Identification and Quantification of Grapefruit Juice Furanocoumarin

Article
pubs.acs.org/JAFC
Identiﬁcation and Quantiﬁcation of Grapefruit Juice Furanocoumarin
Metabolites in Urine: An Approach Based on Ultraperformance
Liquid Chromatography Coupled to Linear Ion Trap-Orbitrap Mass
Spectrometry and Solid-Phase Extraction Coupled to
Ultraperformance Liquid Chromatography Coupled to Triple
Quadrupole-Tandem Mass Spectrometry
Jorge Regueiro,† Anna Valverdú-Queralt,‡,§ Noelia Negreira,∥ Jesús Simal-Gándara,†
and Rosa M. Lamuela-Raventós*,‡,§
†
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology,
Ourense Campus, University of Vigo, 32004 Ourense, Spain
‡
Nutrition and Food Science Department, Food Technology Reference Net (XaRTA), Nutrition and Food Safety Research Institute
(INSA), Pharmacy School, University of Barcelona, Avinguda Joan XXIII s/n, 08028 Barcelona, Spain
§
Spanish Biomedical Research Centre in Physiopathology of Obesity and Nutrition (CIBERobn), Instituto de Salud Carlos III
(ISCIII), Avenida Monforte de Lemos, 5, 28029 Madrid, Spain
∥
Water and Soil Quality Research Group, Department of Environmental Chemistry, Institute of Environmental Assessment and
Water Research (IDAEA), Spanish National Research Council (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
ABSTRACT: Grapefruit is a rich source of ﬂavonoids but also contains furanocoumarins, which are known to strongly interact
with a variety of medications. Thus, characterization of grapefruit furanocoumarin metabolites may help in a better understanding
of grapefruit−drug interactions. In the present work, identiﬁcation of the main metabolites of grapefruit juice furanocoumarins in
urine was performed by ultraperformance liquid chromatography (UPLC) coupled to linear ion trap-Orbitrap mass spectrometry
(LTQ-Orbitrap). Glucuronides of 6′,7′-dihydroxybergamottin and a hydroxybergamottin-like metabolite were identiﬁed for the
ﬁrst time as grapefruit juice metabolites. Afterward, a fast and sensitive method based on solid-phase extraction (SPE) and UPLC
coupled to triple quadrupole-tandem mass spectrometry (QqQ-MS/MS) was developed for determination of the identiﬁed metabolites
in urine. The proposed method was applied to urine samples of ﬁve volunteers after intakes of moderate doses of grapefruit, lemon, and
orange juices. Furanocoumarin metabolites were only detected in urines after consumption of grapefruit juice.
KEYWORDS: bergaptol, biomarker, furanocoumarins, grapefruit, UPLC−LTQ-Orbitrap, urine
■
INTRODUCTION
Grapefruit (Citrus paradisi Macfad.) is a rich source of potential
health-promoting components, such as dietary ﬁber, vitamin C,
limonoids, ﬂavonoids, β-carotene, and lycopene (pink and red
varieties).1,2 In fact, their consumption seems to be associated
with a lower risk of several chronic diseases, such as cancer3,4 and
cardiovascular disease.5,6 Despite these potential health beneﬁts,
grapefruit also contains important amounts of furanocoumarins,
a family of compounds that are known to strongly inhibit
intestinal cytochrome P450 (CYP) enzymes, namely,
CYP3A4.7,8 As a result, grapefruit juice (GFJ) can interact with
a variety of orally administered drugs by increasing their
bioavailability. Examples of medications aﬀected by consumption
of GFJ include cardiovascular drugs, such as calcium channel
blockers, statins, benzodiazepines, antihistamines, and immunosuppressants.9 Among GFJ furanocoumarins, 6′,7′-dihydroxybergamottin (DHBMT) has been reported as the main
furanocoumarin responsible for the GFJ−drug interactions,10
but also bergamottin (BMT), 6′,7′-epoxybergamottin, and
several furanocoumarin dimers, commonly known as paridisins,
have been shown to be active CYP3A4 inhibitors.11,12
© XXXX American Chemical Society
With regard to their concentration in GFJ, bergaptol (BT),
BMT, and DHBMT have been reported as the major furanocoumarins,13−15 although signiﬁcant diﬀerences in their levels can be observed
between grapefruit varieties15,16 and also, depending upon the
agronomic practices, post-harvest treatments, processing, and
storage.17 Messer et al.13 found that BT was the major furanocoumarin
in commercial juices, followed by BMT and DHBMT, whereas the
reverse order was observed in freshly prepared juices.13
A major limiting factor to study the interactions between GFJ
and drugs in clinical trials is precisely the variability in the
concentration of furanocoumarins in grapefruit. In this regard,
the discovery of new biomarkers that enable a more accurate and
objective assessment of the intake of grapefruit furanocoumarins
may be very useful in this kind of study. However, very scarce
information is still available on the metabolism of grapefruit
furanocoumarins. To the best of our knowledge, only one study
Received: December 19, 2013
Revised: February 15, 2014
Accepted: February 17, 2014
A
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Because of the lack of commercial standards of furanocoumarin
metabolites, a pooled urine sample, prepared by mixing equal volumes of
urine from two volunteers after GFJ consumption, was employed for the
method development. To ensure concentration levels high enough to
allow for the determination of major and minor metabolites, the two
volunteers drank a rather large volume (1 L) of freshly prepared GFJ.
