Article pubs.acs.org/JAFC
Determination of Grayanotoxins in Honey by Liquid Chromatography Tandem Mass Spectrometry Using Dilute-andShoot Sample Preparation Approach Muammer Kaplan,* Elmas O. Olgun, and Oznur Karaoglu Food Institute, TUBITAK Marmara Research Centre, P.O. Box 21, 41470 Gebze, Kocaeli, Turkey ABSTRACT: A simple and rugged method of analysis for grayanotoxins I and III in honey using liquid chromatography triple quadrupole mass spectrometry with electrospray ionization was developed. This paper describes the first LC−MS/MS method for the quantitation and confirmation of the grayanotoxins in honey using “dilute-and-shoot” sample preparation approach. Honey sample was diluted 10-fold in methanol−water (1:4 v/v) prior to analysis. Chromatographic separation was achieved on a reversed phase HPLC column using a water−methanol gradient with 0.1% acetic acid. The method was fully validated for quantitative purposes. Overall recoveries, selectivity, overall intraday and interday repeatability, decision limit, and detection capability of the analytes was determined. The matrix effects, ruggedness, and analyte stability in standards and samples were studied. Ten real honey samples were successfully analyzed using the developed method. All the samples were found to contain residues of GTXs ranging from 0.1 to 39 mg/kg. KEYWORDS: grayanotoxin, mad honey, LC−MS/MS, dilute-and-shoot
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methods11 were used for the analysis of grayanotoxins in plant materials and honey. Grayanotoxins in honey was carried out by gas chromatography (GC) with stainless steel capillary columns following trimethylsilyl derivatization.12 A two-dimensional TLC was used for the qualitative analysis of GTXs in plant materials, gastrointestinal contents, and feces at a concentration of 0.2 μg/g.13 Quantitative determination of GTXs in biological samples was performed using a liquid chromatography combined with an ion trap tandem mass spectrometry.14 In a recent study, liquid chromatography coupled to high-resolution mass spectrometry was used to analyze plant toxins including GTX III at 50 μg/kg detection level in honey.15 To the best of our knowledge, there is only one method published in the literature using liquid chromatography tandem mass spectrometry (LC−MS/MS) for determination of GTXs in honey.16 In this method, honey samples were mixed with methanol and loaded onto a C18 SPE cartridge for cleanup, and the filtrate was diluted with water and analyzed. However, the method is relatively expensive and timeconsuming due to SPE sample preparation step. SPE cleanup step is also prone to errors in sample preparation. Therefore, there is a great need for low cost, simple, and high-throughput analytical procedures for prompt detection and quantitative analysis of toxic GTXs that may coexist in honey. Recently, LC−MS/MS has become the most popular analytical tool for accurate and reliable determination of different residues and contaminants because of high specificity, selectivity, and sensitivity.17−19 In this study, we present the first triple quadrupole LC−MS/ MS method developed and optimized for simultaneous
INTRODUCTION Grayanotoxins (GTXs) are a group of chemical compounds found naturally in plants from the Ericaceous family which includes Rhododendrons. There are over 60 grayanotoxinrelated compounds.1 From these, grayanotoxins I and III are reported to be the most toxic. 2 Grayanotoxins cause intoxication by increasing the membrane permeability to sodium ions.3 Grayanotoxin-containing Rhodendron species are grown naturally in different countries including United States, Brazil Spain, Portugal, Turkey, Nepal, and Japan.4 Toxic Rhododendron species, Rhododendron ponticum, native to the Black Sea region of Turkey, is commonly used as folk medicine to treat various health problems including colds, edema, and pains.5 Honey produced from pollen and nectar of these flowers is known as “mad honey”. Mad honey is also used as folk medicine to treat gastritis, ulcers, hyperglycemia, hypertension, arthritis, sexual impotence, and performance enhancement.3,6 It is largely produced in the Black Sea region and marketed as folk medicine. The consumption of “mad honey” causes intoxications in humans, and the severity of symptoms may vary from mild to life-threatening depending on the amount consumed.3 Grayanotoxin poisoning remains to be one of the most frequently encountered food toxicant in Turkey.7 Cases of GTX poisoning have also been reported from Austria, Korea, Nepal, and a few other countries.8 Moreover, recently, concerns about poisoning by mad honey were raised by the German Federal Institute for Risk Assessment (BfR- Bundesinstitut für Risikobewertung), calling for more systematic control of honey from specific regions.9 Because of GTXs’ unseen dangerous potential for public health, there is a great need for the development of rapid and reliable methods of analysis enabling the detection of GTXs in honey. In the scientific literature, there are only very few analytical methods published for grayanotoxin determination in honey samples. Thin-layer chromatography10 and direct spectrometric © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5485
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determination of grayanotoxins (GTX I and GTX III) in honey samples. The proposed method is rapid and very sensitive, using a methanol−water extraction for sample preparation. The avoidance of a cleanup step together with the optimized chromatography simplified the sample pretreatment and reduced the laboratory costs and the overall analysis time compared to prior methodology. The developed method was fully validated for grayanotoxin analysis in honey, and it is used routinely in our laboratory.
