**4. Identification of derivatized amino acids and their isotopomer analysis with gas chromatography-mass spectrometry**

Derivatization involves reactions with one or more reagents to change the chemical nature of the analyte to make it more suitable for analysis. As chemical reactions,[41] derivatizations are efficient chemical processes between the analyte and the reagent, such as reactions forming acyl, alkyl or aryl derivatives, silylation reactions, adding to carbon-hetero multiple bonds, formation of cyclic compounds, etc. These reactions result in a replacement of active hydrogens in an analyte in functional groups such as OH, COOH, SH, NH, CONH.

The purpose of derivatization varies depending on the analyte, the matrix of the sample, and the analytical method to be applied.[41] Some derivatizations are used in the sample cleanup or concentration process. Much more frequently, they are done to change the analyte properties for the chromatographic separation, to achieve better thermal stability, better detectability and improve separation in GC analysis. In GC-MS analytical technique, derivatization may help in spectra identification. For HPLC using liquid mobile phase, derivatization is performed mainly to increase detectability and improve the separation.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis 239

moieties),[42] and has been employed in the derivatization of Arg and Gln for analysis by gas liquid chromatography (GLC)[43]. Mawhinney et al.[4] reported an analytic method which employs the tBDMS derivatives of amino acids for their separation and quantification in a single GLC analysis. The hydrochloride salts of the amino acids, dissolved in dimethylformamide, are derivatized in a single step using MTBSTFA. As the tBDMS-amino acid derivatives, the neutral and acidic amino acids are stable for over 24 h and the basic amino acids are stable for 6 h. Mass spectroscopy is probably the most powerful tool used for compound identification purposes. The mass spectrum for each tBDMS-amino acid is relatively simple, being dominated by a unique and unambiguous mass minus 57 [M - 57] fragment ion which for many of the amino acids serves as the base fragment ion. Employing amino acid standards, a linear response curve in the range 1-100 nmol was obtained for each neutral and acidic amino acid using a flame ionization detector. The basic amino acids lysine and arginine demonstrated a linear response curve in the range 2-150 nmol. Histidine (His) displayed a linear response curve in the range of 5-150 nmol. In contrast with the results of my experiments (data not published) employing amino acid standards, a linear response curve in the range of 10-30 nmol can be obtained only for Leu, Ser, Asp, Cys; Met; Thr, and Tyr, but not for His, Arg, Gln and Pro. These last four amino acids produced a non-linear

curve with GC-MS in a Trace 2000 gas chromatograph (Thermo Electron).

**Figure 3. The GC-MS total ion chromatogram (TIC) of amino acid mixture in the ERM of A**M**fungi**. Ala= alanine; Gly = glycine; Val = valine; Leu = leucine; Ile = isoleucine; Pro = proline; Ser = serine; Thr = threonine; Phe = phenylalanine; Asp = aspartate; Glu = glutamate; Orn= ornithine; Asn = asparagine;

In our experiments, identities of amino acids were confirmed by comparison with mass spectra of authentic standards (Fig.3). The mass isomer distribution for each derivatized

Gln= glutamine; Arg= arginine; Tyr = tyrosine.

The trimethylsilyl (TMS) derivatives are obtained in one-step derivatization procedure, whereas almost all other derivatives are formed in two or more reaction steps. The derivatization with the formation of silylated derivatives is applied to replace the active hydrogens in an analyte in groups such as OH, SH, NH, CONH, POH, and SOH. The purpose of silylation is to reduce the polarity of the analyte, increase its stability and improve detectability. Although the TMS derivatives are by far the most commonly used for analytic purposes, TBDMS is used when compounds more resistant to hydrolysis are required. The thermal stability of TBDMS derivatives is better than that of TMS derivatives. The TBDMS derivatives give reproducible results in amino acid analysis. Gehrke[36] pointed out that the best foundation for a successful amino acid analysis by GC is (a) reproducible and quantitative conversion of amino acids to suitable derivatives; and (b) separation and quantitative elution of the derivatives from the chromatographic column. For satisfactory analysis of amino acids by GC, a complete derivatization is essential. In my own experiments, free amino acids samples were derivatized with MTBSTFA containing 1% Nmethyl-N-t-butyldimethylchlorosilane; such derivatized Arg and ornithine (Orn) are shown in Fig.2. [30]