Sample Preparation. On the day of the analysis, the urine samples
were thawed on ice, vortexed for 1 min, and centrifuged at 15000g for
5 min at 4 °C. Supernatants (990 μL) were then spiked with 10 μL of the
internal standard solution (4-MU, 30 μg/mL in methanol) and 175 μL
of 2 M sodium acetate buﬀer (pH 4.80) and vortexed for 30 s.
Sample cleanup was carried out by SPE using Oasis WAX 96-well
plates (30 mg, 30 μm). The optimized protocol involved conditioning
the cartridges with 1 mL of methanol, equilibrating with 1 mL of 300 mM
sodium acetate buﬀer (pH 4.80), loading 1 mL of urine sample, washing
with 1 mL of 300 mM sodium acetate buﬀer (pH 4.80) followed by 1 mL
of 15% methanol in 300 mM sodium acetate buﬀer (pH 4.80), and then
eluting the analytes with 1.5 mL of 2.5% (v/v) ammonium hydroxide in
methanol. The eluate was brought to dryness under a nitrogen ﬂow and
then reconstituted with 200 μL of 0.1% formic acid in water. The extract
was passed through a 0.20 μm polytetraﬂuoroethylene (PTFE) syringe
ﬁlter and analyzed by UPLC−QqQ-MS/MS. All samples were analyzed
in triplicate.
UPLC−MS Analysis. Sample analyses were performed on a Waters
Acquity ultraperformance liquid chromatography (UPLC) system
(Waters, Milford, MA) consisting of a binary pump, a vacuum degasser,
an autosampler, and a thermostatted column compartment. Chromatographic separation was performed on a reversed-phase column Acquity
UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) also from Waters, maintained
at 25 °C. Mobile phases A and B were 0.1% formic acid in water and
0.1% formic acid in acetonitrile, respectively. The following linear
gradient was used: 0 min, 25% B; 0.20 min, 25% B; 3.5 min, 65% B; 4.0 min,
100% B; 4.20 min, 100% B; 4.80 min, 25% B, and 6.0 min, 25% B. The ﬂow
rate was set to 550 μL/min, and the injection volume was 10 μL.
For qualitative characterization of the GFJ furanocoumarin metabolites,
the UPLC system was coupled to a linear trap quadrupole-Orbitrap mass
spectrometer LTQ-Orbitrap Velos from Thermo Fisher Scientiﬁc (San
Jose, CA) equipped with an electrospray ionization (ESI) source. The ESI
was operated in both polarity modes under the following speciﬁc
conditions: spray voltage, −3.5 kV for negative mode and +3.8 kV for
positive mode; sheath gas, 40 arbitrary units; auxiliary gas, 10 arbitrary units;
sweep gas, 10 arbitrary units; and capillary temperature, 320 °C. Nitrogen
(>99.98%) was employed as sheath, auxiliary, and sweep gas. The scan cycle
used a full-scan event at a resolution of 60 000 (at m/z 400) and three datadependent MS/MS events acquired at a resolving power of 30 000. The
most intense ions detected in the full-scan spectrum were selected for datadependent scan. Parent ions were fragmented by high-energy C-trap
dissociation (HCD) with a normalized collision energy of 45% and an
activation time of 100 ms. Additionally, MS3 experiments were performed
on the most intense MS/MS fragments of each metabolite using collisioninduced dissociation (CID) with a normalized collision energy of 35%.
