Article pubs.acs.org/JAFC
Identification and Quantitation of Furocoumarins in Popularly Consumed Foods in the U.S. Using QuEChERS Extraction Coupled with UPLC-MS/MS Analysis Melissa M. Melough,† Sang Gil Lee,‡ Eunyoung Cho,§ Kijoon Kim,†,∥ Anthony A. Provatas,⊥ Christopher Perkins,⊥ Min Kyung Park,§ Abrar Qureshi,§ and Ock K. Chun*,† †
Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut 06269, United States Department of Family and Consumer Sciences, North Carolina Agricultural and Technical State University, Greensboro, North Carolina 27411, United States § Department of Dermatology, Warren Alpert Medical School of Brown University, Providence, Rhode Island 02903, United States ∥ BOM Institute of Nutrition and Natural Medicine, Seoul National University, Gwanak-gu, Seoul, Republic of Korea 08826 ⊥ Center for Environmental Sciences and Engineering, University of Connecticut, Storrs, Connecticut 06269, United States ‡
ABSTRACT: Furocoumarins are a class of photoactive compounds found in several plant species and may be responsible for the observed association between consumption of citrus products and the risk of skin cancer. Furocoumarin contents of several foods have been reported previously, but no comprehensive database of furocoumarin content of foods is currently available. Therefore, this study aimed to determine the distribution of furocoumarins in popularly consumed foods in the U.S. Samples of three varieties of each of 29 foods known or suspected to contain furocoumarins were purchased, prepared for analysis using a solid phase extraction method, and analyzed using UPLC−MS/MS for the presence of seven major furocoumarins. Most foods measured contained more than one furocoumarin, and some contained all seven of the furocoumarins examined. Total furocoumarin concentration was greatest in fresh parsley (23215 ng/g), grapefruits (21858 ng/g), lime juice (14580 ng/g), grapefruit juice (95341 ng/g), and limes (9151 ng/g). Bergamottin was found in the greatest proportion of foods sampled (23 of 29), followed by bergapten (19 of 29) and 6′7′-dihydroxybergamottin (16 of 29). These measurements will enable more accurate estimation of dietary furocoumarin exposure and will strengthen future epidemiological work investigating the relationships between furocoumarin intake and health outcomes. KEYWORDS: furocoumarins, vegetables, fruits, QuEChERS, UPLC-MS/MS
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squamous cell carcinoma, and basal cell carcinoma.20,21 In support of this apparent relationship, several animal studies have shown that furocoumarins including 8-methoxypsoralen (8-MOP) can be found in the skin following oral administration,11,22−24 leading to the appearance of tumors there.20,24−26 In light of this evidence, it is of high importance to improve current understanding of dietary furocoumarin exposure and associated health consequences. One longstanding obstacle to studying dietary furocoumarin exposure is the lack of a database of furocoumarin contents of foods. Therefore, only a very limited number of published studies have attempted to estimate dietary furocoumarin intake,26−28 and the resulting estimates exhibit considerable variation. These reports relied on previously published furocoumarin measurements in foods, drawing upon data from various laboratories using different procedures, spanning from the 1960s through the 1990s. A limited number of foods have been measured and reported in the literature, and for each of these foods, a limited number of distinct furocoumarin
INTRODUCTION Furocoumarins are a class of organic compounds synthesized by many plants as a natural chemical defense against predators including fungi, bacteria, and insects.1,2 When irradiated with UV light, furocoumarins can undergo photoactivation, putting them into an excited and highly reactive triplet state.3−7 In this state, certain furocoumarins can form adducts with DNA, induce protein denaturation, form cycloadducts with saturated fatty acids, and react with ground state oxygen to form reactive oxygen species that can cause cellular damage.7−13 Furthermore, exposure to furocoumarins in high doses, even in the absence of UV light, has been shown to exert toxic effects in multiple tissue types in mice.14 While certain insect species that feed on plants rich in furocoumarins have efficient routes for the detoxification of furocoumarins, humans lack similar metabolic pathways and are believed to clear furocoumarins relatively inefficiently from the body.15 Therefore, much work has been conducted to investigate the effects of ingested furocoumarins in humans.9,16−19 Two recently published studies that prospectively analyzed participants from the Nurses’ Health Study and the Health Professionals Follow-Up Study found that higher consumption of citrus products, which are known to be particularly rich in furocoumarins, was associated with increased risk of melanoma, © 2017 American Chemical Society
