Highly sensitive quantitative analysis of nicotianamine using LC/ESI-TOF-MS with an internal standard

Article abstract of DOI:10.1271/bbb.60496

A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N^ε-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA.

Full text of DOI:10.1271/bbb.60496

Biosci. Biotechnol. Biochem., 71 (2), 435–441, 2007
Highly Sensitive Quantitative Analysis of Nicotianamine
Using LC/ESI-TOF-MS with an Internal Standard
1
2
1
1
Yasuaki WADA, Isomaro YAMAGUCHI, Michiko TAKAHASHI, Hiromi NAKANISHI,
1
1;3;y
Satoshi MORI, and Naoko K. NISHIZAWA
1
Graduate School of Agricultural and Life Sciences, The University of Tokyo,
-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
1
2
Faculty of Technology, Maebashi Institute of Technology, 460-1 Maebashi-shi, Gunma 371-0816, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science
3
and Technology Corporation (JST)
Received September 12, 2006; Accepted October 17, 2006; Online Publication, February 7, 2007
[doi:10.1271/bbb.60496]
A highly sensitive quantitative method for analyzing
studies on the biosynthetic pathways of MAs, we have
isolated many NAS genes from barley, Arabidopsis, rice,
and maize.
nicotianamine (NA) by liquid chromatography/electro-
spray ionization time-of-flight mass spectrometry (LC/
ESI-TOF-MS) is reported. Fluorenylmethoxycarbony-
lation of nicotianamine reduced its polarity and enabled
its retention in a reversed-phase column. The adoption
1
4–17)
The quantification of endogenous NA in plants is
critical for elucidating the detailed function of NA in
plants. In terms of its antihypertensive effect, quantify-
ing NA in various foods is very important for determin-
ing those that are abundant in NA and that are expected
to have an antihypertensive effect. Nicotianamine is
usually quantified by measuring the UV absorption of
its o-phthalaldehyde derivative that is obtained by post-
labeling after high-performance liquid chromatography
"
of N -nicotyllysine (NL) as an internal standard ensured
reliable quantification by giving a linear calibration
curve drawn between the NA/NL molar ratios of
standard solutions injected and the NA/NL area ratios
in mass chromatograms. The high sensitivity of this
analytical method allowed us to measure the amount
of NA. This analytical method has applications to all
research concerning NA.
1
8–20)
(HPLC),
and its detection limit is ca. 10 pmol (3.0
ng) for identification and 100 pmol (30.3 ng) for quanti-
fication. However, the concentration of NA in plants
examined so far is so low that the measurements are
always hampered by the need for a relatively large plant
sample (e.g., the endogenous NA concentration in the
leaves of wild-type tobacco (Nicotiana tabacum
Key words: 9-fluorenylmethoxycarbonyl (FMOC) deri-
vatization; liquid chromatography/electro-
spray ionization time-of-flight mass spec-
trometry (LC/ESI-TOF-MS); nicotianamine
2
1)
L. cv SR1) is 8:2 ꢀ 0:6 mg/g fresh weight). Conse-
quently, the establishment of a high-sensitivity system to
measure NA has long been desired. Weber et al. have
reported the analysis of endogeneous NA in Arabidopsis
using liquid chromatography/electrospray ionization
Nicotianamine (NA; Fig. 1A) was originally found in
tobacco leaves,1) and has been detected in all higher
2
,3)
plants that have been examined.
Nicotianamine
chelates many kinds of metal cations and is thought to
play an important role in the internal transport of metals
2
2)
time-of-flight mass spectrometry (LC/ESI-TOF-MS),
but their system was for a qualitative and not quanti-
tative analysis of NA.
4
,5)
nutrients including iron, zinc, and copper. In grami-
naceous plants, NA is also an essential intermediate of
mugineic acid family phytosiderophores (MAs), which
are natural Fe chelators secreted by the roots to
In this study, we developed a highly sensitive
quantitative method for NA analysis using LC/ESI-
TOF-MS, and demonstrated the quantification of en-
dogenous NA in rice seed and young tobacco leaves.
The adoption of an internal standard validated this
quatification.
