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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Phytochem Anal. 2013 Jun 18;24(6):10.1002/pca.2449. doi: 10.1002/pca.2449

Rapid Quantitation of Furanocoumarins and Flavonoids in Grapefruit Juice using Ultra Performance Liquid Chromatography

Karen M VanderMolen a, Nadja B Cech a, Mary F Paine b, Nicholas H Oberlies a,✉
PMCID: PMC3855432  NIHMSID: NIHMS496575  PMID: 23780830

Abstract

Introduction

Grapefruit juice can increase or decrease the systemic exposure of myriad oral medications, leading to untoward effects or reduced efficacy. Furanocoumarins in grapefruit juice have been established as inhibitors of cytochrome P450 3A (CYP3A)-mediated metabolism and P-glycoprotein (P-gp)-mediated efflux, while flavonoids have been implicated as inhibitors of organic anion transporting polypeptide (OATP)-mediated absorptive uptake in the intestine. The potential for drug interactions with a food product necessitates an understanding of the expected concentrations of a suite of structurally diverse and potentially bioactive compounds.

Objective

Develop methods for the rapid quantitation of two furanocoumarins (bergamottin and 6′,7′-dihydroxybergamottin) and four flavonoids (naringin, naringenin, narirutin, and hesperidin) in five grapefruit juice products using ultra performance liquid chromatography (UPLC).

Methodology

Grapefruit juice products were extracted with ethyl acetate; the concentrated extract was analyzed by UPLC using acetonitrile:water gradients and a C18 column. Analytes were detected using a photodiode array detector, set at 250 nm (furanocoumarins) and 310 nm (flavonoids). Intraday and interday precision and accuracy and limits of detection and quantitation were determined.

Results

Rapid (<5.0 min) UPLC methods were developed to measure the aforementioned furanocoumarins and flavonoids. R2 values for the calibration curves of all analytes were >0.999. Considerable between-juice variation in the concentrations of these compounds was observed, and the quantities measured were in agreement with the concentrations published in HPLC studies.

Conclusion

These analytical methods provide an expedient means to quantitate key furanocoumarins and flavonoids in grapefruit juice and other foods used in dietary substance-drug interaction studies.

Keywords: Grapefruit, furanocoumarin, flavonoid, quantitation, UPLC

INTRODUCTION

Grapefruit juice has been shown to increase the systemic exposure of a diverse array of oral medications that undergo extensive pre-systemic metabolism by cytochrome P450 3A (CYP3A) in the intestine, including felodipine, lovastatin, and cyclosporine (Paine et al., 2006; Seden et al., 2010; Hanley et al., 2011; Won et al., 2012). The increase in systemic drug exposure can be sufficient to cause untoward effects, ranging from relatively mild (e.g., hypotension and dizziness with some calcium channel blockers) to potentially severe (e.g., nephrotoxicity with some immunosuppressants). The mechanism underlying these interactions is irreversible inhibition of intestinal CYP3A activity by grapefruit juice (Bailey et al., 1998; Paine et al., 2006; Paine & Oberlies, 2007; Hanley et al., 2011). Furanocoumarins, a class of compounds present in grapefruit juice, have been established as major CYP3A inhibitors in human volunteers (Paine et al., 2006). This effect may be augmented if the “victim” drug also is a substrate for P-glycoprotein (P-gp), a transmembrane efflux transport protein located on the apical membranes of numerous cell types, including enterocytes (Paine & Oberlies, 2007; Hanley et al., 2011). P-gp demonstrates substrate specificity that overlaps with that of CYP3A substrates (Wacher et al., 1995), and several in-vivo and in-vitro studies have shown an inhibitory effect by grapefruit juice and/or furanocoumarins towards P-gp activity (Lown et al., 1997; Edwards et al., 1999; Ohnishi et al., 2000; De Castro et al., 2007; Paine et al., 2008; Dahan & Amidon, 2009). A more recently discovered mechanism underlying grapefruit juice-drug interactions is inhibition of intestinal organic anion transporting polypeptides (OATPs), which are uptake transporters located on the apical membranes of enterocytes and other cell types (Dresser & Bailey, 2003; Won et al., 2012). Opposite to P-gp, OATPs in the intestine act to facilitate drug absorption. Thus, inhibition of these transport proteins leads to a decrease in systemic exposure of drug substrates, including the antihistamine fexofenadine (Dresser & Bailey, 2003; Dresser et al., 2005) and the antihypertensive agent aliskiren (Vanamala et al., 2005; Avula et al., 2007), with the consequent potential for therapeutic failure. Candidate OATP inhibitors in grapefruit juice include the flavonoids naringin and hesperidin (Bailey et al., 2007). The potential for drug interactions with a widely available food product necessitates an understanding of the expected concentrations of a suite of structurally diverse and potentially bioactive compounds.