Received: Revised: Accepted: Published: 5049
March 21, 2017 May 2, 2017 June 5, 2017 June 5, 2017 DOI: 10.1021/acs.jafc.7b01279 J. Agric. Food Chem. 2017, 65, 5049−5055
Article
Journal of Agricultural and Food Chemistry
of acetonitrile was added to each sample, and then samples were again vortexed for 5 min at 2500 rpm. Next, 7.5 g of QuEChERS powder (MgSO4/NaOAc) was added to each sample and samples were shaken by hand for 5 min. Samples were centrifuged for 3 min at 4000 rpm and then filtered with a 0.22 μm filter into liquid chromatography vials and spiked with the internal standard, 10 000 ng/mL ketoprofen-d3. Samples were then analyzed using ultraperformance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). UPLC and Mass Spectrometric Conditions. Food and beverage samples were analyzed with a Waters Acquity UPLC coupled with an Acquity TQD tandem mass spectrometer (Waters Co., Milford, MA). An Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column, heated to 25 °C and with a sample injection volume of 5 μL, was utilized for analyte separation. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), and the column conditions are shown in Table 1. Total
compounds have been measured, likely leading to substantial underestimation of furocoumarin content of the diet. No published study has measured and reported furocoumarin concentrations in a wide variety of commonly consumed products. Rather, existing research has largely focused on variability in furocoumarin concentrations within single plant species induced by changed conditions or processing.19,29−36 Other studies have measured only single food items for the purposes of measuring metabolic parameters or validating measurement methods,25,37−41 and others have examined furocoumarins in particular plant oils, supplements, and oral Chinese medicines not popularly consumed in the United States.42−45 Therefore, the objective of this study was to measure the concentrations of seven distinct furocoumarin compounds in foods and beverages commonly consumed in the United States that are believed to contain furocoumarins. In this way, we aim to create a database that can be used for more accurate assessment of dietary furocoumarin exposure.
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Table 1. Column Conditions, Solvent Composition, and Gradient Profile for Ultra-Performance Liquid Chromatography Analysis column
MATERIALS AND METHODS
Selection and Procurement of Foods. Through a review of the literature using PubMed and Web of Science, foods known or suspected to contain furocoumarins were identified. Of the foods identified, 29 distinct foods were selected for analysis, prioritizing foods popularly consumed in the United States and those that were likely to contribute significantly to the population’s total furocoumarin intake. For each of the 29 foods, three samples were purchased, each from a different retailer and/or brand. For foods that are commercially processed or packaged, selection of samples was based on popularity and availability. Foods were purchased between the months of June and September of 2016 from 17 different grocery stores in central and eastern Connecticut. Fresh foods were refrigerated at 4 °C until the time of analysis, which was no more than 2 days after purchase. Frozen foods were kept frozen at −18 °C for no more than 3 months until the time of analysis. Materials and Instrumentation. On the basis of previous work examining furocoumarin content of foods, 2,25,30,41,45−48 seven furocoumarin standards were selected, based on their overall contribution to the total furocoumarin content of individual foods and total diets. Of these standards, six (bergaptol, psoralen, 8-MOP, bergapten, epoxybergamottin, and bergamottin) were purchased from Herboreal Ltd. (Edinburgh, U.K.) and 6′,7′-dihydroxybergamottin (6′,7′-DHB) was purchased from Cayman Chemical (Ann Arbor, MI). Warfarin, used as a surrogate, and ketoprofen-d3, used as an internal standard, were both purchased from Sigma-Aldrich (St. Louis, MO). QuEChERS kits were purchased from Agilent (Palo Alto, CA). A Mettler Toledo XS105 (Columbus, OH) analytical balance was used for the measurement of sample weights. A Fisher Scientific digital