6
–8)
solubilize iron in the soil.
received attention as an antihypertensive substrate.
Nicotianamine is synthesized by the trimerization of S-
Nicotianamine has recently
9
–12)
1
3)
adenosylmethionine by NA synthase (NAS). In the
y
To whom correspondence should be addressed. Fax: +86-3-5841-7514; E-mail: annaoko@mail.ecc.u-tokyo.ac.jp
Abbreviations: FMOC-Cl, 9-fluorenylmethoxycarbonyl chloride; HPLC, high-performance liquid chromatography; LC/ESI-TOF-MS, liquid
chromatography/electrospray ionization time-of-flight mass spectrometry; MAs, mugineic acid family phytosiderophores; NA, nicotianamine; di-
"
FMOC NA, di-9-fluorenylmethoxylcarbonylated NA; NL, N -nicotyllysine; di-FMOC NL, di-9-fluorenylmethylcarbonylated NL

436
Y. WADA et al.
Materials and Methods
35 min, 100% eluent B. The flow rate was kept at 0.2
ml/min.
Synthesis of the internal standard. Nicotic acid (492
mg, 4 mmol) was dissolved in 10 ml of dry C5H5N–
CH3CN mixture (1:5). N-hydroxysuccinimide (460 mg,
4 mmol) dissolved in CH3CN (5 ml) was added to the
0
nicotinic acid solution and thoroughly mixed. N,N -
Derivatization with FMOC-Cl. Each aqueous sub-
strate solution (2.5 ml) was finely mixed with 2.5 ml of
50 mM EDTA 2Na and 5 ml of sodium borate (1 M,
.
pH 8.0). Ten ml of 25 mM 9-fluorenyl methoxycarboxyl
chloride (FMOC-Cl; dissolved in CH3CN) was added
with immediate vortexing. The sample was allowed to
dicyclohexylcarbodiimide (824 mg, 4 mmol) dissolved
in CH CN (5 ml) was added to this mixture, which was
3
ꢃ
kept at ambient temperature overnight and then centri-
0
react at 30 C for 60 min. Ten ml of 50 mM 3-amino-1-
fuged to remove the precipitate of N,N -dicyclohexyl-
propanol was added to the sample with vortexing to
consume excess FMOC-Cl. An aliquot (5 ml) of each
reaction mixture (30 ml) was injected for the LC/ESI-
TOF-MS analysis.
urea. The supernatant was concentrated and purified by
Sephadex LH-20 chromatograpy (1.5 cm i.d. ꢁ 30 cm),
swelled and eluted with 1-BuOH:deionized water (6:
1
00) in 10 ml fractions. The fractions showing a single
spot on TLC (Merck GF254) in EtOAc:CHCl3:AcOH
20:8:1) were combined and concentrated to give crys-
tals of an N-hydroxysuccinimidyl active ester (495 mg,
Reproducibility of the measurement by adopting an
internal standard. A solution (25 ml) of authentic NA
(1 nmol) and NL (1 nmol) was prepared. An aliquot
(2.5 ml) of the solution was FMOC derivatized and
subjected to the LC/ESI-TOF-MS analysis (triplicate).
The peak area of the mass chromatograms for the ½M þ
(
5
0
3% yield). The active ester of nicotic acid (93 mg,
.42 mmol) was dissolved in DMF (1 ml) and added to a
0
mixture of D,L-lysine-HCl (182 mg, 1 mmol) and p-N,N -
dimethylaminopyridine (122 mg, 1 mmol) in deionized
water (5 ml). After an overnight reaction at ambient
temperature, the reaction mixture was concentrated in
vacuo and purified by Sephadex LH-20 chromatography
as already described. The fractions showing a spot at Rf
þ
þ
Hꢂ ions of di-FMOC NA, that for the ½M þ Hꢂ ions of
di-FMOC NL, and their peak area ratio (NA/NL) were
calculated.