Instrument control and data acquisition were performed with Xcalibur 2.0.7
software (Thermo Fisher Scientiﬁc). An external calibration for mass
accuracy was carried out the day before the analysis according to the
guidelines of the manufacturer.
Quantiﬁcation was carried out on a triple quadrupole mass
spectrometer API 3000 (Applied Biosystems, Foster City, CA)
equipped with a TurboIonspray ionization source. The mass
spectrometer was operated in the negative ESI mode under the
following speciﬁc conditions: ion spray voltage (IS), −4.5 kV; source
temperature (TEM), 350 °C; curtain gas (CUR), 12 arbitrary units;
nebulizer gas (NEB), 10 arbitrary units; entrance potential (EP), −10 V;
cell exit potential (CXP), −15 V; focusing potential (FP), −200 V; and
collisionally activated dissociation (CAD) gas, 4 arbitrary units.
Nitrogen (>99.98%) was employed as curtain, nebulizer, and collision
gas. The detection was performed in the multiple reaction monitoring
(MRM) mode. Analyst v1.4 software (Applied Biosystems) was used for
data acquisition and control of all system components.
Statistical Analysis. Statistical calculations were made using the
software package GraphPad Prism, version 5.0 (GraphPad Software,
dealing with the grapefruit furanocoumarin metabolites has been
conducted in humans.13 Authors reported that the consumption of
900 mL of commercial GFJ led to urinary excretion of BT and its
conjugated forms, which were evaluated by enzymatic hydrolysis.
Although this procedure allowed for the quantiﬁcation of metabolites
as BT equivalents, information about structures, concentration
proﬁles, and overall distribution of metabolites was lost.
Thus, the main objective of the present study was to provide a
further qualitative and quantitative characterization of furanocoumarin metabolites in urine after GFJ consumption. Identities
of main metabolites of GFJ furanocoumarins were elucidated by
accurate-mass measurements and multi-stage mass experiments
(MSn) using high-resolution/accurate-mass (HR/AM) hybrid
linear trap quadrupole-Orbitrap mass spectrometry (LTQOrbitrap). To improve the detection limits for the determination
of the identiﬁed metabolites, a rapid and sensitive methodology
based on solid-phase extraction (SPE) and ultraperformance
liquid chromatography coupled to triple quadrupole-tandem
mass spectrometry (UPLC−QqQ-MS/MS) was developed.
Finally, to demonstrate the applicability of the proposed method
and the potential of furanocoumarin metabolites as speciﬁc
biomarkers of GFJ consumption, urine samples of ﬁve volunteers
were analyzed after intakes of moderate doses of diﬀerent citrus
fruit juices: grapefruit, lemon, and orange juices.
■
MATERIALS AND METHODS
Reagents and Materials. BT, BMT, and bergapten were purchased
from Extrasynthese (Genay, France), whereas DHBMT was supplied
by Cayman Europe (Tallinn, Estonia). The internal standard
4-methylumbelliferone (4-MU) was obtained from Sigma (Madrid,
Spain). Formic acid (∼98%) and sodium hydroxide (≥98%) were
purchased from Panreac (Barcelona, Spain). Ammonium hydroxide
(32%, w/w), acetic acid glacial (100%), hydrochloric acid (37%, w/v),
sodium acetate anhydrous, acetonitrile, acetone, and methanol were
obtained from Merck (Barcelona, Spain). Solvents were high-performance
liquid chromatography (HPLC)-grade, and all other chemicals were
analytical-reagent-grade. Ultrapure water was obtained from a Milli-Q
gradient water puriﬁcation system (Millipore, Bedford, MA). Individual
stock solutions of each analyte and a mixture of them were prepared in
methanol. Working standard solutions were made by appropriate dilution in
80% methanol in water acidiﬁed with 0.1% formic acid and then stored in
amber glass vials at −20 °C.
Sweet orange (Citrus sinensis L. Osbeck, cv. Navelate), lemon
(Citrus limon L., cv. Fino), and grapefruit (C. paradisi Macfad., cv. Star Ruby)
of diﬀerent Spanish cultivars were purchased from a local market at maturity.