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Table 1. Optimized MS/MS Conditions Used for the Determination of Grayanotoxins precursor ion (m/z)
fragment ions (m/z)a
declustering potential (V)
collision energy (eV)
grayanotoxin I
435
Q: 375 C: 357 C: 91
111 111 111
29 35 109
grayanotoxin III
335
Q: 299 C: 91 C: 316
81 81 81
15 75 9
analyte
MATERIALS AND METHODS
Reagents and Chemicals. Glacial acetic acid (AA) (Emprove, 100%) and methanol (MeOH) (Lichrosolve purity ≥99.9) were obtained from Merck (Darmstadt, Germany). Grayanotoxin I was purchased from AnalytiCon Discovery GmbH, Potsdam, Germany, and grayanotoxin III was provided from Sigma-Aldrich. Pure water was obtained using a Milli-Q Plus system (Millipore, Billerica, MA). Standard Solutions. Individual grayanotoxin stock solutions (1000 mg/kg) were prepared in methanol. A mixed intermediate standard solution (10 mg/kg) was obtained by dilution of GTX I and GTX III stock solutions in methanol. Working standard mixed solutions (0.01, 0.1, 0.5, 1, and 2 mg/kg) were prepared by diluting intermediate solution in MeOH−water (1:4 v/v). Stock and intermediate standard solutions were kept in amber flasks at 4 °C. They were stable more than 2 months under these conditions. Working standard solutions were prepared fresh daily. Sample Preparation. Honey samples, upon arrival at our laboratory, were homogenized with a wooden spoon and were kept at room temperature (+22 ± 4 °C) until analysis. An aliquot of approximately 0.25 g of honey sample was weighed into a 15 mL glass tube. Then 2.5 mL of MeOH−water (1:4 v/v) with 0.1% AA was added, and the sample was vortexed vigorously for 1 min. The extract was filtered through a 0.45 μm filter prior to LC−MS/MS analysis. Honey samples obtained from local market were analyzed, and the ones containing no detectable amount of grayanotoxins were used as control blanks. Real mad honey samples were randomly acquired in local honey producers in different provinces from the Black Sea Region in Turkey between July and September 2013. Of the samples, five were from Duzce, two from Sinop, one from Rize, and two from Tokat province. The honey samples (about 500 g) were stored in their original packaging at room temperature until analysis. LC−MS/MS Analysis. The HPLC system used for chromatographic analyses consisted of a binary pump (Shimadzu UFLC LC20AD model), Shimadzu automatic injector (Auto Sampler SIL-20A HT model), and a column oven (CTO-20AC). Analytical columns, Kinetex 100 mm × 2.1 mm, 2.6 μm (Phenomenex, Torrance, USA), and Waters X Terra C18 150 mm × 2.1 mm, 5 μm (Waters, Milford, MA) were used. Chromatographic separation of grayanotoxins was performed using a Waters X Terra C18 column. The method used a binary gradient with mobile phases consisted of water (A) and methanol (B) containing 0.1% acetic acid. Optimized separation of all analytes was obtained using a gradient program as follows: 10−80% solution B in10 min, kept at 80% solution B for 10 min. The column was then returned back to its initial gradient conditions. Finally, the system was equilibrated to initial mobile phase conditions of 10% B and 90% A for 10 min before the next injection. A 10 μL sample was injected into the column maintained at 40 °C. The flow rate was 0.3 mL/min. The mass spectrometer used was an API 4000 Q-TRAP (Applied Biosystems, Foster City, CA) operating in the triple quadrupole mode. The MS/MS parameters were as follows: curtain gas 20 mL/min, exit potential 10 V, and drying and nebulizing gases (GS1 and GS2) were set to 50 mL/min. The ion spray voltage and turbo spray temperature were set to 5500 V and 550 °C, respectively. The mass spectra were acquired using positive ion ESI mode. Three transitions were monitored for each analyte. The selected molecular ion and optimized collision voltages of product ions used for quantification and confirmation are summarized in Table 1. For data acquisition and processing, Analyst version 1.6 (Applied Biosystems)
a
Q, transition used for quantification; C, transition used for confirmation.
