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 Vallverdú-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 © 2014 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: Revised: Accepted: Published: 2134

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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. 2135

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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 2136

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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 2137

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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

2138

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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.



REFERENCES

(1) Chaudhary, P. R.; Jayaprakasha, G. K.; Patil, B. S.; Porat, R. Grapefruit degreening influence on health promoting limonoids and flavonoids. Acta Hortic. 2012, 939, 113−120. (2) Chebrolu, K. K.; Jayaprakasha, G. K.; Jifon, J.; Patil, B. S. Production system and storage temperature influence grapefruit vitamin C, limonoids, and carotenoids. J. Agric. Food Chem. 2012, 60, 7096− 7103.

amount is excreted as free BT. The proposed method for the determination of these metabolites might be useful for studying the grapefruit−drug interactions in dietary studies. In addition, these results demonstrate that these metabolites can be selective enough to be considered as potential biomarkers of GFJ consumption, because they were not detected in urine after the 2139

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf405701a | J. Agric. Food Chem. 2014, 62, 2134−2140

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.

Grapefruit is a rich source of flavonoids but also contains furanocoumarins, which are known to strongly interact with a variety of medications. Thus,...
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