Egyptian Journal of Forensic Sciences (2014) xxx, xxx–xxx Contents lists available at ScienceDirect Egyptian Journal of Forensic Sciences journal homepage: www.ejfs.org SHORT REPORT A way of handling Taxus baccata intoxications in forensic laboratories Marek Dziadosz a b c a,b,* , Ruădiger Lessig b,c , Heidemarie Bartels b Institute of Legal Medicine, Hannover Medical School (MHH), Carl-Neuberg-Str 1, D-30625 Hannover, Germany Institute of Forensic Medicine, Otto-von-Guericke-University, Leipziger Str 44, D-39120 Magdeburg, Germany Institute of Forensic Medicine, Martin-Luther-University, Franzosenweg 1, D-06112 Halle, Germany Received 21 March 2013; revised 24 July 2013; accepted 11 December 2013 KEYWORDS Taxus; Taxine B; Isotaxine B; 3,5-Dimethoxyphenol Abstract We here describe a possibility to handle Taxus baccata intoxications by the use of an LC–MS/MS method for the direct confirmation of taxine B A simple and fast preparation procedure of a methanol yew leaves’ extract was applied This extract made the LC–MS/MS taxine B/isotaxine B optimisation possible by the use of its continuous infusion The analyses were performed with an Agilent 1100 Series HPLC-API 2000ä LC–MS/MS system The following mass-transitions were monitored in the positive MRM mode: 584.4/194.1 and 584.4/107.2 A Zorbax SB-C18, 4.6 · 50 mm, micron column maintained at 45 ± °C was used for the separation 20 lL of a prepared sample was injected into the HPLC and eluted with a mobile phase consisting of A: CH3OH and B: 50 mL 0.1 M CH3COONH4 + 50 mL CH3CN + 900 mL H2O + mL CH3COOH (A:B = 7:3, v/v) The flow rate was 0.3 mL/min and a single LC–MS/MS run was completed in 20 The biological material was extracted with ExtrelutÒ NT extraction tubes In fatalities, where a T baccata intoxication was indicated by the presence of 3,5-dimethoxyphenol in biological material, taxine B/isotaxine B was also identified in the appropriate post-mortem specimens ª 2014 Production and hosting by Elsevier B.V on behalf of Forensic Medicine Authority Introduction * Corresponding author at: Institute of Legal Medicine, Hannover Medical School (MHH), Carl-Neuberg-Str 1, D-30625 Hannover, Germany Tel.: +49 5115324558 E-mail addresses: analytiker@chemist.com, Dziadosz.Marek@mh-hannover.de (M Dziadosz) Peer review under responsibility of Forensic Medicine Authority Production and hosting by Elsevier Taxus is a genus of small trees and shrubs in the family Taxaceae The most common species in western, central and southern Europe is Taxus baccata, which is also known as the yew tree The Taxus genus plays an important role both in the medicine and in the toxicology The bark of Taxus species contains small amounts of paclitaxel, which has a powerful effect on breast, ovarian and lung cancers Paclitaxel is very rare and expensive, so huge efforts have been made to develop different sources of this important compound A common way to obtain paclitaxel is a semi-synthesis route from 10-deacetylbaccatin 2090-536X ª 2014 Production and hosting by Elsevier B.V on behalf of Forensic Medicine Authority http://dx.doi.org/10.1016/j.ejfs.2013.12.001 Please cite this article in press as: Dziadosz M et al A way of handling Taxus baccata intoxications in forensic laboratories Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001 III, which is located mainly in leaves of the same plant in relatively large amounts.1 The toxicity of T baccata is well known and associated with taxine B (Fig 1), the main pseudo-alkaloid of the taxine fraction.2 Taxine B is relatively highly concentrated in leaves of the plant, which were very often used to induce abortion in ancient times.3 The ingestion of extracted plant material and yew leaves is responsible for T baccata intoxications in Europe.3–5 The reported clinical signs are dizziness, nausea, diffuse abdominal pain, mydriasis, unconsciousness, weak breathing and brief ventricular flutter.6 With the onset of intoxication tachycardia occurs Terminal rhythm is bradycardia with a drop of the blood pressure followed by death caused by respiratory arrest and diastolic cardiac standstill These symptoms result from the taxine B sodium and calcium transport inhibition across the cell membrane in the heart.7 The widespread distribution of T baccata and suicidal guidance in the internet5 make the yew tree very attractive for people with suicidal tendencies In the forensic medicine/toxicology the confirmation of T baccata intoxications is very important The examination of the gastrointestinal tract is a common proceeding However, in many cases the plant material is not present in the stomach Its degradation or the ingestion of an extracted plant material is responsible for this fact.8 Thus, the determination of taxine B in biological material is necessary and the analytical toxicology should be equipped with an appropriate analytical method Since taxine B is not available commercially, an indirect confirmation of T baccata intoxication has been proposed by Mußhoff et al.9 3,5-dimethoxyphenol the aglycon of the Figure Isomerisation of taxine B to isotaxine B due to acetyl group migration M Dziadosz et al Taxus ingredient taxicatin was used as an intoxication