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Home , Aconitine, Aflatoxin B1, Batrachotoxin, Chaconine, Cinobufagin, Domoic acid

Forensic (2020) 38:232–242 https://doi.org/10.1007/s11419-019-00506-w

SHORT COMMUNICATION

Development and application of a forensic toxicological library for identifcation of 56 natural toxic substances by liquid –quadrupole time‑of‑fight

Tadashi Ogawa1,2 · Kei Zaitsu3 · Tetsuo Kokaji4 · Kayako Suga4 · Fumio Kondo1,5 · Masae Iwai1,2 · Takayoshi Suzuki1,2 · Akira Ishii3 · Hiroshi Seno1,2

Received: 7 July 2019 / Accepted: 28 October 2019 / Published online: 17 November 2019 © The Author(s) 2019

Abstract Purpose The present study aims to develop a forensic toxicological library to identify 56 natural toxic substances by liquid chromatography–quadrupole time-of-fight tandem mass spectrometry (LC–QTOF-MS/MS). Methods For setting up the library of product ion spectra, individual substances (31 , 7 mushroom toxins, 5 marine toxins, 5 , 4 , and 4 substances derived from ) were analyzed by LC–QTOF-MS/MS with positive and negative ionization. The product ion spectra were acquired at the collision energies (CEs) of 20, 35, and 50 eV in single enhanced product ion mode and then in collision energy spread mode in which the CE ramp range was set to 35 ± 15 eV. Results To test the performance of the library, human blood plasma samples were spiked with a mixture of lycorine and , extracted by acetonitrile deproteinization and analyzed by LC–QTOF-MS/MS. Identifcation by our library search could be achieved for these toxins at the purity scores of 79.1 and 67.2, respectively. The method was also applied to postmortem blood from a death case with an aconite intake, and showed that four toxins in an aconite could be identifed in the blood sample at the purity scores of 54.6–60.3. Conclusions This library will be more efective for the screening of natural toxic substances in routine forensic toxicologi- cal analysis. To our knowledge, there are no reports dealing with development of library for natural toxic substances by LC–QTOF-MS/MS.

Keywords Natural toxic substances · Forensic toxicological library · Screening and identifcation · · and amanitin · LC–QTOF-MS/MS

Introduction

Natural toxins are chemicals that are naturally produced by Electronic supplementary material The online version of this living organisms such as some plants, mushrooms, marine article (https​://doi.org/10.1007/s1141​9-019-00506​-w) contains animals, and so on [1]. These toxins are not harmful to the supplementary material, which is available to authorized users.

* Tadashi Ogawa 4 SCIEX, 4‑7‑35 Kitashinagawa, Shinagawa‑ku, ogawatd@aichi‑med‑u.ac.jp Tokyo 140‑0001, Japan 5 Department of Biomedical Sciences, Chubu University 1 Department of Legal Medicine, Aichi Medical College of Life and Health Sciences, 1200 Matsumoto‑cho, University School of Medicine, 1‑1 Yazakokarimata, Kasugai 487‑8501, Japan Nagakute 480‑1195, Japan 2 Analysis Center, Aichi Medical University School of Medicine, 1‑1 Yazakokarimata, Nagakute 480‑1195, Japan 3 Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku, Nagoya 466‑8550, Japan