Calibration curve with an internal standard. Standard
solutions (25 ml) containing 0.25 nmol, 0.5 nmol, 1.0
nmol, 2 nmol, and 4 nmol of NA and 1 nmol of NL were
prepared. An aliquot (2.5 ml) of each solution was
FMOC derivatized and subjected to the LC/ESI-TOF-
MS analysis (triplicate). The peak area ratios of the mass
0
.05 on TLC in the upper phase of 1-BuOH:AcOH:
deionized water (4:1:5) were collected and concentrated
in vacuo to give a gum (ca. 220 mg). An aliquot (4.5 mg)
of this concentrate was subjected to PEGASIL ODS-
HPLC (10 mm i.d. ꢁ 30 cm, Senshu Scientific Co.,
Tokyo, Japan), eluted with 10% MeOH containing
þ
chromatograms for the ½M þ Hꢂ ions of di-FMOC NA
0.05% AcOH at 2.0 ml/min. A peak at tR 6.5 min was
"
collected and gave ca. 1.4 mg of N -nicotyllysine (NL).
and di-FMOC NL were adopted for the calibration
curve.
HR-MS (LC/ESI-TOF-MS): obs. m=z 252.13746
þ
(
252.13482 calcd. for ½M þ Hꢂ ), m=z 274.11678
Validating the quantification. Solutions (25 ml) of
authentic NA (1 nmol) and NL (1 nmol), endogenous
NA in rice seed and NL (1 nmol), and endogenous NA
supplemented with authentic NA (1 nmol) and NL
(1 nmol) were prepared. An aliquot (2.5 ml) of each
solution was FMOC derivatized and subjected to the
LC/ESI-TOF-MS analysis (triplicate). The peak area
ratios were determined as described in Calibration curve
with an internal standard.
þ
1
(
274.11676 calcd. for ½M þ Naꢂ ). H-NMR [ꢀ-value,
D2O contaminated with C5H5N]: 1.36 (5-CH2, tt, J ¼ 7,
7
Hz), 1.58 (4-CH2, m), 1.73 and 1.83 (1H and 1H, 3-
CH2, m), 2.86 (6-CH2, t, J ¼ 7 Hz), 4.3 (2-CH, q, J ¼
0
0
2
:5, 4 Hz), 7.7 (5 -H, q, J ¼ 6:5, 10 Hz), 8.4 (4 -H, dt,
0
J ¼ 1:7, 8.3 Hz), 8.62 (6 -H, overlapped with C H N),
5
5
0
8.9 (2 -H, br.s). A JMS-T100LC AccuTOF (Jeol, Tokyo,
Japan) was used for the LC/ESI-TOF-MS analysis, and
1
a JNM-500 (Jeol, Tokyo, Japan) for the H-NMR
analysis.
Quantification of NA in a rice grain. One grain of
dry, mature, unpolished rice seed (Oryza sativa L. cv
Tsukinohikari; about 20 mg) was finely homogenized
with a Multi-Beads Shocker (Yasui Kikai, Osaka,
Japan), weighed, and suspended in 400 ml of deionized
water. The sample was then centrifuged at 15000 ꢁ g for
10 min and the supernatant was collected. This extrac-
tion was conducted three times, and all the supernatants
were collected in one tube. After adding 1 nmol of NL
(1 ml of a 1 mM solution) to the supernatant, the sample
was concentrated to approximately 300 ml in Micro Vac
(Tomy Seiko, Tokyo), passed through a 10000 NMWL
Filter Unit (Millipore, MS, USA), further concentrated
Liquid chromatography-electrospray ionization time-
of-flight mass spectrometry. A JSM-T100LC AccuTOF
(
Jeol) was used a Synergi Hydro RP column (4 mm, 80A,
1
50 ꢁ 2:0 mm; Phenomenex, CA, USA) in the positive
ionization mode and in the mass range of m=z 200–800.
The column was equilibrated with 100% eluent A (0.5%
formic acid, 20% water, and 79.5% CH3CN) for 25 min,
and developed in the following conditions: 0–5 min,
1
00% eluent A; 5–15 min, gradient elution with increas-
ing the concentration of eluent B (0.5% formic acid and
9.5% CH CN) from 0 to 100% in eluent A; and 15–
9
3

New Quantitative LC/ESI-TOF-MS Method for Analyzing Nicotianamine
437
to dryness. The dried sample was dissolved in 25 ml of
A. Nicotianamine and its FMOC derivative
deionized water. Three concentrated extracts were
prepared from three independent grains. An aliquot (2.5
ml) of each concentrated extract was FMOC derivatized
and subjected to LC/ESI-TOF-MS analysis. The quan-
tification was carried out once for each grain sample.