**Figure 2. Molecular structure of ions of N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)-derivatized Arg and Orn**. This figure gives the structure of the ion remaining after a 70 eV impact on the MTBSTFA derivative of Arg and Orn. It demonstrates that derivatized Arg has an *m*/*z* of 442, which is the M-188 (molecular ion minus 188) fragment arising from losing the guanido Ns and a tbutyl group. This fragmentation is different from that of Orn, which only loses a t-butyl group (M-57) in its fully derivatized form.[30]

MTBSTFA has been reported as a very powerful tBDMS silyl donor capable of tertbutyldimethylsilylating active protic functions (hydroxyl, amino, carboxylic and thiol

in Fig.2. [30]

its fully derivatized form.[30]

derivatization may help in spectra identification. For HPLC using liquid mobile phase, derivatization is performed mainly to increase detectability and improve the separation.

The trimethylsilyl (TMS) derivatives are obtained in one-step derivatization procedure, whereas almost all other derivatives are formed in two or more reaction steps. The derivatization with the formation of silylated derivatives is applied to replace the active hydrogens in an analyte in groups such as OH, SH, NH, CONH, POH, and SOH. The purpose of silylation is to reduce the polarity of the analyte, increase its stability and improve detectability. Although the TMS derivatives are by far the most commonly used for analytic purposes, TBDMS is used when compounds more resistant to hydrolysis are required. The thermal stability of TBDMS derivatives is better than that of TMS derivatives. The TBDMS derivatives give reproducible results in amino acid analysis. Gehrke[36] pointed out that the best foundation for a successful amino acid analysis by GC is (a) reproducible and quantitative conversion of amino acids to suitable derivatives; and (b) separation and quantitative elution of the derivatives from the chromatographic column. For satisfactory analysis of amino acids by GC, a complete derivatization is essential. In my own experiments, free amino acids samples were derivatized with MTBSTFA containing 1% Nmethyl-N-t-butyldimethylchlorosilane; such derivatized Arg and ornithine (Orn) are shown

**Figure 2. Molecular structure of ions of N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)-derivatized Arg and Orn**. This figure gives the structure of the ion remaining after a 70 eV impact on the MTBSTFA derivative of Arg and Orn. It demonstrates that derivatized Arg has an *m*/*z* of 442, which is the M-188 (molecular ion minus 188) fragment arising from losing the guanido Ns and a tbutyl group. This fragmentation is different from that of Orn, which only loses a t-butyl group (M-57) in

MTBSTFA has been reported as a very powerful tBDMS silyl donor capable of tertbutyldimethylsilylating active protic functions (hydroxyl, amino, carboxylic and thiol moieties),[42] and has been employed in the derivatization of Arg and Gln for analysis by gas liquid chromatography (GLC)[43]. Mawhinney et al.[4] reported an analytic method which employs the tBDMS derivatives of amino acids for their separation and quantification in a single GLC analysis. The hydrochloride salts of the amino acids, dissolved in dimethylformamide, are derivatized in a single step using MTBSTFA. As the tBDMS-amino acid derivatives, the neutral and acidic amino acids are stable for over 24 h and the basic amino acids are stable for 6 h. Mass spectroscopy is probably the most powerful tool used for compound identification purposes. The mass spectrum for each tBDMS-amino acid is relatively simple, being dominated by a unique and unambiguous mass minus 57 [M - 57] fragment ion which for many of the amino acids serves as the base fragment ion. Employing amino acid standards, a linear response curve in the range 1-100 nmol was obtained for each neutral and acidic amino acid using a flame ionization detector. The basic amino acids lysine and arginine demonstrated a linear response curve in the range 2-150 nmol. Histidine (His) displayed a linear response curve in the range of 5-150 nmol. In contrast with the results of my experiments (data not published) employing amino acid standards, a linear response curve in the range of 10-30 nmol can be obtained only for Leu, Ser, Asp, Cys; Met; Thr, and Tyr, but not for His, Arg, Gln and Pro. These last four amino acids produced a non-linear curve with GC-MS in a Trace 2000 gas chromatograph (Thermo Electron).