was used. Quantification was made by comparison with a five-point calibration (0.01, 0.1, 0.5, 1, and 2 mg/kg). Validation Study. The EU Decision 2002/657/EC20 criteria were used for the validation of the analytical method developed for determination of GTX I and GTX III in honey. The parameters studied during validation of the method were selectivity, sensitivity, linearity, accuracy, and precision (intraday and interday reproducibility). Identification of grayanotoxins was achieved using retention time data and relative ion intensities of each compound. The Decision 2002/657/EC requires at least four identification points (IPs) for unequivocal confirmation of an analyte. Thus, 5.5 IPs were earned for each grayanotoxin by monitoring one precursor ion and the three most abundant fragment ions. The Decision Limits (CCα) and Detection Capability (CCβ). The decision limit and detection capability that are the new parameters introduced by Commission Decision 2002/657/EC aimed at replacing the LOD and LOQ. In Decision 2002/657/EC, the CCα is described as “the limit at and above which it can be concluded with an error probability of α that a sample is non-compliant”. CCβ is defined as “the smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β”.20 Ten blank honey samples were analyzed for the calculation of CCα and CCβ values. The CCα was calculated as three times the signal-to-noise ratio determined at the elution time of the analyte. The results obtained from the analyses of ten honey blanks spiked at CCα concentration level were used for the calculation of CCβ. The value obtained for CCα was added up to a value of 1.64 and multiplied by the corresponding standard error. Stability. The stability of the grayanotoxins in both standard solution and honey sample was assessed. Stability in solution was evaluated for individual intermediate solutions prepared in methanol at 10 mg/L concentration level. The solutions were stored at room temperature under daylight and at −20 °C and +4 °C in darkness. Evaluation study was performed by comparing the original stored solution with a working solution prepared fresh daily. The stability of the GTXs in honey matrix was performed using mad honey sample containing residues of GTX I (1.5 mg/kg) and GTX III (0.25 mg/kg). Matrix Effect. Matrix effects were evaluated using standard solvent and matrix matched calibration curves. The calibration curves were established using peak areas of the SRM transitions chosen for quantification. The slopes of the curves obtained with sample matrix and pure solvent were compared for the assessment of matrix-induced effects. Ruggedness. Ruggedness of the method was extensively investigated. As the dilute and shoot approach involved injection of diluted honey samples directly to the LC−MS/MS system, it is possible to face column problems, accuracy, and precision loss and reduction in detection sensitivity. Therefore, the method ruggedness was studied using calibration standards and real mad honey sample and honey blanks prepared with honey purchased from a local market. Moreover, we have investigated the likelihood of sample carryover from one sample to the other. 5486
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Figure 1. SRM chromatograms obtained from a honey blank (A) and honey spiked with 0.01 mg/kg of GTX I and GTX III (B) using dilute-andshoot sample preparation.
