Chemicals and reagents
Target natural toxic substances were selected on the basis of previously reported poisoning cases as follows: 31 plant toxins (coniine, lycorine, galantamine, atropine, picrotoxinin, scopolamine, picrotin, strychnine, colchicine, veratramine, cyclopamine, jervine, amygdalin, aconine, cymarin, convallatoxin, cucurbitacin E, oleandrin, benzoylmesaconine, benzoylaconine, tubocurarine, hypaconitine, mesaconitine, 14-anisoylaconine, aconitine, jesaconitine, digitoxin, digoxin, α-chaconine, α-solanine, and dioscin), 7 mushroom toxins (muscimol, ibotenic acid, muscarine, phalloidin, γ-amanitin, α-amanitin, and β-amanitin), 4 mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2), 5 marine toxins (domoic acid, tetrodotoxin, okadaic acid, dinophysistoxin-1, and brevetoxin b), and 5 frog venoms (bufotenine, resibufogenin, bufalin, cinobufagin, and batrachotoxin). In addition to the natural toxins, berberine, cinchonidine, diosgenin, and quinine were selected as target substances, which are considered to be important materials of herbal medicines. Scopolamine, aflatoxin B1, aflatoxin B2, tetrodotoxin, quinine, aflatoxin G1, aflatoxin G2, resibufogenin, bufalin, colchicine, cyclopamine, diosgenin, cinobufagin, amygdalin, benzoylmesaconine, tubocurarine, mesaconitine, 14-anisoylaconine, jesaconitine, okadaic acid, and dinophysistoxin-1 were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan); muscimol, coniine, ibotenic acid, muscarine, bufotenine, cinchonidine, strychnine, cymarin, convallatoxin, cucurbitacin E, oleandrin, digitoxin, digoxin, and α-solanine from Sigma-Aldrich (St. Louis, MO, USA); picrotoxinin, picrotin, domoic acid, γ-amanitin, and β-amanitin from Abcam Biochemicals (Cambridge, UK); berberine, aconine, and benzoylaconine from Cayman Chemical (Ann Arbor, MI, USA); galantamine, atropine, and jervine from Tokyo Chemical Industry (Tokyo, Japan); α-chaconine and dioscin from Extrasynthese (Lyon, France); batrachotoxin and aconitine from Latoxan (Valence, France); phalloidin and α-amanitin from Merck Millipore (Billerica, MA, USA); lycorine, hypaconitine, brevetoxin b, and veratramine from Enzo Life Sciences (New York, NY, USA); Kishida Chemical (Osaka, Japan), LKT Laboratories (St. Paul, MN, USA), and Toronto Research Chemicals (Toronto, Canada), respectively. The stock solutions of all substances were prepared at a concentration of 10–1000 μg/mL. Muscarine, lycorine, cinchonidine, scopolamine, tetrodotoxin, quinine, berberine, resibufogenin, cinobufagin, amygdalin, hypaconitine, and α-amanitin were dissolved in distilled water (DW). Aconitine and benzoylaconine were dissolved in acetonitrile. Other toxins were dissolved in methanol solution. Stock solutions were stored at − 80 °C until analysis. Methanol, acetonitrile and DW of the HPLC grade were purchased from Kanto Chemical (Tokyo, Japan). Other common chemicals used were of the highest purity commercially available. Human whole blood was obtained from Tennessee Blood Services (Memphis, TN, USA).
