[ A APPLICATIONPPLICATION NOTENOTE ] DETERMINATION OF ACONITINE IN BODY FLUIDS BY LC/MS/MS Justus Beike1, Lara Frommherz1, Michelle Wood2, Bernd Brinkmann1 and Helga Köhler1 1 Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse, Münster, Germany 2 Clinical Applications Group, Waters Corporation, Simonsway, Manchester M22 5PP, UK. INTRODUCTION The method was fully validated for the determination of aconitine from whole blood samples and applied in two cases of fatal poisoning. Plants of the genus Aconitum L (family of Ranunculaceae) are known to be among the most toxic plants of the Northern Hemisphere and are widespread across Europe, Northern Asia and North America. Two plants from this genus are of particular importance: the blue-blooded Aconitum napellus L. (monkshood) which is cultivated as an ornamental plant in Europe and the yellow-blooded Aconitum vulparia Reich. (wolfsbane) which is commonly used in Asian herbal medicine1 (Figure 1). Many of the traditional Asian medicine preparations utilise both the aconite tubers and their processed products for their pharmaceutical properties, which include anti-inflammatory, analgesic and cardio- Figure 1: Aconitum napellus (monkshood) (A) and tonic effects2-4. These effects can be attributed to the presence of Aconitum vulparia (wolfsbane) (B). the alkaloids; the principal alkaloids are aconitine, mesaconitine, hypaconitine and jesaconitine. The use of the alkaloids as a homicidal agent has been known for METHODS AND INSTRUMENTATION more than 2000 years. Although intoxications by aconitine are rare in the Western Hemisphere, in traditional Chinese medicine, the Sample preparation use of aconite-based preparations is common and poisoning has Biological samples were prepared for LC/MS/MS by means of a been frequently reported. Poisoning has occurred both during clini- solid-phase extraction (SPE) procedure. Blood and tissue samples cal use and also as consequence of accidental ingestion (0.5 g each) were mixed with 3 mL of 0.15 M phosphate buffer pH e.g. by eating plant material or Aconitum preparations5, 6. The 6.0, homogenised and centrifuged at 5000 g for 10 min. The super- use of aconite tubers for suicide and homicide purposes has also natants were decanted and loaded on a prepared SPE cartridge. been reported7. Cartridges were pre-conditioned with 3 mL methanol, 3 mL water and 1 mL of 0.15 M phosphate buffer pH 6.0. Samples were allowed The first symptoms of aconitine poisoning appear ~20 min to 2 to pass through the cartridge under gravity, before an initial wash hours after oral uptake and include paraesthesia, sweating and step (3 mL water followed by 1 mL 0.01 M HCl) was performed. nausea. This leads to severe vomiting, colicky diarrhea, intense pain and then paralysis of the skeletal muscles. Following the onset Two further washing steps i.e. 2 mL dichloromethane, followed by of life-threatening arrhythmia, including ventricular tachycardia 2 mL methanol, were performed before elution of the aconitine. and ventricular fibrillation, death finally occurs as a result of Cartridges were dried under vacuum between each of the 3 wash respiratory paralysis or cardiac arrest5-7. steps. Aconitine was eluted (2 x 1.5 mL) with a mixture of dichlo- romethane:2-propanol:25% aqueous ammonia (80:20:2). Eluents Clearly in the case of suspected aconitine intoxication there is a were pooled and evaporated to dryness under a stream of nitrogen need for rapid analytical techniques to enable prompt diagnosis at 40 ˚C before reconstitution with 100 μL LC mobile phase. and treatment. To this end we have developed a simple LC/MS/MS method for the determination of aconitine in various body fluids8. [ A APPLICATIONPPLICATION NOTENOTE ] LC/MS/MS gram of the blood sample of aconite victim no 2. At the time of autopsy the body was already in an advanced state of putrefaction. A Quattro micro™ tandem mass spectrometer fitted with ZSpray™ Despite these difficult circumstances, the chromatogram shows a ion interface was used for all analyses. Ionisation was achieved strong signal for aconitine. using electrospray in the positive ionisation mode (ES+). Detection of aconitine was performed using multiple reaction monitoring (MRM). The transition MRM transition m/z 646.4 > m/z 586.5 was