Vol:.(1234567890)1 3 Forensic Toxicology (2020) 38:232–242 233 organisms themselves but they may be toxic to other crea- 5 marine toxins (domoic acid, tetrodotoxin, , tures, including humans, when eaten [1]. For example, tetro- dinophysistoxin-1, and b), and 5 frog venoms dotoxin in puferfsh and some marine animals is a powerful (bufotenine, resibufogenin, bufalin, , and batra- blocker in excitable tissues such as nerves chotoxin). In addition to the natural toxins, berberine, cin- and muscles, and is about 10,000 times more lethal than chonidine, diosgenin, and quinine were selected as target by weight [2]. Forensic toxicology is a part of the substances, which are considered to be important materi- pharmacological science, which is concerned with the iden- als of herbal medicines. , afatoxin B1, afa- tifcation/quantifcation and efects of various drugs and poi- B2, tetrodotoxin, quinine, afatoxin G1, afatoxin G2, sons in human beings [3]. Natural toxins are very important resibufogenin, bufalin, , cyclopamine, diosgenin, analytical targets in forensic toxicology [4–6]. It is difcult cinobufagin, , benzoylmesaconine, tubocurarine, to attribute a cause of death to natural toxin(s) in routine mesaconitine, 14-anisoylaconine, jesaconitine, okadaic acid, toxicological analysis because there is currently no efective and dinophysistoxin-1 were purchased from Fujiflm Wako routine screening method for a variety of natural toxins [7]. Pure Chemical (Osaka, Japan); , , ibotenic In the last decade, liquid chromatography–tandem mass acid, , bufotenine, cinchonidine, , cyma- spectrometry (LC–MS/MS) has been developing its impor- rin, convallatoxin, cucurbitacin E, , , tance for the screening of drugs and/or , most of , and α- from Sigma-Aldrich (St. Louis, MO, which is based on triple quadrupole mass spectrometers USA); picrotoxinin, picrotin, domoic acid, γ-amanitin, and employing multiple reaction monitoring survey scan fol- β-amanitin from Abcam Biochemicals (Cambridge, UK); lowed by a product ion scan by electrospray ionization berberine, aconine, and benzoylaconine from Cayman [8–11]. The application of this method is limited because of Chemical (Ann Arbor, MI, USA); galantamine, , disturbances by high matrix burden and co-eluting peaks, and jervine from Tokyo Chemical Industry (Tokyo, Japan); indicating that analytes can be detected only if they are α- and dioscin from Extrasynthese (Lyon, France); abundantly contained in the samples and that the method and aconitine from Latoxan (Valence, France); can easily lead to false positive/negative detection [12]. and α-amanitin from Merck Millipore (Billerica, This drawback prompts the need for additional approaches MA, USA); lycorine, hypaconitine, brevetoxin b, and vera- achieving unambiguous identifcation of analytes. tramine from Enzo Life Sciences (New York, NY, USA); Recently, liquid chromatography–quadrupole time-of- Kishida Chemical (Osaka, Japan), LKT Laboratories (St. fight tandem mass spectrometry (LC–QTOF-MS/MS) has Paul, MN, USA), and Toronto Research Chemicals (Toronto, been utilized to develop libraries of compounds relevant to Canada), respectively. The stock solutions of all substances clinical and forensic toxicology [13–21]. However, to the were prepared at a concentration of 10–1000 μg/mL. Mus- best of our knowledge, there are no reports on development carine, lycorine, cinchonidine, scopolamine, tetrodotoxin, of library for natural toxic substances by LC–QTOF-MS/ quinine, berberine, resibufogenin, cinobufagin, amygdalin, MS. In this paper, we describe the development and appli- hypaconitine, and α-amanitin were dissolved in distilled cation of forensic toxicological library of 56 natural toxic (DW). Aconitine and benzoylaconine were dissolved substances using LC–QTOF-MS/MS. in acetonitrile. Other toxins were dissolved in solu- tion. Stock solutions were stored at − 80 °C until analysis. Methanol, acetonitrile and DW of the HPLC grade were Materials and methods purchased from Kanto Chemical (Tokyo, Japan). Other com- mon chemicals used were of the highest purity commercially Chemicals and reagents available. Human whole blood was obtained from Tennessee Blood Services (Memphis, TN, USA). Target natural toxic substances were selected on the basis of previously reported cases as follows: 31 plant tox- LC–QTOF‑MS (/MS) conditions ins (coniine, lycorine, galantamine, atropine, picrotoxinin, scopolamine, picrotin, strychnine, colchicine, veratramine, Sciex Triple TOF 5600 mass spectrometer (Sciex, Framing- cyclopamine, jervine, amygdalin, aconine, , con- ham, MA, USA) and Shimadzu NexeraX2 LC system (Shi- vallatoxin, cucurbitacin E, oleandrin, benzoylmesaconine, madzu Co., Kyoto, Japan) were used for analysis. The col- benzoylaconine, tubocurarine, hypaconitine, mesaconi- umn used for chromatographic separation was L-column tine, 14-anisoylaconine, aconitine, jesaconitine, digitoxin, ODS (150 × 1.5 mm i.d., particle size 5.0 μm; Chemicals digoxin, α-chaconine, α-solanine, and dioscin), 7 mush- Evaluation and Research Institute, Sugito, Saitama, Japan). room toxins (muscimol, , muscarine, phalloi- The column temperature was maintained at 40 °C, and the din, γ-amanitin, α-amanitin, and β-amanitin), 4 mycotoxins gradient system was used with a mobile phase (A) 10 mM (afatoxin B1, afatoxin B2, afatoxin G1 and afatoxin G2), ammonium formate in 5% methanol aqueous solution and

