Herbal Medicine Must Be Treated with Care—A Case Report on Aconitine

: Pathologists usually only request a screening for natural toxic substances if plant material has been observed during autopsy or if there exists a hint in the police investigation ﬁle. This situation is aggravated by the fact that most toxins are not covered by typical immunoassays and gas chromatography–mass spectrometry (GC–MS) proﬁling systems. In addition, only a few forensic toxicological libraries based on liquid chromatography coupled to high-resolution tandem mass spectrometry (LC–HRMS / MS) exist. In the following case, femoral blood and urine were applied to systematic toxicological analysis (STA). However, the concentrations determined in blood did not lead to death. Consequently, a liquid chromatography high-resolution tandem mass spectrometry (LC–HRMS / MS) screening approach was applied. Aconitine was quantitated in all specimens taken during autopsy and urine and bile ﬂuid screened for aconitine metabolites. Aconitine, jesaconitine, hypaconitine, and mesaconitine were found in the root piece collected from the duodenum. Apart from aconitine, no other alkaloids were detected in the urine or in the femoral blood sample. The highest concentrations of aconitine were found in gastric content (55.2 µ g / mL), bile ﬂuid (11.7 µ g / mL), and liver (9.14 µ g / g), and least in femoral blood (0.15 µ g / mL) and cerebrospinal ﬂuid (0.07 µ g / mL). The liver / peripheral blood ratio amounted to 61 L / kg and indicated that aconitine undergoes postmortem redistribution. During our metabolism investigation, we found 3-dehydrogen-aconitine in the urine and bile ﬂuid sample and N -deethyl-aconitine only in the bile ﬂuid sample. If the routine GC–MS screening approach does not come up with a toxin, then LC–HRMS / MS proﬁling could represent the method of choice. In this case aconitine was identiﬁed. The concentrations determined were compared to those reported in literature and clearly indicate that the deceased died due to an aconitine overdose.


Introduction
Aconitum plants belong to the Ranunculaceae family and are frequently employed as herbal medicine in Asian countries [1,2]. As the tuber is well-known for its pharmaceutical properties due to its anti-inflammatory, analgesic, and cardiotonic effects [3], it has been used as a homicidal and suicidal agent in the past [4]. The tuber contains the toxic diesterditerpene-type aconitum alkaloids aconitine (C 34 H 47 NO 11 ), mesaconitine (C 33 H 45 NO 11 ), hypaconitine (C 33 H 45 NO 10 ), and jesaconitine (C 35 H 49 NO 12 ) [5,6]. However, the composition of the terpenoid alkaloids depends on species, place of origin, time of harvest, and method of processing [7]. These alkaloids can cause conduction blocking and paralysis through action on the sodium channel in the axons [8]. The neurotoxin aconitine produces prolonged depolarization and thus prevents repolarization of the excitable membrane [9].
As a consequence, a combination of local (tingling and numbness in mouth and tongue), systemic sensory (in the extremities), and motor (weakness in the extremities) symptoms is expected [8]. Even a low dose (1-2 mg) is sufficient to cause an intoxication. The elimination half-life is 3-18 h [8].
First symptoms occur within the first hour after ingestion [10] as this compound is rapidly absorbed by the upper gastrointestinal tract. They include paresthesia, sweating, and nausea. These symptoms are followed by severe vomiting, colicky diarrhea, intense pain, paralysis of the skeletal muscles, and life-threatening arrhythmia. Finally, death occurs as a result of respiratory paralysis and cardiac arrest [4,11].
This report presents a screening method for the analysis of naturally occurring toxic substances in urine. As proof of concept, a fatal aconitine poisoning is presented, in which concentrations in biological fluids, tissues, and gastrointestinal content are summarized. The values determined exceed those reported in literature. The high concentration of aconitine determined might be explained by the fact that the root material was soaked in ethanol. We assume that the deceased had drunk the aconite tincture and eaten some of the root pieces, respectively.