Reported analytical methods for measuring furanocoumarins in fruit juices typically require separation times of one hour or more (Fukuda et al., 2000; Ross et al., 2000; Manthey & Buslig, 2005; Vanamala et al., 2005; De Castro et al., 2006; Avula et al., 2007). For example, in 2005, a study of the distribution of furanocoumarins in grapefruit juice fractions utilized a 65-min high performance liquid chromatography (HPLC) method (Manthey & Buslig, 2005). The same year, an exhaustive analysis of several furanocoumarins and furanocoumarin dimers in 58 juices employed a similar 65-min method (Widmer & Huan, 2005). The following year, a 45-min HPLC method was utilized to determine the concentrations of bergamottin and 6′,7′-dihydroxybergamottin in grapefruit juice (De Castro et al., 2006). In 2009, a shorter HPLC method was developed for the determination of five furanocoumarins (bergaptol, psoralen, bergapten, bergamottin, and 6′,7′-dihydroxybergamottin) in citrus juices that required a run time of 23 min, which appears to be the most rapid method published to date (Lin et al., 2009). As with the furanocoumarins, analytical procedures for measuring flavonoids often employ methods of one hour or more. A survey of 9 commercial grapefruit juices and associated flavonoid concentrations published in 2000 utilized a 60 min method (Ross et al., 2000). Six years later, a 65 min method in a similar survey of orange and grapefruit juices was published (Vanamala et al., 2005), followed the next year by another 60 min method for the simultaneous analysis of adrenergic amines and flavonoids in fruit jams and juices (Avula et al., 2007). De Castro et al. utilized a 70 min separation to determine the concentrations of naringin and naringenin (De Castro et al., 2006). The most rapid HPLC analyses of flavonoids reported in the literature was a 45 min method published in 2008 (Fujita et al., 2008). A handful of UPLC analyses of flavonoids in food, supplements and traditional Chinese medicines have appeared in the literature in the last few years as well. The majority of these studies (Baranowska & Magiera, 2011; Medina-Remon et al., 2011; Cao et al., 2012; Huang et al., 2012) utilize a C18 column and UV detection, though one expansive study quantifying 39 phenolic compounds in apples (De Paepe et al., 2013) additionally used electrospray ionization mass spectrometry (ESI-MS) to confirm the identity of the analytes. The most rapid of these UPLC studies quantified 11 flavonoids (including those listed in this paper) in three citrus fruit extracts, and had a run time of 5.5 min (Cao et al., 2012).

Ultra performance liquid chromatography (UPLC) offers significant advantages in sensitivity and speed compared to conventional high performance liquid chromatography (HPLC). After overcoming the difficulties presented by working with very high pressures (MacNair et al., 1997; MacNair et al., 1999; Wu et al., 2001), early studies demonstrated enhanced resolution and sensitivity, reduced solvent consumption, and rapid analyses (Gerber et al., 2004; Swartz, 2005; Kumar et al., 2012) with UPLC compared to that which can be achieved with HPLC. With greater integration in diverse research areas, UPLC offers the opportunity to streamline quantitative determinations, such as those described herein, reducing cost and expediting research.