vortex mixer (Pittsburgh, PA) was used for sample mixing, and an Eppendorf 5810R centrifuge (Hauppauge, NY) was used for sample centrifugation. Extraction of Furocoumarins. Food samples were analyzed in the form purchased, with the exception of turnips and parsnips, which were both briefly cooked in boiling water for 12 and 7 min, respectively, before analysis. Citrus fruits were peeled prior to analysis in order to obtain the edible flesh, and the skin was also removed from the fresh figs. Frozen items were allowed to thaw to room temperature before analysis. All samples were prepared by thoroughly blending with a kitchen blender and a laboratory homogenizer to obtain a uniform consistency. Agilent’s QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) kit was then used to extract furocoumarins from food and beverage samples. The details of this method have been described in our previous publications.49,50 Briefly, five grams of each sample was spiked with 100 μL of 100 000 ng/mL warfarin and 100 μL of the quality control solution. The quality control solution contained the targeted furocoumarin compounds in concentrations of 100 000 ng/mL. Samples were vortexed at 2500 rpm for 5 min, 10 mL
column temperature solvent A solvent B
Acquity Ultra-Performance Liquid Chromatography BEH C18, 1.7 μm, 2.1 mm × 50 mm 25 °C 0.1% formic acid in water 0.1% formic acid in acetonitrile
time (min) solvent A % flow rate (mL/min)
initial 90.0 0.500
0.10 90.0 0.500
7.50 2.0 0.500
7.80 2.0 0.500
8.00 90.0 0.500
run time was 8 min with a constant flow rate of 0.5 mL/min. The detection and quantification of analytes, surrogate, and internal standard were performed in positive electrospray ionization mode (ESI+) with multiple reaction monitoring (MRM) using the Waters, Inc. IntelliStart software for analyte signal optimization. Statistical analysis for obtaining calibration and quantification results for all compounds were performed using Waters QuanLynx, which was included in the MassLynx software v.4.2. Parameters for the mass spectrometer were set as follows: capillary voltage, 3.8 kV; cone voltage, 45 V; desolvation temperature, 350 °C; source temperature, 125 °C; desolvation gas flow, 400 L/h; collision gas flow, 0.2 mL/min. Warfarin was used as the surrogate standard for sample preparation evaluation, and ketoprofen-d3 was used as the internal standard to account for any instrumentation variations. Multiple-reaction monitoring parameters for the identification and quantification of furocoumarins, internal standard, and surrogate with their retention times and correlation coefficients (R2) of their calibration curves are shown in Table 2.
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RESULTS AND DISCUSSION Using the methods described, the seven targeted furocoumarins were detectable and quantifiable in the food and beverages sampled with a high degree of sensitivity, as indicated by their limits of quantification and detection (Table 3). Average recovery rate of the surrogate from samples was 95.0% (standard deviation of 7.2%). The limits of detection and quantification observed, along with the calculated recoveries, indicate this method’s suitability for the extraction of furocoumarins from the samples analyzed. Mean and standard deviation of furocoumarin concentrations from three samples of each food are displayed in Table 4, and sample MRM chromatograms for parsley and lemon juice are shown in Figures 1 and 2, respectively. In 27 of the 29 foods analyzed, at least one of the targeted furocoumarins was detected, and multiple furocoumarins were detected in 25 5050
DOI: 10.1021/acs.jafc.7b01279 J. Agric. Food Chem. 2017, 65, 5049−5055
Article
Journal of Agricultural and Food Chemistry
Table 2. Multiple-Reaction Monitoring Parameters for the Identification and Quantification of Furocoumarins, Internal Standard, and Surrogate with Their Respective Retention Times and Correlation Coefficients (R2) of Their Calibration Curves at a Concentration Range from 25 ng mL−1 to 10 000 ng mL−1
a
compound
retention time (min)
precursor ion (m/z)
fragment ion (m/z)
dwell (s)
collision (V)
R2
bergaptol psoralen 8-MOPa bergapten 6′,7′-DHBb epoxybergamottin bergamottin ketoprofen-d3 (internal standard) Warfarin (surrogate)
2.32 2.69 2.82 3.11 3.37 4.81 6.13 3.48 3.83
203 187 217 217 373 355 339 258 309
159 131 202 202 203 203 203 212 163
0.075 0.075 0.075 0.075 0.075 0.075 0.075 0.075 0.075
32.0 32.0 28.0 31.0 10.0 10.0 12.0 5.0 21.0
0.9993 0.9995 0.9946 0.9959 0.9986 0.9988 0.9928 N/A 0.9994
8-MOP: 8-methoxypsoralen. b6′,7′-DHB: 6′,7′-dihydroxybergamottin.