Case History
A 19-year-old man (height 168 cm, weight 62 kg, BMI 21.97 kg/m 2 ) was found dead at home by his parents. He was lying in bed in supine position and his eyes were widely opened. High-proof alcoholic beverages, several medications (ibuprofen and Vomex A), material resembling controlled substances, and a bowl filled with root pieces with a star-shaped cross section ( Figure 1) were secured. In addition, vomit including root pieces was located in the area surrounding him. During autopsy 1630 g brain, 330 g heart, 1515 g liver, 40 mL gastric content, 625 g left lung, 680 g right lung, 250 g spleen, 125 g right kidney, 125 g left kidney, and 440 mL urine were obtained. Thereof, 24 g femoral blood, 26 g urine, 40 g gastric contents, 11 g bile fluid, 60 g heart blood, 80 g liver, and 11 g cerebrospinal fluid were available for toxicological analysis. The deceased person did not have any chronic disease related to liver, kidney, or heart. Only a minimal heart enlargement was determined. However, three yellow-brown colored pieces looking like the root pieces from the bowl were found in the duodenum (Figure 1). Signs of central death, such as blood-filled inner organs, pulmonary edema, cerebral swelling, a frothy secretion in the airways, and a filled urinary bladder were observed. From the root pieces in the duodenum, the widely opened eyes, the blood-filled inner organs, and the situation in which the body was found, we assumed an intoxication.

Systematic Toxicological Analysis (STA)
Ethanol was determined with a Clarus 580 gas chromatograph-flame ionization (GC-FID) system from Perkin Elmer (Shelton, CT, USA). The presence of barbiturates, benzodiazepines, opiates, cocaine, cannabinoids, methadone, amphetamine, and salicylate was checked with the help of cloned enzyme donor immunoassay (CEDIA TM ) Drugs of Abuse Assays using the Indiko instrument from Thermo Fisher Scientific (Waltham, MA, USA). The downstream systematic toxicological analysis was conducted with a Finnigan Trace GC 2000 system equipped with a programmed temperature vaporization (PTV) injector (Thermo Quest, Manchester, UK). The capillary column used was a Zebron ZB-5MSi (Phenomenex, Torrance, CA, USA) and exhibited the following quality parameters: 30 m × 0.25 mm × 0.25 µm. Medications were quantitated with an Agilent Technologies 1290 Infinity high-performance liquid chromatography system coupled to a diode array detector (HPLC-DAD) (Agilent, Waldbronn).

Screening Urine and Blood for Naturally Occurring Toxic Substances
Qualitative LC-HRMS/MS analysis was conducted on a Nexera XR LC system (Shimadzu, Duisburg, Germany) coupled to a Sciex X500R QTOFMS instrument (Sciex, Darmstadt, Germany). The LC conditions were equal to those described for the HPLC-4000 QTRAP ® system. The general mass spectrometry settings were as follows: SWATH ® , polarity positive, ion source gas 1: 40 psi; ion source gas 2: 60 psi; curtain gas: 35 psi; CAD gas: 7 psi; temperature: 600 • C, spray voltage 5500 volts, and those for the MS as follows: m/z 105-1000; accumulation time: 200 ms; declustering potential 60 volts; collision energy 10 volts, and those for the MS/MS as follows: m/z 50-1000; accumulation time: 70 ms; charge state 1; declustering potential 60 volts; collision energy 35 volts; collision energy spread 15 volts, with 36 windows overlapping by 1 Da.

Systematic Toxicological Analysis Does Not Come Up with a Toxin
Ethanol, ibuprofen, diphenhydramine, trimethoprim, and 8-chlortheophylline were found in the urine sample. Finally, 1.1‰ ethanol, 3.5 µg/mL ibuprofen, 0.1 µg/mL diphenhydramine, 1.8 µg/mL trimethoprim, and 1 µg/mL 8-chlortheophylline were quantitated in femoral blood. As these concentrations can be settled within the therapeutic range, the urine sample was profiled by LC-HRMS/MS. For this purpose, a dedicated method for the analysis of natural toxic substances was compiled (Table 1).   This method is not restricted to the substances listed. It can be applied to any substance exhibiting a molecular weight within the set mass range and a concentration in the low µg/mL range.

