Egyptian Journal of Forensic Sciences (2014) xxx, xxx–xxx

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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,*, Rudiger Lessig b,c, Heidemarie Bartels b

a Institute of Legal Medicine, Hannover Medical School (MHH), Carl-Neuberg-Str. 1, D-30625 Hannover, Germanyb Institute of Forensic Medicine, Otto-von-Guericke-University, Leipziger Str. 44, D-39120 Magdeburg, Germanyc 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

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KEYWORDS

Taxus;

Taxine B;

Isotaxine B;

3,5-Dimethoxyphenol

Corresponding author at: I

edical School (MHH), Car

ermany. Tel.: +49 51153245-mail addresses

ziadosz.Marek@mh-hannove

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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 proce-

dure of a methanol yew leaves’ extract was applied. This extract made the LC–MS/MS taxine B/iso-

taxine B optimisation possible by the use of its continuous infusion. The analyses were performed

with an Agilent 1100 Series HPLC-API 2000� 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, 5 micron column maintained at 45 ± 1 �C was used for the separation. 20 lL of a pre-

pared 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+ 1 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 min. The biological material was extracted with Extrelut� NT 3 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.

of Legal Medicine, Hannover

-Str. 1, D-30625 Hannover,

analytiker@chemist.com,

Dziadosz).

nsic Medicine Authority.

g by Elsevier

ing by Elsevier B.V. on behalf of F

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ziadosz M et al. A way of hand.org/10.1016/j.ejfs.2013.12.001

1. Introduction

Taxus is a genus of small trees and shrubs in the family Taxa-

ceae. The most common species in western, central and south-ern Europe is Taxus baccata, which is also known as the yewtree. The Taxus genus plays an important role both in the med-

icine and in the toxicology. The bark of Taxus species containssmall amounts of paclitaxel, which has a powerful effect onbreast, ovarian and lung cancers. Paclitaxel is very rare and

expensive, so huge efforts have been made to develop differentsources of this important compound. A common way to obtainpaclitaxel is a semi-synthesis route from 10-deacetylbaccatin

orensic Medicine Authority.

ling Taxus baccata intoxications in forensic laboratories Egypt

2 M. Dziadosz et al.

III, which is located mainly in leaves of the same plant inrelatively large amounts.1

The toxicity of T. baccata is well known and associated

with taxine B (Fig. 1), the main pseudo-alkaloid of the taxinefraction.2 Taxine B is relatively highly concentrated in leaves ofthe plant, which were very often used to induce abortion in an-

cient times.3 The ingestion of extracted plant material and yewleaves is responsible for T. baccata intoxications in Europe.3–5

The reported clinical signs are dizziness, nausea, diffuse

abdominal pain, mydriasis, unconsciousness, weak breathingand brief ventricular flutter.6 With the onset of intoxicationtachycardia occurs. Terminal rhythm is bradycardia with adrop of the blood pressure followed by death caused by respi-

ratory arrest and diastolic cardiac standstill. These symptomsresult from the taxine B sodium and calcium transport inhibi-tion 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 suicidaltendencies.

In the forensic medicine/toxicology the confirmation ofT. baccata intoxications is very important. The examinationof 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 materialis responsible for this fact.8 Thus, the determination of taxineB in biological material is necessary and the analytical toxicol-

ogy should be equipped with an appropriate analytical meth-od. Since taxine B is not available commercially, an indirectconfirmation of T. baccata intoxication has been proposed

by Mußhoff et al.9 3,5-dimethoxyphenol the aglycon of the

Figure 1 Isomerisation of taxine B to isotaxine B due to acetyl

group migration.

Please cite this article in press as: Dziadosz M et al. A way of handJ Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001

Taxus ingredient taxicatin was used as an intoxication marker.Recently toxicological relevant data were presented by Pietschet al.10 An HPLC-UV method was used for the 3,5-dimethoxy-

phenol quantification in five fatalities and an indirect confir-mation of the ingestion of extracted plant material or yewleaves was given. A semi-quantitative LC–MS method for

the detection of the pseudo alkaloids of T. baccata was devel-oped by Beike et al.8 and an LC–MS/MS method for the quan-titative determination of taxine B in body fluids by Frommherz

et al.2 These methods used a complicated and time consumingTaxus leaves’ extraction/purification procedure to obtainquantitative and semi-quantitative data. Thus, the applicationof 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 detec-tion in biological material is very important. Since this pseudo-alkaloid of the taxine fraction is directly responsible for fatal

intoxications, an appropriate LC–MS/MS method for thequalitative confirmation was developed and applied. Thisstrategy can be used in addition to the 3,5-dimethoxyphenol

quantification with a different method.9 We have already ap-plied the Extrelut� NT 3 tubes for imatinib extraction fromhuman serum.11 The achieved results and universal applicationof this extraction method made it possible to use the Extrelut�NT 3 tubes also for the taxine B extraction from post-mortemspecimens.

2. Materials and methods

Extrelut� NT 3 extraction tubes and all other chemicals/sol-vents (analytical grade) were purchased from Merck KGaA

(Darmstadt, Germany). Nanosep� MF 0.45 lm CentrifugalDevices were purchased from Pall GmbH (Dreieich,Germany).

