**3. Determination of amino acid concentrations with high performance liquid chromatography (HPLC)**

Since amino acids are non-volatile compounds and most of them show low UV absorbance, they have been commonly analyzed by liquid chromatography (LC) methods with precolumn or post-column derivatization using UV chromophore or fluorophore reagents. The use of HPLC analysis is extremely common because this technique has no specific analyte volatility or thermal stability restrictions.[38-39] Derivatization can make the analysis more sensitive, gives a linear detection response and avoids specific interference. The common

approach to the preparation of derivatized samples for HPLC and GC analysis is to replace the active hydrogens to form a desired physical property. However, in HPLC, the elimination of all active hydrogens from the analyte is not usually necessary. Some derivatizations requiring only the attachment of a chromophore or fluorophore group to the analyte use one of the functional groups (such as phenyl isothiocyanate, O-phthalaldehyde or 9-fluorenylmethyl chloroformate) which react only at the amino group. Other derivatizations of amino acids involve both NH2 and COOH groups; for example, when isothiocyanates are used to form thiohydantoins. The quantification of derivatized amino acids, such as phenylthiocarbamoyl (PTC) derivatives, is commonly used prior to their analysis by HPLC. Phenyl isothiocyanate (PITC) reacts both with the primary and secondary amino-groups, at room temperature, within 5-20 min; PITC-amino acids are very stable in dried samples, and their elution and detection only requires a binary gradient pump and a UV detector.[39] The disadvantages of the method are its limited sensitivity (because of the lack of fluorogenic derivatives) and the need for removal of excess reagent. Therefore, in our experiments to determine the levels of free amino acids in the ERM and mycorrhizal root tissues, we used a Waters Pico-Tag amino acid analyzer (HPLC), employing the Pico-Tag method. As in the study by Endres & Mercier[40], the amount of each amino acid was measured by high-performance liquid chromatography (HPLC) of the PITC derivatives. The extracted amino acids were dissolved in 0.1 M HCl and vacuum-dried in a Pico-Tag workstation, then ethanol/ water/triethylamine mixture was added and evaporated by vacuum-drying. 20 ml of ethanol/ water/triethylamine/ phenylisothiocyanate (7 : 1 : 1 : 1) was added to derivatize the amino acids at 23°C for 20 min. The samples were then dried under vacuum and re-dissolved in 100 μl of Pico-Tag sample diluent. 20 μl of each sample was loaded onto a reverse-phase C18 column (3.9 mm ID X 150 mm long) using a Waters 510 autosampler. An eluent gradient consisting of 38 ml Pico-Tag Eluent A (0.05 M sodium acetate) and Eluent B (0.1 M sodium acetate/acetonitrile/methanol (46:44:10) was used as mobile phase. The flow rate was 1.0 ml min−1, with the proportion of Eluent B rising from 0– 100%. The elution was monitored at 254 nm with a Waters 486 tunable absorbance detector. The concentrations of the amino acids were calculated by comparing the integrated peak area with those for standard amino acids at known concentrations using Waters MILLENIUM software (Waters Chromatography Division). The threshold for detection of amino acids in standard solutions was 30 pM of each amino acid per assay, corresponding to <10 nmol g-1 of dry weight of tissue.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis 237

**Table 1.** Concentration of free amino acids, established using HPLC, in the root compartment tissue and extraradical mycelium of AM fungus *G. intraradices* after culturing for 1 or 3 weeks in two-

**4. Identification of derivatized amino acids and their isotopomer analysis** 

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

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,

hydrogens in an analyte in functional groups such as OH, COOH, SH, NH, CONH.

compartment Petri dishes, with15NH4Cl labeling in fungal compartment .\*

**with gas chromatography-mass spectrometry** 

\* Mean *±* standard deviation.