Aconitine Might Be the Toxin
Besides ibuprofen, diphenhydramine, trimethoprim, and 8-chlortheophylline, the alkaloid aconitine was detected (Figure 2 [12]. Aconitine was also found in the extract of the interior root piece (27 µg/g). In addition, jesaconitine (m/z 676.33), hypaconitine (m/z 616.31), and mesaconitine (m/z 632.31) were identified. The relative amount of aconitine was 89%, that of mesaconitine, hypaconitine, and jesaconitine 5%, 4%, and 2%, respectively. The minor occurring alkaloids were only detected in the root piece, not in the urine or in the femoral blood sample.
Tandem mass spectrometry (MS/MS) spectra of both compounds can be found in Figure 3.

Discussion
The medication (ibuprofen, diphenhydramine, trimethoprim) found in femoral blood was not overdosed. In order to find the toxin, the urine sample was profiled by LC-HRMS/MS. As aconitine is readily distributed in the human body, it is not routinely detected by common GC/MS-based systematic toxicological analysis.
Besides GC/SIM [15], LC-MS/MS is a valid and precise method for the analysis of the alkaloid content in blood and urine [4]. To explore unexplained deaths, it is worth considering LC-MS/MS for the profiling of specimens taken during autopsy. Table 2. Concentration distribution of aconitine among various specimens taken during autopsy. n.d.: not determined. Hofman [17] only detected the alkaloid aconitine. Nitsu [16] found aconitine and mesaconitine in the specimens investigated. The values for mesaconitine are given in brackets. Maximum values are highlighted in red bold letters.

Specimen
Our However, the qualitative and quantitative composition depends on plant species and ecological factors. Mizugaki et al. detected mesaconitine, aconitine, hypaconitine, and their hydrolysis products in serum [5]. Niitsu et al. presented three autopsy cases of suicide by aconite poisoning. They found jesaconitine in the first autopsy case, mesaconitine in the second autopsy case, and mesaconitine as well as aconitine in the third autopsy case [16].
In addition, a low margin of safety is assumed between the therapeutic and toxic doses. According to Moritz et al. [11], a 21-year-old man had taken one capsule Aconitum napellus a day to calm his anxiety. A few weeks later he decided to increase the effect by ingesting three capsules a day, with the result that he arrived in the emergency department; however, cardiovascular symptoms and neurological symptoms disappeared several hours after ingestion [11]. A similar phenomenon was observed in eight patients, who suffered from accidental intoxication after ingesting chuanwu and caowu. First symptoms began 0.5 to 1.5 h after drinking the herbal broth and lasted up to 30 h. Most subjects experienced a combination of cardiovascular, gastrointestinal, and neurological symptoms [1]. In our study, aconitine reached relatively high concentrations in the gastric contents, bile fluid, and liver (Table 2). Nitsu et al. [16] also found the highest aconitine concentration in the liver and least concentration in femoral blood. This also holds true for Hofmann et al. [17]. Interestingly, the amount detected in gastric content was comparable to that in the cerebrospinal fluid.
Regardless of the mode of intake (eating aconite leaves and roots, drinking liquor in which roots were soaked), aconitum alkaloids were found predominantly in the liver, kidney, and bile [8]. In our autopsy case, the concentration of aconitine was high in all specimens taken during autopsy compared to other autopsy cases reported [16][17][18][19]. Thus, we conclude that an overdose of aconitine led to death.

Conclusions
The LC/HRMS-based screening approach contributed to the clarification of the case. However, from our experiments we cannot conclude whether the young man suffered from a derailed intoxication attempt or chose suicide. From literature it is known that aconitum is a very poisonous plant. Consequently, it might be expected that ingesting contents of a bottle filled with root pieces soaked in ethanol would end in death, in particular if one considers that ethanol increases the absorption of aconitine. Furthermore, aconitum plants possess a low therapeutic index. Thus, aconite extracts are surely known to present an unpredictable danger.
In case the GC/MS-based screening approach fails and clear evidence related to intoxication is found during autopsy and the investigative work, we recommend applying LC-HRMS/MS. This approach is not limited to the analysis of plant toxins. Any putative toxin occurring within the set mass range at a sufficient concentration will be detected.