1 3 234 Forensic Toxicology (2020) 38:232–242

(B) 10 mM ammonium formate in 95% methanol solution. on chromatographic and mass spectrometric information, Linear gradient elution was started from 100% A to 100% including RT error, mass error, isotope matching, and library B over 15 min. The 100% B was held for 5 min. It was then search results. The product ion for library search could be returned to 100% A over 10 min for the next run. The autosa- chosen from four spectra by CID energies of (±) 20, 35, 50, mpler was maintained at 4 °C and the injection volume was and 35 ± 15 eV, automatically. 10 µL. Electrospray ionization was used in both positive and negative modes. The optimal MS parameters were decluster- Limits of detection and recovery rates ing potential at 80 V and information dependent acquisition (IDA) criteria set at over 50 cps. The LC–QTOF-MS sys- To determine the limits of detection (LODs), 5 plots with tem allowed the acquisition of highly sensitive full scan MS diferent concentrations of each substance spiked into blank spectra with high resolution and mass accuracy. In addition, blood plasma were used. The LODs were defned as the IDA can be used to collect MS/MS spectra for compound concentrations giving a signal-to-ratio of 3:1. The recov- identifcation based on MS/MS library searching. ery rates were calculated by the ratio of peak area obtained This LC–QTOF-MS (/MS) method had several advan- from a target substance spiked into ante-extraction matrix to tages for accurate detection of natural toxic substances. For that obtained from the substance spiked into post-extraction instance, the mass spectrometer used in this study, triple matrix. TOF 5600, had high throughput which enabled very fast MS/MS acquisition rates at as low as 20 ms accumulation Analysis of spiked samples time in IDA mode. To fully leverage the instrument speed and obtain the best depth of coverage, the IDA workfow was The blood plasma samples spiked with lycorine (1 µg/mL) optimized such that software overhead is minimized. The and domoic acid (10 µg/mL) were prepared. A 100-μL vol- IDA method consisted of a high-resolution TOF-MS survey ume of blood plasma containing the target substances was scan could follow up to 50 MS/MS ions. The combined use mixed with 100 μL methanol and 300 μL acetonitrile. The of high-resolution MS and IDA were extremely efective mixture was then mixed by vortexing for 30 s and centri- for the simultaneous detection of natural toxic substances fuged at 15,000 g for 10 min. The supernatant was trans- in forensic samples. The instrument gave the resolution of ferred to another tube and evaporated with a centrifugal 35,000. evaporator (CVE-2000; Tokyo Rikakikai, Tokyo, Japan). Data acquisition and processing were performed by The residue was reconstituted in 100 μL of 10 mM ammo- Analyst software and Peak View incorporated with the nium formate in 5% methanol solution and mixed by vortex- XIC Manager application (Sciex). The XIC Manager can ing for 1 min. A 10-µL of the extract solution was analyzed be used for targeted processing of high-resolution MS and by LC–QTOF-MS/MS using our newly developed library. MS/MS data allowing for screening and identifcation with the highest confdence based on retention time (RT), mass Application to forensic autopsy samples error of molecular ion, isotopic pattern, and automatic MS/ MS library searching. A 45-year-old male with groan was found at his home. He was taken to hospital by an ambulance, but died shortly Construction of library of natural toxic substances afterward. Beside the body in the room, there were dried by LC–QTOF‑MS (/MS) roots of an aconite plant. Femoral vein and right and left heart blood samples were collected at autopsy performed All target substances were analyzed to investigate their in our laboratory and stored at − 80 °C until analysis. The retention properties, isotopic ratios and high-resolution blood samples were treated and analyzed in the same way MS/MS spectra obtained by collision induced dissociation as spiked samples described above. (CID) with the injected amount of each compound of 0.1 µg. The four spectra were acquired at the collision energy (CE) at 20, 35, and 50 eV in single enhanced product ion (EPI) Results mode together with collision energy spread (CES) mode, in which the CE ramp range was set to 35 ± 15 eV. The CES Development of library of natural toxic substances parameter, in conjunction with the CE, determined the col- lision energy applied to the precursor ion in a product ion Registered data consisted of 56 natural toxic substances with scan. The CE is ramped from low to high energies. The compound name, source, formula, exact mass, polarity, exact selection ranges of the precursor ion and RT of each com- mass of precursor ion, ion form, RT, LOD, and recovery pound for acquiring the library search were 20 mDa and rate (Table 1). Extracted ion chromatograms of simultaneous 4.0 min, respectively. Compound identifcation was based determination of 56 substances are shown in Fig. 1. Product

