Determination Of Aconitine In Body Fluids By LC/MS/MS

[ APPLICATION NOTE ]
D E T E RM I NAT IO N O F ACO N I T I N E I N BO DY F LU I D S BY L C / M S / M S
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.
INT RODUC T ION
Plants of the genus Aconitum L (family of Ranunculaceae)
The method was fully validated for the determination of aconitine from
whole blood samples and applied in two cases of fatal poisoning.
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 cardiotonic effects2-4. These effects can be attributed to the presence of
Figure 1: Aconitum napellus (monkshood) (A) and
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
MET HODS AND INST RUMENTAT ION
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
The first symptoms of aconitine poisoning appear ~20 min to 2
hours after oral uptake and include paraesthesia, sweating and
nausea. This leads to severe vomiting, colicky diarrhea, intense
and 1 mL of 0.15 M phosphate buffer pH 6.0. Samples were allowed
to pass through the cartridge under gravity, before an initial wash
step (3 mL water followed by 1 mL 0.01 M HCl) was performed.
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-
Clearly in the case of suspected aconitine intoxication there is a
need for rapid analytical techniques to enable prompt diagnosis
and treatment. To this end we have developed a simple LC/MS/MS
method for the determination of aconitine in various body fluids8.
romethane:2-propanol:25% aqueous ammonia (80:20:2). Eluents
were pooled and evaporated to dryness under a stream of nitrogen
at 40 ˚C before reconstitution with 100 μL LC mobile phase.
[ APPLICATION NOTE ]
LC /MS/MS
gram of the blood sample of aconite victim no 2. At the time of
A Quattro micro™ tandem mass spectrometer fitted with ZSpray™
ion interface was used for all analyses. Ionisation was achieved
using electrospray in the positive ionisation mode (ES+). Detection
autopsy the body was already in an advanced state of putrefaction.
Despite these difficult circumstances, the chromatogram shows a
strong signal for aconitine.
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
i.e. m/z 646.4 > m/z 526.4 and m/z 646.4 > m/z 368.4 were
SUMMA RY
We have developed a rapid and sensitive method for the quantifica-
monitored for confirmatory purposes.
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®
RP 8 pre-column (2.1 x 10 mm, 3.5 μm) and a XTerra ® RP 8
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™
program (Waters).
Aconitine concentration
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.
RESULT S
A series of calibrators (0.1 – 25 ng/g) were prepared in duplicate
by adding aconitine standards to control blood. Samples were
4.80
MRM of 3 Channels ES+
100
646.4>586.5
then extracted, using the SPE method described above, prior to
646.4>526.4
646.4>368.4
LC/MS/MS analysis.
2.37e3
%
Following analysis, the areas under the specific MRM chromatograms 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.
In two forensic cases of suspected aconitine intoxication, aconitine
was detected in the blood samples and also in the stomach content
and urine of the deceased (Table 1). Figure 3 shows the chromato-
Figure 2: MRM chromatograms for a blood calibrator spiked at 0.1 ng aconitine/g blood.
Peak intensity is given in the top right-hand corner of the trace.
[ APPLICATION NOTE ]
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
2.00
4.00
6.00
8.00
Time
10.00
Chinese herbal medicine: a report of two cases. Forensic Sci Int
67: 55 – 58.
Figure 3: MRM chromatograms of the blood sample from the victim in case 2, with
12.1 ng aconitine/g. The chromatograms show no interferences although the body
was in an advanced state of putrefaction at the time of the autopsy.
6. Chan TY, Tomlinson B, Tse LK, Chan JC, Chan WW, Critchley JA
(1994). Aconitine poisoning due to Chinese herbal medicines: a
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.
REF E RENC ES
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.
Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07, Australia 61 2 9933 1777, Belgium 32 2 726 1000,
Brazil 55 11 5094 3788, Canada 1 800 252 4752 x2205, China 86 10 8586 8899, CIS/Russia 7 095 336 7000, Czech Republic 420 2 617 1 1384
Denmark 45 46 59 8080, Finland 358 9 5659 6288, France 33 1 30 48 72 00, Germany 49 6196 400600, Hong Kong 852 29 64 1800
Hungary 36 1 350 5086, India and India Subcontinent 91 80 2837 1900, Ireland 353 1 448 1500, Italy 39 02 27 421 1, Japan 81 3 3471 7191
Korea 82 2 820 2700, Mexico 52 55 5200 1860, The Netherlands 31 76 508 7200, Norway 47 6 384 60 50, Poland 48 22 833 4400
Puerto Rico 1 787 747 8445, Singapore 65 6273 1221, Spain 34 93 600 9300, Sweden 46 8 555 11 500, Switzerland 41 62 889 2030
Taiwan 886 2 2543 1898, United Kingdom 44 208 238 6100
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