Determination of ferric iron chelators by high-performance liquid chromatography using luminol chemiluminescence detection

Journal of Chromatography B, 1014 (2016) 75–82

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Determination of ferric iron chelators by high-performance liquid chromatography using luminol chemiluminescence detection Tomoko Ariga, Yuki Imura, Michio Suzuki, Etsuro Yoshimura ∗ Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan

a r t i c l e

i n f o

Article history: Received 11 November 2015 Received in revised form 27 January 2016 Accepted 30 January 2016 Available online 5 February 2016 Keywords: Chemiluminescence HPLC Luminol Mugineic acids Nicotianamine Organic acids Phytosiderophores

a b s t r a c t Iron is an essential element for higher plants, and its acquisition and transportation is one of the greatest limiting factors for plant growth because of its low solubility in normal soil pHs. Higher plants biosynthesize ferric iron [Fe(III)] chelator (FIC), which solubilizes the iron and transports it to the rhizosphere. A high-performance liquid chromatography (HPLC) post-column method has been developed for the analysis of FICs using the luminol/H2 O2 system for chemiluminescence (CL) detection. A size-exclusion column was the most suited in terms of column efficiency and CL detection efficiency. Mixing of the luminol with H2 O2 in a post-column reaction was feasible, and a two-pump system was used to separately deliver the luminol and H2 O2 solutions. The luminol and H2 O2 concentrations were optimized using Fe(III)–EDTA and Fe(III)–citrate (Cit) solutions as analytes. A strong CL intensity was obtained for Fe(III)-Cit when EDTA was added to the luminol solution, probably because of an exchange of Cit with EDTA after separation on the HPLC column; CL efficiency was much higher for Fe(III)–EDTA than for Fe(III)–Cit with the luminol/H2 O2 system. The present method can detect minute levels of Fe(III)–FICs; the detection limits of Fe(III)–EDTA, Fe(III)–Cit and Fe(III)–nicotianamine were 0.77, 2.3 and 1.1 pmol, respectively. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Iron (Fe) is an essential element for all higher plants. While it is the fourth-most abundant element in the earth’s crust [1], Fe deficiency, a severe nutrient problem for higher plants, may occur when Fe ions exist exclusively as Fe(III). This oxidation state is only sparingly soluble in normal soil pHs. For this reason, higher plants have developed Fe acquisition systems. Two such systems have been identified, which are known as Strategy I and Strategy II. Non-graminaceous plants adopt Strategy I, where plant roots secrete phenolic compounds such as protocatechuic acid and chlorogenic acid to chelate and solubilize Fe(III) [3–5]. Graminaceous plants, on the other hand, adopt Strategy II, where the plants secret a series of Fe(III)-chelating compounds known as mugineic acids (MAs) from their roots to cope with the problem [2]. Thus, ferric ion chelators (FICs) play a pivotal role in Fe acquisition in higher plants. Furthermore, after absorption from the roots, Fe is translocated to the shoots via the xylem and also to developing organs via the phloem. Xylem sap is only slightly acidic (pH 5.0–6.0) while phloem sap is alkaline (pH 8.0),

∗ Corresponding author. E-mail address: [email protected] (E. Yoshimura). http://dx.doi.org/10.1016/j.jchromb.2016.01.048 1570-0232/© 2016 Elsevier B.V. All rights reserved.

in which the solubility of Fe(III) ion is very low. Thus, Fe(III) is believed to be transferred as FIC complexes [6–11]. Nicotianamine (NA;2(S), 3 (S), 3 (S)-N-[N-(3-amino-3-carboxypropyl)-3-amino3-carboxypropyl]-azetidine-2-carboxylic acid; Fig. 1A) is an FIC and also a precursor for the biosynthesis of MAs in graminaceous plants [12]. In contrast to MAs, NA is merely involved in the internal translocation of Fe in higher plants, especially in phloem sap [13–15]. High-performance liquid chromatography (HPLC) has been frequently applied to the analysis of FICs. For example, lowmolecular-weight organic acids were directly detected by monitoring the ultraviolet (UV) absorbance near 214 nm attributed to their carboxylic acid groups [16–18], and ethylenediamine-N,N,N ,N tetraacetic acid (EDTA; Fig. 1B) was detected at 258 nm [19]. The detection limit of citric acid (Cit) and EDTA reported in these papers was 210 and 1.71 pmol, respectively [18,19]. The organic acids were also labeled either in a pre-column or a post-column fashion, where the primary or secondary amine groups were target sites. Derivatization with phenacyl [20,21], naphthacyl [22] or p-nitrobenzyl [23,24] prior to the UV detection gave detection limits of 44, 67, 15 and 39 pmol for oxalic acid, oxamic acid, malonic acid and adipic acid, respectively [17,23]. MAs were mainly analyzed using post-column derivatization with o-phthaldialdehyde [25,26] or pre-column derivatization with 9-fluorenylmethyl chloroformate