COOH
COOH
COOH
N
N
R
NH
R
R = H: Nicotianamine (NA), R = FMOC: di-FMOC NA
Comparison of the NA content of a plant sample
determined by LC/ESI-TOF-MS and by HPLC. Approx-
imately 1 g of dry, mature, unpolished rice seed
ε
B. N-nicotyl-D,L-lysine and its FMOC derivative
O
COOH
(
approximately 50 grains) was finely homogenized with
a Multi-Beads Shocker (Yasui Kikai). Three lots of
0 mg of the homogenate (approximately the same
N
R
NH
R
2
N
weight as one rice grain) were prepared and used for the
LC/ESI-TOF-MS analysis. The extract from each lot
was FMOC derivatized and analyzed once. The remain-
ing homogenate was used for the HPLC analysis as
ε
R = H: N-nicotyl-D,L-lysine (NL), R = FMOC: di-FMOC NL
"
Fig. 1. The Chemical Structures of Nicotianamine (NA), N -Nicotyl-
D,L-lysine (NL), and Their FMOC Derivatives.
1
8–20)
described previously.
One extract was prepared
from all the remaining homogenate, and the measure-
ment was executed three times.
However, the preparation of the stable isotope-labeled
"
One g of fresh young tobacco leaves (Nicotiana
tabacum L. cv SR) was finely homogenized with a
mortar and a pestle. The homogenate was suspended
in 20 ml of deionized water. The sample was then
centrifuged at 15000 ꢁ g for 10 min and the supernatant
was collected. This extraction was conducted three
times, and all the supernatants were collected in one
tube. Three lots of an aliquot (120 ml; corresponding to
NA is too laborious, so we prepared N -nicotyllisine
"
(NL, Fig. 1B). N -nicotyllysine was expected to behave
similarly to NA on HPLC when they were FMOC
derivatized, because both of their FMOC derivatives still
carry a basic amino group and carboxyl group(s).
Furthermore, the difference between their molecular
weights (MW) is in a reasonable range (MW 251 for NL
and MW 303 for NA). Followed by FMOC derivatiza-
tion, NL was detected at tR 17.9 min as a di-FMOC
2
mg of the homogenate) of the supernatant were, after
þ
adding 1 nmol of NL, was concentrated to approximate-
ly 300 ml in Micro Vac (Tomy Seiko), passed through
derivative showing the ion of m=z 696 (½M þ Hꢂ ).
þ
þ
In this study, we selected the ½M þ Hꢂ ions of di-
1
0000 NMWL Filter Unit (Millipore), further concen-
trated to dryness. Each dried sample was dissolved in
5 ml of deionized water (1/10 the concentration in case
FMOC NA (m=z 748) and the ½M þ Hꢂ ions of di-
FMOC NL (m=z 696) for the quantification of NA. The
mass chromatograms of the ions at m=z 748 and 696
were shown in Fig. 2A. A sufficient supply of FMOC-Cl
for derivatization was confirmed by detecting the FMOC
derivative of 3-amino-1-propanol, a quencher, as the ion
2
of the rice seed). The three concentrated extracts were
subjected to the LC/ESI-TOF-MS analysis, and the
remaining extract was used for the HPLC analysis as just
described.
þ
of m=z 298 (½M þ Hꢂ ) at tR 15.9 min.