marker Recently toxicological relevant data were presented by Pietsch et al.10 An HPLC-UV method was used for the 3,5-dimethoxyphenol quantification in five fatalities and an indirect confirmation of the ingestion of extracted plant material or yew leaves was given A semi-quantitative LC–MS method for the detection of the pseudo alkaloids of T baccata was developed by Beike et al.8 and an LC–MS/MS method for the quantitative determination of taxine B in body fluids by Frommherz et al.2 These methods used a complicated and time consuming Taxus leaves’ extraction/purification procedure to obtain quantitative and semi-quantitative data Thus, the application of these methods is associated with a big effort and leads to difficult interpretable quantitative data In this work we wanted to present a simple and fast method for the direct confirmation of T baccata intoxications From the point of view of the forensic toxicology, the taxine B detection in biological material is very important Since this pseudoalkaloid of the taxine fraction is directly responsible for fatal intoxications, an appropriate LC–MS/MS method for the qualitative confirmation was developed and applied This strategy can be used in addition to the 3,5-dimethoxyphenol quantification with a different method.9 We have already applied the ExtrelutÒ NT tubes for imatinib extraction from human serum.11 The achieved results and universal application of this extraction method made it possible to use the ExtrelutÒ NT tubes also for the taxine B extraction from post-mortem specimens Materials and methods ExtrelutÒ NT extraction tubes and all other chemicals/solvents (analytical grade) were purchased from Merck KGaA (Darmstadt, Germany) NanosepÒ MF 0.45 lm Centrifugal Devices were purchased from Pall GmbH (Dreieich, Germany) Analyses were performed with an Agilent 1100 Series HPLC and with an API 2000ä LC–MS/MS system The positive electrospray ionisation and the multiple-reaction monitoring mode (MRM) were used (monitored mass-transitions: 584.4/194.1 and 584.4/107.2) Data acquisition and integration were performed by the Analyst 1.5 software A Zorbax SBC18, 4.6 · 50 mm, micron column (Agilent Technologies Deutschland GmbH, Boăblingen, Germany) maintained at 45 °C was used for the separation 20 lL of a prepared sample was injected into the HPLC and eluted with a mobile phase consisting of A: CH3OH and B: 50 mL 0.1 M CH3COONH4 + 50 mL CH3CN + 900 mL H2O + mL CH3COOH (A:B = 7:3, v/v) The flow rate was 0.3 mL/min and a single LC–MS/MS run was completed in 20 To realise the compound optimisation, extraction of T baccata leaves was performed with a pointed flask and a Dimroth condenser g of leaves was boiled in 15 mL methanol for an hour The extract was cooled and 500 lL was filtered with a NanosepÒ MF 0.45 lm Centrifugal Device The purified T baccata leaves’ extract was diluted with methanol (1:1000, v/ v) and used to perform the LC–MS/MS taxine B/isotaxine B optimisation Tissue samples (brain, liver and kidney) and stomach content were processed with a StomacherÒ 80 biomaster lab system (Seward Limited, Worthing, UK) g of analysed material was added to a Standard StomacherÒ Bag with Please cite this article in press as: Dziadosz M et al A way of handling Taxus baccata intoxications in forensic laboratories Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001 A way of handling Taxus baccata intoxications in forensic laboratories Table Taxine B/isotaxine B MS/MS settings Mass –transition Time [ms] DPa [V] FPb [V] EPc [V] CEPd [V] CEe [V] CXPf [V] 584.4/194.1 584.4/107.2 150 150 26 16 370 370 12 12 22 22 35 75 14 a b c d e f Declustering potential Focusing potential Entrance potential Cell entrance potential Collision energy Cell exit potential 10 mL 0.1 M phosphate buffer (pH = 7.6) The bag content was centrifuged after the StomacherÒ was operated at normal speed and at high speed mL of supernatant was transferred into an ExtrelutÒ NT extraction tube After 10 min, the tube was eluted with 15 mL of diethyl ether/ethyl Figure case) acetate (50:50, v/v), and next with 15 mL of chloroform/isopropanol/ammonia (84:15:1, v/v/v) The eluates were collected in a reaction tube and evaporated until dry under water-jet pump pressure and by heating the tube in a heating block (60 °C) The dry residue was redissolved in 100 lL methanol MRM chromatograms of: (a) the Taxus baccata leaves’ methanol extract and (b) a femoral blood sample (real intoxication Please cite this article in press as: Dziadosz M et al A way of handling Taxus baccata intoxications in forensic laboratories Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001 M Dziadosz et al Cardiac/femoral blood (1.5 mL blood +1.5 mL distilled water) and urine (3 mL) were directly added to the extraction tubes and processed afterwards in the same way Funding Results and discussion Conflict of interest The prepared T baccata leaves’ methanol extract was used for taxine B/isotaxine B LC–MS/MS compound optimisation by the use of continuous infusion experiments Specific masstransitions of this compound (584.4/194.1 and 584.4/107.2) published by Frommherz et al.2 were identified in this extract Thus, the presence of this