LC–QTOF-MS (/MS) conditions
Sciex Triple TOF 5600 mass spectrometer (Sciex, Framingham, MA, USA) and Shimadzu NexeraX2 LC system (Shimadzu Co., Kyoto, Japan) were used for analysis. The column used for chromatographic separation was L-column ODS (150 × 1.5 mm i.d., particle size 5.0 μm; Chemicals Evaluation and Research Institute, Sugito, Saitama, Japan). The column temperature was maintained at 40 °C, and the gradient system was used with a mobile phase (A) 10 mM ammonium formate in 5% methanol aqueous solution and (B) 10 mM ammonium formate in 95% methanol solution. Linear gradient elution was started from 100% A to 100% B over 15 min. The 100% B was held for 5 min. It was then returned to 100% A over 10 min for the next run. The autosampler was maintained at 4 °C and the injection volume was 10 µL. Electrospray ionization was used in both positive and negative modes. The optimal MS parameters were declustering potential at 80 V and information dependent acquisition (IDA) criteria set at over 50 cps. The LC–QTOF-MS system allowed the acquisition of highly sensitive full scan MS spectra with high resolution and mass accuracy. In addition, IDA can be used to collect MS/MS spectra for compound identification based on MS/MS library searching.
This LC–QTOF-MS (/MS) method had several advantages for accurate detection of natural toxic substances. For instance, the mass spectrometer used in this study, triple TOF 5600, had high throughput which enabled very fast MS/MS acquisition rates at as low as 20 ms accumulation time in IDA mode. To fully leverage the instrument speed and obtain the best depth of coverage, the IDA workflow was optimized such that software overhead is minimized. The IDA method consisted of a high-resolution TOF-MS survey scan could follow up to 50 MS/MS ions. The combined use of high-resolution MS and IDA were extremely effective for the simultaneous detection of natural toxic substances in forensic samples. The instrument gave the resolution of 35,000.
Data acquisition and processing were performed by Analyst software and Peak View incorporated with the XIC Manager application (Sciex). The XIC Manager can be used for targeted processing of high-resolution MS and MS/MS data allowing for screening and identification with the highest confidence based on retention time (RT), mass error of molecular ion, isotopic pattern, and automatic MS/MS library searching.
Construction of library of natural toxic substances by LC–QTOF-MS (/MS)
All target substances were analyzed to investigate their retention properties, isotopic ratios and high-resolution MS/MS spectra obtained by collision induced dissociation (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) mode together with collision energy spread (CES) mode, in which the CE ramp range was set to 35 ± 15 eV. The CES parameter, in conjunction with the CE, determined the collision energy applied to the precursor ion in a product ion scan. The CE is ramped from low to high energies. The selection ranges of the precursor ion and RT of each compound for acquiring the library search were 20 mDa and 4.0 min, respectively. Compound identification was based on chromatographic and mass spectrometric information, including RT error, mass error, isotope matching, and library search results. The product ion for library search could be chosen from four spectra by CID energies of (±) 20, 35, 50, and 35 ± 15 eV, automatically.
Limits of detection and recovery rates
To determine the limits of detection (LODs), 5 plots with different concentrations of each substance spiked into blank blood plasma were used. The LODs were defined as the concentrations giving a signal-to-ratio of 3:1. The recovery rates were calculated by the ratio of peak area obtained from a target substance spiked into ante-extraction matrix to that obtained from the substance spiked into post-extraction matrix.
Analysis of spiked samples
The blood plasma samples spiked with lycorine (1 µg/mL) and domoic acid (10 µg/mL) were prepared. A 100-μL volume of blood plasma containing the target substances was mixed with 100 μL methanol and 300 μL acetonitrile. The mixture was then mixed by vortexing for 30 s and centrifuged at 15,000 g for 10 min. The supernatant was transferred to another tube and evaporated with a centrifugal evaporator (CVE-2000; Tokyo Rikakikai, Tokyo, Japan). The residue was reconstituted in 100 μL of 10 mM ammonium formate in 5% methanol solution and mixed by vortexing for 1 min. A 10-µL of the extract solution was analyzed by LC–QTOF-MS/MS using our newly developed library.
Application to forensic autopsy samples
A 45-year-old male with groan was found at his home. He was taken to hospital by an ambulance, but died shortly afterward. Beside the body in the room, there were dried roots of an aconite plant. Femoral vein and right and left heart blood samples were collected at autopsy performed in our laboratory and stored at − 80 °C until analysis. The blood samples were treated and analyzed in the same way as spiked samples described above.