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Problems of Forensic Sciences 2006, LXVI, 131–139
ISSN 1230-7483
DETERMINATION OF TRAZODONE AND ITS METABOLITE
1-(3-CHLOROPHENYL)PIPERAZINE (mCPP) IN HAIR BY THE HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY METHOD
COUPLED WITH MASS SPECTROMETRY (HPLC-ESI-MS)
Roman STANASZEK, Wojciech LECHOWICZ
Institute of Forensic Sciences, Krakow, Poland
Abstract
The aim of this paper was to develop, optimise and validate an analytical procedure for determining the popular antidepressant
drug trazodone and its active metabolite 1-(3-chlorophenyl)piperazine (mCPP) in hair. mCPP, being a derivative of piperazine, is
considered to be a so-called designer drug. It exhibits stimulating and hallucinogenic activity similar to MDMA. Hair samples
(50 mg) were extracted by 1-chlorobutane after prior alkaline hydrolysis (0.5 M NaOH, 1 h, 70°C), and the obtained extracts were
analysed by high performance liquid chromatography coupled with electrospray ionisation mass spectrometry (HPLC-ESI-MS).
Applying the technique of direct introduction of the sample into the MS, optimisation of ESI-MS parameters was carried out and
the following values were obtained: capillary voltage 3 kV, fragmentor voltage 45 V, ion source temperature 100°C, desolvation
temperature 300°C, and desolvation gas flow 600 l/h. Quantitative analysis was carried out in the selected ion monitoring (recording) mode (SIR). Quantitative analysis was carried out on the basis of analytical signals obtained for positive pseudomolecular ions of the determined substances (m/z – 197 for mCPP, m/z – 372 for trazodone) and deuterated ketamine (m/z – 242),
which was used as an internal standard. Additionally, 2 ions each were monitored for analytes: m/z 199, 154 for mCPP and 374,
176 for trazodone, which were used for identification together with pseudomolecular ions. For mCPP, the limit of detection was
0.1 ng/mg of hair, and for trazodone it was 0.02 ng/mg. The linearity for both compounds was confirmed in the range 0 to
5 ng/mg. The obtained values of other validation parameters fulfilled criteria required for this type of analysis and were:
intra-group precision for a concentration of 0.5 ng/mg: mCPP – CV 6.5%, trazodone – CV 14.0%; for a concentration of 2 ng/mg:
mCPP – CV 5.3% and CV 10.1% for trazodone; recovery: 70% for mCPP and 129% for trazodone at a concentration of 2 ng/mg.
The developed method was applied to determination of trazodone and mCPP in 7 authentic samples of hair. In the case of a hair
sample taken from a person one month after the person had consumed a single dose of 75 mg trazodone, the following concentrations were ascertained: segment 1 (1 cm) trazodone – 5.93 ng/mg and mCPP – 0.1 ng/mg; segment 2 (1 cm) trazodone –
0.04 ng/mg, mCPP not detected. The six remaining samples were negative. The developed method may be applied to differentiating between consumption of the hallucinogenic mCPP and consumption of its precursor trazodone.
Key words
Trazodone; mCPP; Hair; LC-ESI-MS.
Received 7 November 2006, accepted 27 December 2006
1. Introduction
Trazodone is an antidepressant from the group of
triazolopyridine derivatives (Figure 1), a selective se-
rotonin reuptake inhibitor and also an antagonist of
5-HT2 receptors, exhibiting antidepressant, soporific
and sedative activity. It is applied in the treatment of
depression, and also disturbances linked with aggres-
132
R. Stanaszek, W Lechowicz
sive behaviour, panic attacks, in states of tension and
anxiety, in alcoholism, and also in the treatment of cocaine withdrawal. In the liver, this medicine is metabolised via hydroxylation, dealkylation and N-oxidation.
N
The aim of this work was to develop, optimise and
validate an analytical procedure for determining the
popular antidepressant – trazodone – and its active metabolite, 1-(3-chlorophenyl)piperazine (mCPP) in hair,
allowing detection of an individual dose of this substance.
N
N
Cl
2. Material and methods
O
N
N
Fig. 1. Chemical structure of trazodone.
Cl
HN
N
Fig. 2. Chemical structure of 1-(3-chlorophenyl)piperazine
(mCPP).
1-(3-chlorophenyl)piperazine, known by the acronym mCPP (Figure 2), is an active metabolite formed
as a result of transformations (in the organism) of
trazodone and also other antidepressant medicines,
such as nefazodone, etoperidone, cloperidone, enziprazole and fefazodone, and also the analgesic mepiprazole [3]. The active metabolite mCPP is formed as
a result of N-dealkylation of nitrogen in the piperazine
ring. mCPP itself, as a derivative of piperazine, is classified as a so-called designer drug and is still a legal
substance not yet subject to anti-narcotics legislation
in most countries, including Poland. The mechanism
of pharmacological activity of mCPP consists in interaction with various serotonin receptors, and also
adrenergic and dopaminergic receptors, which leads to
release of serotonin [4]. This activity is analogical to
that of MDMA, hence on the narcotics market, mCPP
is usually distributed in the form of tablets mimicking
ecstasy tablets. Introduction of mCPP into the organism leads to broad neuroendocrinological, physiological and psychological effects described by Murphy
et al. [5]. Cases are known of detection of mCPP in biological material in persons who had taken this narcotic earlier.