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COOH

COOH

N

COOH

N

NH2

(A) Nicotianamine (NA)

HOOC

COOH

HOOC

HOOC

N

N

COOH N

COOH

HOOC

COOH

COOH

(B) EDTA

(C) DTPA COOH

HOOC HOOC

N

N

N

O

O

N

COOH COOH

(D) GEDTA

N

COOH

N

COOH COOH

(E) CyDTA Fig. 1. Structures of (A) nicotianamine and (B)–(E) synthetic ferric iron chelators (FICs) added to the post-column solution A (PCS-A).

[27] prior to fluorescence detection, and using pre-column derivatization with phenylisothiocyanate [28] prior to UV detection. The detection limits of MAs reported in these papers were 370 (mugineic acid), 70 (2 -deoxymugineic acid), 100 (3-epihydroxymugineic acid) and 500 pmol (nicotianamine) [27,26]. A novel detection system for FIC was presented, with which the dequenching of Fe(III)–Calcein Blue (CB; 4-methylumbelliferone8-methyleneiminodiacetic acid) was exploited. This system has the feature that the signal intensity correlates with the binding strength of the FICs to Fe(III) [29]. Using this methodology, the detection limit of Cit was 72 pmol. Chemiluminescence (CL) has been widely exploited as an HPLC detector and in flow-injection analysis because of its high sensitivity, wide linear range and relatively simple instrumentation. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is a reagent frequently employed in CL spectrometry because it provides an intense CL emission near 425 nm when it is oxidized in the presence of oxidants such as dioxygen, hydrogen peroxide, hexacyanoferrate(III) and permanganate [30]. In the reaction, a co-oxidant is required for the oxidation to occur. As a Fe(III)–FIC complex may function as a co-oxidant, a novel detection system can be devised in which Fe(III)–FIC complexes alone are specifically detected. This paper reports analytical instrumentation for the detection of FICs based on an HPLC post-column derivatization with CL detection using a luminol/H2 O2 system. 2. Material and methods 2.1. Reagents Luminol, hydrogen peroxide, 1 M ammonium acetate solution (HPLC grade), acetic acid (HPLC grade), 25% ammonia solution, potassium chloride and Cit monohydrate were purchased from WAKO Pure Chemical Industries (Tokyo, Japan). Ammonium bicarbonate (LC–MS grade) was purchased from Sigma–Aldrich

(St. Louis, MO, USA). Ethylenediamine-N,N,N ,N -tetraacetic acid iron(III) [Fe(III)–EDTA] sodium salt trihydrate, ethylenediamineN,N,N ,N -tetraacetic acid (EDTA) disodium salt dihydrate, diethylenetriamine-N,N,N ,N ,N -pentaacetic acid (DTPA; Fig. 1C), acid o,o -bis(2-aminoethyl)ethyleneglycol-N,N,N ,N -tetraacetic (GEDTA; Fig. 1D) and trans-1,2-diaminocyclohexane-N,N,N’,N’tetraacetic acid (CyDTA; Fig. 1E) monohydrate were obtained from Dojindo Laboratories (Kumamoto, Japan). Ferric chloride hexahydrate and boric acid were purchased from Kokusan Chemical (Tokyo, Japan). Synthetic NA was kindly provided by Professor Emeritus Satoshi Mori at The University of Tokyo. All other reagents were of analytical grade. Milli-Q Ultrafree water (Merck Millipore, Darmstadt, Germany) was used to dissolve and dilute the reagents. 2.2. HPLC analytical system Fig. 2 shows the HPLC analysis system used in this study. The system consisted of pumps 1 (PU-980, Jasco, Tokyo, Japan), 2 and 3 (PU-2080, Jasco), a dynamic mixer (MX-2080-32, Jasco), a three-line degasser (DG-2080-53, Jasco), an automatic sampler (AS-950-10, Jasco), two column ovens (column oven 1 (CO1), CO965(Jasco); and column oven 2 (CO2), SSC-2120(Senshu Scientific Co., Ltd., Tokyo, Japan)) and a CL detector (CL-2027, Jasco). An eluent with a pH of 5.5 was prepared by adding dilute acetic acid to a 10 mM ammonium acetate solution. An eluent with a pH of 8.0 was made by adding a diluted ammonia solution to a 10 mM ammonium bicarbonate solution. The pH values corresponded to the pHs of xylem sap (pH 5.5) and phloem sap (pH 8.0). The Fe(III)–FIC complex was separated by isocratic elution of a degassed eluent that was prepared daily and delivered by pump 1 at a flow rate of 0.5 mL/min. Twenty microliters of a sample were loaded on a size-exclusion column (8.0 mm × 300 mm, Superdex Peptide 10/300 GL, GE Healthcare UK Ltd., Buckinghamshire, UK) at a temperature of 39 ◦ C in oven CO1. After separation on the column, the Fe(III)–FIC complex was reacted in the reaction coil in oven CO2