Analyses were performed with an Agilent 1100 SeriesHPLC and with an API 2000� LC–MS/MS system. The posi-tive electrospray ionisation and the multiple-reaction monitor-

ing mode (MRM) were used (monitored mass-transitions:584.4/194.1 and 584.4/107.2). Data acquisition and integrationwere performed by the Analyst 1.5 software. A Zorbax SB-C18, 4.6 · 50 mm, 5 micron column (Agilent Technologies

Deutschland GmbH, Boblingen, Germany) maintained at45 ± 1 �C was used for the separation. 20 lL of a preparedsample was injected into the HPLC and eluted with a

mobile phase consisting of A: CH3OH and B: 50 mL 0.1 MCH3COONH4 + 50 mL CH3CN + 900 mL H2O + 1 mLCH3COOH (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 min.To realise the compound optimisation, extraction of T. bac-

cata leaves was performed with a pointed flask and a Dimrothcondenser. 2 g of leaves was boiled in 15 mL methanol for an

hour. The extract was cooled and 500 lL was filtered with aNanosep� 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 Boptimisation.

Tissue samples (brain, liver and kidney) and stomach

content were processed with a Stomacher� 80 biomaster labsystem (Seward Limited, Worthing, UK). 4 g of analysedmaterial was added to a Standard Stomacher� Bag with

ling Taxus baccata intoxications in forensic laboratories Egypt

Table 1 Taxine B/isotaxine B MS/MS settings.

Mass –transition Time [ms] DPa [V] FPb [V] EPc [V] CEPd [V] CEe [V] CXPf [V]

584.4/194.1 150 26 370 12 22 35 8

584.4/107.2 150 16 370 12 22 75 14

a Declustering potential.b Focusing potential.c Entrance potential.d Cell entrance potential.e Collision energy.f Cell exit potential.

A way of handling Taxus baccata intoxications in forensic laboratories 3

10 mL 0.1 M phosphate buffer (pH = 7.6). The bag contentwas centrifuged after the Stomacher� was operated 2 min atnormal speed and 2 min at high speed. 3 mL of supernatantwas transferred into an Extrelut� NT 3 extraction tube. After

10 min, the tube was eluted with 15 mL of diethyl ether/ethyl

Figure 2 MRM chromatograms of: (a) the Taxus baccata leaves’ m

case).

Please cite this article in press as: Dziadosz M et al. A way of handJ Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001

acetate (50:50, v/v), and next with 15 mL of chloroform/iso-propanol/ammonia (84:15:1, v/v/v). The eluates were collectedin a reaction tube and evaporated until dry under water-jetpump pressure and by heating the tube in a heating block

(60 �C). The dry residue was redissolved in 100 lL methanol.

ethanol extract and (b) a femoral blood sample (real intoxication

ling Taxus baccata intoxications in forensic laboratories Egypt

4 M. Dziadosz et al.

Cardiac/femoral blood (1.5 mL blood +1.5 mL distilledwater) and urine (3 mL) were directly added to the extractiontubes and processed afterwards in the same way.

3. Results and discussion

The prepared T. baccata leaves’ methanol extract was used for

taxine B/isotaxine B LC–MS/MS compound optimisation bythe use of continuous infusion experiments. Specific mass-transitions 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 taxinefraction was confirmed. The performed optimisation

procedure led to the MS/MS settings presented in Table 1.The optimised settings were used for the extract analysis

with liquid chromatography-tandem mass spectrometry. The

appropriate chromatograms are presented in Fig. 2a. Thepresence of two peaks can be explained by the taxine Bisomerisation 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 testedpositive for 3,5-dimethoxyphenol (other quantitative

method),9 gave also positive taxine B/isotaxine B findings inour LC–MS/MS analyses. The obtained chromatograms werecomparable to the chromatograms of the plant extract. Two

peaks with reproducible retention times, associated with taxineB and isotaxine B were observed (Fig. 2b).

The presented LC–MS/MS method is very useful for the di-rect confirmation of T. baccata intoxication in cases when this

fact is indirectly suggested by the presence of 3,5-dimethoxy-phenol in post-mortem specimens.9 Because a taxine B/isotax-ine B standard is not available commercially, the T. baccata

leaves’ methanol extract was used for a simple validation ofthis qualitative method. The performed taxine B/isotaxine BLC–MS/MS peak high comparison (Fig. 2) between the ana-

lysed T. baccata leaves’ extract and the post-mortem samplesshowed, that the signal intensity was much lower for the ex-tract diluted with methanol (1:1000, v/v). Thus, the sensitivity

of the presented LC–MS/MS method is sufficient for the qual-itative confirmation of a taxine B/isotaxine B intoxication.

To produce quantitative results, extraction of yew leavesand afterwards an appropriate purification procedure is neces-

sary. Some presented methods gave semi-quantitative andquantitative 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 qualitativedetermination of this compounds is more important. Underthese circumstances a quantification method based on a

self-prepared standard, would not be the best choice for manyforensic laboratories. Therefore, the presented qualitative con-firmation of taxine B/isotaxine B in post-mortem material, to-gether with the 3,5-dimethoxyphenol quantification9 (standard

available commercially) is a good way to handle T. baccataintoxications.

Please cite this article in press as: Dziadosz M et al. A way of handJ Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.001

Funding

None.

Conflict of interest

None declared.

Ethical approval

Necessary ethical approval was obtained from the instituteethics committee.

Informed consent

Informed consent were enrolled for the study.

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