The goal of this study was to develop rapid (< 5.0 min) methods for the quantitation of two furanocoumarins (bergamottin and 6′,7′-dihydroxybergamottin) and four flavonoids (naringin, naringenin, narirutin, and hesperidin) in grapefruit juice using UPLC (Fig. 1). An additional goal was to apply these methods to determine the concentrations of the aforementioned analytes in five grapefruit juices used in previous clinical interaction studies.

Figure 1.

Figure 1

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Structures of the furanocoumarins bergamottin (1) and 6′,7′-dihydroxybergamottin (2) and the flavonoids naringin (3), narirutin (4), naringenin (5), and hesperidin (6).

EXPERIMENTAL

Instrumentation

UPLC analyses were performed using a Waters Acquity UPLC system (Milford, MA) equipped with an autosampler, photodiode array detector (PDA), column manager, and binary solvent manager. Data were collected and analyzed using Empower software. An HSS C18 column (50 mm × 2.1 mm i.d., 1.8 µm, Waters, Milford, MA) was used for all chromatographic analyses.

Materials

Bergamottin (purity ≥ 96.9%) was purchased from ChromaDex (Irvine, CA, USA); narirutin (purity ≥ 99.0%) was purchased from Indofine (Hillsborough, NJ, USA); naringin (purity ≥ 96.8%), naringenin (purity ≥ 99.9%), hesperidin (purity ≥ 97.0%), and 6′,7′-dihydroxybegamottin (purity ≥ 97.2%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Purity of standards are reported as determined by HPLC by the manufacturers. Methanol, ethyl acetate, and acetonitrile were purchased from Pharmco-Aaper (Shelbyville, KY, USA).

Juices A and B were used in clinical studies with felodipine (Paine et al., 2006), cyclosporine (Paine et al., 2008), and fexofenadine (Won et al., 2010). Juice A was a commercially available product. Juice B was the same juice, but the furanocoumarin fractions were removed using a series of food-grade solvents and absorption resins and column chromatography (Paine et al., 2006). Juice C was a commercially available concentrated grapefruit juice (Minute Maid Premium 100% Pure Frozen Concentrated Grapefruit Juice with added calcium). A dilution of Juice C was used in a clinical study examining the effect of grapefruit juice on the systemic exposure of loperamide and the CYP3A-mediated metabolite, desmethylloperamide (Wolf et al., 2011). Juice D (Florida’s Natural Original Ruby Red 100% Pure Florida Grapefruit Juice) and Juice E (Simply Orange Juice Co. Simply Grapefruit 100% Pure Squeezed Grapefruit Juice) were commercially available not-from-concentrate juices and were purchased from a local grocery store.

Standards preparation

Bergamottin and 6′,7′-dihydroxybergamottin were dissolved in methanol to produce a 1.03 mM (bergamottin) and 1.00 mM (6′,7′-dihydroxybergamottin) multi-standard stock solution. This solution was used to prepare seven standard solutions at concentrations ranging from 10.3 to 659 µM (bergamottin) and from 10.0 to 640 µM (6′,7′-dihydroxybergamottin). Naringin, narirutin, naringenin, and hesperidin were dissolved separately in methanol to produce stock solutions of 10.0 mM, 7.92 mM, 10.2 mM, and 9.97 mM, respectively. These stock solutions were used to prepare standard solutions for each compound (six for naringin, five for each of the other flavonoids) at concentrations ranging from 0.649 to 5.00 mM (naringin), 0.198 to 3.17 mM (narirutin), 3.18 to 814 µM (naringenin), and 3.12 to 798 µM (hesperidin).