Results
Reproducibility of the measurement by adopting an
internal standard
General aspects
We first examined the effect of the internal standard
on the reliability of the measurement. A solution of
authentic NA and NL were FMOC derivatized and
subjected to the LC/ESI-TOF-MS analysis. The peak
areas of mass chromatograms for the ½M þ Hꢂþ ions of
di-FMOC NA varied somewhat in each measurement as
often occurs in LC/ESI-MS. A similar problem hap-
pened to the peak areas of mass chromatograms for the
Since NA (Fig. 1A) is not retained in a reversed-
phase column, NA was FMOC derivatized to form di-
FMOC derivative (di-FMOC NA), which was less polar
and could be retained in the column. The di-FMOC NA
was eluted at tR 16.9 min under the HPLC conditions
described in the Materials and Methods, and showed the
ions of m=z 748 (½M þ Hꢂ ) and 770 (½M þ Naꢂ ) at the
ratio of 100:5. The LC/ESI-TOF-MS analysis devel-
oped in this study enabled us to quantify as little as
pmol (0.3 ng) of NA, while HPLC required 100 pmol
30.0 ng) to clearly detect and quantify NA as its o-
phthalaldehyde derivative even though it could detect
0 pmol (3.0 ng) of the NA derivative.
It is preferable to use an internal standard whose
þ
þ
þ
½M þ Hꢂ ions of di-FMOC NL (Table 1). However, the
þ
ratios of peak areas of the ½M þ Hꢂ ions of NA and NL
1
(
(NA/NL ratios) were similar in each measurement and
reproducible (Table 1). We therefore adopted the NA/
NL ratios to evaluate the quantification of NA.
1
8–20)
1
Calibration curve with an internal standard
We next investigated the relationship between the
molar ratios (NA/NL) and the peak area ratios (NA/
chemical property is similar to that of NA, and NA
labeled with a stable isotope could be an ideal choice.

438
Y. WADA et al.
2
peak area ratio; R ¼ 0:99). We therefore adopted the
A
regression line for quantification of NA.
Authentic NA and NL solution
m/z 748
m/z 696
Intensity
Intensity
Validating the quantification
Generally, EDTA is not used for FMOC derivatiza-
tion.23,24) However, when a concentrated extract of rice
3
2
00000
3
2
1
0000
00000
0000
0000
0
100000
0
seed was subjected to the derivatizing reaction without
EDTA, the derivatization efficiency of NA in the extract
was much lower than that of authentic NA in deionized
water. Since NL in the concentrated extract was
derivatized almost quantitatively, it was thought that
this inefficient derivatization occured specifically to
NA. We circumvent the problem by adding sufficient
amounts of EDTA to FMOC deivatizing solution (NA/
NL ratio without EDTA, 0.06 (n ¼ 3); with EDTA, 0.19
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
tR (min)
tR (min)
B
Extract of rice seed
m/z 748
m/z 696
Intensity
Intensity
3
2
1
00000
3
2
1
00000
00000
00000
00000
0
(
n ¼ 3)).
00000
0
In order to validate the quantification, solutions (25
ml) of authentic NA (1 nmol) and NL (1 nmol), of
endogenous NA in rice seed and NL (1 nmol), and of
endogenous NA supplemented with authentic NA
(1 nmol) and NL (1 nmol) were prepared. The solution
of endogenous NA in rice seed was adjusted to be a
similar concentration as that of the authentic NA
solution based on the result of a preliminary experiment.
An aliquot was FMOC derivatized and subjected to the
LC/ESI-TOF-MS analysis. The increase in peak area
ratio derived from the addition of authentic NA
approximated the peak area ratio calculated when
equimolar authentic NA was derivatized and analyzed
(Fig. 4). This indicated that the derivatizing efficiency of
NA in the extracts of rice seed was similar to that in
deionized water. We concluded that the regression line
depicted in Fig. 3 could be applied to quantify NA in a
plant extract.
0
2
4
6
8
10
12
14
16
18
0
2
4
6
8
10
12
14
16
18
tR (min)
tR (min)
Fig. 2. Mass Chromatograms of Ions at m=z 748 and 696.
A, The peak of the ½M þ Hꢂþ ion of di-FMOC NA (m=z 748) was
detected at 16.9 min, and that of di-FMOC NL (m=z 696) was
detected at 17.9 min. The other peaks apparent in both the mass
chromatograms were irrelevant to di-FMOC NA and di-FMOC NL.
