Determination of Grayanotoxins from Rhododendron brachycarpum in

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Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by LC-Q-TOF-MS and LC-MS/MS Taeik Hwang, Eunyoung Noh, Ji Hye Jeong, Sung-Kwan Park, Dongwoo Shin, and Hoil Kang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05054 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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Journal of Agricultural and Food Chemistry

Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by LC-QTOF-MS and LC-MS/MS

Taeik Hwang, Eunyoung Noh, Ji Hye Jeong, Sung-Kwan Park, Dongwoo Shin, 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.

Corresponding Author (Tel: +82-43-719-5301; Fax: +82-43-719-5300; E-mail: [email protected])

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Abstract

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A sensitive and specific high-performance liquid chromatography–quadrupole–time-of-flight mass spec-

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trometry (LC-QTOF-MS) method combined with liquid chromatography–tandem mass spectrometry

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(LC-MS/MS) was developed for the determination of grayanotoxins I and III in dietary supplements and

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homemade wine. Grayanotoxins I and III were successfully extracted using solid-phase extraction car-

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tridges, characterized by LC-QTOF-MS, and quantitated by LC-MS/MS. The LC-MS/MS calibration

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curves were linear over concentrations of 10–100 ng/mL (grayanotoxin I) and 20–400 ng/mL (grayano-

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toxin III). Grayanotoxins I and III were found in 51 foodstuffs, with quantitative determinations reveal-

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ing total toxin concentrations of 18.4–101000 ng/mL (grayanotoxin I) and 15.3–56000 ng/mL (grayano-

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toxin III). The potential of the validated method was demonstrated by successful quantitative analysis of

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grayanotoxins I and III in dietary supplements and homemade wine; the method appears suitable for the

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routine detection of grayanotoxins I and III from Rhododendron brachycarpum.

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Keywords: grayanotoxin, LC-MS/MS, homemade wine, Rhododendron brachycarpum, LC-QTOF-MS

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Introduction

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Grayanotoxins are a group of chemical compounds found naturally in the plant family Ericaceous,

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which includes the Rhododendron species. Over 60 grayanotoxin-related compounds exist,1 of which

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grayanotoxin I, 1, and grayanotoxin III, 2 (Figure 1) are the most toxic.2 The consumption of grayano-

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toxins contained in mad honey, leaves, nectar, and flowers causes grayanotoxin poisoning, because of

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the widespread belief in the medicinal properties of these plant-based products. Grayanotoxin poisoning

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is often caused by misidentification of the plant species. More specifically, the ingestion of flowers,

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flower juice, wine, or boiled extracts of R. schlippenbachii and R. brachycarpum has led to grayanotox-

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in poisoning in Korea,3–6 as R. schlippenbachii (which contains grayanotoxins) seemingly resembles R.

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mucronulatum. Indeed, R. mucronulatum flowers have been usually applied for preparing vegetable

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pancakes, tea, and wine, in addition to being used as a herbal medicine for the treatment of the symp-

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toms of the common cold, and as a diuretic.7 Grayanotoxins cause intoxication by increasing the mem-

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brane permeability to sodium ions.8 Grayanotoxin-containing Rhododendron species grow naturally in

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various countries, including the United States, Brazil, Spain, Portugal, Turkey, Nepal, and Japan.9

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Interestingly, the toxic Rhododendron species, R. ponticum, which is native to the Black Sea region of

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Turkey, is commonly used as a folk medicine to treat various health problems, including colds, edemas,

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and general aches and pains.10 In addition, mad honey, which is produced from the pollen and nectar of

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these flowers, is also used as a folk medicine to treat gastritis, ulcers, hyperglycemia, hypertension, ar-

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thritis, and sexual impotence.11 However, as indicated above, the consumption of such products can

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cause intoxication in humans, and the severity of symptoms may vary from mild to life-threatening, de-

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pending on the quantity consumed. Indeed, grayanotoxin poisoning remains one of the most frequently

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encountered causes of food intoxication in Turkey, although cases have also been reported in Austria,

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Korea, and Nepal.12-14

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Recently, concerns regarding poisoning from Rhododendron species were raised by the German Federal

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Institute for Risk Assessment, which called for a greater systematic control of honey from specific re3 ACS Paragon Plus Environment


