Occurrence of pipecolic acid and pipecolic acid betaine (homostachydrine) in Citrus genus plants

Article abstract of DOI:10.1021/jf204286r

The presence of pipecolic acid and pipecolic acid betaine, also known as homostachydrine, is herein reported for the first time in Citrus genus plants. Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and bergamot (Citrus bergamia Risso et Poit). As homostachydrine was not commercially available, as a comparative source, extracts of alfalfa leaves (Medicago sativa L.) were used, in which homostachydrine is present at high concentration. Then, the results where confirmed by comparison with an authentic standard synthesized and purified starting from pipecolic acid. The synthesized standard was characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When subjected to MS/MS fragmentation in positive ion mode, homostachydrine, unlike its lower homologue proline betaine (also known as stachydrine), showed a pattern of numerous ionic fragments that allowed unambiguous identification of the compound. For the quantitation in the plant sources, high sensitivity and specificity were achieved by monitoring the transition (158→72), which is absent in the fragmentation patterns of other major osmolytes commonly used as markers for studies of abiotic stress. As for the metabolic origin of homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the hypothesis that it could act as a homostachydrine precursor through direct methylation.

Full text of DOI:10.1021/jf204286r

Article
pubs.acs.org/JAFC
Occurrence of Pipecolic Acid and Pipecolic Acid Betaine
(Homostachydrine) in Citrus Genus Plants
Luigi Servillo,*,† Alfonso Giovane,† Maria Luisa Balestrieri,† Giovanna Ferrari,‡ Domenico Cautela,§
and Domenico Castaldo#,⊥
†Dipartimento di Biochimica e Biofisica, II Universita
̀
degli Studi di Napoli, via L. De Crecchio 7, 80138, Naples, Italy
‡Dipartimento di Ingegneria Industriale e ProdAl scarl, Universita
Italy
̀
degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA),
§Stazione Sperimentale per le Industrie delle Essenze e dei Derivati dagli Agrumi (SSEA), Azienda Speciale C.C.I.A.A., Reggio
Calabria, via Tommaso Campanella 12, 89125 Reggio Calabria, Italy
#Ministero dello Sviluppo Economico (MiSE), via Molise 2, Roma, Italy
ABSTRACT: The presence of pipecolic acid and pipecolic acid betaine, also known as homostachydrine, is herein reported for
the first time in Citrus genus plants. Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and bergamot
(Citrus bergamia Risso et Poit). As homostachydrine was not commercially available, as a comparative source, extracts of alfalfa
leaves (Medicago sativa L.) were used, in which homostachydrine is present at high concentration. Then, the results where
confirmed by comparison with an authentic standard synthesized and purified starting from pipecolic acid. The synthesized
standard was characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When subjected to MS/MS
fragmentation in positive ion mode, homostachydrine, unlike its lower homologue proline betaine (also known as stachydrine),
showed a pattern of numerous ionic fragments that allowed unambiguous identification of the compound. For the quantitation in
the plant sources, high sensitivity and specificity were achieved by monitoring the transition (158→72), which is absent in the
fragmentation patterns of other major osmolytes commonly used as markers for studies of abiotic stress. As for the metabolic
origin of homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the hypothesis that it could act as a
homostachydrine precursor through direct methylation.