1 3 Forensic Toxicology (2020) 38:232–242 235 75.5 88.4 98.6 68.2 92.8 69.1 70.2 81.4 71.7 90.7 88.5 70.5 71.0 75.0 73.8 69.0 73.3 96.0 71.1 78.6 76.1 83.1 75.5 83.4 87.5 79.8 82.0 69.1 79.9 90.8 78.6 97.5 79.2 87.2 86.3 86.0 Recovery Recovery (%) rate 100 500 100 5 10 0.5 0.5 5 0.5 0.05 0.05 10 10 0.05 5 10 0.1 100 50 0.05 5 5 5 100 1 0.05 1 10 1 1 5 0.1 0.05 50 1 500 LOD (ng/mL) LOD 1.9 2.1 2.1 3.2 4.2 6.2 8.4 9.0 9.5 9.6 9.7 10.2 10.2 10.7 10.7 11.0 11.1 11.2 11.5 11.7 12.8 12.9 12.9 12.9 13.0 13.1 13.4 13.6 13.6 13.8 13.8 13.9 14.1 14.1 14.1 14.1 Retention Retention time (min) - + ] 4 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M] [M+H] [M+NH [M+H] [M+H] [M+H] [M+H] [M] [M+H] [M-H] [M+H] [M+H] [M] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+HCOO] Ion form 205.1335 128.1434 920.3455 919.3615 903.3665 295.1805 789.3236 320.1088 115.0502 159.0400 174.1489 312.1442 475.1922 500.2854 288.1230 288.1594 290.1751 609.2959 304.1543 309.0980 293.1020 335.1754 336.1230 604.3116 331.0812 590.2960 329.0656 551.2851 634.3222 315.0863 325.1911 400.1755 410.3054 426.3003 539.3116 825.4278 Extracted mass (ion - Extracted calculated ized form) Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Polarity 204.1263 127.1361 919.3382 918.3542 902.3593 294.1732 788.3163 319.1016 114.0429 158.0328 174.1494 311.1369 457.1584 499.2781 287.1158 287.1521 289.1678 609.2965 303.1471 310.1053 292.0947 334.1681 336.1236 603.3044 330.0740 589.2887 328.0583 550.2778 633.3149 314.0790 324.1838 399.1682 409.2981 425.2930 538.3043 780.4296 Exact mass (Da) Exact S S S S 14 13 8 15 6 2 11 2 6 6 11 9 4 3 3 4 4 10 10 11 6 2 3 O O 2 4 2 O O O O O O O O O 3 2 10 9 2 10 7 6 2 2 8 7 7 10 6 2 2 14 O O 2 2 N NO N NO NO NO NO N N NO N N NO O O N NO N NO N O NO O O NO O N NO NO NO N O NO N 17 N N 21 16 27 41 17 21 54 53 23 41 54 21 18 16 22 18 22 45 48 14 43 12 42 47 14 24 25 39 39 42 64 6 6 20 17 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H 11 4 5 9 15 12 20 8 25 16 17 39 39 17 37 39 17 15 15 21 20 19 32 35 17 31 17 29 33 17 20 22 27 27 31 41 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C Molecular formula C Amanita pantherinaAmanita pantherinaAmanita pantherinaAmanita Chondria armata gargarizans Bufo Prunus armeniaca Conium maculatum radiata Lycoris Lycoris radiata Lycoris phalloides Amanita phalloides Amanita metel Datura Chondrodendron tomentosum Chondrodendron phalloides Amanita metel Datura Anamirta cocculus Anamirta cocculus nux-vomica japonica Coptis pubescens Cinchona Aconitum Amanita phalloides Amanita favus Aspergillus Aconitum favus Aspergillus majalis Convallaria Aconitum Aspergillus favus Aspergillus pubescens Cinchona autumnale Colchicum Veratrum album Veratrum terribilis Phyllobates lanata Source Registered data in a forensic toxicological library for natural toxic substances for liquid chromatography–quadrupole time-of-fight mass spectrometry listed according to the retention the to according retention spectrometrymass time-of-fight listed chromatography–quadrupole liquid substances for toxic natural library for toxicological data forensic a in Registered Muscimol acid Ibotenic Muscarine Domoic acid Bufotenine Amygdalin Coniine Aconine Lycorine Galantamine α-Amanitin β-Amanitin Atropine Tubocurarine γ-Amanitin Scopolamine Picrotin Picrotoxinin Strychnine Berberine Cinchonidine Benzoylaconine Phalloidin G2 Afatoxin Benzoylmesaconine G1 Afatoxin Convallatoxin 14-Anisoylaconine Afatoxin B2 Afatoxin Quinine Colchicine Veratramine Jervine Batrachotoxin Digoxin Compound name Compound Tetrodotoxin 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 1 Table No times 1