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RESULTS AND DISCUSSION LC and MS Conditions. The initial stage of the method development was optimization of the mass spectrometer conditions for the detection of GTXs. MS/MS parameters were optimized by infusing GTX standard solutions individu-
ally. In the course of instrument tuning both positive (ESI+) and negative (ESI−) ionization modes were tested, and the mobile phase composition was compared. Satisfactory results were obtained only in the positive ionization mode. For each analyte, GTX I and GTX III, three most intensive transitions 5487
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0.01, 0.5, 1, and 2 mg/kg. The results obtained for the method accuracy and precision (Table 2) conform to the values given in
were chosen in this study. The most intensive fragment ion was selected for the quantitative determination and the remaining two ions were used for the confirmation of GTXs (Table 1). Two LC columns with different particle sizes and lengths were assessed for the optimization of chromatographic separation and the signal intensity of grayanotoxins. Both Waters X Terra (150 mm × 2.1 mm, 5 μm) and Kinetex (100 mm × 2.1 mm, 2.6 μm) columns showed similar separation efficiency when MeOH−water solution containing 0.1% AA was used as the mobile phase. X Terra column produced slightly better peak intensities and therefore was chosen for the validation study. Under optimized gradient conditions good separation of GTXs in 7 min was achieved (Figure 1). Development of Sample Pretreatment. Grayanotoxins are often extracted from the suspected products by classic extraction methods using methanol or dichloromethane.13 These extraction solvents co-extract a range of other substances along with grayanotoxins. The ionization efficiency of the analytes is affected by the co-extracts especially when working with mass spectrometry in electrospray ionization mode. Therefore, to obtain a robust LC−MS/MS method, it is crucial to remove or minimize their presence. In this study, to overcome the matrix effects, the samples were prepared using dilute-and-shoot approach. For sample dilution, various solvents including water, MeOH, and MeOH−water solutions (1:2 and 1:4 v/v) were tested. The dilution solvents were acidified using acetic acid (0.1, 0.5 and 0.1%, v/v) in order to obtain the maximum signal response for the analytes of interest. Of these, MeOH−water solution (1:4 v/v) containing 0.1% AA gave the best results for both analytes and used for the dilution of honey samples. Furthermore, the efficacy of sample dilutions was examined. To maximize robustness of the procedure, the amount of honey injected per analysis was minimized. For this purpose, the signals obtained after sequential dilution of honey samples (1:4, 1:10, and 1:20) were compared with the signals obtained from the standard solutions prepared in pure solvent. Equally good peak responses were obtained for all dilutions, however, to reduce the instrument cleaning cycle and to meet the required method sensitivity, a 1:10 dilution with 10 μL injection volumes was selected and used for the method validation study. A typical SRM ion chromatogram obtained from a blank honey spiked with 0.01 mg/kg GTX I and GTX III is shown in Figure 1. The sample was prepared by diluting 0.25 g of honey in 2.5 mL of MeOH−water (1:4 v/v) with 0.1% AA. A 10 μL aliquot of this solution was injected. Method Validation. Selectivity. The method selectivity was assessed by duplicate analysis of seven honey blanks from different floral sources (pine, chestnut, and flower honey). No peaks of interfering compounds were observed in the blank honey samples at the retention time of the target GTXs. Typical chromatograms obtained from the analysis of blank and spiked honey samples are given in Figure 1. Linearity. Linearity was evaluated from the calibration curves by triplicate analyses of blank honey samples fortified with the analytes at five (0.01, 0.1, 0.5, 1, and 2 mg/kg) concentration levels. Linearity was expressed as the coefficient of linear correlation (r) and from the slope of the calibration curve. The correlation coefficients obtained over the concentration range studied was higher than 0.998 for both analytes, GTX I and GTX III. Accuracy and Precision. The method accuracy and precision were assessed through recovery measurements analyzing honey blanks fortified at concentration levels of
Table 2. Method Performance in Spiked Honey Samples at Concentration Levels Ranged between 0.01 and 2 mg/kg spiked level (mg/kg)
average recovery (%) (n = 18)
intraday precision (RSD%, n = 6)
interday precision (RSD%, n = 18)
grayanotoxin I
0.01 0.10 0.50 1.00 2.00
86.0 95.6 98.0 102.0 105.0
8.3 7.7 5.9 4.5 3.9
13.8 12.4 12.1 9.3 7.2
grayanotoxin III
0.01 0.10 0.50 1.00 2.00
87.2 98.0 97.0 103.7 95.5
8.4 7.0 6.1 5.8 3.7
15.9 14.7 13.3 10.1 9.5
analytes
Decision 2002/657/EC. Thus, the mean accuracy values obtained in the recovery tests were between 86 and 105%. The precision of the method was determined in two stages: intraday and interday repeatability. The repeatability of the results was expressed as RSDs calculated from six replicates analyzed using the same equipment with the same operator. The interday repeatability was expressed by the RSD of the results obtained from 18 measurements carried out on three different days, six analyses/day, using the same equipment by the same operator. The intraday and interday RSD values calculated for grayanotoxins analyzed using the developed method ranged between 3.7 and 8.3% and 7.2 and 15.9%, respectively (Table 2). Decision Limits and Detection Capabilities. The decision limit (CCα) and detection capability (CCβ) values were calculated as 12 and 17 μg/kg for GTX I and 10 and 13 μg/kg for GTX III, respectively. Then, a preliminary experiment was conducted to check if all compounds were detected when spiked at their CCα level (Figure 1). As can be seen from Figure 1, very satisfactory S/N ratios were obtained for all analytes at CCα level. Stability. The stability of GTX I and GTX III was assessed after storage in methanol solutions at room temperature under daylight and at −20 °C and +4 °C in darkness. Under these conditions, individual grayanotoxin solutions were stable following 8 weeks of storage. Real honey sample containing GTX I and GTX III at concentrations of 0.2 and 1.5 mg/kg, respectively, was stored at room temperature over 12 months period for stability study. GTXs were found to be very stable in honey matrix after 12 months of storage. Matrix Effects. Evaluation of matrix effect is important during validation of analytical methods using the LC−MS/MS technique. Matrix interferences may affect the ionization efficiency of the analytes in ESI source. The degree of ion suppression or signal enhancement were evaluated using the calibration curves established with and without matrix. The slopes of these curves were used for the assessment of matrixinduced effects using the following formula: matrix effect (ME) = 1 − (amatrix/astandard) × 100, where amatrix and astandard are the slopes for the calibration curves obtained in pure standard and matrix-matched honey, respectively. The matrix-matched curves were constructed using honey blanks (0.1 g/mL matrix 5488
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equivalent) spiked with GTX I and GTX III (0.1, 0.5, 1, and 2 mg/kg) prepared in MeOH−water solution with 0.1% AA. Figure 2 shows the calibration curves for GTX I and GTXIII.