KEYWORDS: pipecolic acid betaine, homostachydrine, pipecolic acid, citrus fruit, betaine biosynthesis, food analysis,
citrus fruit composition
4-hydroxy-L-prolinebetaine (betonicine), and N,N-dimethyl-L-
proline (stachydrine).8−10 Also present at much lower
concentrations are other osmolytes such as N-methylnicotinic
acid (trigonelline) and choline. Despite the presence of
moderate levels of γ-aminobutyric acid, γ-aminobutyric acid
betaine was never detected.11−13
Pipecolic acid and its betaine can be considered as the higher
homologues of proline and proline betaine and, for this reason,
they are also called homoproline and homostachydrine,
respectively. Their presence has been reported at moderate
levels in some vegetal species.14−18 However, the presence of
pipecolic acid betaine has never been reported so far in woody
plants, but it has been detected at noticeable concentrations in
the Medicago and Achillea genera,19,20 in which it was found
that pipecolic acid betaine accumulates, under stress conditions,
together with proline betaine, which is the most represented
betaine in the Citrus genus.8,10,11,21 Moreover, it was also
reported that some halophilic plants and sand dune plants15
accumulate both pipecolic acid and proline, which is the
supposed precursor of proline betaine in the Citrus genus. On
INTRODUCTION
■
The cytoplasmic accumulation of betaines is a common
biochemical and physiological plant response to stress
phenomena, which are induced in large part by adverse
environmental conditions, such as sudden changes in temper-
ature, high salinity, dryness of the soil, and drought.1,2 These
substances, biochemically inert in the cell, are synthesized from
some specific amino acids such as serine, alanine, methionine,
the nonprotein amino acid γ-aminobutyric acid, and some
cyclic amino acids such as proline and piperidine-2-carboxylic
acid (pipecolic acid). The biosynthesis of betaines in the
cytoplasm under abiotic stress conditions is mainly due to the
action of methyltransferases, which utilize S-adenosylmethio-
nine as methyl group donor.3−5 The betaines resulting from the
amino acid methylation are permanently charged compounds,
highly soluble in water. They are also commonly defined as
compatible osmolytes because they do not negatively interfere
with the cellular metabolism even at molar concentrations. On
the contrary, they exert a protective action on the proteins and
nucleic acid structures, contributing to maintain osmotic
homeostasis during variations of soil osmotic potential (water
stress) or specific ionic effects (salt stress).6,7
Received: October 20, 2011
Revised: December 14, 2011
Accepted: December 14, 2011
Published: December 29, 2011
In the Citrus genus, osmoregulation in stress conditions is
mainly accomplished through accumulation of proline and its
methylated derivatives such as N-methylproline (hygric acid),
© 2011 American Chemical Society
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and the edible part the fruit. The recovered seeds were washed with
water (Milli-Q grade), drained, and finally dried on filter paper.
Subsequently, 10 g was homogenized in a blender with 90 mL of 0.2%
formic acid in Milli-Q grade water. The homogenate was then kept
under stirring for about 3 h and finally centrifuged at 18000g for 30
min. The supernatant was stored in 20 mL vials at −20 °C.
the basis of these studies, taking into account what was
hypothesized (Figure 1) for proline betaine formation in this
Citrus Leaf Extracts. The citrus leaves were washed with distilled
water and dried with filter paper. Then 25 g of product, finely
chopped, was homogenized in a blender with 100 mL of 0.2% formic
acid in Milli-Q grade water and then kept under stirring for 3 h. The
homogenate was finally centrifuged at 18000g for 30 min, and the
supernatant was stored in 20 mL vials at −20 °C.
Citrus Juices. The juices of lemon, orange, and bergamot were
obtained manually with a squeezer starting from 3 kg of each type of
fruit. The juice yield was about 40% in each case. The pulp was
removed by centrifugation at 18000g for 30 min. The supernatant was
stored in 50 mL vials at −20 °C until used.
Alfalfa (Medicago sativa L.) Extracts. The seeds of Medicago sativa
L. (Garisenda variety, N. reg. 1416 Reg ref: EMG 41) were provided
by the Agricultural Consortium of Reggio Calabria and produced by
the Juffray Drilland Co. (France). The seeds were germinated in
styrofoam flats containing a mixture of peat moss and sand (1:2). After
10 days, the seedlings were transferred on a vermiculite−sand (2.1, v/
v) bed and received an adequate amount of fertilizers. Leaves were
harvested for analyses after 4 weeks. The leaves (200 g) were washed
with distilled water, dried with filter paper, homogenized (1:1 w/v)
with 0.2% formic acid in a mixer, and then kept under stirring for 3 h.
The homogenate was centrifuged at 18000g for 30 min. The
supernatant was recovered and stored in 20 mL vials at −20 °C.