B, The peak of the ½M þ Hꢂþ ion of di-FMOC NA (m=z 748) was
detected at 16.9 min and di-FMOC NL (m=z 696) at 17.9 min.
NL). Five standard solutions (molar ratios of 0.25, 0.5,
1
, 2, and 4) were prepared. An aliquot (5 ml) of each
solution was FMOC derivatized and subjected to the
LC/ESI-TOF-MS analysis. Linearity was apparent
between the molar ratios and the peak area ratios
(
0
Fig. 3), and the formula for the regression line was y ¼
:0977x þ 0:0125 (x is the molar ratio and y is the
Table 1. Drift of Peak Areas and Peak Area Ratios
Di-FMOC NA peak area
(
Di-FMOC NL peak area
(intensityꢄsec)
NA/NL
ratio
Solution
intensityꢄsec)
1
2
3
444254
306893
378588
2978483
1991742
2571624
0.149
0.154
0.147
0
0
.4
.2
0
0
1
2
3
4
5
NA/NL molar ratio
Fig. 3. Calibration Curve with an Internal Standard.
The formula representing the regression line was y ¼ 0:977x þ 0:0125; (x is the molar ratio, y is the peak area ratio; R ¼ 0:99).
2

New Quantitative LC/ESI-TOF-MS Method for Analyzing Nicotianamine
.4
439
0
0
0
0
.3
.2
.1
0
Authentic NA
Endogeneous NA
Endogeneous NA
+
Authentic NA
Fig. 4. Validating the Quantification.
Authentic NA and NL, endogenous NA in rice grains and NL, and endogenous NA supplemented with authentic NA and NL were prepared,
and aliquots were subjected to the LC/ESI-TOF-MS analysis.
Quantification of NA in a rice grain
leaves calculated in the LC/ESI-TOF-MS analysis was
slightly higher than that in the HPLC analysis.
Using the regression line in Fig. 3, we measured NA
in small plant using our analytical method. Concentrated
extracts from one rice grain were FMOC derivatized and
subjected to the LC/ESI-TOF-MS analysis. The mass
chromatograms of ions at m=z 748 and 696 were shown
in Fig. 2B. The NA content in one rice grain was
calculated to be 21:2 ꢀ 0:9 mg/g dry weight.
Discussion
We have described a new analytical method for
quantifying NA using LC/ESI-TOF-MS. Nicotianamine
was stably retained on a reversed phase column when
derivatized with FMOC-Cl. Much higher sensitivity was
achieved for detecting NA in this analytical method than
Comparison of the NA contents of a plant sample
determined by LC/ESI-TOF-MS and by HPLC
Using the same plant sample, the NA content
calculated by the LC/ESI-TOF-MS analysis was com-
2
1)
in the HPLC analysis. This analytical method was
capable of quantifying NA (Fig. 3 and 4), and we could
determine sensitively the content of NA in a minute
plant sample (e.g., one grain of unpolished rice seed;
21:2 ꢀ 0:9 mg/g dry weight). The values of NA content
calculated in the LC/ESI-TOF-MS analysis and the
HPLC analysis were almost the same.
1
8–20)
pared with the result from the HPLC analysis.
Since
large amounts of a plant sample would be needed for the
HPLC analysis because of its lower sensitivity, approx-
imately 1 g of dry, mature, unpolished rice seed
(
approximately 50 grains) was homogenized finely. This
As mentioned in the Results, for resolving the
inefficient derivatization relevant specifically to NA,
we added EDTA to the FMOC deivatizing solution. Our
hypothesis was as follows: NA is a strong chelator of
many kinds of transition metal cations in the neutral and
alkaline pH range, at which FMOC derivatization is
generally performed (pH 8.0 in this study). Consequent-
ly, NA would chelate metal cations derived from the
plant sample at pH 8.0 in the reaction mixture of the
concentrated extract, and that the formation of NA-metal
complex should make itself inert to the derivatization.