Figure 3. Plots of peak areas against injection number obtained from the sequential analysis of GTX I and GTX III in honey using diluteand-shoot sample preparation methodology.
that there was no contamination or carryover from one sample to another. Grayanotoxin Residues in Real Samples. The developed method was used for the analysis of 10 mad honey samples purchased from local honey producers in four different provinces of the Black Sea region. Table 3 shows the results Figure 2. Calibration curves obtained for GTX I (A) and GTX III (B) prepared in pure solvent and fortified honey.
Table 3. Concentrations of GTX I and GTX III Detected in Honey Samples Collected from Different Provinces in the Black Sea Region, Turkey
The upper curve represents the analyte signals in pure solvent, whereas the lower one shows the signals in a honey spiked with GTXs. The calibration curves prepared in the spiked samples have lower slope, indicating negligible ion suppression in GTX I ( 91
335 > 316
0.01 0.10 0.50 1.50 2.00
100:16:89 100:12:64 100:14:62 100:13:64 100:12:64
GTX I
435 > 375
435 > 357
435 > 91
0.01 0.10 0.50 1.50 2.00
100:23:2 100:32:2 100:29:2 100:23:2 100:22:2
(13) Holstege, D. M.; Francis, T.; Puschner, B.; Booth, M. C.; Galey, F. D. Multiresidue screen for cardiotoxins by two-dimensional thinlayer chromatography. J. Agric. Food Chem. 2000, 48, 60−64. (14) Holstege, D. M.; Puschner, B.; Le, T. Determination of grayanotoxins in biological samples by LC−MS/MS. J. Agric. Food Chem. 2001, 49, 1648−1651. (15) Mol, H. G. J.; Van Dam, R. C. J.; Zomer, P.; Mulder, P. P. J. Screening of plant toxins in food, feed and botanicals using full-scan high-resolution (Orbitrap) mass spectrometry. Food Addit. Contam., Part A 2011, 28, 1405−1423. (16) Lee, S.; Choi, Y.; Lee, K.; Cho, T.; Kim, J.; Son, Y.; Park, J.; Im, S.; Choi, H.; Lee, D. Determination and monitoring of grayanotoxins in honey using LC−MS/MS. Korean J. Food Sci. Technol. 2008, 40, 8− 14. (17) Malik, A. K.; Blasco, C.; Picó, Y. Liquid chromatography−mass spectrometry in food safety. J. Chromatogr., A 2010, 1217, 4018−4040. (18) Lohne, J. J.; Andersen, W. C.; Casey, C. R.; Turnipseed, S. B.; Madson, M. R. Analysis of stilbene residues in aquacultured finfish using LC−MS/MS. J. Agric. Food Chem. 2013, 61, 2364−2370. (19) Kaplan, M.; Olgun, E. O.; Karaoglu, O. A rapid and simple method for simultaneous determination of triphenylmethane dye residues in rainbow trouts by liquid chromatography tandem mass spectrometry. J. Chromatogr., A 2014, 1349, 37−43. (20) European Commission Decision 2002/657/EC. Off. J. Eur. Communities 2002, L221, 8−36.
throughput methodology and well suited for use in the routine laboratory.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +90-262-6773241. Fax: +90-262-6432109. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
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