1 3 236 Forensic Toxicology (2020) 38:232–242 86.3 90.4 76.8 85.9 80.1 75.6 82.6 80.1 72.3 93.9 68.6 97.4 83.1 72.1 73.0 94.7 87.2 82.4 84.0 89.8 Recovery Recovery (%) rate 1 0.1 50 5 0.5 1 10 0.5 50 5000 5000 100 5 5 100 100 50 50 5000 100 LOD (ng/mL) LOD 14.2 14.6 14.8 14.9 14.9 14.9 15.0 15.1 15.3 15.9 16.0 16.7 16.8 16.9 17.5 17.9 18.6 18.9 19.8 24.3 Retention Retention time (min) - - - - + ] 4 + + + + + + + + + + + + + + - [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+H] [M+NH [M+HCOO] [M+HCOO] [M-H] [M+H] [M+H] [M+HCOO] [M+H] [M+H] [M+H] [M+HCOO] [M+H] Ion form 313.0707 616.3116 412.3210 632.3065 646.3222 676.3328 549.3058 387.2530 574.3374 912.4962 896.5013 575.3226 443.2428 385.2373 809.4329 805.4733 819.4889 895.4838 913.4802 415.3207 Extracted mass (ion - Extracted calculated ized form) Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Pos Pos Neg Pos Pos Pos Neg Pos Polarity 312.0634 615.3044 411.3137 631.2993 645.3149 675.3255 548.2985 386.2457 556.3036 867.4980 851.5031 576.3298 442.2355 384.2301 764.4347 804.4660 818.4816 894.4766 868.4820 414.3134 Exact mass (Da) Exact 10 2 11 11 12 15 14 6 9 4 8 9 6 4 13 13 13 14 16 3 O NO NO NO NO NO O O O NO NO O O O O O O O O O 12 45 41 45 47 49 44 34 44 73 73 48 34 32 64 68 70 70 72 42 H H H H H H H H H H H H H H H H H H H H 17 33 27 33 34 35 30 24 32 45 45 32 26 24 41 44 45 50 45 27 C C C C C C C C C C C C C C C C C C C Molecular formula C Aconitum Veratrum album Veratrum Aconitum Aconitum Aconitum ramosa Adonis gargarizans Bufo siceraria Lagenaria tuberosum Solanum tuberosum Nerium oleander Nerium gargarizans Bufo gargarizans Bufo purpurea Digitalis okadai Halichondria fortii Dinophysis brevis Karenia quinqueloba Dioscorea quinqueloba Dioscorea Source Aspergillus favus Aspergillus (continued) Hypaconitine Cyclopamine Mesaconitine Aconitine Jesaconitine Cymarin Bufalin Cucurbitacin E α-Solanine α-Chaconine Oleandrin Cinobufagin Resibufogenin Digitoxin Okadaic acid Dinophysistoxin-1 b Brevetoxin Dioscin Diosgenin Compound name Compound Afatoxin B1 Afatoxin 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 1 Table values LOD Each measurements. rate and recovery (LOD) the limit of detection for substances except of 56 toxic mass measurements accurate used for (100 ng standards were each) Reference 0.1–10 µg/mL measurement, substance rate spiked each recovery blank blood plasma. For into substance spiked in plasma for obtained with of each concentrations using 5 plots diferent was used matrices was and post-extraction ante-extraction into No 37