As shown in Table1, our HPLC analysis of free amino acid levels reveals that Arg is by far the most abundant fungal amino acid (between 50 and 200mM depending on developmental stage), representing *c*. 90% of the total free amino acids in the ERM. Arg levels are also substantially higher in colonized than in un-colonized roots (54.2 ± 19.3% versus 10.9± 4.8% of free amino acids). Johansen *et al*. [12] have observed, without reporting absolute levels, that Arg is the dominant free amino acid in extraradical mycelium of *Glomus claroideum*. However, they have not measured Arg levels in *G. intraradices* because of the problems with derivatization and decomposition of the silated product.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis 237


\* Mean *±* standard deviation.

236 Chromatography – The Most Versatile Method of Chemical Analysis

to <10 nmol g-1 of dry weight of tissue.

versus 10.9± 4.8% of free amino acids). Johansen *et al*.

problems with derivatization and decomposition of the silated product.

approach to the preparation of derivatized samples for HPLC and GC analysis is to replace the active hydrogens to form a desired physical property. However, in HPLC, the elimination of all active hydrogens from the analyte is not usually necessary. Some derivatizations requiring only the attachment of a chromophore or fluorophore group to the analyte use one of the functional groups (such as phenyl isothiocyanate, O-phthalaldehyde or 9-fluorenylmethyl chloroformate) which react only at the amino group. Other derivatizations of amino acids involve both NH2 and COOH groups; for example, when isothiocyanates are used to form thiohydantoins. The quantification of derivatized amino acids, such as phenylthiocarbamoyl (PTC) derivatives, is commonly used prior to their analysis by HPLC. Phenyl isothiocyanate (PITC) reacts both with the primary and secondary amino-groups, at room temperature, within 5-20 min; PITC-amino acids are very stable in dried samples, and their elution and detection only requires a binary gradient pump and a UV detector.[39] The disadvantages of the method are its limited sensitivity (because of the lack of fluorogenic derivatives) and the need for removal of excess reagent. Therefore, in our experiments to determine the levels of free amino acids in the ERM and mycorrhizal root tissues, we used a Waters Pico-Tag amino acid analyzer (HPLC), employing the Pico-Tag method. As in the study by Endres & Mercier[40], the amount of each amino acid was measured by high-performance liquid chromatography (HPLC) of the PITC derivatives. The extracted amino acids were dissolved in 0.1 M HCl and vacuum-dried in a Pico-Tag workstation, then ethanol/ water/triethylamine mixture was added and evaporated by vacuum-drying. 20 ml of ethanol/ water/triethylamine/ phenylisothiocyanate (7 : 1 : 1 : 1) was added to derivatize the amino acids at 23°C for 20 min. The samples were then dried under vacuum and re-dissolved in 100 μl of Pico-Tag sample diluent. 20 μl of each sample was loaded onto a reverse-phase C18 column (3.9 mm ID X 150 mm long) using a Waters 510 autosampler. An eluent gradient consisting of 38 ml Pico-Tag Eluent A (0.05 M sodium acetate) and Eluent B (0.1 M sodium acetate/acetonitrile/methanol (46:44:10) was used as mobile phase. The flow rate was 1.0 ml min−1, with the proportion of Eluent B rising from 0– 100%. The elution was monitored at 254 nm with a Waters 486 tunable absorbance detector. The concentrations of the amino acids were calculated by comparing the integrated peak area with those for standard amino acids at known concentrations using Waters MILLENIUM software (Waters Chromatography Division). The threshold for detection of amino acids in standard solutions was 30 pM of each amino acid per assay, corresponding

As shown in Table1, our HPLC analysis of free amino acid levels reveals that Arg is by far the most abundant fungal amino acid (between 50 and 200mM depending on developmental stage), representing *c*. 90% of the total free amino acids in the ERM. Arg levels are also substantially higher in colonized than in un-colonized roots (54.2 ± 19.3%

absolute levels, that Arg is the dominant free amino acid in extraradical mycelium of *Glomus claroideum*. However, they have not measured Arg levels in *G. intraradices* because of the

[12] have observed, without reporting

**Table 1.** Concentration of free amino acids, established using HPLC, in the root compartment tissue and extraradical mycelium of AM fungus *G. intraradices* after culturing for 1 or 3 weeks in twocompartment Petri dishes, with15NH4Cl labeling in fungal compartment .\*