Preparations of Standards. The standard stock solutions of L-
Proline, N-methyl-L-proline, N,N-dimethyl-L-proline, 4-hydroxy-L-pro-
line betaine, γ-aminobutyric acid betaine, choline, N-methyl nicotinic
acid, and pipecolic acid betaine were prepared at a concentration of
2000 ng/mL. Additional calibration levels (400, 200, 100, 50, and 25
ng/mL) were prepared by serial dilution with water containing 0.1%
formic acid. The calibration curves were built using these standard
solutions. The linear regression analysis was carried out by plotting the
peak areas of the monitored fragment ions versus the concentrations of
the analyte standard solutions. The linearity of the instrumental
response was demonstrated by a correlation coefficient (r2) of >0.99
for all analytes.
Figure 1. Chemical structures of proline, proline betaine, pipecolic
acid, and pipecolic acid betaine.
plant genus,22−24 we sought to investigate the presence of
pipecolic acid betaine with a sensitive mass spectrometry
technique, which we previously employed to quantitate proline
derivatives in citrus fruits.10,11
MATERIALS AND METHODS
■
Reagents. L-Proline, N-methyl-L-proline, 4-hydroxy-L-proline, γ-
aminobutyric acid betaine, choline, N-methylnicotinic acid (trigonel-
line), and methyl iodide were from Sigma-Aldrich (Milan, Italy). N,N-
Dimethyl-L-proline (stachydrine) and 4-hydroxy-L-proline betaine
(betonicine) were purchased from Extrasynthese (Genay, France).
The 0.1% solution of formic acid in water used for the LC-ESI-MS
analyses was from Sigma-Aldrich. The standard mixture of L-amino
acids, containing Ala, Arg, Asp, Glu, His, Iso, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Tyr, Val, and Cys at 2.5 mM concentration in 0.01 M HCl,
was from Pierce. Milli-Q water was used for all of the preparations of
solutions and standards. AccQ FLuor reagent was from Waters (Milan,
Italy).
Sample Preparations for HPLC ESI-MS/MS Analyses. The
determination of pipecolic acid betaine and other betaines in the
samples was performed by HPLC ESI-MS/MS according to the
method of Servillo et al.10,11 by subjecting the centrifuged samples to a
passage on a (5 × 1 cm) column filled with Bio-Rad AG 50WX8-(H+)
resin. In brief, the column, after loading 1 mL of extract, was washed
with 5 volumes of Milli-Q water and then one step eluted with 10 mL
of 12% ammonia solution, followed by 5 mL of water. The pooled
eluates were dried in a rotavapor and reconstituted with 1 mL of 0.1%
formic acid in water.
HPLC ESI-MS/MS and FIA ESI-MS/MS Analyses of Pipecolic
Acid Betaine. The optimization of the instrumental parameters for
pipecolic acid betaine analyses was performed by continuous infusion
of 5 μM standard solution in 0.1% formic acid. The mass cutoff and
the fragmentation amplitude were optimized to obtain the most
efficient MS/MS transitions from the positively charged precursor ion
[M + H]+ to the fragment ions. Successively, the substance was
analyzed by HPLC ESI-MS/MS, as described for the dosage of proline
derivatives.10 Briefly, the chromatography, isocratically conducted with
0.1% formic acid in water, was performed with a Supelco Discovery-C8
column, 100 × 3.0 mm, particle size = 5 μm, at flow rate of 100 μL/
min. Aliquots of 20 μL of standard solutions or samples were injected.
An Agilent 1100 series liquid chromatograph equipped with an online
degasser and an automatic injector was employed. The ESI-MS/MS
analyses were performed, both for FIA and for HPLC, with an Agilent
LC-MSD SL quadrupole ion trap, in positive ion mode, utilizing
nitrogen as the nebulizing and drying gas. The instrumental conditions
were as follows: nebulizer pressure, 30 psi; drying temperature, 350
°C; drying gas, 7 L/min. The ion charge control (ICC) was applied
Synthesis and Purification of Pipecolic Acid Betaine. For the
conversion of pipecolic acid into its betaine, we used, with some
modifications, the procedure proposed by Chen and Benoiton,25,26
which is based on a heterogeneous phase reaction employing methyl
iodide as methylating agent in the presence of KHCO3. In short, about
200 mg of pipecolic acid was dissolved in 20 mL of methanol, and 1 g
of KHCO3 was added; subsequently, 10 mL of methyl iodide (CH3I)
was added. The mixture was stirred for 12 h at room temperature. The
addition of methyl iodide (10 mL) and KHCO3 (1 g) was repeated
twice more. Finally, the mixture was centrifuged and the supernatant
collected and evaporated to dryness at 40 °C in a rotavapor. The
residue, containing the pipecolic acid betaine, was dissolved in 10 mL
of Milli-Q grade water and applied on a 10 cm column, filled with a
mixed-bed resin of Dowex-1-OH− and Biorex-70-H+ (1:1 v/v) able to
retain amino acids but not betaines.27 The aqueous wash from this
column was then applied to a 10 × 2 cm column with AG50WX8-H+
resin and washed with 20 mL of water. The pipecolic acid betaine was
finally eluted with 30 mL of 6 M NH4OH and evaporated to dryness
under a stream of air.