Sample preparation

Juice extracts were prepared by transferring 25.0 mL of each juice to a 50-mL conical polypropylene tube. After adding 20.0 mL of ethyl acetate, the contents were shaken vigorously and centrifuged (2500 × g for 30 min at 25 °C). The resulting organic layer was transferred by Pasteur pipette to a 250 mL round-bottom flask. An additional 20.0 mL of ethyl acetate were added to the remaining contents of the tube, shaken and centrifuged, and the resultant organic layer was combined with the first organic layer. This extraction procedure was repeated a third time, and the combined organic layers were evaporated in vacuo. The residue in the round-bottom flask was transferred quantitatively to a 2 mL vial, using methanol as a rinse, and evaporated to dryness under air. This material was resuspended with 5.00 mL of methanol, yielding a 5-fold concentrated extract of each juice.

Chromatographic conditions

Quantitative analysis of furanocoumarins (bergamottin and 6′,7′-dihydroxybergamottin)

A 6 µL volume of each 5-fold concentrated juice extract was injected and analyzed at a wavelength of 250 nm. Chromatographic separations were carried out with a mobile phase consisting of HPLC-grade acetonitrile (solvent A) and nanopure water (solvent B) at a flow rate of 0.6 mL/min. The following linear gradient was used: 0.0 min, 30% A; 4.0 min, 100% A; 4.5 min, 100% A. The column temperature was 30 °C.

Quantitative analysis of flavonoids (naringin, narirutin, naringenin, and hesperidin)

A 6 µL volume of each 5-fold concentrated juice extract was injected and analyzed at a wavelength of 310 nm. The following linear gradient was used: 0.0 min, 10% A; 1.0 min, 20% A; 2.0 min, 20% A; 2.5 min, 40% A; 3.5 min, 100% A; 4.0 min, 100% A. Solvents A and B, the flow rate, and column temperature were the same as described in the previous section.

Method Validation

The identities of analyte peaks in the juice extracts were confirmed by comparing the UV spectra of the peaks and their standards, as well as, in the case of the flavonoids, by co-injection of the standards with the juices (see Supporting Information, Supplement 1 and 2).

Linearity of the calibration curves was assessed by least-squares analysis. Precision and accuracy were determined by calculating the relative standard deviation (RSD) and relative error (RE), defined as the percent difference between the mean observed concentration and the nominal concentration, of three replicate analyses of the standards. All analyses were performed in triplicate in a single day. Interday RSD and RE were determined by analyzing the standard solutions in triplicate on three separate days. The limit of detection (LOD) was defined as the concentration corresponding to the signal detection limit, which was defined as b + 3 sy, where b is the y-intercept of the calibration curve, and sy is the standard deviation of the vertical deviations (Harris, 2007). The limit of quantitation (LOQ) was defined as b + 10 sy.

RESULTS AND DISCUSSION

Method Validation

All standard curves exhibited coefficients of determination (r2) greater than 0.999 (Table 1). Baseline resolution of all analytes (Fig. 1), both as standards and in the juice extracts, was achieved (Figs. 2 and 3). Precision and accuracy data are summarized in Table 2. RSD of the furanocoumarin standards (1 and 2) was below 0.33% (intraday) and 3.1 % (interday) for all standard concentrations. The intraday RSD values of analytes 3, 4, and 5 were below 1.5%, 2.6%, and 2.9%, respectively, while the interday RSD averages for each analyte remained below 1.0%. For analyte 6, the largest RSD value was 4.7% at the lowest concentration of that standard, while the interday RSD was 3.1%. For the furanocoumarins (1 and 2), intraday RE remained below 5.7% (1) and 2.8% (2). Interday RE values predominantly remained under 3.0%, except for the lowest concentrations of 1 and 2, the highest variation being 21% for the lowest concentration of 1. The RE values (both intraday and interday) of the flavonoids (3–6) were predominantly below 5%, and all below 15%. The only exception was the lowest of the standard concentrations of 5 (3.18 µM), which had much higher RE values (32%); thus, this concentration was below the LOQ as defined above. For each analyte measured, all standard concentrations above the LOQ resulted in an RE value of ≤ 15%. LOD values for analytes 1, 2, 5, and 6 were all below 5 µM. LOD values for analytes 3 and 4 were higher (18 and 69 µM, respectively).

Table 1.

Calibration curve data for analytes 1–6.