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Sample

of the complex that gave a signal whose peak height was three times higher than the noise level. CO2

CO1

Pump 1 M

V

RC

CL

3. Results and discussion Waste

C AS

PC solution

0.5 mL/min

0.5 mL/min

77

DM

Pump 2 PCS-A

Pump 3

0.5 mL/min

PCS-B

Fig. 2. Schematic diagram of the high-performance liquid chromatography (HPLC) detection system. M: mobile phase (10 mM ammonium acetate buffer at pH 5.5); PCS-A: luminol solution containing luminol and pH buffer; post-column solution B (PCS-B): H2 O2 solution; PC solution: 1:1 mixture of PCS-A and PCS-B; pump 1: PU-980 (Jasco); pumps 2 and 3: PU-980 (Jasco); AS: automatic sampler (AS950-10, Jasco); DM: dynamic mixer (MX-2080-32, Jasco); C: size-exclusion column (Superdex peptide 10/300 GL, 10 mm × 300 mm); V: three-way valve; RC: reaction coil [PEEK tubing (3 m long × 0.25 mm i.d.)]; CO1: oven containing the column (CO965, Jasco); CO2: oven containing the reaction coil (SSC-2120, Senshu Scientific); CL: chemiluminescence detector (CL-2027, Jasco).

with a post-column (PC) solution that consisted of a 1:1 mixture of post-column solution A (PCS-A) and post-column solution B (PCSB), which were delivered by pumps 2 and 3, respectively, at a flow rate of 0.5 mL/min and then mixed online by the dynamic mixer. PCS-A contained luminol with the pH being buffered by 100 mM H3 BO4 /NaOH, while PCS-B contained H2 O2 . The 30-mm-diameter reaction coil was made of PEEK tubing (3 m × 0.25 mm i.d.) and its temperature was kept constant in oven CO2. The CL signals were monitored by the CL detector CL-2027. 2.3. Sample preparation Stock solutions of 1 mM Fe(III)–EDTA, Cit and NA were prepared by dissolving each reagent in the eluent at pH 5.5 or pH 8.0. A stock solution of 1 mM FeCl3 was prepared by dissolving iron(III) chloride hexahydrate in 1 mM HCl. The Fe(III)–Cit and Fe(III)–NA complexes were prepared by adding 1 mM stock solution of each FIC to an equal volume of 1 mM FeCl3 stock solution, and diluting the mixtures with the HPLC eluents to the appropriate concentrations. 2.4. Optimization of a PC solution composition and other reaction conditions A PC solution composition and other reaction conditions were optimized using 10 ␮M Fe(III)–EDTA and 100 ␮M Fe(III)–Cit as analytes with an HPLC eluent of pH 5.5. The analytes (20 ␮L) were analyzed. Because the CL noise level of the chromatogram changed significantly with the reaction conditions used, the optimization was based on the ratio of the peak height to the noise level (S/N ratio) of the chromatogram. The noise level was represented by one standard deviation of the signal intensities at retention times from 3.0 to 4.0 min, during which time no peak appeared. The signals were sampled every 0.5 s. 2.5. Determination of the detection limit of each FIC The detection limit of each FIC was determined with eluents at pH 5.5 and pH 8.0. The Fe(III)–EDTA, Fe(III)–Cit or Fe(III)–NA standard (20 ␮L) was injected into the column and the peak heights were determined. The detection limits were defined as the amount