Plant Materials. Citrus fruits (≈3 kg) and fully expanded leaves
(≈50 g) were taken from a single tree of each species or cultivar. Trees
were 10−20 years old. The sampling (fruits and leaves from orange
and lemon) was conducted in February and April 2011 at SSEA Citrus
Arboretum in Reggio Calabria (Calabria, Italy). Bergamot fruits and
leaves were harvested in the “Pellaro” area near Reggio Calabria
(Calabria, Italy).
Citrus Seed Extracts. Citrus fruits were first washed with water to
remove dust and pollutants from the exocarp. Then flavedo was
separated manually from albedo (endocarp), which contained seeds
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Figure 2. Chromatographic separation of some proline-derived compounds and pipecolic acid betaine by HPLC ESI-MS/MS. For each compound,
the extracted ion chromatogram (EIC) at the indicated transition (MS2) is reported. The insets indicated by the arrows above proline betaine and
pipecolic acid betaine peaks report the respective fragmentation patterns and the fragment relative intensities. Pro, proline; HypBet, 4-hydroxy-L-
proline betaine; NmePro, N-methyl-L-proline; ProBet, proline betaine; PipBet, pipecolic acid betaine.
with target set at 30000 and maximum accumulation time at 20 ms.
The measurements were performed from the peak area of the
extracted ion chromatogram (EIC). The quantitation was achieved by
comparison with the calibration curves obtained with standard
solutions. The retention time (min) and peak areas of the monitored
fragment ions were determined by Agilent software Chemstation
version 4.2.
fact, in the optimized MS/MS instrumental conditions show
the relative abundance of parent ions by far higher than that of
fragment ions (Figure 2). Harsher MS/MS fragmentation
conditions did not improve the fragment yield but brought
about uniform intensity decrease of both parents and
fragments.
The most useful fragments for pipecolic acid betaine
determination in complex matrices are at m/z 112, 102, and
72. The highest specificity and sensitivity are accomplished by
monitoring the fragment at m/z 72, which is absent in the
fragmentation patterns of proline betaine and other methylated
proline derivatives that occur in citrus juices.10,11
RESULTS AND DISCUSSION
■
Pipecolic Acid Betaine Synthesis and MS/MS Charac-
terization. As pipecolic acid betaine was not commercially
available, we first synthesized it according to the Chen and
Benoiton method.25,26 The purified substance was subjected to
mass spectrometry analysis to find the best instrumental
conditions for detection and quantitation. In particular,
amplitude and cutoff, which are the main parameters for an
ionic trap mass spectrometer to achieve the most effective
collision-induced dissociation (CID) of the parent ion toward
its ionic fragments, were optimized. This study was performed
in positive ion mode by direct infusion of 5 μM pipecolic acid
betaine solution in 0.1% formic acid. The pipecolic acid betaine
and proline betaine fragmentation patterns are shown in Figure
2. It is interesting to note that pipecolic acid betaine shows a
MS/MS spectrum richer in fragments than proline betaine.