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gions. Thus, due to the unseen danger to public health from grayanotoxins, the development of rapid

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and reliable analytical methods for the detection of grayanotoxins in Rhododendron-containing dietary

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supplements and homemade wines is of particular importance. However, to date, only a few analytical

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methods have been reported for the determination of grayanotoxin in honey samples and plant materials,

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such as thin-layer chromatography,15–17 direct spectrometric methods,18 gas chromatography (GC) fol-

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lowing trimethylsilyl derivatization,19 and high-performance liquid chromatography–tandem mass spec-

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trometry.20-23 However, no analytical methods have been described for the determination of grayanotox-

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ins in dietary supplements and homemade wine.

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Herein, we present the development and optimization of the first LC-QTOF-MS and triple quadrupole

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LC–MS/MS method for the simultaneous determination of grayanotoxin I and grayanotoxin III in die-

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tary supplements and homemade wine. The proposed method employs a solid-phase extraction (SPE)

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step for sample cleanup, and therefore, it is expected to exhibit excellent sensitivity. We propose that

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these chromatographic methods will allow accurate and reliable determination of such compounds, and

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method was fully validated for grayanotoxin I and grayanotoxin III analysis in dietary supplements and

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homemade wine for routine laboratory use.

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Materials and Methods

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Chemicals and reagents

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Grayanotoxin I was kindly provided by the Kangwon National University (Kangwondo, Korea).

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Grayanotoxin III was purchased from the National Development Institute of Korean Medicine

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(NIKOM). The structures of grayanotoxins I and III are shown in Figure 1. Formic acid (analytical

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grade) and acetic acid (analytical grade) were purchased from Sigma-Aldrich (St Louis, MO). Methanol

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(HPLC grade) and acetonitrile (HPLC grade) were obtained from Merck (HPLC grade, Darmsradt,

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Germany).

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Preparation of the standard solutions

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Standard stock solutions were prepared by dissolving grayanotoxin I or grayanotoxin III (10 mg) in

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methanol (10 mL). Working standard solutions were prepared daily by diluting the appropriate volumes

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of the grayanotoxin I (10, 20, 40, 60, 80, and 100 ng/mL) and grayanotoxin III (20, 40, 80, 100, 200,

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and 400 ng/mL) stock solutions in methanol.

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Sample preparation

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51 dietary supplement and homemade wine samples made from R. brachycarpum or R. mucronulatum

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flowers were purchased from a local market and from four different provinces of Korea. Samples con-

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sisted of both solids (two of the powder type, four of the capsule type, seven of the pill type, nine of the

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tablet type, four of the tea type), and liquid (25 types) products. The contents from capsules, tablets,

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pills, powder, and liquid products were vortex mixed briefly to obtain a uniform sample. For example,

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for capsules, the ingredients present inside were used, and tablets and pills were broken into pieces and

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ground to obtain a powder, so that all the samples were homogenized. To determine the grayanotoxin

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contents present in the dietary supplements, the following sample preparation method was employed to

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obtain analytical samples for injection. A portion of the liquid or solid sample (1 g) was dissolved in

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methanol (10 or 50 mL, respectively). After mixing for 30 min under ultrasonication, the resulting solu-

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tion was employed for the SPE cleanup step. Specifically, Oasis HLB LP cartridge, 6 cc, 500 mg (Wa-

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ters, Milford, MA) were conditioned with methanol (5 mL) and distilled water (5 mL) prior to sample

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loading. After loading a portion (2 mL) of the desired sample onto the SPE cartridge, elution of

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grayanotoxins I and III was carried out using methanol (2 × 5 mL), and the eluted fractions were made

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up to 20 mL in a volumetric flask using methanol. Finally, the sample extracts were filtered using a

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0.2 µm membrane filter (VWR, Darmstadt, Germany) prior to analysis.

