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Feb 13, 2018 - Standard stock solutions were prepared by dissolving grayanotoxin I or III (10 mg) in methanol (10 mL). Working standard solutions were...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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