Subjects and Study Design. Five healthy Caucasian male
volunteers, ranging between 20 and 35 years old, were recruited for
this study. The participants had no history of cardiovascular, hepatic, or
renal disease and had stable alimentary habits. They had not adhered to
any special diet for at least 4 weeks prior to the consumption; therefore,
the obtained results can be attributed to a normal dietary pattern. The
study protocol was approved by the Ethics Committee of Clinical
Investigation of the University of Barcelona (IRB00003099). The study
was explained to subjects through verbal and written instructions, and
written informed consent was obtained from all participants.
Before each intervention, participants followed a 7-day wash-out
period, in which they were requested not to consume citrus fruit or their
processed products. After this period, subjects collected their blank
urines. The feeding study consisted of three interventions, involving the
consumption at dinner of a glass of juice (250 mL) of orange, lemon, and
grapefruit. Fresh juices were prepared on the day of each intervention
using a home juicer machine. Participants were asked to collect the ﬁrst
morning urine, 10 h after juice intake. All samples were collected in
100 mL random coded sterile specimen containers and immediately
stored at 4 °C. Upon receipt of each sample, four aliquots of 1.0 mL were
transferred to individual 1.5 mL capped Eppendorf tubes and stored
at −80 °C until the analyses.
B
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Table 1. Identiﬁcation of GFJ Metabolites in Human Urine
a
compound
tR
(min)
formula
measured
mass (Da)
Δm
(ppm)a
[M − H]−
(m/z)
MS/MS fragments (m/z)
NAR-diglcU 1
NAR-glcU-SO3 1
NAR-SO3 1
NAR-diglcU 2
BT-glcU
0.84
0.87
0.97
1.00
1.10
C27H28O17
C21H20O14S
C15H12O8S
C27H28O17
C17H14O10
624.1324
528.0567
352.0249
624.1326
378.0583
−0.35
−1.21
−0.99
−0.06
−1.14
623.1254
527.0497
351.0179
623.1256
377.0513
447.093, 271.061, 227.071, 151.002, 119.049
447.093, 351.018, 331.122, 271.061, 151.002,
271.061, 177.018, 151.002, 119.049, 107.012
447.093, 271.061, 227.071, 151.002, 119.049
201.018, 173.023, 145.028, 117.033
BT-SO3
1.27
C11H6O7S
281.9833
−0.57
280.9763
201.018, 173.023, 145.028, 117.033
NAR-glcU
NAR-glcU-SO3 2
NAR-SO3 2
BTb
DHBMT-glcU
1.44
1.47
1.62
2.10
2.32
C21H20O11
C21H20O14S
C15H12O8S
C11H6O4
C27H32O12
448.1002
528.0572
352.0250
202.0257
548.1890
−0.85
−0.28
−0.91
−4.45
−0.78
447.0932
527.0502
351.0180
201.0187
547.1820
271.061, 177.018, 151.002, 113.023, 119.049
271.061, 254.981, 175.024, 96.959
271.061, 177.018, 151.002, 119.049, 107.012
191.034, 173.023, 163.039, 145.028, 117.033
371.148, 345.154, 201.018, 173.023
NARb
HBMT-glcU 1
2.38
2.66
C15H12O5
C27H30O11
272.0683
530.1787
−0.74
−0.21
271.0613
529.1717
177.018, 151.003, 119.049, 107.012
353.138, 201.018, 173.023
HBMT-glcU 2
2.79
C27H30O11
530.1783
−1.02
529.1713
353.138, 201.018, 173.023
MS/MS/MS fragments
(m/z)
447 > 429, 271, 175
351 > 271, 151
271 > 151, 177, 119
447 > 429, 271, 175
201 > 191, 173, 163, 147,
145, 119, 117
201 > 191, 173, 163, 147,
145, 119, 117
271 > 151, 177, 119
271 > 151, 177, 119
271 > 151, 177, 119
173 > 163, 145, 117
201 > 191, 173, 163, 147,
145, 119, 117
151 > 107, 83, 64
201 > 191, 173, 163, 147,
145, 119, 117
201 > 191, 173, 163, 147,
145, 119, 117
Δm = mass measurement error. bPositively identiﬁed.
San Diego, CA). Unless otherwise speciﬁed, data are presented as the
mean ± standard deviation (SD). Statistical signiﬁcance was determined
by one-way analysis of variation (ANOVA) followed by Dunnett’s
multiple comparison tests when comparing more than two groups and
two-way ANOVA followed by Bonferroni’s test when comparing two
factors.