1 3 Forensic Toxicology (2020) 38:232–242 237

Fig. 1 Extracted ion chromatograms (XICs) of simultaneous determination of 56 natural toxic substances by liquid chromatography–quadrupole time-of-fight mass spectrometry (LC–QTOF-MS). The reference standards (100 ng each) were injected to the instrument

1 3 238 Forensic Toxicology (2020) 38:232–242

Fig. 1 (continued)

1 3 Forensic Toxicology (2020) 38:232–242 239

Fig. 2 Product ion spectra for tetrodotoxin at collision energy of a 20 eV, b 35 eV, c 50 eV, or d collision energy spread (CES), obtained by LC– QTOF-MS/MS ion spectra of all substances were obtained by four diferent strychnine (compounds no. 20) is 335.1754, but 6 ions were CE settings (see supplementary material Fig. S1). As an observed in the range of 335–337 (336.2 could be the iso- example the four spectra obtained for tetrodotoxin, which is topic ion) (supplementary material Fig. S1, no. 20). This is one of the important toxins in food poisoning cases in Japan, also observed in the spectra of berberine (no. 21), afatoxin are shown in Fig. 2. The [M + H]+ (m/z 320.1088) was the G1 (no. 27), colchicine (no. 32), veratramine (no. 33), jer- most abundant ion at 20 eV (Fig. 2a), while it remarkably vine (no. 34), afatoxin B1 (no. 37), cyclopamine (no. 39), decreased at 35 eV (Fig. 2b) and became a very small peak and Aconitum (nos. 9, 23, 26, 29, 38, and 40–42). at 50 eV (Fig. 2c), and the number and intensity of frag- Unfortunately, the sources of the ions are still unknown. In ment ions increased instead (Fig. 2a–c). Product ion spectra future, it is necessary to analyze the assignment of these obtained by CES mode showed both [M + H]+ and fragment ions. ions (Fig. 2d). CES mode can collect an average MS spec- With respect to the chromatographic separation, water/ trum of diferent CE values in one single EPI scan, resulting methanol both containing 10 mM ammonium formate in a full scan spectrum with both molecular and fragment ion was used. The used conditions provided RTs ranging from information that can be used in library search-based iden- 1.9 min to 24.3 min (Table 1). Although some hydrophilic tifcation with increased confdence. Digoxin, α-solanine, substances like ibotenic acid, musimol, and muscarine eluted α-chaconine, digitoxin and dioscin provided [M + HCOO]−, quickly, it had no problems to detect them by high-resolution + and amygdalin and cucurbitacin E showed [M + NH4] MS analysis. instead of [M + H]+ (Table 1, supplementary material Fig. S1, nos. 36, 46, 47, 51, 57, 7, and 45, respectively); there- Analysis of spiked samples fore, it is necessary to pay attention to this phenomenon. Several precursor ions accompany unknown ions with The present library was applied for the identifcation of strange mass defects. For example, the m/z of [M + H]+ of lycorine and domoic acid spiked into blank blood plasma.

