a way of handling taxus baccata intoxications in forensic laboratories

44, D-39120 Magdeburg, Germany c Institute of Forensic Medicine, Martin-Luther-University, Franzosenweg 1, D-06112 Halle, Germany Received 21 March 2013; revised 24 July 2013; accepted 1

SHORT REPORT

A way of handling Taxus baccata intoxications

in forensic laboratories

Marek Dziadosz a,b,* , Ru¨diger Lessig b,c, Heidemarie Bartels b

a

Institute of Legal Medicine, Hannover Medical School (MHH), Carl-Neuberg-Str 1, D-30625 Hannover, Germany

b

Institute of Forensic Medicine, Otto-von-Guericke-University, Leipziger Str 44, D-39120 Magdeburg, Germany

c

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 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

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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 yew tree The Taxus genus plays an important role both in the med-icine 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

* 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

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

Contents lists available atScienceDirect

Egyptian Journal of Forensic Sciences

journal homepage: www.ejfs.org

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.2Taxine B is relatively highly concentrated in leaves of

the plant, which were very often used to induce abortion in

an-cient times.3The 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

respi-ratory arrest and diastolic cardiac standstill These symptoms

result from the taxine B sodium and calcium transport

inhibi-tion across the cell membrane in the heart.7The 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 baccataintoxications 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.8Thus, the determination of taxine

B 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 indirect

confirmation of T baccata intoxication has been proposed

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

Taxus ingredient taxicatin was used as an intoxication marker Recently toxicological relevant data were presented by Pietsch

et al.10An 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 yew leaves 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.8and an LC–MS/MS method for the quan-titative determination of taxine B in body fluids by Frommherz

et al.2These 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 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 the qualitative confirmation was developed and applied This strategy can be used in addition to the 3,5-dimethoxyphenol quantification with a different method.9We have already ap-plied the Extrelut NT 3 tubes for imatinib extraction from human serum.11The achieved results and universal application

of this extraction method made it possible to use the Extrelut

NT 3 tubes also for the taxine B extraction from post-mortem specimens

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 Centrifugal Devices were purchased from Pall GmbH (Dreieich, Germany)

Analyses were performed with an Agilent 1100 Series HPLC 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 integration were performed by the Analyst 1.5 software A Zorbax SB-C18, 4.6· 50 mm, 5 micron column (Agilent Technologies Deutschland GmbH, Bo¨blingen, Germany) maintained at

45 ± 1C 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 + 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

To realise the compound optimisation, extraction of T bac-cataleaves was performed with a pointed flask and a Dimroth condenser 2 g of leaves was boiled in 15 mL methanol for an hour The extract was cooled and 500 lL was filtered with a Nanosep MF 0.45 lm Centrifugal Device The purified T baccataleaves’ 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 Stomacher 80 biomaster lab system (Seward Limited, Worthing, UK) 4 g of analysed material was added to a Standard Stomacher Bag with

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

group migration

10 mL 0.1 M phosphate buffer (pH = 7.6) The bag content

was centrifuged after the Stomacher was operated 2 min at

normal speed and 2 min at high speed 3 mL of supernatant

was transferred into an Extrelut NT 3 extraction tube After

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

acetate (50:50, v/v), and next with 15 mL of chloroform/iso-propanol/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 (60C) The dry residue was redissolved in 100 lL methanol

Table 1 Taxine B/isotaxine B MS/MS settings

Mass –transition Time [ms] DP a [V] FP b [V] EP c [V] CEP d [V] CE e [V] CXP f [V]

a Declustering potential.

b

Focusing potential.

c

Entrance potential.

d

Cell entrance potential.

e

Collision energy.

f

Cell exit potential.

Figure 2 MRM chromatograms of: (a) the Taxus baccata leaves’ methanol extract and (b) a femoral blood sample (real intoxication case)

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

3 Results and discussion

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

mass-transitions of this compound (584.4/194.1 and 584.4/107.2)

published by Frommherz et al.2were 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 inTable 1

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

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.9Because a taxine

B/isotax-ine 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

ana-lysed T baccata leaves’ extract and the post-mortem samples

showed, 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 leaves

and afterwards an appropriate purification procedure is

neces-sary 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 known2a 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

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 baccata

intoxications

Funding None

Conflict of interest None declared

Ethical approval

Necessary ethical approval was obtained from the institute ethics committee

Informed consent Informed consent were enrolled for the study

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