Apart from grapefruits and grapefruit juice, scarce data exist for comparison of our measurements to previous reports. One study published in 1986, which examined carrots using an ultrasensitive bioassay combined with HPLC analysis, detected concentrations of 8-MOP and bergapten about 10 times higher than what was found in the current study.31 In addition to these compounds, our analysis detected psoralen, 6′7′-DHB, epoxybergamottin, and bergamottin in carrots. Interestingly, an older report failed to detect any targeted furocoumarins in carrot samples.54 In the current study, celery was found to contain five of our targeted furocoumarins (psoralen, 8-MOP, bergapten, 6′7′-DHB, and bergamottin), three of which were previously reported in concentrations approximately 10 times greater than our levels.2 Variability in natural products is expected and likely explains part of the variation between measurements of the same food item, both between studies and between samples within a study. We noted considerable variation between samples in our study, especially in fresh foods such as clementines, turnips, carrots, and dill. It is also well established that factors including growing conditions, maturity, temperature, harvest season, storage, and processing can affect the levels of furocoumarins in fruits and vegetables.30,32,34 Importantly, our data also indicate that furocoumarin concentrations differ between raw foods and their cooked or processed derivatives. For example, raw carrots had a total furocoumarin concentration 4.9 times higher than carrot juice and more than 90 times higher than cooked canned carrots. Additionally, grapefruit had a total furocoumarin concentration 2.3 times higher than grapefruit juice. Grapefruit juice contained bergaptol in concentrations over 14 times greater than that of grapefruit, whereas grapefruit was much higher in 6′7′-DHB, epoxybergamottin, and bergamottin than grapefruit juice. The database of furocoumarin measurements we compiled represents the most comprehensive information on furocoumarins in popular foods and beverages available. Importantly, the quantitation of seven major furocoumarins does not constitute a complete examination of all furocoumarins, and some previous studies have found small amounts of other furocoumarins in these foods including isopimpinellin and paradisin A and C.7,15,26,30,48 Additionally, due to the variability between and within studies of furocoumarins in food products, it is important that these procedures be repeated to increase accuracy and better understand factors that may impact furocoumarin stability in foods. Furthermore, a thorough understanding of the varying levels of phototoxicity of individual furocoumarins will be required in order to assess
Table 3. Limit of Detection (LOD) and Limit of Quantification (LOQ) for Seven Furocoumarins analyte
LOD (ng/g)
LOQ (ng/g)
bergaptol psoralen 8-MOPa bergapten 6′,7′-DHBb epoxybergamottin bergamottin
5.91 0.36 0.76 0.76 1.42 0.45 0.13
19.69 1.21 2.55 2.54 4.74 1.50 0.44
a
8-MOP: 8-methoxypsoralen. b6′,7′-DHB: 6′,7′-dihydroxybergamottin.
foods. Bergamottin was found in the greatest proportion of foods sampled (23 of 29), followed by bergapten (19 of 29) and 6′7′-DHB (16 of 29). Bergamottin contributed most significantly to the entire sampling of foods (38.5% of total furocoumarin concentration among all foods), followed by 6′7′DHB (19.1%) and bergapten (16.0%). Total furocoumarin concentration, calculated by summing the concentrations of individual compounds, was greatest in fresh parsley (23215 ng/g), grapefruits (21858 ng/g), lime juice (14580 ng/g), grapefruit juice (95341 ng/g), limes (9151 ng/g), and lemon juice (1561 ng/g). Other highly concentrated foods included root vegetables such as celeriac (396 ng/g), parsnips (335 ng/g), and carrots (68 ng/g). Much research related to furocoumarins has been focused on grapefruit and its juice, as these are known to be major dietary contributors of furocoumarins25,30,32,36 and are well studied due to their interference with drug metabolism at cytochrome P450.15,18,40,51 Consistent with previous reports, our data indicate that the principal furocoumarins in both grapefruit and g r a p ef r u i t j ui c e w er e 6 ′7 ′ - D H B a n d b e r g a m o t tin.15,25,26,30,32−34,36,45,49,50,52,53 In our samples, bergaptol and epoxybergamottin were additionally detected in relatively high concentrations in grapefruit. Bergaptol was also detected in grapefruit juice in the current study as well as others.25,30,45,49,50 Concentrations of furocoumarins detected in grapefruit and grapefruit juice in previous reports have shown considerable variation. For example, 6′7′-DHB has been observed in whole grapefruit in concentrations ranging from 1000 ng/g to over 45000 ng/g in previous reports.30,34,49,50 The concentrations of furocoumarins detected in our grapefruit and grapefruit juice samples fell within the range of other reports and often tended to fall at the higher end of the ranges. 5051
DOI: 10.1021/acs.jafc.7b01279 J. Agric. Food Chem. 2017, 65, 5049−5055
5052
ND ND
Other Fruits fig (dried)e fig (fresh)
ND 5.7 (8.1) ND ND ND
740.4 (432.7) ND ND ND ND ND
Vegetables celeriac parsnipf celery carrots carrot juicee cooked carrot (canned)e turnip (fresh)g turnip (frozen)e
Herbs and Spices parsley cilantro dill cuminh corianderh mustard seedh 5386.0 (7461.3) 258.2 (51.1) 9.5 (11.3) 3.9 (0.6) ND ND
1.7 (0.5) 4.0 (3.0) 13.7 (11.4) 0.1 (0.1) ND ND ND ND
13.5 (7.4) 0.1 (0.0)
NDd 14.0 (3.5) 2.0 (1.2) 0.1 (0.1) 0.1 (0.1)