3.1. Optimization of the analytical system 3.1.1. Selection of the column Selection of the column is fundamental to an HPLC analytical system. Optimal selection of the eluent is important for the best separation power and because the CL detection efficiency is sensitive to the composition of the eluent. A resin ion exclusion column has a high separation efficiency for organic acids. However, a strongly acidic eluent is incompatible with CL detection using the luminol/H2 O2 system because the CL reactions only occur in the pH range of 9–11 [31]. Although a resin anion exchange column (TSKgel DEAE-5PW, Tosoh Corporation, Tokyo, Japan) using gradient elution with increasing ionic strength could separate organic acids, the increased ionic strength resulted in a baseline drift of a chromatogram using CL detection and an increase in the noise level when 10 ␮M Fe(III)–EDTA was examined (Fig. S1A). A column for hydrophilic interaction liquid chromatography (HILIC) may be a candidate for FIC analysis [32–34]. With the ZIC-HILIC column (Millipore Corporation), however, an eluent with a high (50–95%) organic solvent content was required, which resulted in decreased peak heights when 10 ␮M Fe(III)–EDTA was examined (Fig. S1B). Consequently, to avoid problems with CL detection, the Superdex Peptide 10/300 GL size-exclusion column was selected for this study (Fig. S1C). This column is designed for gel filtration separation of peptides and other small biomolecules having molecular weights between 100 and 7000 in aqueous solution at pH 1–14. 3.1.2. Post-column solutions PCS-A and PCS-B were prepared and delivered separately by their respective pumps and mixed by an HPLC dynamic mixer prior to reaction with the eluate. This is because a considerable decrease in the S/N ratio was observed when PCS-A and PCS-B were mixed in advance: 10 ␮M Fe(III)–EDTA gave a CL signal that was 30% lower than the initial value after 1 h of mixing (Fig. S2). Optimization of the H2 O2 concentration in PCS-B was undertaken at a fixed luminol concentration of 100 ␮M in 100 mM H3 BO4 /NaOH buffer at pH 10.5 in PCS-A. Fig. 3A shows that when 10 ␮M Fe(III)–EDTA was examined, the S/N ratio maximized at a H2 O2 concentration of 10 mM. In the case of 100 ␮M Fe(III)–Cit, the S/N ratio also reached a maximum at an H2 O2 concentration of 10 mM (Fig. 3B). Although the maximum peak heights for Fe(III)–EDTA and Fe(III)–Cit occurred at different H2 O2 concentrations (10 and 50 mM, respectively), a steeply elevating noise level as a function of H2 O2 concentration up to 50 mM established the optimum H2 O2 concentration to be 10 mM (Fig. S3). Next, the concentration of luminol in PCS-A was optimized at a fixed H2 O2 concentration of 10 mM. When 10 ␮M Fe(III)–EDTA and 100 ␮M Fe(III)–Cit were examined, 100 ␮M luminol yielded the maximum S/N ratio for both Fe(III) complexes (Fig. 4A and B). The peak heights for Fe(III)–EDTA and Fe(III)–Cit maximized at luminol concentrations of 100 and 250 ␮M, respectively (Fig. S4A and B). An increasing noise level with increasing luminol concentration (Fig. S4C and D), however, led to the optimum luminol concentration of 100 ␮M for both complexes. It appeared from the chromatograms (Fig. 5) that Fe(III)–EDTA had a higher sensitivity than Fe(III)–Cit: the peak height of Fe(III)–Cit was only about 0.8-times higher than that of Fe(III)–EDTA despite the concentration of Fe(III)–Cit being 200 times higher than that of Fe(III)–EDTA (20 ␮M vs.100 nM). This

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6000

(A) 10 μM Fe(III)–EDTA

(B) 100 μM Fe(III)–Cit 200

S/N

4000

100

2000

0

0

20

40

60

80

100

0

0

20

40

60

80

100

H2O2 (mM)

H2O2 (mM)

Fig. 3. The effect of H2 O2 concentration in PCS-B on the ratio of the peak height to the noise level (S/N ratio). Each S/N ratio was obtained from chromatograms of 20 ␮L spikes of (A) 10 ␮M ferric iron [Fe(III)]–ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA) and (B) 100 ␮M Fe(III)–citrate (Cit). The HPLC chemiluminescence (CL) detector was located after the post-column reaction with the 1:1 mixture of 100 ␮M luminol solution and PCS-B. Lines are only guidelines. Error bars indicate standard deviation from triplicate determinations.

result suggested that the CL intensity of the luminol/H2 O2 system depended on the structure of the Fe(III) complex, and a much higher sensitivity could be attained for Fe(III)–FIC when the FIC was replaced by another relevant ligand that formed an Fe(III) complex showing a high CL efficiency with the luminol/H2 O2 system after separation on an HPLC column. Several types of synthetic chelators (i.e., EDTA, DTPA, GEDTA and CyDTA) were examined to ascertain if they enhanced the CL intensity with the luminol/H2 O2 system. For this purpose, a 20-␮L spike of 10 ␮M Fe(III)–Cit was analyzed using PCS-A mixed with 5 ␮M of the synthetic chelators. EDTA and CyDTA enhanced the peak intensity more than DTPA and GEDTA (Fig. S5A), but EDTA and CyDTA also increased the noise level, with CyDTA demonstrating a higher level than EDTA (Fig. S5B). Fig. 6A shows that EDTA provided the greatest enhancement of the S/N ratio, followed by CyDTA, whereas DTPA and GEDTA had little effect. Inclusion of EDTA in PCS–A enhanced the peak heights of a 20␮L spike of 10 ␮M Fe(III)–Cit in a hyperbolic manner with respect to EDTA concentration (Fig. S6A), indicating substitution of Cit by EDTA. The noise level also increased in a manner similar to the peak height (Fig. S6B). Consequently, the S/N ratio increased at an EDTA concentration up to 5 ␮M and then gradually decreased (Fig. 6B). These results indicated that among the synthetic chelators tested,