used for quantification purposes and a further two transitions SUMMARY i.e. m/z 646.4 > m/z 526.4 and m/z 646.4 > m/z 368.4 were monitored for confirmatory purposes. We have developed a rapid and sensitive method for the quantifica- tion of aconitine in biological specimens. The method involves a LC analyses were performed using an Alliance® 2695 separations simple SPE purification prior to analysis using LC/MRM. ® module (Waters). Chromatography was achieved using a XTerra ® RP8 pre-column (2.1 x 10 mm, 3.5 μm) and a XTerra RP8 The utility of the method was demonstrated by its application to analytical column (2.1 x 150 mm, 3.5 μm). The column was authentic samples in 2 fatal cases of suspected aconitine poisoning. maintained at 40 ˚C and eluted isocratically with 0.1 % ammonium Blood, urine and stomach contents were collected during autopsy acetate (adjusted to pH 6.0 with 1 M acetic acid) and methanol and analysed using the developed LC/MS/MS method. Aconitine (50:50) at 200 μL/min. The injection volume was 10 μL and could be detected in the blood of both victims, in the stomach a total run time of 10 min was used. All aspects of system opera- content of one individual and in the urine of the other. tion and data acquisition were controlled using MassLynx™ NT 4.0 software with automated data processing using the QuanLynx™ Aconitine concentration program (Waters). Case no. Blood [ng/g] Stomach content [ng/g] Urine [ng/mL] 1 10.0 3.0 Not available 2 12.1 Not available 180.0 Table 1: Concentrations of aconitine in autopsy samples from two cases of fatal aconite intoxication. RESULTS A series of calibrators (0.1 – 25 ng/g) were prepared in duplicate 4.80 MRM of 3 Channels ES+ by adding aconitine standards to control blood. Samples were 100 646.4>586.5 then extracted, using the SPE method described above, prior to 646.4>526.4 LC/MS/MS analysis. 646.4>368.4 % 2.37e3 Following analysis, the areas under the specific MRM chromato- grams were integrated. The response was linear (r2 = 0.999) over the range investigated. The limit of detection (LOD) of the assay was estimated at 0.1 ng/g blood. Figure 2 shows the responses Time 2.00 4.00 6.00 8.00 10.00 for the quantifier and qualifier ions of aconitine obtained with a calibrator spiked at the LOD. Figure 2: MRM chromatograms for a blood calibrator spiked at 0.1 ng aconitine/g blood. In two forensic cases of suspected aconitine intoxication, aconitine Peak intensity is given in the top right-hand corner of the trace. was detected in the blood samples and also in the stomach content and urine of the deceased (Table 1). Figure 3 shows the chromato- [ A APPLICATIONPPLICATION NOTENOTE ] 3. Desai HK, Hart BP, Caldwell RW, Jianzhong-Huang JH, Pelletier SW (1998). Certain norditerpenoid alkaloids and their 4.80 100 cardiovascular action. J Nat Prod 61: 743 – 748. 4. Ameri A (1998). The effects of Aconitum alkaloids on the central nervous system. Prog Neurobiol 56: 211 – 235. % 5. Dickens P, Tai YT, But PPH, Tomlinson B, Ng HK, Yan KW (1994). Fatal accidental aconitine poisoning following ingestion of Time Chinese herbal medicine: a report of two cases. Forensic Sci Int 2.00 4.00 6.00 8.00 10.00 67: 55 – 58. Figure 3: MRM chromatograms of the blood sample from the victim in case 2, with 6. Chan TY, Tomlinson B, Tse LK, Chan JC, Chan WW, Critchley JA 12.1 ng aconitine/g. The chromatograms show no interferences although the body (1994). Aconitine poisoning due to Chinese herbal medicines: a was in an advanced state of putrefaction at the time of the autopsy. review. Vet Hum Toxicol 36: 452-455. 7. Ito K, Tanaka S, Funayama M, Mizugaki M (2000). Distribution of Aconitum Alkaloids in body fluids and tissues in a suicidal case of aconite ingestion. J Analytical Toxicol 24: 348 – 353. REFERENCES 8. Beike J, Frommherz L, Wood M, Brinkmann B, Köhler H. Determination of aconitine in body fluids by LC-MS-MS. 1. List PH, Hörhammer L (1969). Hagers Handbuch der Int. J. Legal Med. 118: 289-293 (2004). Pharmazeutischen Praxis. Vol II, 1066 – 1082, Springer Berlin, Heidelberg. 2. Hikino H, Konno C, Takata H, Yamada Y, Yamada C, Ohizumi Y, Sugio K, Fujimura H (1980). Antinflammatory principles of Aconitum roots. J Pharmacobiodyn 3: 514 – 525. 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