■
m/z 201.0187 produced characteristic ions at m/z 173.023,
145.028, and 117.033. Glucuronide metabolite of DHBMT
(DHBMT-glcU) was also tentatively identiﬁed (Δm = −0.78 ppm)
at 2.32 min with a [M − H]− ion at m/z 547.1820. Its MS/MS
spectrum (Figure 1d) showed fragment ions at m/z 371.148
(M − H − 176, loss of glucuronic acid), which corresponds to the
deprotonated molecule of DHBMT, m/z 345.154 (M − H −
BT), and also m/z 201.018. In the study conducted by Messer
et al.,13 no DHBMT could be detected after enzymatic hydrolysis
of urine, which authors mainly attributed to the possible transformation into free BT during its metabolism. An explanation to
this disagreement might rely on the hydrolysis step used by the
authors, because enzymatic eﬃciency greatly depends upon the
structure of the metabolites.18 Two isomeric forms of another
metabolite could also be detected at 2.66 and 2.79 min with a
[M − H]− ion at m/z 530.1787. Its fragmentation yielded an ion
at m/z 353.138, corresponding to the loss of a glucuronic acid
moiety, and also the characteristic ion of BT at m/z 201.018. This
pattern suggested that these metabolites might be glucuronides
of a hydroxybergamottin-like compound (HBMT-glcU) (Figure 1e).
To the best of our knowledge, this study provides for the ﬁrst time
evidence of the existence in urine of glucuronides of DHBMT and
HBMT as GFJ furanocoumarin metabolites. No free furanocoumarins, other than BT, could be detected (in both polarity ESI modes) in
urine after the GFJ consumption, which indicated that they are
mainly metabolized to the reported forms.
In addition to these furanocoumarin metabolites, naringenin
(NAR) and its glucuronide and sulfate conjugates were also
identiﬁed in urine (Table 1), which is in agreement with previous
studies dealing with the metabolism of GFJ ﬂavonoids.19,20
SPE Optimization. Once the main furanocoumarin
metabolites present in urine were identiﬁed, the research was
focused on the development of an analytical method sensitive
enough to allow for their detection after the consumption of a
low regular dose of GFJ. To this end, an approach based on the
use of SPE and UPLC coupled to triple quadrupole mass
spectrometry was performed. The speciﬁc MRM conditions for
quantiﬁcation and conﬁrmation are detailed in Table 2.
As known, the sorbent type and solvent composition for
washing and elution steps are crucial parameters for the
RESULTS AND DISCUSSION
Characterization of GFJ Metabolites by HR/AM Mass
Spectrometry. To elucidate the chemical structures of the main
GFJ metabolites, a pooled urine sample was collected 10 h after
the intake of 1 L of freshly prepared GFJ and analyzed by UPLC
coupled to a LTQ-Orbitrap mass spectrometer. Taking
advantage of the HR/AM of this MS instrument, main GFJ
metabolites could be tentatively identiﬁed with mass measurement errors below 4.5 ppm (Table 1). Combining HR/AM
measurement and MSn increased the conﬁdence of the proposed
metabolites. Thus, the availability of chemical formulas for the
precursor and product ions generated from high-resolution datadependent accurate-mass analysis allowed for the speed up of
structural elucidation of metabolites.
After the intake of GFJ, a major metabolite was found at
1.10 min, showing a [M − H]− ion at m/z 377.0513. Its MS/MS
fragmentation produced a base ion at m/z 201.018 (M − H −
176, loss of glucuronic acid moiety), which corresponds to the
deprotonated molecule of BT (Figure 1a). Thus, this compound
was tentatively identiﬁed as a glucuronide metabolite of BT (BTglcU). Another main metabolite with [M − H]− ion at m/z
280.9763 eluted at 1.27 min, yielding again a MS/MS base ion at
m/z 201.018 produced by the loss of 80 Da (Figure 1b). This
characteristic loss of a sulfate group indicated that this metabolite
might be the sulfoconjugate of BT (BT-SO3). On the basis of
data obtained by enzymatic hydrolysis, Messer et al. 13 proposed
BT glucuronide and sulfate as main GFJ furanocoumarin
metabolites in humans. To the best of our knowledge, these
metabolites have been identiﬁed for the ﬁrst time in the present
work using a mass spectrometry approach. Free BT could also be
positively identiﬁed in urine after GFJ consumption, which is in
agreement with the results previously reported.13 As shown in
Figure 1c, the fragmentation by HCD of the [M − H]− ion at
C
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Figure 1. MS/MS spectra obtained for the identiﬁed furanocoumarin metabolites: (a) BT-glcU, (b) BT-SO3, (c) BT, (d) DHBMT-glcU, and (e)
HBMT-glcU.
