Article Cite This: J. Agric. Food Chem. 2018, 66, 1935−1940
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Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by Liquid Chromatography−Quadrupole Time-of-Flight−Mass Spectrometry and Liquid Chromatography−Tandem Mass Spectrometry Taeik Hwang, Eunyoung Noh, Ji Hye Jeong, Sung-Kwan Park, Dongwoo Shin, and Hoil Kang* Division of Advanced Analysis, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju-si, Chungcheongbuk-do 28159, Republic of Korea S Supporting Information *
ABSTRACT: A sensitive and specific high-performance liquid chromatography−quadrupole time-of-flight−mass spectrometry (LC−QTOF−MS) method combined with liquid chromatography−tandem mass spectrometry (LC−MS/MS) was developed for the determination of grayanotoxins I and III in dietary supplements and homemade wine. Grayanotoxins I and III were successfully extracted using solid-phase extraction cartridges, characterized by LC−QTOF−MS, and quantitated by LC−MS/ MS. The LC−MS/MS calibration curves were linear over concentrations of 10−100 ng/mL (grayanotoxin I) and 20−400 ng/ mL (grayanotoxin III). Grayanotoxins I and III were found in 51 foodstuffs, with quantitative determinations revealing total toxin concentrations of 18.4−101 000 ng/mL (grayanotoxin I) and 15.3−56 000 ng/mL (grayanotoxin III). The potential of the validated method was demonstrated by successful quantitative analysis of grayanotoxins I and III in dietary supplements and homemade wine; the method appears suitable for the routine detection of grayanotoxins I and III from Rhododendron brachycarpum. KEYWORDS: grayanotoxin, LC−MS/MS, homemade wine, Rhododendron brachycarpum, LC−QTOF−MS
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INTRODUCTION Grayanotoxins are a group of chemical compounds found naturally in the plant family Ericaceous, which includes the Rhododendron species. Over 60 grayanotoxin-related compounds exist,1 of which grayanotoxin I (1) and grayanotoxin III (2) (Figure 1) are the most toxic.2 The consumption of
Grayanotoxin-containing Rhododendron species grow naturally in various countries, including the United States, Brazil, Spain, Portugal, Turkey, Nepal, and Japan.9 Interestingly, the toxic Rhododendron species, Rhododendron ponticum, which is native to the Black Sea region of Turkey, is commonly used as a folk medicine to treat various health problems, including colds, edemas, and general aches and pains.10 In addition, mad honey, which is produced from the pollen and nectar of these flowers, is also used as a folk medicine to treat gastritis, ulcers, hyperglycemia, hypertension, arthritis, and sexual impotence.11 However, as indicated above, the consumption of such products can cause intoxication in humans, and the severity of symptoms may vary from mild to life-threatening depending upon the quantity consumed. Indeed, grayanotoxin poisoning remains one of the most frequently encountered causes of food intoxication in Turkey, although cases have also been reported in Austria, Korea, and Nepal.12−14 Recently, concerns regarding poisoning from Rhododendron species were raised by the German Federal Institute for Risk Assessment, which called for a greater systematic control of honey from specific regions. Thus, as a result of the unseen danger to public health from grayanotoxins, the development of rapid and reliable analytical methods for the detection of grayanotoxins in Rhododendron-containing dietary supplements
Figure 1. Structures of grayanotoxin I (1) and grayanotoxin III (2).