EDTA showed the greatest enhancement effect on the S/N ratio at the optimum concentration of 5 ␮M. The optimum pH of PCS-A was determined using 100 mM H3 BO4 /NaOH solutions buffered at pH 9.5–11.5 and 12 mM NaOH/KCl solution buffered at pH 12.0. HPLC analysis was undertaken using these PCS-A solutions spiked with 20 ␮L of 10 ␮M Fe(III)–Cit. Fig. 7A shows that the maximum S/N ratio occurred at pH 10.5. Additionally, Fig. 7B shows the effect of the column temperature on the S/N ratio; the S/N ratio was maximized at 50 ◦ C. Fig. S7 shows the effects of the pH of the PCS-A solution and the column temperature on peak height and noise level. The optimized conditions for the FIC analysis are summarized in Table 1. Fig. 8 shows the chromatograms of Fe(III)–EDTA and Fe(III)–Cit obtained using the optimized conditions. To assess the effect of EDTA addition to PCS-A, the sensitivity was defined here as the peak height divided by the concentration of Fe(III)–EDTA. Figs. 5 A and 8 A reveal that the inclusion of EDTA in PCS-A more than doubled the sensitivity for Fe(III)–EDTA (36 vs. 96 mV/␮M). A much greater enhancement of sensitivity by addition of EDTA was apparent for the Fe(III)–Cit analysis, in which the sensitivity increased two orders of magnitude (0.082 vs. 20 mV/␮M) (Fig. 7B and D). This enhancement was attributed to the replacement of Cit in Fe(III)–Cit by EDTA to form Fe(III)–EDTA, which had a higher CL efficiency with

200

(A) 10 μM Fe(III)–EDTA

5000

150

4000 S/N

(B) 100 μM Fe(III)–Cit

3000

100

2000 50

1000 0

0

100

200

300

Luminol (μM)

400

500

0

0

100

200

300

400

500

Luminol (μM)

Fig. 4. The effect of luminol concentration in PCS-A on the S/N ratio. Each S/N ratio was obtained from chromatograms of 20 ␮L spikes of (A) 10 ␮M Fe(III)–EDTA and (B) 100 ␮M Fe(III)–Cit. The HPLC CL detector was located after the post-column reaction with the 1:1 mixture of PCS-A and the 10 mM H2 O2 solution. Lines are only guidelines. Error bars indicate standard deviation from triplicate determinations.

T. Ariga et al. / J. Chromatogr. B 1014 (2016) 75–82

CL intensity (mV)

10

(A) 100 nM Fe(III)–EDTA

7.5 5 2.5 0

10

10 CL intensity (mV)

79

20

30

40

50

40

50

(B) 20 μM Fe(III)–Cit

7.5 5 2.5 0

10

20

30 Retention time (min)

Fig. 5. Chromatograms of a 20 ␮L spike of (A) 100 nM Fe(III)–EDTA and (B) 20 ␮M Fe(III)–Cit. The chromatograms were obtained in the absence of ETDA under the optimal conditions shown in Table 1.

800

(A)

S/N

600

400

200

0

control

800

EDTA

DTPA

GEDTA

CyDTA

(B)

S/N

600

400

200

0

0

10

20

30

40

50

EDTA (μM) Fig. 6. (A) The effect of synthetic Fe(III) chelators in PCS-A on the S/N ratio. The concentration of the chelator was 5 ␮M. (B) Effect of the concentration of EDTA in PCS-A on the S/N ratio. Each S/N ratio was obtained from the chromatograms of 20 ␮L spikes of 10 ␮M Fe(III)–Cit, which were monitored by the HPLC CL detector. Lines are only guidelines. Error bars indicate standard deviation from triplicate determinations.