1 3 240 Forensic Toxicology (2020) 38:232–242

Fig. 3 Identifcation of lycorine (1 μg/mL) and domoic acid (10 μg/mL) spiked into blank blood plasma by LC–QTOF-MS/MS. XICs (left pan- els), TOF-MS spectra (100–1000 Da, middle panels) and TOF-MS/MS spectra (50–1000 Da, right panels) of a lycorine and b domoic acid

Fig. 4 Identifcation of aconitine, jesaconitine, hypaconitine, and and TOF-MS/MS spectra (50–1000 Da, right panels) of a aconitine, b mesaconitine in a forensic autopsy sample by LC–QTOF-MS/MS. jesaconitine, c hypaconitine, and d mesaconitine XICs (left panels), TOF-MS spectra (100–1000 Da, middle panels),

Figure 3 shows the results processed using the automatic performed in our laboratory were analyzed using the pre- extracted ion chromatograms (XICs) and spectra by TOF- sent library. In all samples, aconitine, jesaconitine, hypaco- MS and TOF-MS/MS. In the XICs on the left panels of nitine, and mesaconitine were identifed, and Fig. 4 shows Fig. 3a and b, the peaks corresponded to the target analytes. representative results from the femoral vein sample. In the In the TOF-MS spectra on the middle panels, the measured previous study, Niitsu et al. [22] reported the four poisons as masses were at m/z 288.1233 for lycorine and 312.1443 for major substances detected from blood in the cases of suicide domoic acid, which matched the theoretical masses with by aconite poisoning. In the TOF-MS spectra, the measured errors of 0.9 and 0.5 ppm, respectively. In the TOF-MS/ masses matched the registered masses with mass errors of MS spectra on the right panels, the masses of the fragment 0.5–1.0 ppm. In the TOF-MS/MS spectra, the masses of the ions agreed very well with those of the registered MS/MS fragment ions agree very well with the registered MS/MS spectra. The purity scores were 79.1 and 67.2%, respectively. spectra. The purity score was 54.6–60.3%.

Application to forensic autopsy samples

Femoral vein and right and left heart blood samples col- lected from a 45-year-old male at the forensic autopsy