toxic pseudo-alkaloid of the taxine fraction was confirmed The performed optimisation procedure led to the MS/MS settings presented in Table The optimised settings were used for the extract analysis with liquid chromatography-tandem mass spectrometry The appropriate chromatograms are presented in Fig 2a The presence of two peaks can be explained by the taxine B isomerisation to isotaxine B due to acetyl group migration (Fig 1) These results correspond very well with other presented findings.2,8,12 Extracts of the post-mortem specimens, which were tested positive for 3,5-dimethoxyphenol (other quantitative method),9 gave also positive taxine B/isotaxine B findings in our LC–MS/MS analyses The obtained chromatograms were comparable to the chromatograms of the plant extract Two peaks with reproducible retention times, associated with taxine B and isotaxine B were observed (Fig 2b) The presented LC–MS/MS method is very useful for the direct confirmation of T baccata intoxication in cases when this fact is indirectly suggested by the presence of 3,5-dimethoxyphenol in post-mortem specimens.9 Because a taxine B/isotaxine B standard is not available commercially, the T baccata leaves’ methanol extract was used for a simple validation of this qualitative method The performed taxine B/isotaxine B LC–MS/MS peak high comparison (Fig 2) between the analysed T baccata leaves’ extract and the post-mortem samples showed, that the signal intensity was much lower for the extract diluted with methanol (1:1000, v/v) Thus, the sensitivity of the presented LC–MS/MS method is sufficient for the qualitative confirmation of a taxine B/isotaxine B intoxication To produce quantitative results, extraction of yew leaves and afterwards an appropriate purification procedure is necessary Some presented methods gave semi-quantitative and quantitative results,2,8 however, the appropriate extraction/ purification procedure was very time consuming.12 Since the toxic range of taxine B/isotaxine B is not known2 a qualitative determination of this compounds is more important Under these circumstances a quantification method based on a self-prepared standard, would not be the best choice for many forensic laboratories Therefore, the presented qualitative confirmation of taxine B/isotaxine B in post-mortem material, together with the 3,5-dimethoxyphenol quantification9 (standard available commercially) is a good way to handle T baccata intoxications None declared None Ethical approval Necessary ethical approval was obtained from the institute ethics committee Informed consent Informed consent were enrolled for the study References Ghassempour A, Rezadoost H, Mashouf A, Abdoul-Enein HY, Spengler B, Roămpp A Monitoring of paclitaxel, taxine B and 10deacethylbaccatin III in Taxus baccata L by nano LC-FTMS and NMR spectroscopy Chromatographia 2010;72:833–9 Frommherz L, Kintz P, Kijewski H, Koăhler H, Lehr M, Brinkmann B, et al Quantitative determination of taxine B in body fluids by LC–MS–MS Int J Legal Med 2006;120:346–51 Frohne D, Pribilla O Toădliche Vergiftung mit Taxus baccata Arch Toxicol 1965;21:15062 Czerwek H, Fischer W Toădlicher Vergiftungsfall mit Taxus baccata Arch Toxicol 1960;18:8892 Wehner F, Gawatz O Suizidale Eibenintoxikationen-von Caăsar bis heute-oder Suizidalanleitung im Internet Arch Kriminol 2003;211:19–26 Schulte Th Toădliche Vergiftung mit Eibennadeln (Taxus baccata) Arch Toxicol 1975;34:1538 Tekol Y, Kameyama M Elektrophysiologische Untersuchungen uăber den Wirkungsmechanismus des Eibentoxins Taxin auf das Herz Arzneim Forsch 1987;37:428–31 Beike J, Karger B, Meiners T, Brinkmann B, Koăhler H LC–MS determination of Taxus alkaloids in biological specimens Int J Legal Med 2003;117:335–9 Mußhoff F, Jacob B, Fowinkel C, Daldrup T Suicidal yew leave ingestion – phloroglucindimethylether (3,5-dimethoxyphenol) as a marker for poisoning from Taxus baccata Int J Legal Med 1993;106:45–50 10 Pietsch J, Schulz K, Schmidt U, Andresen H, Schwarze B, Dreßler J A comparative study of five fatal cases of Taxus poisoning Int J Legal Med 2007;121:417–22 11 Dziadosz M, Bartels H Imatinib quantification in human serum for clinical purposes using high-performance liquid chromatography with a diode array detector Acta Chim Slov 2011;58:347–50 12 Jenniskens LHD, van Rozendaal ELM, van Beek TA Identification of six taxine alkaloids from Taxus baccata needles J Nat Prod 1996;59:117–23 Please cite this article in press as: Dziadosz M et al A way of handling Taxus baccata intoxications in forensic laboratories Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001 ... Worthing, UK) g of analysed material was added to a Standard StomacherÒ Bag with Please cite this article in press as: Dziadosz M et al A way of handling Taxus baccata intoxications in forensic laboratories. .. located mainly in leaves of the same plant in relatively large amounts.1 The toxicity of T baccata is well known and associated with taxine B (Fig 1), the main pseudo-alkaloid of the taxine fraction.2... laboratories Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001 A way of handling Taxus baccata intoxications in forensic laboratories Table Taxine B/isotaxine B MS/MS settings