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T. Ariga et al. / J. Chromatogr. B 1014 (2016) 75–82

600

600

(A)

400

200

200

S/N

400

(B)

0

9.5

10

10.5

11

11.5

12

0

40

45

50

55

Temperature (

pH

60

65

)

Fig. 7. The effects of (A) the pH of PCS-A and (B) the reaction temperature on the S/N ratio. Each S/N ratio was obtained from the chromatograms of 20 ␮L spikes of 10 ␮M Fe(III)–Cit, which were monitored by the HPLC CL detector after the post-column reaction with the 1:1 mixture of PCS-A containing 5 ␮M EDTA and PCS-B at each reaction temperature. The temperature of the oven that contained the reaction coil was adjusted for each reaction temperature. Error bars indicate standard deviation from triplicate determinations. Table 1 Optimized conditions for the post-column solutions for Fe(III)–FIC analysis. Parameter

Condition

PCS-A Concentration of luminol Concentration of EDTA pH

100 ␮M 5 ␮M 10.5

PCS-B Concentration of H2 O2

10 mM

PCS-A, post-column solution A; EDTA, ethylenediamine-N,N,N’,N’-tetraacetic acid; PCS-B, post-column solution B.

CL intensity (mV)

20 15 10

3.2. Determination of FICs

5 0

10

500 CL intensity (mV)

the luminol/H2 O2 system than Fe(III)–Cit. However, the replacement may have been incomplete because the chromatogram for Fe(III)–Cit displayed a lower sensitivity than that for Fe(III)–EDTA when EDTA was added to PCS-A (20 vs.96 mV/␮M) (Figs. 5 A and 8 A). Dissociation of Fe(III)–Cit on the column during chromatography may have contributed to the reduced sensitivity, because Cit possesses a lower binding affinity than EDTA for Fe(III) [35].

(A) 100 nM Fe(III)–EDTA

20

30

40

50

40

50

(B) 20 μM Fe(III)–Cit

400 300 200 100 0

10

20

30

Retention time (min) Fig. 8. Chromatograms of 20 ␮L spikes of (A) 100 nM Fe(III)–EDTA and (B) 20 ␮M Fe(III)–Cit. The chromatograms were obtained under the optimal conditions listed in Table 1.

3.2.1. Detection limit of FICs Table 2 shows the detection limits of Fe(III)–FICs that were determined from the concentration of analytes that gave a signal whose intensity was three times the S/N ratio. The detection limit of Fe(III)–Cit was three times higher than that of Fe(III)–EDTA at pH 5.5, probably because of the lower sensitivity of Fe(III)–Cit. The detection limit of Fe(III)–EDTA was dependent on the pH of the eluent: it was 0.77 pmol at pH 5.5 but 0.25 pmol at pH 8.0. Because the affinity of Fe(III) to EDTA increases with the pH of the solution [36], a decreased eluent pH would enhance dissociation of Fe(III)–EDTA during chromatography, leaving Fe(III) ions. The detection limits obtained by this study were considerably lower than those reported elsewhere (Table 2). Previous work established detection limits of 1.71 and 210 pmol for EDTA and Cit based on UV absorptions at 258 and 214 nm, respectively. These values are significantly higher than those obtained in this study. For NA analysis, the present method provided a detection limit of 1.1 pmol, which was substantially lower than the 500 pmol reported for fluorescence detection following post-column derivatization with o-phthaldialdehyde [26]. 4. Conclusion In this paper, an improved analytical method has been proposed for the determination of Fe(III)–FICs with CL detection using the

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81

Table 2 Detection limits of typical Fe(III)–FICs. Substance

pH of eluent

Detection limit (pmol)

Dynamic range (␮M)

R2

Fe(III)–EDTA

5.5 8.0

0.77 0.25

1–15 1–10

0.99 0.99

Fe(III)–Cit

5.5 8.0

2.3 28

1–20 20–150

0.99 0.99

Fe(III)–NA

5.5 8.0

N.D. 1.1

N.D. 1–10

N.D. 0.99

N.D., not determined; Fe(III), ferric iron; EDTA, ethylenediamine-N,N,N’,N’-tetraacetic acid; Cit, citrate; NA, nicotianamine. The detection limit of each FIC was calculated from calibration plots using the peak height in the concentration ranges of 0–10 ␮M for Fe(III)–EDTA and 0–20 ␮M at pH 5.5 or 0–150 ␮M at pH 8.0 for Fe(III)–Cit with a S/N ratio of 3. The analytical conditions were the same as listed in Table 1. R is the correlation coefficient between the peak height and the concentration of Fe(III)–FIC.