This wealth of fragments is of great importance for the
unambiguous MS/MS identification of the substance in a
complex matrix. However, pipecolic acid betaine and proline
betaine also show a common feature, that is, the resistance of
the parent ions to MS/MS fragmentation. Both substances, in
Chromatographic Analyses. In a previous study10,11 was
developed an isocratic chromatographic method that employed
a Supelco Discovery-C8 column, 150 × 3.0 mm, 5 μm, and
0.1% formic acid solution in water, at flow rate of 100 μL/min
as unique eluent. In this way, five proline derivatives and other
betaines present in citrus juices were analyzed in about 10 min
without using any gradient. The use of a gradient, in fact, has
adverse effects both on analysis time, requiring column
equilibration after each analysis, and on the detection response,
the analyte ionization being in the ion source strongly
dependent on the eluent composition. However, we observed
that in these conditions pipecolic acid betaine showed a rather
long retention time (>15 min) with a consequent peak
broadening and sensitivity loss. For this reason in the present
study we employed a shorter column (10 cm). In this new
condition, many marker compounds of abiotic stress (i.e.,
methylated proline derivatives, choline, trigonelline, and γ-
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aminobutyric acid betaine) were still well resolved and easily
identified. Moreover, pipecolic acid betaine eluted at 11.4 min,
resolved from the other methylated proline derivatives, which
showed retention times lower than 9 min (Figure 2). Actually,
elution times were 6.1 min for proline, 6.4 min for
hydroxyproline betaine, 7.2 min for N-methylproline, and 8.8
min for proline betaine. For the sake of clarity, the elution
peaks of γ-aminobutyric acid betaine (7.0 min), choline (8.0
min), and trigonelline (9.2 min) are not reported in Figure 2. It
is worth noting for later considerations that one more
methylene group in the ring noticeably increased the retention
time of pipecolic acid betaine with respect to its lower
homologue proline betaine. Conversely, the introduction of a
hydroxyl group on the ring, as happens in the case of
hydroxyproline betaine, drastically reduces the retention time
on the octyl phase (C8) column. This clearly demonstrates
that, also in the case of these highly hydrophilic substances,
their retention mechanism relies on their different hydrophobic
interaction with the column stationary phase.
3 reports a typical MS/MS EIC chromatogram of a leaf extract.
Proline betaine was monitored through the transition 144→84
and pipecolic acid betaine through the transition 158→72. In
particular, pipecolic acid betaine was easily recognized by the
matching of retention time and fragmentation pattern with the
authentic standard. In agreement with that reported by Wood
et al.19 and Bonham et al.,20 both substances were present at
comparable levels. On the contrary, the situation was
completely different for the extracts of citrus plants. Figure 4
Identification and Quantitation of Pipecolic Acid
Betaine in Plant Extracts. As alfalfa (M. sativa L.) is well-
known to contain high levels of methylated proline derivatives,
particularly proline betaine and pipecolic acid betaine,19,20,23 we
first tested our analytical procedure on this plant species. Figure
Figure 4. Chromatographic separation of a bergamot juice by HPLC
ESI-MS/MS. The extracted ion chromatograms at the MS2 transitions
144.1 → 84.2 (peak 1) and 158.1→72.1 (peak 2) are reported. (Inset
1) Fragmentation pattern of peak 1. (Inset 2) Fragmentation pattern
of peak 2.
depicts the dramatic diversity of levels between the two
betaines in a bergamot juice extract. Notwithstanding the
noticeable difference of the retention times, the proline betaine
peak completely overwhelms that of pipecolic acid betaine.
However, the great resolving power of the mass spectrometry
method, well represented by Figure 4, allows pipecolic acid
betaine to be resolved and quantitated with confidence. It is
worth noting that Wood et al.27 detected the presence of an ion
species at m/z 158, which produced major fragments at m/z
140, 112, 102 and 72 in the ESI mass spectra of Achillea
filipendulina extracts. The authors hypothesized that the
compound was a hydroxy derivative of dehydroprolinebetaine,
but the lack of authentic standard made it impossible to sustain
such a hypothesis. In our case, we can exclude this possibility
Figure 3. Chromatographic separation of a Medicago sativa leaf extract
by HPLC ESI-MS/MS. The extracted ion chromatograms at the MS2
transitions 144.1 → 84.2 (peak 1) and 158.2→72.1 (peak 2) are
reported. (Inset 1) Fragmentation pattern of peak 1. (Inset 2)
Fragmentation pattern of peak 2.