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UPLC-QTOF-MS analysis 5 ACS Paragon Plus Environment


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An ACQUITY ultra performance liquid chromatography (UPLC) system (Waters, Milford, MA) was

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coupled with a QTOF Premier hybrid quadrupole orthogonal acceleration time-of-flight mass spectrom-

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eter (Waters Micromass, Manchester, UK) equipped with an orthogonal Z-spray electrospray ionization

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(ESI) interface operated in both positive and negative ion modes. LC separation was achieved using a

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100 mm × 2.1 mm i.d., 1.7 µm, Waters ACQUITY BEH C18 column maintained at 40 °C. The mobile

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phase was composed of both distilled water (solvent A) and methanol (solvent B) containing 0.1% for-

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mic acid. The gradient elution profile employed as follows: 0–3 min 15% B; 3.0–3.1 min 15–90% B;

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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

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0.3 mL min−1 was employed along with a sample injection volume of 4 µL. Other acquisition conditions

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were as follows: capillary voltage, 3.0 kV; desolvation temperature, 600 °C; desolvation gas flow, 800 L

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h−1; cone gas flow, 30 L h−1. Lock-mass and mass calibration of the Q-TOF-MS were achieved using

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leucine-enkephalin (m/z 556.2771 [M+H]+, m/z 554.2615 [M−H]−) and a sodium formate calibration

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solution (0.1 M sodium hydroxide, 10% formic acid, and 80% acetonitrile). The mass resolution was in

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the range of 10,000–20,000 at m/z 50–1000. Nitrogen gas was used as the collision gas in a hexapole

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collision cell. Two types of acquisition, namely MS/MS and MSE, were performed. For the MS/MS ex-

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periments, the cone voltage and collision energy ramp were optimized individually for each compound.

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For the MSE experiments, two acquisition functions with different collision energies were created,

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namely the low energy (LE) and high energy (HE) functions. The compound-dependent optimized cone

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voltage and collision energy ramp determined for the MS/MS experiments were also employed.

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LC-MS/MS analysis

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Analytes were separated using an Agilent 1200 Series HPLC instrument (Agilent Technologies, Palo

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Alto, CA) consisting of a quaternary pump, a vacuum degasser, and a thermostated autosampler. Sample

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extracts (2 µL) were injected onto a 2.0 mm × 100 mm i.d., 3 µm, Capcell Pak C18 MGII column

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(Shiseido, Tokyo, Japan). The mobile phase was composed of 0.1% formic acid in distilled water (sol6 ACS Paragon Plus Environment

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vent A) and 0.1% formic acid in methanol (solvent B). A flow rate of 0.25 mL min−1 was employed, and

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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;

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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

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were detected using a QTrap5500 linear ion trap quadrupole mass spectrometer (AB SCIEX, Concord,

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ON, Canada). The TurboIonSpray source was employed in the positive ion mode with a source tempera-

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ture of 450 °C, an ion voltage of 5500 V, and a curtain gas pressure of 30 psi. To identify the target

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compounds, the precursors and fragmentation patterns for each analyte were optimized through a series

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of continuous infusion experiments. Initially, >2 multiple reaction monitoring (MRM) transitions were

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monitored for each analyte, and two transitions were selected in the final method, one for quantitation

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and the other for confirmation. To improve the sensitivity and selectivity of the system, the target com-

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pounds were measured using the time-scheduled MRM mode. Analytical instrument control, data acqui-

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sition, and data treatment were performed using the Analyst 1.5.1 software.

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Results and Discussion

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LC-QTOF-MS/MS analysis and proposed fragmentation patterns

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To determine the sensitivity and selectivity of targets, standard solutions were infused directly into the

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ESI source that is operated in the positive ion mode by varying the different MS parameters. MS condi-

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tions were optimized and investigated for the quantitation of both grayanotoxin I and III, for which the

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MS/MS product ion spectra are shown in Figure 2. In the case of grayanotoxin I, a prominent sodium

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adduct of the molecular ion [M+Na] + was observed at m/z 435.22 in the Q1 full-scan mass spectrum,

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although the most abundant signal was observed at m/z 375.21, which corresponded to

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[M+Na−CH3COOH−H2O]+. More specifically, grayanotoxin I fragmented readily by loss of an acetyl

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group and formed a more stable structure, which resembled grayanotoxin III (Figure 3A). We therefore

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selected the m/z 435.22→375.21 ion transition for grayanotoxin I, although it should be noted that an



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additional product ion at m/z 357.21 was also formed by the loss of another water molecule to afford