Analyte Slope (± SD) r2 LOD a (µM) LOQ b (µM)
1 4.338 × 103 (± 9) 1.000 0.78 2.6
2 3.784 × 103 (± 5) 1.000 1.1 3.8
3 9.04 × 102 (± 7) 0.999 69 230
4 9.98 × 102 (± 7) 0.999 18 58
5 1.626 × 103 (± 6) 1.000 2.5 8.4
6 8.95 × 102 (± 5) 1.000 3.9 13
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a

Limit of detection

b

Limit of quantitation

Figure 2.

Figure 2

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UPLC separation of furanocoumarins. Numbered peaks correspond to the compounds in Fig. 1. (A) Furanocoumarin standards (659 and 640 µM, respectively) and (B) Juice A.

Figure 3.

Figure 3

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UPLC separation of flavonoids. Numbered peaks correspond to the compounds in Fig. 1. (A) Flavonoid standards (1080, 500, 800, and 800 µM, respectively) and (B) Juice A.

Table 2.

Precision (RSD) and accuracy (RE) for analysis of analytes 1–6.

Analytes Concentration of
Standard Solution
Injected (µM)		 Intraday Interday
RSD (%) RE (%) RSD (%) RE (%)
1 10.3 0.33 5.7 3.1 21.
20.6 0.27 4.4 0.48 8.5
41.2 0.091 1.6 0.31 2.4
82.4 0.19 0.21 0.53 1.1
165 0.16 0.03 0.26 2.1
330 0.29 1.2 0.16 0.83
659 0.32 0.29 0.32 0.33
2 10 0.15 2.8 1.2 9.2
20 0.11 1.1 0.67 2.7
40 0.017 0.94 0.62 0.69
80 0.21 0.10 0.56 2.1
160 0.16 0.64 0.59 2.5
320 0.26 0.70 0.61 2.1
640 0.31 0.13 0.53 1.4
3 649 1.5 15. 0.84 17.
1081 1.3 4.6 0.14 5.7
1802 1.1 0.81 0.38 0.79
3003 0.36 3.3 0.45 3.0
5005 0.72 3.6 0.29 2.1
10010 0.99 1.0 0.28 1.9
4 198 1.6 13. 0.67 7.7
396 2.6 1.4 0.72 2.1
792 1.5 1.6 0.43 1.3
1584 1.3 2.5 0.26 2.0
3168 0.72 0.66 0.78 0.55
5 3.18 1.4 32. 0.03 32.
12.7 2.9 9.7 0.32 5.3
50.9 1.4 0.4 0.23 1.4
203 0.23 1.6 0.35 0.66
814 1.2 0.094 0.18 0.39
6 3.12 4.7 2.4 3.1 3.7
12.5 0.64 2.8 0.96 8.3
49.9 0.93 3.5 0.69 9.0
199 0.71 4.1 0.47 8.1
798 1.6 3.0 0.72 1.4
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Rapidity of Method

Compared to HPLC analyses of furanocoumarins and flavonoids in the literature, the UPLC method described above markedly reduces the time required for the analyses of these compounds in grapefruit juice. A run time of 4.5 min represents an 80% reduction in the time required to analyze furanocoumarin content when compared with the most rapid published method (Lin et al., 2009), whereas a run time of 4.0 min represents an order of magnitude less time required for the analysis of flavonoids (Fujita et al., 2008). Such methods facilitate the determination of furanocoumarin and flavonoid concentrations in several grapefruit juices in a single day, reducing costs associated with both labor and materials. Moreover, the time saved expedites the characterization of juices used in in vitro or in vivo studies, thus accelerating the evaluation and determination of active constituents.