**Figure 3. The GC-MS total ion chromatogram (TIC) of amino acid mixture in the ERM of A**M**fungi**. Ala= alanine; Gly = glycine; Val = valine; Leu = leucine; Ile = isoleucine; Pro = proline; Ser = serine; Thr = threonine; Phe = phenylalanine; Asp = aspartate; Glu = glutamate; Orn= ornithine; Asn = asparagine; Gln= glutamine; Arg= arginine; Tyr = tyrosine.

In our experiments, identities of amino acids were confirmed by comparison with mass spectra of authentic standards (Fig.3). The mass isomer distribution for each derivatized amino acid was determined by measuring the M-57 ions which result from the loss of a tbutyl group (i.e., [M - C(CH3)3]) from the molecules of MTBSTFA derivatives, except for Arg whose m-188 ion was used. The Arg ion examined had a *m*/*z* of 442, which corresponds to M-188 (molecular ion minus 188) fragment arising from the loss of one guanido nitrogen together with a tBDMS and DMS group from the tetra-substituted *tert*butyldimethylsilyl(tBDMS)-derivatized Arg (Fig. 2). When using [guanido-2-15N] Arg, we observed an ion at an *m*/*z* of 443 (M-189, molecular ion minus 189). This isotopomer corresponds to the derivatized [guanido-2-15N]Arg because the ion loses one of the guanido nitrogens by fragmentation at 70 eV.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis 241

13CU6 arginine was added to the ERM. After 6 weeks, MS analysis of MTBSTFA-derivatized Arg isotopomer revealed that 34% of the free Arg in the ERM and 33% of the free Arg in the colonized roots showed 13CU6 labeling (M+6, Fig. 4). The mass spectra showed that the free Arg molecules in the colonized roots are either completely unlabeled (natural abundance mass isomer distribution) or labeled in all six carbon positions, thus indicating that Arg is

Chromatographic and mass spectrometry analysis of amino acids, in combination with

up, assimilated, and incorporated into Arg by the ERM. The accumulated Arg as well as polyP is then bidirectionally translocated along the coenocytic fungal hyphae from the ERM to the IRM or from the mycorrhizal compartment tissue to the ERM. Arg is catabolized through catabolic arm of the urea cycle ( utilizing arginase and urease activities) in the IRM, releasing NH3/NH4+ in the arbuscules. The NH3/NH4+ acquired by the plant is either transported into adjacent cells or immediately incorporated into AAs, as shown in Fig.1

However, although Ala, Gly, Val, Leu, Ileu, Pro, Ser, Thr, Phe, Asn, Asp, Glu, Orn, Gln, Arg and tyrosine(Tyr) are detected, GC-MS of samples from AM fungal tissues performed after MTBSTFA-derivatized amino acids cleaned up on a cation exchange column (DOWEX 50 \*4- 200, hydrogen form) does not detect some of S-containing amino acids and basic amino acids lysine (Lys) and His. Some amino acids cannot be quantified with chromatographic MS due to non-linear response. Nevertheless, analysis of PITC-derivatized amino acids with HPLC shows excellent linear relationship between the molar concentrations of amino acids and peak areas in the chromatogram, and thereby can be used for effective quantification. Although many techniques are already in use in this field, we will need some novel methods, yet to be developed, to achieve a simultaneous measurement and identification of

*College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, China* 

GC: gas chromatography; GLC: gas liquid chromatography; MS: mass spectrometry; HPLC: high performance liquid chromatography; MTBSTFA: N-methyl-N-(t-butyldimethylsilyl) trifluoroacetamide; tBDMS: *tert*-butyldimethylsilyl; TMS: trimethylsilyl; PTC: phenylthiocarbamoyl; PITC: phenyl isothiocyanate; SPME: solid-phase microextraction.

Ala: alanine; Gly : glycine; Val: valine; Leu: leucine; Ile: isoleucine; Pro: proline; Ser: serine; Thr: threonine; Phe: phenylalanine;Asp: aspartate; Glu: glutamate;Orn: ornithine; Asn:

, NH4+, amino acids) are taken

transported intact from ERM to IRM.

(modified from Govindarajulul et al.[29]).

all free amino acids in biological tissues.

**Author details** 

**Abbreviations** 

Jin HaiRu and Jiang Xiangyan

isotopic tracing, shows that various forms of N sources (NO3-

**6. Conclusion** 