1 3 Forensic Toxicology (2020) 38:232–242 241

Discussion compounds using the purity scores, their distinct criteria have not been established [16, 25–27]. According to our In this article, we have created a forensic toxicologi- results on the spiked and forensic autopsies (Figs. 3, 4), the cal library including 56 natural toxic substances. The purity scores more than 50% seems to be acceptable prior to drugs of abuse originated from plants, such as Δ9- considering the matches of a mass error and RT. and have been excluded. To When the present library of natural toxic substances our knowledge, only one trial to construct libraries specifc by LC–QTOF-MS were created, low-resolution MS/MS to natural toxic substances has been published [6]; but they spectra of 54 natural toxic substances were also recorded, used low-resolution LC–MS/MS instrument unable to make except for picrotoxinin and diosgenin (unpublished obser- estimation of the molecular formulae, using its accurate vation). The low-resolution MS/MS spectra at CEs of 20, mass numbers, which are very useful for tentative identif- 35, and 50 eV, and one spectrum in the CES mode were cation of an unknown substances. Recently, Wang et al. [23] acquired; the low-resolution MS/MS spectra were similar to reported high-throughput screening of more than 200 toxic the high-resolution MS/MS spectra in this study. Therefore, substances including narcotic drugs, psychotropic drugs, the detailed high-resolution MS/MS spectra of natural toxic , natural toxins, and other drugs; however, in their substances located in the electronic supplementary material collection, only 3 substances were in common with those in the present article (Fig. 2, Fig. S1) seems to be also useful in our article. Moreover, they did not use a high-resolution in routine forensic toxicological screening by low-resolution MS instrument, but a low-resolution linear ion trap quadru- LC–MS/MS. pole MS coupled with a homemade extractive electrospray ionization. Broecker et al. [12] reported an article on devel- opment and application of a library for CID accurate mass Conclusions spectra of more than 2500 toxic compounds by LC–QTOF- MS/MS. However, the readers cannot get access to the MS/ We have developed a forensic toxicological library for iden- MS spectra only with their paper. In the present article, the tifcation of 56 natural toxic substances by LC–QTOF-MS/ readers can readily gain access to the detailed high-resolu- MS. The applicability of the library was exemplifed by tion MS/MS spectra of 56 natural toxic substances located identifying four plant toxins in blood samples collected from in the electronic supplementary material. an autopsy. This library may be efective for the screening of Martin et al. [24] compared the performance of three natural toxic substances and can become a powerful tool for types of LC–QTOF-MS/MS platforms created by three dif- searching natural toxic substances in routine forensic toxi- ferent manufacturers including the Sciex Triple TOF 5600 cological analysis. To our knowledge, this is the frst trial to system used in the present study. There are usually three develop a toxicological library for natural toxic substances parameters for compound identifcation by LC–QTOF-MS/ using high-resolution LC–MS/MS. MS: mass errors not greater than 4 or 5 ppm, RT difer- Acknowledgements ences with 0.2–0.5 min, and similarities of MS/MS spectrum This work was supported by JSPS KAKENHI (Grant no. 18H03064). profles. The former two parameters were common to the three types of the instruments. For the similarity of the MS/ Compliance with ethical standards MS profles, one manufacturer did not incorporate such a parameter as of 2014. Another manufacturer provided MS/ Conflict of interest The authors declare that they have no confict of MS libraries at three collision energies for matching. The interest. system of our instrument takes into consideration the pres- Ethical approval ence/absence of all MS/MS spectral peaks and their rela- The analysis of blood samples from deceased subjects was requested by the judicial authorities. tive abundance, which are compared to those of the MS/MS library record, calculating the purity score. In addition, the Open Access system also includes the CES mode, in which the CE ramp This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat​iveco​ range is set to 35 ± 15 eV, in which a small parent peak and mmons.org/licen​ ses/by/4.0/​ ), which permits unrestricted use, distribu- important small product ions are magnifed automatically. tion, and reproduction in any medium, provided you give appropriate Therefore, we presented three MS/MS spectra at CEs of 20, credit to the original author(s) and the source, provide a link to the 35, and 50 eV, and one spectrum in the CES mode (Fig. 2, Creative Commons license, and indicate if changes were made. Fig. S1). Such algorithms adopted by Sciex for compari- son of MS/MS spectrum profles seem most sophisticated and thus reliable in current LC–QTOF-MS arena. Although some previous studies described identifcation of target

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