luminol/H2 O2 CL reaction. The method can selectively detect an FIC according to its Fe(III) binding ability, regardless of its structural characteristics. The detection limits of Fe(III)–EDTA and Fe(III)–Cit, which are typical FICs, and Fe(III)–NA, which is one of the bestknown FICs derived from plants, were quite low. It can therefore be concluded that the present method has greatly improved the detection limit of Fe(III)–FICs compared with previous methods. Additionally, the method requires no complicated pretreatments or special reagents. By these merits, the method is expected to be widely applicable to detect a range of trace Fe(III)–FICs. Acknowledgment We thank Dr. Satoshi Mori for supplying the synthetic NA. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016. 01.048. References [1] P.A. Cox, The Elements on Earth, New York, Oxford University Press Inc, 1995, pp. 29–34. [2] M. Yoshino, K. Murakami, Interaction of iron with polyphenolic compounds: application to antioxidant characterization, Anal. Biochem. 257 (1998) 40–44. [3] Y. Ishimaru, Y. Kakei, H. Shimo, K. Bashir, Y. Sato, Y. Sato, N. Uozumi, H. Nakanishi, N.K. Nishizawa, A rice phenolic efflux transporter is essential for solubilizing precipitated apoplasmic iron in the plant stele, J. Biol. Chem. 286 (2011) 24649–24655. [4] K. Bashir, Y. Ishimaru, H. Shimo, Y. Kakei, T. Senoura, R. Takahashi, Y. Sato, Y. Sato, N. Uozumi, H. Nakanishi, N.K. Nishizawa, Rice phenolics efflux transporter 2 (PEZ2) plays an important role in solubilizing apoplasmic iron, Soil Sci. Plant Nutr. 57 (2011) 803–812. [5] L.L. Barton, J. Abadía, Iron Nutrition in Plants and Rhizospheric Microorganisms, Springer, Dordrecht, 2006, pp. 289–309. [6] Y. Ando, S. Nagata, S. Yanagisawa, T. Yoneyama, Copper in xylem and phloem saps from rice (Oryza sativa): the effect of moderate copper concentrations in the growth medium on the accumulation of five essential metals and a speciation analysis of copper-containing compounds, Funct. Plant Biol. 40 (2013) 89–100. [7] R. Nishiyama, M. Kato, S. Nagata, S. Yanagisawa, T. Yoneyama, Identification of Zn–nicotianamine and Fe–2 -deoxymugineic acid in the phloem sap from rice plants (Oryza sativa L.), Plant Cell Physiol. 53 (2012) 381–390. [8] R. Rellán-Álvarez, J. Giner-Martínez-Sierra, J. Orduna, I. Orera, J.A. Rodríguez-Cstrillón, J.I. García-Alonso, J. Abadía, Identification of a tri-iron(iii): tri-citrate complex in the xylem sap of iron-deficient tomato resupplied with iron: new insights into plant iron long-distance transport, Plant Cell Physiol. 51 (2010) 91–102. [9] A.F. López-Millán, F. Morales, A. Andaluz, A. Abadía, Abadía, Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport, Plant Physiol. 124 (2000) 873––884. [10] L.O. Tiffin, Iron translocation II. Citrate/iron ratios in plant stem exudates, Plant Physiol. 41 (1966) 515–518. [11] M. Nikolic, V. Römheld, Mechanism of Fe uptake by the leaf symplast: is Fe inactivation in leaf a cause of Fe deficiency chlorosis? Plant Soil 215 (1999) 229–237.