grayanotoxins contained in mad honey, leaves, nectar, and flowers causes grayanotoxin poisoning as a result of the widespread belief in the medicinal properties of these plantbased products. Grayanotoxin poisoning is often caused by misidentification of the plant species. More specifically, the ingestion of flowers, flower juice, wine, or boiled extracts of Rhododendron schlippenbachii and Rhododendron brachycarpum has led to grayanotoxin poisoning in Korea,3−6 because R. schlippenbachii (which contains grayanotoxins) seemingly resembles Rhododendron mucronulatum. Indeed, R. mucronulatum flowers have been usually applied for preparing vegetable pancakes, tea, and wine, in addition to being used as a herbal medicine for the treatment of the symptoms of the common cold and as a diuretic.7 Grayanotoxins cause intoxication by increasing the membrane permeability to sodium ions.8 © 2018 American Chemical Society
Received: Revised: Accepted: Published: 1935
November 6, 2017 January 29, 2018 February 13, 2018 February 13, 2018 DOI: 10.1021/acs.jafc.7b05054 J. Agric. Food Chem. 2018, 66, 1935−1940
Article
Journal of Agricultural and Food Chemistry
Figure 2. LC−QTOF−MS/MS spectra of (A) grayanotoxin I and (B) grayanotoxin III. grade] and acetonitrile (HPLC grade) were obtained from Merck (Darmstadt, Germany). Preparation of the Standard Solutions. Standard stock solutions were prepared by dissolving grayanotoxin I or III (10 mg) in methanol (10 mL). Working standard solutions were prepared daily by diluting the appropriate volumes of grayanotoxin I (10, 20, 40, 60, 80, and 100 ng/mL) and grayanotoxin III (20, 40, 80, 100, 200, and 400 ng/mL) stock solutions in methanol. Sample Preparation. A total of 51 dietary supplement and homemade wine samples made from R. brachycarpum or R. mucronulatum flowers were purchased from a local market and from four different provinces of Korea. Samples consisted of both solids (two of the powder type, four of the capsule type, seven of the pill type, nine of the tablet type, and four of the tea type), and liquid (25 types) products. The contents from capsules, tablets, pills, powder, and liquid products were vortex-mixed briefly to obtain a uniform sample. For example, for capsules, the ingredients present inside were used, and tablets and pills were broken into pieces and ground to obtain a powder, so that all of the samples were homogenized. To determine the grayanotoxin contents present in the dietary supplements, the following sample preparation method was employed to obtain analytical samples for injection. A portion of the liquid or solid sample (1 g) was dissolved in methanol (10 or 50 mL, respectively). After mixing for 30 min under ultrasonication, the resulting solution was employed for the SPE cleanup step. Specifically, Oasis HLB LP cartridges, 6 cm3, 500 mg (Waters, Milford, MA, U.S.A.), were conditioned with methanol (5 mL) and distilled water (5 mL) prior to sample loading. After a portion (2 mL) of the desired sample was loaded onto the SPE cartridge, elution of grayanotoxins I and III was carried out using methanol (2 × 5 mL), and the eluted fractions were made up to 20 mL in a volumetric flask using methanol. Finally, the sample extracts were filtered using a 0.2 μm membrane filter (VWR, Darmstadt, Germany) prior to analysis. Ultra Performance Liquid Chromatography (UPLC)−QTOF− MS Analysis. An ACQUITY UPLC system (Waters, Milford, MA, U.S.A.) was coupled with a QTOF Premier hybrid quadrupole
and homemade wine is of particular importance. However, to date, only a few analytical methods have been reported for the determination of grayanotoxin in honey samples and plant materials, such as thin-layer chromatography,15−17 direct spectrometric methods,18 gas chromatography (GC) following trimethylsilyl derivatization,19 and high-performance liquid chromatography−tandem mass spectrometry.20−23 However, no analytical methods have been described for the determination of grayanotoxins in dietary supplements and homemade wine. Herein, we present the development and optimization of the first liquid chromatography−quadrupole time-of-flight−mass spectrometry (LC−QTOF−MS) and triple quadrupole liquid chromatography−tandem mass spectrometry (LC−MS/MS) method for the simultaneous determination of grayanotoxins I and III in dietary supplements and homemade wine. The proposed method employs a solid-phase extraction (SPE) step for sample cleanup, and therefore, it is expected to exhibit excellent sensitivity. We propose that these chromatographic methods will allow for accurate and reliable determination of such compounds, and the method was fully validated for grayanotoxins I and III analysis in dietary supplements and homemade wine for routine laboratory use.