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[M+Na−CH3COOH−2H2O]+. Similarly, the sodium adduct of the molecular ion [M+Na]+ was observed

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at m/z 393.22 for grayanotoxin III in the Q1 full-scan mass spectrum. Successive loss of water produced

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signals at m/z 375.21, 357.20, and 317.21, which corresponded to [M+H−H2O]+, [M+H−2H2O]+, and

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[M+H−3H2O−rearrangement(C13-C16)]+, respectively (Figure 3B). It was assumed that the loss of H2O

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renders the base ion more stable, with the number of H2O molecules lost being dependent on the number

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of hydroxyl substituents present in the structure. Additional product ions at m/z 299.20, 289.17, and

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246.16 corresponded to the stepwise loss of H2O, C2H4, and C2H3O from the m/z 357.20 ion.

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LC-MS/MS Analysis

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The initial stage of method development for LC-MS/MS analysis involved optimization of the mass

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spectrometer conditions for the detection of the grayanotoxin I and grayanotoxin III. For this purpose,

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the MS/MS parameters were optimized by individually infusing standard solutions of grayanotoxin.

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Both positive (ESI+) and negative (ESI−) ionization modes were tested, although only the positive mode

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gave satisfactory results. For each analyte (i.e., grayanotoxin I and grayanotoxin III), the two most in-

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tense transitions were selected for quantitative determination of the components, and the remaining ions

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were used for the confirmation of grayanotoxin I and grayanotoxin III. As the grayanotoxin III structure

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fragmented readily to lose two molecules of H2O, this precursor ion was selected for detection by LC-

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TQ/MS. Therefore, the precursor ion of m/z 335.0 was selected for the quantitation of grayanotoxin III,

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as the optimized MS/MS patterns of the LC-MS/MS and LC-QTOF-MS techniques produced different

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precursor and product ions. Under the optimized solvent gradient conditions, good separation of

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grayanotoxin I and grayanotoxin III was achieved within 4 min.

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Method Validation

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Specificity 8 ACS Paragon Plus Environment

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Method selectivity was then assessed by duplicate analysis of blank samples from different dietary sup-

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plements, and no peaks corresponding to interfering compounds were observed in the blank samples

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close to the retention times of the target grayanotoxin I and grayanotoxin III. Typical chromatograms

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obtained from the analysis of the blank and spiked blank samples are given in Figure 4.

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The specificity was then determined using independent blank samples from the different analytes. Typi-

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cal chromatograms of the blank samples spiked with 100 ng mL−1 of the grayanotoxin I and grayanotox-

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in III are shown in Figure 4. More specifically, as shown in Figures 4B and D, no significant interfer-

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ence was observed at the retention times of the analytes (i.e., 4.71 and 4.78 min for grayanotoxins I and

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III, respectively). These results indicated that this method is both specific and selective, and therefore, it

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is suitable for application in the analysis of dietary supplement samples.

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Linearity and the limit of quantitation (LOQ)

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Calibration curves for grayanotoxin I and III were constructed by plotting the peak area ratio (y) of each

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compound against the nominal concentration (x) using weighted (1 x−1) least-squares linear regression

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analysis. Linearity was determined over the concentration ranges of 10–100 ng mL−1 (grayanotoxin I)

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and 20–400 ng mL−1 (grayanotoxin III), and the mean regression equations of the calibration curves

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were derived as: y = 1040x − 368.68, r2 = 0.999 for grayanotoxin I, and y = 6663x − 27352, r2 = 0.999

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for grayanotoxin III. In addition, the limits of quantitation (LOQs) for grayanotoxin I and grayanotoxin

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III were 7.5 and 15 ng mL−1, respectively, which are sufficiently low to render this method suitable for

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their determination in dietary supplements.

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Precision and accuracy

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The intra- and inter-day precision and accuracy values for grayanotoxin I and grayanotoxin III were de-

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termined. These values were determined by analyzing quality control (QC) samples at the LOQ, and at

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low, medium, and high concentrations in triplicate over one day (intra-day) and over three separate days

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(inter-day). Thus, the intra- and inter-day precisions of these analytes were

Determination of Grayanotoxins from Rhododendron brachycarpum in

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