Table 2. Speciﬁc MRM Conditions for the Determination of GFJ Furanocoumarin Metabolites
a
compound
tR (min)
parent ion
MRM1 (m/z, quantiﬁer)
CE1 (eV)a
MRM2 (m/z, qualiﬁer)
CE2 (eV)a
BT-glcU
BT-SO3
4-MU (IS)b
BT
DHBMT-glcU
HBMT-glcU 1
HBMT-glcU 2
1.10
1.27
1.71
2.10
2.32
2.66
2.79
[M − H]−
[M − H]−
[M − H]−
[M − H]−
[M − H]−
[M − H]−
[M − H]−
377 > 201
281 > 201
175 > 133
201 > 117
547 > 201
529 > 201
529 > 201
−25
−22
−30
−40
−35
−25
−25
377 > 173
281 > 173
175 > 119
201 > 145
547 > 345
529 > 353
529 > 353
−28
−34
−35
−35
−32
−23
−23
CE = collision energy. bIS = internal standard.
groups of sorbent positively charged (pKa ∼ 6). Thus, urine was
buﬀered to pH 4.8 prior to the loading step, which guarantees a
deprotonation rate of glucuronides higher than 98%. Working
under these pH conditions, diﬀerent percentages of methanol
(0−15%) were tested, aiming to improve the cleanup step
(Figure 2). This study may be especially critical for the neutral
compounds, such as the BT, because they are only retained by a
reversed-phase mechanism. No reduction in the amounts of any
of target metabolites was observed in the studied range;
therefore, 15% methanol was ﬁnally selected.
Another critical step is the selection of a suitable phase for the
quantitative elution of the retained compounds. When dealing
with weak anion-exchange mechanisms, two diﬀerent approaches are usually possible to achieve the elution. The most
common one consists of the protonation of the acidic
compounds by using an elution solvent with a pH low enough.
Nevertheless, sulfoconjugates are no able to elute under these
optimization of any SPE procedure. Taking into account the
ionizable nature of major furanocoumarin metabolites, mainly
glucuronide and sulfate derivatives, it was hypothesized with the
use of an ionic-exchange mechanism. Glucuronide metabolites
can be easily ionized because of the presence of the carboxylic
group on the glucuronic acid moiety, which usually shows pKa
values around 3,21 whereas sulfate conjugates carry a permanent
negative charge (pKa < 1). Therefore, a mixed-mode weak anionexchange (WAX)/reversed-phase polymeric sorbent was
evaluated for the extraction and preconcentration of furanocoumarin metabolites from urine. As a result of their dual properties,
this kind of sorbent has been used to eﬃciently retain both
neutral and acidic metabolites from biological samples.22 Stramm
et al.23 were able to selectively retain glucuronides and sulfate
conjugates of steroids using Oasis WAX SPE cartridges.
To obtain maximal retention, it is necessary to deprotonate
glucuronides while maintaining the piperazine anion-exchange
D
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of 3 using the less sensitive MS/MS transition, were in the range
of 0.9−1.5 ng/mL.
Feeding Study. The proposed method was applied to the
analysis of urine samples from ﬁve volunteers (U1−U5) after the
consumption at dinner of a glass (250 mL) of freshly prepared
juice of orange, lemon, and grapefruit. Because no standards were
available, only BT could be accurately quantiﬁed in the urine
samples. For the rest of the metabolites, concentrations were
estimated as equivalents of BT, which provide semi-quantitative
information about their levels in urine.
The subjects were requested not to consume citrus fruits or
derivatives during the 7 days previous to each intervention. First
morning urines were collected the day before the intake and in
the morning following the intervention, 10 h after juice
consumption. No furanocoumarin metabolites were detected
in the urine samples after the wash-out periods or after the
consumption of orange and lemon juices. Nevertheless,
important concentrations of metabolites were found in the
urine samples of all of the volunteers after the intake of a glass of
GFJ (Table 3). Figure 4 shows the MRM chromatograms for the
quantiﬁcation MS/MS transitions obtained for U2 after the
intake of 250 mL of GFJ.
