cu wire at 550 • C. The nitrogen isotopic composition is expressed as the per mil (‰) deviation from the standard (vs. Air), as conventionally defined by the following equation: ␦ 15 N = [( 15 N/ 14 N) sample /( 15 N/ 14 N) standard -1] × 1000. The standard deviation (1 ) of the analytical precision was estimated to be within ±0.4‰ based on the repeated injection of laboratory working standards [15].  Results are shown for representative alkyl (e.g., C2-C6; Gly, Ala, ␣-ABA, Leu), acidic (e.g., Glu), sulfur containing (e.g., Met), heterocyclic (e.g., Pro), and aromatic (e.g., Phe) amino acids. The gray layer (zero, normalized as 15 N) represents the 2 range (>95% of the mean values) for the precision of the GC/C/IRMS in this verification. The analytical scale of the GC/C/IRMS analysis was approximately 30 ng (ca. 2 nmol N as injected quantity of nitrogen) [15]. The raw data are also shown in Table 3. measurement of the nitrogen isotopic composition of individual amino acids. To assess the reproducibility of the isotope measurement and obtain the amino acid isotopic composition, reference mixtures of 9 amino acids (alanine, glycine, leucine, isoleucine, aspartic acid, methionine, glutamic acid, phenylalanine, and hydroxyproline; Fig. 1c) with known ␦ 15 N values (ranging from −26.1‰ to +45.7‰, Indiana University, USA and SI science Co., Tokyo Japan) were analyzed after every four to six samples runs.

Separation and identification of underivatized amino acids by ion-pair LC
We previously reported the nitrogen isotopic consistency [21] and practical applications using a cation-exchange resin (Bio-Rad Laboratories AG 50W-X8; 200-400 mesh; Tokyo, Japan) to eliminate possible matrix effects and to purify the amino acid fraction [22][23][24]. The workflow can be adapted according to the amount of impurities present in a sample. Representative extracted ion chromatograms (positive ion mode, [M+H] + ; Fig. 2) of underivatized amino acids (adjusted to pH 1) are shown for the LC/ESI-MS and other detectors (corona CAD and DAD using a UV absorbance at 260 nm). The response of the corona CAD is independent of the chemical structure and constant among amino acids (e.g., [10,11]). Within the protein amino acids, only phenylalanine and tyrosine have sufficient UV absorbance, i.e., DAD is not helpful for other amino acid detection. We confirmed that corona CAD detection is Table 3 Comparison of the nitrogen isotopic composition of underivatized amino acids (␦ 15 N ‰ vs. Air) before and after LC injection to validate 15 N (the difference between ␦ 15 N before and ␦ 15 N after LC separation).   Table 3.
useful for detecting and fraction collection of underivatized amino acids for further compound-specific analysis. ESI-MS spectra are useful for the rapid identification, screening, and diagnosis of underivatized amino acids using their retention times and corresponding product ion(s) (Fig. 3 and Supplementary Information). In the ESI-MS analysis of neutral alkyl amino acids (e.g., Val, Norval, Isoval, Leu and Norleu), abundant protonated molecule and their product ions [M+H − 46] which corresponds to neutral formic acid loss ( HCOOH) were observed. Ammonia product for [M+H − 17] was also observed in basic amino acids (e.g., Lys, Asn and Gln). Furthermore, loss of water [M+H − 18] was observed for some acidic amino acids including Ser, Thr, Asp and Glu. Tracing the retention time and specific ion transitions, each underivatized amino acid was identifiable on the ion-pair LC/ESI-MS. A co-injection of threonine ([M+H] + = 120) and phenylalanine ([M+H] + = 166) for [M+H − 46] + (= 120) showed the same ion (m/z 120) at differing retention times. Table 1 summarizes the elution order, retention times, and fragment ions of ESI-MS for protein and non-protein amino acids.

Nitrogen isotopic compositions of amino acids before and after LC separation
The difference of nitrogen isotopic composition of amino acids before and after the ion-pair LC, where 15 N represents the difference between ␦ 15 N before and ␦ 15 N after LC separation are shown in Fig. 4. The injection volume of underivatized amino acids ranged from 1 to 100 nmol on the ion-pair LC. The recovery average during the LC was 91.7 ± 4.3% (n = 3) for the injection of representative amino acids (Table 2). Fig. 4a represents the consistency of nitrogen isotopic compositions for alkyl amino acids including Gly (C 2 ), Ala (C 3 ), ␣-ABA (C 4 ), and Leu (C 6 ). We also confirmed this consistency for acidic (Glu, C 5 ), sulfur-containing (Met, C 5 ), heterocyclic (Pro, C 5 ), and aromatic amino acids (Phe, C 9 ) (Fig. 4b). Consequently, the nitrogen isotopic composition of these underivatized amino acids is independent of injection volume onto the ion-pair LC column. Hare et al. (1991) reported a large isotopic variability (␦ 15 N > 30‰) within a chromatographic peak of glycine during LC separation with a resin (St. John Associates, Adelphi, MD, USA) [25]. Therefore, to precisely determine the nitrogen isotopic composition, baseline resolution of amino acids and careful isolation of the entire peak should be required prior to further GC analysis ( Table 3).
The comparison of nitrogen isotopic compositions of underivatized amino acids between before and after the ion-pair LC separation (>1.25 nmol) indicated good correlation with the mean of difference ( 15 N) within −0.1% (R 2 = 0.997 in 14 amino acids; Fig. 5). Given this close correlation, the nitrogen isotope compositions for the amino acids are independent of the chemical structure of the investigated amino acids. The present ion-pair LC and offline IRMS method can compensate a 15 N assessment of minor amino acids (e.g., Met, Pro) and the resolution of some amino acids (e.g., Asp, almost co-elution with Thr on an Ultra-2 capillary column; [15]) by GC.

Implication and perspectives
This analytical procedure using LC × GC/C/IRMS is applicable to the high-precision analysis of amino acids obtained from microbial, ecological, and biogeochemical materials [26][27][28][29], if high-resolution fingerprinting of amino acids is necessary. The method is also applicable to fossil materials preserved in hard tissues, as commonly examined in archeological and paleo-dietary research [30][31][32][33]. Amino acids contain chiral center for their d-and l-enantiomers with some exceptions (e.g., glycine in protein type and ␣-aminoisobutyric acid in non-protein type). The preparative isolation procedure demonstrated here is thus useful for the accurate evaluation of d-and l-enantiomers when chiral separation by further GC or LC analysis is employed. In this manner, accurate evaluation of d-and l-enantiomers of amino acids originated from pristine abiotic processes [34][35][36][37][38] could be possible for opening up the possibility of high-precision enantiomer-specific isotope analysis (ESIA). The present results contribute to the refinement of nitrogen isotope analysis in determining the biotic or abiotic origin of amino acids.