Concentrations of Analytes in Grapefruit Juices

Five different grapefruit juices, four of which were commercially available products, were analyzed to measure the concentrations of selected furanocoumarins and flavonoids (Fig. 1). Samples were analyzed in the same run with the standards to eliminate concerns of systematic error introduced by day-to-day variability in instrument response. Each juice was analyzed using a 5-fold concentrated extract, and the reported values (Table 3) have been adjusted to reflect the concentration in the original juices. Considerable variation in the concentration of each compound was observed. Regarding the furanocoumarins, the concentrations of 1 and 2 in four of the juices ranged from 7.48 (± 0.17) to 24.73 (± 0.16) µM and 7.65 (± 0.17) to 89.03 (± 0.17) µM, respectively. These values were consistent with those reported in the literature using HPLC (Widmer & Huan, 2005; De Castro et al., 2006). The highest concentration of 2 (Juice C) was slightly higher than the ranges reported by De Castro et al. (De Castro et al., 2006), though this was to be expected, as Juice C was a juice concentrate. Regarding the flavonoids, the concentrations of 3 and 4 ranged from 309 (± 15) to 1182 (± 16) µM and 73.4 (± 4.8) to 286.5 (± 3.8) µM, respectively, which again were consistent with literature values using HPLC (Ross et al., 2000; De Castro et al., 2006). The concentration of 6 ranged from below LOQ (although detected in all juices) to 39.48 (± 0.83) µM. The concentration of 5 ranged from below LOQ to 34.72 (± 0.55) µM, which agrees with literature values (Wilson et al., 2000; Wanwimolruk & Marquez, 2006). As with the furanocoumarins, the highest concentrations of the flavonoids were in Juice C. The concentrations of 1–4 in Juices A and B were measured previously using an HPLC method (Paine et al., 2006; Won et al., 2010). The concentration of the furanocoumarins (1 and 2) were consistent between methods (within 30 and 7%, respectively), whereas the concentrations of the flavonoids (3 and 4) by HPLC were approximately 2-fold higher than those by UPLC. Reasons for this difference remains unexplored, as a between-laboratory validated study was beyond the scope of this work. The extracts used in this study were analyzed at a 5-fold concentration. In the future, the accuracy of the measurements for analytes present in very low concentrations could be improved by using a more concentrated extract.

Table 3.

Concentrations of analytes 1–6 in grapefruit juices.

Juice Concentrationa (µM) ± SD
Bergamottin (1) DHBb (2) Naringin (3) Narirutin (4) Naringenin (5) Hesperidin (6)
A 11.76 ± 0.16 41.31 ± 0.16 412 ± 15 100.5 ± 4.5 18.89 ± 0.66 NQ
B NDc ND 309 ± 15 73.4 ± 4.8 ND NQ
C 24.73 ± 0.16 89.03 ± 0.17 1182 ± 16 286.5 ± 3.8 34.72 ± 0.55 39.48 ± 0.83
D 7.48 ± 0.17 7.65 ± 0.17 371 ± 15 85.2 ± 4.7 NQd NQ
E 12.26 ± 0.16 11.58 ± 0.16 381 ± 15 103.5 ± 4.5 5.60 ± 0.79 NQ
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1

Each juice was analyzed in triplicate using a 5-fold concentrated extract, and the values reported have been adjusted to reflect the concentration in the juices, not in the extract.

b

6′,7′-dihydroxybergamottin

c

Not detected

d

Indicates that the analyte was detected but was below the limit of quantitation

Wide between-juice variation in the concentrations of each grapefruit juice constituent was observed, as would be expected for natural products, and the concentrations agreed well with values reported in the literature using HPLC. This marked variation between commercially available brands undoubtedly contributes to the large between-study differences in effect size (i.e., change in drug area under the curve [AUC]) associated with clinical grapefruit juice-drug interaction studies (Won et al., 2012). Quantitation of one or more constituents in a given juice would provide a means for between-study comparisons of clinical, as well as in vitro, data. The UPLC methods developed in the current work offer a rapid means for the quantitation of representative constituents. While the methods were applied to grapefruit juices, they should be applicable to other foods, including other fruit juices.

Supplementary Material

Supplement

ACKNOWLEDGMENTS

This research was supported by the National Institutes of Health/National Institute of General Medical Sciences via grant R01 GM077482.

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