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Table 1. Ranges of Pipecolic Acid Betaine (PipBet) and Pipecolic Acid (Pip) Levels in Citrus Plants Estimated by HPLC ESI-
MS/MS
bergamot
seed
lemon
seed
orange
seed
leaves
juice
1−2
trace−3
leaves
juices
leaves
juice
PipBet (mg/kg)
Pip (mg/kg)
8−10
16−24
1−2
8−12
1−3
6−8
1−2
4−8
max 0.5
max 1
1−2
3−6
max 0.5
trace−2
8−11
trace−2
Figure 5. (A) Chromatogram of the derivative of pipecolic acid with AccQ (Pip-AccQ) standard solution while monitoring the MS2 transition 300→
130. On the right is reported the peak fragmentation pattern and, above, the pipecolic acid derivatization reaction with AccQ. (B) Chromatogram of
a bergamot leaf extract derivatized with AccQ while monitoring the MS2 transitions 300→130 and 300→171. On the right is reported the
fragmentation pattern of the peak with retention time 2.3 min.
with certainty. First, the retention time and fragmentation
pattern of the ion at m/z 158 in the citrus extracts were
identical to those of the authentic pipecolic acid betaine
standard. Furthermore, for the reason discussed above, a
putative hydroxyproline derivative should have shown in our
chromatographic procedure a much shorter retention time,
close to that of hydroxyproline betaine.
Pipecolic acid betaine levels were examined in leaves, seeds,
and juices of lemon, bergamot, and orange (Table 1). It is
evident that pipecolic acid betaine, although at low levels, is
present in all parts of each type of plant (Table 1). The highest
levels were found in bergamot, whereas orange was the species
with the lowest contents. Moreover, it also appears that leaves
contain the highest and juices the lowest pipecolic acid betaine
levels (Table 1).
amino acid.22,24 The most exhaustive literature data on the
presence of pipecolic acid in vegetal matrices are reported by
Fujita et al.,16 who investigated the origin of D- and L-pipecolic
acid in human physiological fluids. The authors reported the
content of pipecolic acid in 17 edible plants, except the citrus
plants, showing that some of them, such as common bean,
broccoli, cabbage, and cauliflower, contained fair levels of
pipecolic acid, in the range of 10−50 mg/kg, with higher
contents of the L-isomer than the D-isomer. From an analytical
point of view, the ESI-MS/MS determination of pipecolic acid
in vegetal matrices presents several problems due to the
simultaneous presence at much higher concentrations of many
free amino acids and other substances that heavily interfere
with the quantitation. Studies on the amino acid determination
by ESI-MS/MS in biological fluids28−30 have pointed out that
some amino acids, particularly glutamic acid, glutamine, lysine,
and pyro-L-glutamic acid, represent important interfering
compounds in the ESI-MS/MS determination of pipecolic
acid. More recently, we showed that N-methylproline,
represented at moderate levels in citrus matrices, constitutes
Identification and Quantification of Pipecolic Acid in
Plant Extracts. The occurrence of pipecolic acid betaine in
citrus plants raises the question if it may originate from a
biosynthetic pathway analogous to that proposed for proline
betaine formation, that is, the methylation of the corresponding
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the main potentially interfering compound having the same
molecular mass of pipecolic acid and, in our chromatographic
conditions, the closest retention time to it.
produced by the action of methyltransferase(s), which employ
adenosylmethionine as a methyl donor, although such enzymes
have never been isolated and characterized in citrus plants so
far. Recently, we quantitated, with the same HPLC ESI-MS/
MS procedure employed in this study, several osmolytes in fruit
juices of numerous commercially important citrus plants such
as yellow orange, blood orange, lemon, mandarin, bitter orange
(Citrus aurantium), chinotto (Citrus myrtifolia), and grape-
fruit.10,11 It was found that the most represented osmolytes in
the juices, that is, proline, proline betaine, and hypdroxyproline
betaine, showed in each case concentrations well above 100
mg/kg. Therefore, the levels of pipecolic acid betaine detected
in this study for the first time in the citrus matrices are
comparatively too low to infer a relevant role as osmolyte in the
plant. However, the finding of pipecolic acid in the same
matrices allows the hypothesis that pipecolic acid betaine might
be a sort of byproduct of proline betaine biosynthesis. In fact,
proline and pipecolic acid have very similar chemical structures
(Figure 1); thus, it could occur that the same methyltransferase-
(s) which methylate proline recognize also pipecolic acid as a
substrate. In this case, the much lower levels of pipecolic acid
betaine than proline betaine, which we observed, could be
ascribed to both the much lower concentration of pipecolic acid
than proline and a likely lower affinity of the enzyme(s) for
pipecolic acid than for proline. Obviously, the validity of this
hypothesis requires further studies.