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MATERIALS AND METHODS
Chemicals and Reagents. Grayanotoxin I was kindly provided by the Kangwon National University (Kangwondo, Korea). Grayanotoxin III was purchased from the National Development Institute of Korean Medicine (NIKOM). The structures of grayanotoxins I and III are shown in Figure 1. Formic acid (analytical grade) and acetic acid (analytical grade) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Methanol [high-performance liquid chromatography (HPLC) 1936
DOI: 10.1021/acs.jafc.7b05054 J. Agric. Food Chem. 2018, 66, 1935−1940
Article
Journal of Agricultural and Food Chemistry
Figure 3. MS/MS fragmentation pathways of [M + Na]+ ion for (A) grayanotoxin I and (B) grayanotoxin III. LC−MS/MS Analysis. Analytes were separated using an Agilent 1200 Series HPLC instrument (Agilent Technologies, Palo Alto, CA, U.S.A.) consisting of a quaternary pump, a vacuum degasser, and a thermostated autosampler. Sample extracts (2 μL) were injected onto a 2.0 × 100 mm inner diameter, 3 μm, Capcell Pak C18 MGII column (Shiseido, Tokyo, Japan). The mobile phase was composed of 0.1% formic acid in distilled water (solvent A) and 0.1% formic acid in methanol (solvent B). A flow rate of 0.25 mL min−1 was employed, and the gradient elution profile was as follows: 0−4 min, 5−40% B; 4− 6.5 min, 40−70% B; 6.5−7 min, 70% B; 7−7.5 min, 70−95% B; 7.5−8 min, 95% B; 8−8.1 min, 95−5% B; and 8.1−10 min, 5% B. The analytes were detected using a QTrap5500 linear ion trap quadrupole mass spectrometer (AB SCIEX, Concord, Ontario, Canada). The TurboIonSpray source was employed in the positive-ion mode with a source temperature of 450 °C, an ion voltage of 5500 V, and a curtain gas pressure of 30 psi. To identify the target compounds, the precursors and fragmentation patterns for each analyte were optimized through a series of continuous infusion experiments. Initially, >2 multiple reaction monitoring (MRM) transitions were monitored for each analyte, and two transitions were selected in the final method, one for quantitation and the other for confirmation. To improve the sensitivity and selectivity of the system, the target compounds were measured using the time-scheduled MRM mode. Analytical instrument control, data acquisition, and data treatment were performed using the Analyst 1.5.1 software.
orthogonal acceleration time-of-flight mass spectrometer (Waters Micromass, Manchester, U.K.) equipped with an orthogonal Z-spray electrospray ionization (ESI) interface operated in both positive- and negative-ion modes. LC separation was achieved using a 100 × 2.1 mm inner diameter, 1.7 μm, Waters ACQUITY BEH C18 column maintained at 40 °C. The mobile phase was composed of both distilled water (solvent A) and methanol (solvent B) containing 0.1% formic acid. The gradient elution profile was employed as follows: 0−3 min, 15% B; 3.0−3.1 min, 15−90% B; 3.1−8.0 min, 90% B; 8.0−8.1 min, 90−15% B; and 8.1−10 min, 15% B. A mobile phase flow rate of 0.3 mL min−1 was employed along with a sample injection volume of 4 μL. Other acquisition conditions were as follows: capillary voltage, 3.0 kV; desolvation temperature, 600 °C; desolvation gas flow, 800 L h−1; and cone gas flow, 30 L h−1. Lock-mass and mass calibration of QTOF−MS were achieved using leucine−enkephalin (m/z 556.2771 [M + H]+ and m/z 554.2615 [M − H]−) and a sodium formate calibration solution (0.1 M sodium hydroxide, 10% formic acid, and 80% acetonitrile). The mass resolution was in the range of 10 000− 20 000 at m/z 50−1000. Nitrogen gas was used as the collision gas in a hexapole collision cell. Two types of acquisition, namely, MS/MS and MSE, were performed. For the MS/MS experiments, the cone voltage and collision energy ramp were optimized individually for each compound. For the MSE experiments, two acquisition functions with different collision energies were created, namely, the low-energy (LE) and high-energy (HE) functions. The compound-dependent optimized cone voltage and collision energy ramp determined for the MS/ MS experiments were also employed. 1937
DOI: 10.1021/acs.jafc.7b05054 J. Agric. Food Chem. 2018, 66, 1935−1940
Article
Journal of Agricultural and Food Chemistry
Figure 4. Extracted ion chromatograms of the grayanotoxins: blank samples of (A) grayanotoxin I and (B) grayanotoxin III and blank samples spiked with 100 ng/mL of (C) grayanotoxin I and (D) grayanotoxin III.