[12] N.K. Shojima, S. Nishizawa, S. Fushiya, T. Nozoe, Biosynthesis of phytosiderophores. In vitro biosynthesis of 2 -deoxymugineic acid from l-methionine and nicotianamine, Plant Physiol. 93 (1990) 1497–1503. [13] Pich, I. Scholz, Translocation of copper and other micronutrients in tomato plants (Lycopersicon esculentum Mill.): nicotianamine-stimulated copper transport in the xylem, J. Exp. Bot. 47 (1996) 41–47. [14] U.W. Stephan, I. Schmidke, A. Pich, Phloem translocation of Fe, Cu Mn, and Zn in Ricinus seedlings in relation to the concentration of nicotianamine, an endogenous chelator of divalent metal ions, in different seedling parts, Plant Soil. 165 (1994) 181–188. [15] U.W. Stephan, I. Schmidke, V.W. Stephan, G. Scholz, The nicotianamine molecule is made-to-measure for complexation of metal micronutrients in plants, Biometals 9 (1996) 84–90. [16] R.M. Marcé, M. Calull, R.M. Manchobas, F. Borrull, F.X. Rius, An optimized direct method for determination of carboxylic acids in beverages by HPLC, Chromatographia 29 (1990) 54–58. [17] L. Yang, L. Liu, B.A. Olsen, M.A. Nussbaum, The determination of oxalic acid oxamic acid, and oxamide in a drug substance by ion-exclusion chromatography, J. Pharm. Biomed. Anal. 22 (2000) 487–493. [18] V. Nour, I. Trandafir, M.E. Ionica, HPLC organic acid analysis in different citrus juices under reversed phase conditions, Not. Bot. Horti Agrobot. 38 (2010) 44–48. [19] S. Loyaux-Lawniczak, J. Douch, P. Behra, Optimisation of the analytical detection of EDTA by HPLC in natural waters, Fresen. J. Anal. Chem. 364 (1999) 727–731. [20] E. Mentasti, M.C. Gennaro, C. Sarzanini, C. Baiocchi, M. Savigliano, Derivatization, identification and separation of carboxylic acids in wines and beverages by high-performance liquid chromatography, J. Chromatogr. A 322 (1985) 177–189. [21] F. Caccamo, G. Carfagnini, A.D. Corcia, R. Samperi, Improved high-performance liquid chromatographic assay for determining organic acids in wines, J. Chromatogr. A 362 (1986) 47–53. [22] M.J. Cooper, M.W. Anders, Determination of long chain fatty acids as 2-naphthacyl esters by high pressure liquid chromatography and mass spectrometry, Anal. Chem. 46 (1974) 1849–1852. [23] E. Grushka, H.D. Durst, E.J. Kikta Jr., Liquid chromatographic nanogram quantities of carboxylic acids, J. Chromatogr. A 112 (1975) 673–678. [24] R. Badoud, G. Pratz, Improved high-performance liquid chromatographic analysis of some carboxylic acids in food and beverages as their p-nitrobenzyl esters, J. Chromatogr. A 360 (1986) 119–136. [25] S. Mori, N. Nishizawa, S. Kawai, Y. Sato, S. Takagi, Dynamic state of mugineic acid and analogous phytosiderophores in Fe-deficient barley, J. Plant Nutr. 10 (1987) 1003–1011. [26] C. Neumann, Improved HPLC method for determination of phytosiderophores in root washings and tissue extracts, J. Plant Nutr. 22 (1999) 1389–1402. [27] L.I. Wheal, W.A. Heller, Reversed-phase liquid chromatographic determination of phytometallophores from strategy II Fe-uptake species by 9-fluorenylmethyl chloroformate fluorescence, J. Chromatogr. A 942 (2002) 177–183. [28] J.A. Howe, Y.H. Choi, R.H. Loeppert, L.C. Wei, S.A. Senseman, A.S.R. Juo, Column chromatography and verification of phytosiderophores by phenylisothiocyanate derivatization and UV detection, J. Chromatogr. A 841 (1999) 155–164. [29] T. Ariga, K. Ito, Y. Imura, E. Yoshimura, High-performance liquid chromatography method for ferric iron chelators using a post-column reaction with Calcein Blue, J. Chromatogr. B 985 (2015) 48–53. [30] Y. Rakicio˘glu, J.M. Schulman, S.G. Schulman, Application of ˜ W.R.G. chemiluminescence in organic analysis, in: A.M. García-CAmpana, Baeyens (Eds.), Chemiluminescence in Analytical Chemistry, Marcel Dekker, New York, 2001 (Chapter 5). [31] J.L. Burguera, A. Townshend, Determination of manganese(II) by a chemiluminescence reaction, Talanta 28 (1981) 731–735. [32] G. Weber, N. von Wirén, H. Hayen, Hydrophilic interaction chromatography of small metal species in plants using sulfobetaine and phosphorylcholine-type zwitterionic stationary phases, J. Sep. Sci. 31 (2008) 1615–1622. [33] Y. Xuan, E.B. Scheuermann, A.R. Meda, H. Hayen, N. von Wirén, G. Weber, Separation and identification of phytosiderophores and their metal

82

T. Ariga et al. / J. Chromatogr. B 1014 (2016) 75–82

complexes in plants by zwitterionic hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry, J. Chromatogr. A 1136 (2006) 73–81. [34] M. Tsednee, Y.W. Mak, Y.R. Chen, K.C. Yeh, A sensitive LC-ESI-Q-TOF-MS method reveals novel phytosiderophores and phytosiderophore-iron complexes in barley, New Phytol. 195 (2012) 951–961.

[35] A.E. Martell, R.M. Smith, Critical Stability Constants, vols. 1,3, Plenum Press, New York, 1977. [36] N. von Wirén, S. Klair, S. Bansal, J.F. Briat, H. Khodr, T. Shioiri, R.A. Leigh, R.C. Hider, Nicotianamine chelates both FeIII and FeII . Implications for metal yransport in plants, Plant Physiol. 119 (1999) 1107–1114.