Actually, glutamic acid, glutamine, and lysine should not
interfere, having higher molecular mass than pipecolic acid.
Instead, these substances easily undergo in-source CID, losing
ammonia. Therefore, they are also detected at m/z 130, the
same m/z value of pipecolic acid. Furthermore, when subjected
to fragmentation, the three compounds produce the same
intense fragment at m/z 84 as pipecolic acid does.29,30 Another
reason for concern arises from the consideration that in citrus
matrices those amino acids are represented at high levels. The
AIJN reports for citrus juices mean concentrations of 400 mg/L
for Glu, 75 mg/L for Gln, and 70 mg/L for Lys.31 For these
reasons, after having unsuccessfully tried to detect with
certainty pipecolic acid by direct HPLC ESI-MS/MS of plant
extracts, we tested a procedure to overcome the presence of
those interfering compounds. This procedure is based on the
derivatization of the free amino acids in the samples with AccQ
(6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) according
to the van Wandelen and Cohen method,32 which is usually
employed for the determination of the free amino acids by
HPLC with fluorescence detection in the quality control of fruit
juices. AccQ reacts with primary and secondary amino groups
of amino acids, forming stable highly fluorescent derivatives. In
particular, Lys, Gln, and Glu form derivatives with molecular
masses of 317 Da (Lys-AccQ), 317 Da (Gln-AccQ), and 318
Da (Glu-AccQ). Interestingly, AccQ does not react with amide
and tertiary amine groups; therefore, it is unable to derivatize
pyroglutamic acid and N-methylproline. The derivatized
pipecolic acid (Pip-AccQ), which has a molecular mass of
299 Da, was characterized by FIA ESI-MS/MS and HPLC ESI-
MS/MS in positive ion mode. Because of the high hydro-
phobicity of Pip-AccQ, the retention time on the 10 cm C8
column was too long. Therefore, the analyses were isocratically
conducted with a shorter column of the same type, that is, a
Supelco Discovery-C8, 20 × 3.0 mm, 5 μm, using as eluent a
mixture of 0.1% formic acid in water and methanol (75:25, v/v)
at flow rate of 100 μL/min. In these conditions, Pip-AccQ
elutes at 2.3 min. As expected, the EIC chromatogram shows
transitions at m/z 300→171 and 300→130 (Figure 5 A). These
two transitions are very specific as they can arise only from Pip-
AccQ and not from the other interfering compounds
mentioned above.
The analyses on citrus plant extracts were performed on
samples subjected to a previous purification step on AG
50WX8-(H+) resin, according to the standard procedure for
amino acid determination in citrus juices and then derivatized
according to the van Wandelen and Cohen method.32 Figure
5B reports the MS/MS EIC chromatogram obtained on a
bergamot leaf extract for the 300→171 and 300→130
transitions. Pipecolic acid was detected in all samples examined
(Table 1). In leaves, the highest pipecolic acid concentration
was found for bergamot and the lowest, for lemon. In the juices
the concentration resulted in each case much lower than in
leaves.
It is well assessed that plants in the course of evolution have
developed defense mechanisms toward abiotic stress based on
the production of osmolyte compounds in patterns which are
characteristic of the various genera. Proline and its methylated
derivatives appear to characterize and to be of great
physiological relevance in the Citrus genus. It has been
hypothesized that the methylated proline derivatives are
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +390815665865. Fax: +390815665863. E-mail: luigi.
servillo@unina2.it.
Present Address
⊥
Dipartimento di Ingegneria Industriale e ProdAl scarl,
̀
Universita degli Studi di Salerno, via Ponte Don Melillo, I-
84084 Fisciano (SA), Italy.
ACKNOWLEDGMENTS
■
We acknowledge the assistance and skill of Francesco Siano in
carrying out numerous MS measurements during his thesis
work.
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