The concentration of BT showed considerable variation
among the ﬁve volunteers, ranging from 382 to 2670 ng/mL
(median of 595 ng/mL). Messer et al.13 found BT in free and
conjugated forms, as the only furanocoumarin excreted in urine
within 6 h after consumption of 900 mL of GFJ. On the basis of
results of enzymatic hydrolysis, they suggested that the majority
of BT was excreted as conjugate forms. Our data suggest a similar
pattern with estimated concentrations for the total BT in
conjugate forms between 30- and 130-fold higher than those of
free BT. Among the furanocoumarin metabolites, BT-glcU was
in all cases the major urinary metabolite, showing a median
concentration around 33 μg/mL (Figure 5). BT-SO3 and
DHBMT-glcU were also found at concentrations in the
microgram per milliliter level. As shown, the glucuronides of
HBMT were the least abundant conjugates, with a median
concentration of 621 ng/mL.
Our ﬁndings suggest that GFJ furanocoumarins are mainly
excreted as glucuronide and sulfate conjugates, whereas a lower
Figure 2. Eﬀect of the proportion of methanol during the SPE washing
step.
conditions, because they are not prone to neutralization.
Therefore, elution could only be carried out by neutralizing the
piperazine anion-exchange function of the sorbent. Three
diﬀerent proportions of NH4OH in methanol were evaluated
for the elution of furanocoumarin metabolites (Figure 3).
Whereas no diﬀerences were observed between the use of 2.5 and
5% NH4OH, the amounts of metabolites eluted were statistically
lower when using 1% NH4OH. The only exception to this
behavior was found for BT, which is the only metabolite retained
exclusively by the reversed-phased mechanism. Consequently, its
elution should not be pH-dependent. Therefore, 2.5% NH4OH
in methanol was chosen for the elution of furanocoumarin
metabolites from the WAX sorbent.
Because of the lack of commercial standards for the
furanocoumarin metabolites, the recovery of the SPE procedure
could only be evaluated for BT. A blank urine sample was spiked
at concentrations of 50, 500, and 5000 ng/mL, and the recovery
was calculated by dividing the diﬀerence between the measured
concentrations for spiked and non-spiked samples by the added
concentrations. Quantitative recoveries, in the range of 94−
102%, were obtained for all concentration levels. The precision of
the method was evaluated by calculating the relative standard
deviation (RSD) at the same three concentration levels. Satisfactory
results were obtained with RSD values below 7% in all cases. Limits
of detection (LODs), calculated for a signal-to-noise ratio (S/N)
Figure 3. Inﬂuence of the percentage of ammonia solution during the SPE elution step. (∗) p < 0.05 compared to 2.5% NH4OH and 5% NH4OH.
Table 3. Urinary Concentrations of Furanocoumarin Metabolites after Consumption of 250 mL of GFJ
concentration ± SD (ng/mL, n = 3)
volunteer
BT
BT-glcU
BT-SO3
DHBMT-glcU
∑HBMT-glcU
U1
U2
U3
U4
382 ± 17
334 ± 20
595 ± 22
2679 ± 136
32649 ± 1210
32913 ± 1954
38702 ± 2326
69229 ± 3815
11294 ± 540
9833 ± 428
24551 ± 456
8524 ± 475
4037 ± 251
3826 ± 263
4780 ± 298
6353 ± 344
461 ± 14
1045 ± 46
1091 ± 36
562 ± 43
E
dx.doi.org/10.1021/jf405701a | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 4. MRM chromatograms obtained for a real urine sample (U2 in Table 3) after the intake of 250 mL of GFJ.
wash-out periods or after consumption of orange and lemon
juices.
■
AUTHOR INFORMATION
Corresponding Author
*Telephone: +34934034843. Fax: +34934035931. E-mail:
[email protected].
Funding
The authors express their gratitude for ﬁnancial support from the
Interdepartmental Committee of Science and Technology
(CICYT) (AGL2010-22319-C03) from the Spanish Ministry
of Science and Innovation (MICINN) and from CIBERobn, an
initiative of the ISCIII, Spain. Jorge Regueiro acknowledges
MICINN for his Juan de la Cierva contract.
Notes
The authors declare no competing ﬁnancial interest.
Figure 5. Box plots of the metabolite concentration in urine from ﬁve
volunteers after the intake of 250 mL of GFJ.
■
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