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RESULTS AND DISCUSSION LC−QTOF−MS/MS Analysis and Proposed Fragmentation Patterns. To determine the sensitivity and selectivity of targets, standard solutions were infused directly into the ESI source that is operated in the positive-ion mode by varying the different MS parameters. MS conditions were optimized and investigated for the quantitation of both grayanotoxins I and III, for which the MS/MS product ion spectra are shown in Figure 2. In the case of grayanotoxin I, a prominent sodium adduct of the molecular ion [M + Na] + was observed at m/z 435.22 in the Q1 full-scan mass spectrum, although the most abundant signal was observed at m/z 375.21, which corresponded to [M + Na − CH3COOH − H2O]+. More specifically, grayanotoxin I fragmented readily by loss of an acetyl group and formed a more stable structure, which resembled grayanotoxin III (Figure 3A). We therefore selected the m/z 435.22 → 375.21 ion transition for grayanotoxin I, although it should be noted that an additional product ion at m/z 357.21 was also formed by the loss of another water molecule to afford [M + Na − CH3COOH − 2H2O]+. Similarly, the sodium adduct of the molecular ion [M + Na]+ was observed at m/z 393.22 for grayanotoxin III in the Q1 full-scan mass spectrum. Successive loss of water produced signals at m/z 375.21, 357.20, and 317.21, which corresponded to [M + H − H2O]+, [M + H − 2H2O]+, and [M + H − 3H2O − rearrangement (C13−C16)]+, respectively (Figure 3B). It was assumed that the loss of H2O renders the base ion more stable, with the number of H2O molecules lost being dependent upon the number of hydroxyl substituents present in the structure. Additional product ions at
m/z 299.20, 289.17, and 246.16 corresponded to the stepwise loss of H2O, C2H4, and C2H3O from the m/z 357.20 ion. LC−MS/MS Analysis. The initial stage of method development for LC−MS/MS analysis involved optimization of the mass spectrometer conditions for the detection of grayanotoxins I and III. For this purpose, the MS/MS parameters were optimized by individually infusing standard solutions of grayanotoxin. Both positive (ESI+) and negative (ESI−) ionization modes were tested, although only the positive mode gave satisfactory results. For each analyte (i.e., grayanotoxins I and III), the two most intense transitions were selected for quantitative determination of the components, and the remaining ions were used for the confirmation of grayanotoxins I and III. As the grayanotoxin III structure fragmented readily to lose two molecules of H2O, this precursor ion was selected for detection by LC−TQ/MS. Therefore, the precursor ion of m/z 335.0 was selected for the quantitation of grayanotoxin III, because the optimized MS/MS patterns of the LC−MS/MS and LC−QTOF−MS techniques produced different precursor and product ions. Under the optimized solvent gradient conditions, good separation of grayanotoxins I and III was achieved within 4 min.
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METHOD VALIDATION Specificity. Method selectivity was then assessed by duplicate analysis of blank samples from different dietary supplements, and no peaks corresponding to interfering compounds were observed in the blank samples close to the retention times of the target grayanotoxins I and III. Typical 1938
DOI: 10.1021/acs.jafc.7b05054 J. Agric. Food Chem. 2018, 66, 1935−1940
Article
Journal of Agricultural and Food Chemistry Table 1. Recoveries of Grayanotoxins I and III from the Various Blank Samples (n = 3) compound grayanotoxin I
grayanotoxin III
concentration (ng/mL) 10.0 60.0 100.0 20.0 200.0 400.0
powder (mean ± RSD, %) 78.31 98.99 97.64 115.51 105.29 118.26
± ± ± ± ± ±
pill (mean ± RSD, %)
0.77 0.44 0.52 1.41 0.34 0.44
102.91 73.39 75.21 102.16 102.45 105.39
chromatograms obtained from the analysis of the blank and spiked blank samples are given in Figure 4. The specificity was then determined using independent blank samples from the different analytes. Typical chromatograms of the blank samples spiked with 100 ng mL −1 of the grayanotoxins I and III are shown in Figure 4. More specifically, as shown in panels B and D of Figure 4, no significant interference was observed at the retention times of the analytes (i.e., 4.71 and 4.78 min for grayanotoxins I and III, respectively). These results indicated that this method is both specific and selective, and therefore, it is suitable for application in the analysis of dietary supplement samples. Linearity and the Limit of Quantitation (LOQ). Calibration curves for grayanotoxins I and III were constructed by plotting the peak area ratio (y) of each compound against the nominal concentration (x) using weighted (x−1) least squares linear regression analysis. Linearity was determined over the concentration ranges of 10−100 ng mL −1 (grayanotoxin I) and 20−400 ng mL−1 (grayanotoxin III), and the mean regression equations of the calibration curves were derived as y = 1040x − 368.68, with r2 = 0.999, for grayanotoxin I and y = 6663x − 27352, with r2 = 0.999, for grayanotoxin III. In addition, the LOQs for grayanotoxins I and III were 7.5 and 15 ng mL−1, respectively, which are sufficiently low to render this method suitable for their determination in dietary supplements. Precision and Accuracy. The intra- and interday precision and accuracy values for grayanotoxins I and III were determined. These values were determined by analyzing quality control (QC) samples at the LOQ and at low, medium, and high concentrations in triplicate over 1 day (intraday) and over 3 separate days (interday). Thus, the intra- and interday precisions of these analytes were