The restoration of reaction efficiency by adding EDTA
suggested that NA chelated the metal cations in the
extracts and the formation of the NA-metal complex
hampered derivatizing NA. Although one study quanti-
homogenate was used for both the LC/ESI-TOF-MS
analysis and the HPLC analysis. The NA content in rice
seed was calculated to be 20:7 ꢀ 2:1 mg/g dry weight in
the LC/ESI-TOF-MS analysis and 15:3 ꢀ 0:3 mg/g dry
weight in the HPLC analysis. The value of the calculated
NA content was slightly higher in the LC/ESI-TOF-MS
analysis than in the HPLC analysis.
Using another plant sample, we also executed a
comparison of the NA contents determined by the LC/
ESI-TOF-MS analysis and by the HPLC analysis. One g
of fresh young tobacco leaves was homogenized finely.
This homogenate was used for both the LC/ESI-TOF-
MS analysis and the HPLC analysis. The LC/ESI-TOF-
MS analysis gave the mass chromatograms of ions at
m=z 748 and 696 similar to those of the rice grain
extracts (data not shown). The NA content in tobacco
leaves was calculated to be 48:4 ꢀ 4:8 mg/g fresh weight
in the LC/ESI-TOF-MS analysis and 41:9 ꢀ 0:4 mg/g
fresh weight in the HPLC analysis. As in the case of the
rice seed, the value of the NA content in the tobacco
2
2)
fied NA using FMOC derivatization
measured other natural metal chelator MAs (derivatives
and another
2
5)
of NA) through derivatization with FMOC-Cl, neither
study mentioned ways to improve the reaction efficien-
cy. Therefore, the precise quantification of NA or MAs
might not have been accomplished in those studies.

440
Y. WADA et al.
The NA content of each plant sample obtained by the
Biological Chemistry, The University of Tokyo, for his
technical advices in the LS/ESI-TOF-MS analysis and
measuring HR-MS.
LC/ESI-TOF-MS analysis differed slightly from that of
the same sample by the HPLC analysis, the former
method giving a slightly higher value. The adoption of
the internal standard in the LC/ESI-TOF-MS analysis
should reasonably compensate for the loss during the
purification process, while such compensation did not
work in the HPLC analysis without an internal standard.
This could be one of the reasons why the LC/ESI-TOF-
MS analysis gave a higher value than the HPLC analysis
for the NA content of the same sample.
References
1
)
Noma, M., Noguchi, M., and Tamaki, E., A new amino
acid, nicotianamine, from tobacco leaves. Tetrahedron
Lett., 22, 2017–2020 (1971).
2
)
Noma, M., and Noguchi, M., Occurrence of nicotian-
amine in higher plants. Phytochemistry, 15, 1701–1702
We should refer to the analysis of NA in tobacco
leaves, for which we had to pay careful attention to the
amount of FMOC-Cl used for drivatization. In the initial
analysis of the tobacco leaves (20 mg), the amount of
FMOC-Cl, which was sufficient for 20 mg of the rice
seed, was insufficient, because the FMOC derivative of 3-
amino-1-propanol was not detected and the derivatization
efficiency of NA in the extract was much lower than those
of authentic NA in deionized water. This insufficiency of
FMOC-Cl would have been due to large amounts of
consumers like other amines and amino acid, in tobacco
leaves. When the extract was diluted to 10 times the
volume and the diluted extract was used for the LC/ESI-
TOF-MS analysis, the FMOC derivative of 3-amino-1-
propanol was detected, and the derivatization efficiency
of NA was restored. We concluded that a sufficient
supply of FMOC-Cl was essential for the derivatization
and smaller aliquot of the sample should be applied at the
onset of analyzing a novel sample. Since this LC/ESI-
TOF-MS analytical method allowed us to measure the
content of NA in a plant sample very abundant in
contaminats such as tobacco leaves, we concluded that
this method could be applied to various plant samples.
For scientists studying the metal nutrition of gramina-
ceous plants, the quantification of MAs is of major
interest in addition to the quantification of NA. In this
study, FMOC-derivatized deoxymugineic acid was
detected in an extract of rice seed (data not shown).
This suggested that our analytical method for NA might
be applicable for the highly sensitive quantification of
MAs. We plan to investigate whether this method can be
used to quantify several kinds of MAs in detail.
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Acknowledgment
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