Taking of trazodone may result in side-effects in the
form of, e.g. priapism in men, arrhythmia, headaches,
disturbed speech or diarrhoea. Trazodone also negatively influences psychomotor performance of drivers.
Hair constitutes a valuable diagnostic material due
to the non-invasiveness of collection of samples, their
durability, and also the fact that analysis of hair provides information on the subject of recent and longterm history of taking a given substance, including dependence or abstinence [1].
2.1. Hair samples
Hair samples used to prepare a bland sample and
also calibration samples were collected from children
not exposed to the action of the studied compounds.
Six real hair samples were obtained from patients
undergoing substitution therapy with methadone in the
Clinic for Persons Addicted to Psychoactive Substances, at the Psychiatric Centre of the “Zdroje” Independent Public Specialist Department of Health Care in
Szczecin who were known, from previous studies to
also take narcotics from the amphetamines derivatives
group, especially ecstasy tablets. One sample of hair of
length 3 cm was collected from a patient, who, a month
earlier had taken one 75 mg dose of trazodone. Hair samples were collected from the occipital part of the head,
as recommended by the Society of Hair Testing [8].
2.2. Standard substances
Standards: 1-(3-chlorophenyl)piperazine hydrochloride (mCPP) and its isomer, 1-(4-chlorophenyl)piperazine (pCPP) were bought from Sigma Aldrich (United
States) in the form of solid substances. An internal
standard in the form of ketamine-d4 (1 mg/ml) was
bought from Cerillian (United States).
2.3. Chemical reagents
Acetonitrile (Gradient Grade for HPLC), n-butyl
chloride (for liquid chromatography) and also methanol (analytical grade) were bought from Merck (Germany). Formic acid was bought from Ubichem (Great
Britain).
2.4. Preparation of samples and hair analysis
Each sample was subjected to the process of decontamination, which encompassed washing hair using
a sequence of solvents: dichloromethane, water, methanol. In order to carry out segmental analysis (analysis
of particular sections of hair), hair samples that had
been dried earlier were divided into one or two centi-
Problems of Forensic Sciences 2006, LXVI, 131–139
133
Determination of trazodone and its metabolite...
metre segments, beginning from the place of cutting of
hair, up to the end of the strand. In order to preliminarily break up the hair sample, it was cut with scissors
into pieces of length approx 1 mm, and then ground in
a ball mill. 50 mg of ground hair originating from each
segment was weighed out and subjected to successive
stages of analysis.
In order to draw up calibration curves and carry out
validation of the method, 50-miligram ground samples
of hair that did not contain analytes were weighed out.
Next, working solutions of trazodone and mCPP were
added to them in appropriate quantities, and also 5 ml
ketamine-d4 solution at a concentration of 10 mg/ml.
The final concentration of the internal standard in each
sample was 1 ng/mg.
Hair samples together with added standard substances were subjected to alkaline hydrolysis. To each
sample was added 0.4 ml 0.5 M sodium hydroxide solution and incubated for 1 hour at a temperature of
70°C. In order to isolate analytes from the obtained solution, liquid-liquid extraction was applied with use of
1 ml n-butyl chloride for 5 min. The solvent was evaporated in a stream of nitrogen after previous addition
of 100 ml 0.025 M HCl.
Analysis of amphetamines in hair was carried out
using a liquid chromatograph coupled with a quadrupole mass detector LC-MS produced by Waters
(United States) of the Alliance series with an electrospray ionisation (ESI) unit. Nitrogen produced from
a Whatman (United States) nitrogen generator was applied as a nebulising gas. Positive ions were analysed
in selected ion recording (SIR) mode. The liquid chromatograph was equipped with a LiChroCART 125 ´ 3
column with Purospher 60 RP-18e filling, 5 mm (Merck,
Germany) thermostated at 35°C. The mobile phase
consisted of a mixture of re-distilled water with addition of formic acid (1000 ml/litr) and acetonitryl with
addition of formic acid (1000 ml/litre). The mobile
phase flow was 0.8 ml/min and took place in a gradient
system: 0 min – 10% acetonitrile (AcCN), 8 min
40% AcCN, 9 min 10% AcCN and 14 min 10% AcCN.
Injection was performed by an autosampler, and the
volume of the injection was 20 ml.
Analysis was performed in selected ion recording
mode. The values of the working parameters of the
mass spectrometer, which were applied in the course
of performed studies, are presented in Table I. Total
time of acquisition was 17 min.
TABLE I. OPTIMAL ESI-MS OPERATING PARAMETERS
Parameter
Value
Cone voltage
45 V
Capillary voltage
3000 V
Source temperature
100°C
Desolvation temperature
300°C
Desolvation gas flow rate
600 l/min
Multiplier voltage
500 V
3. Results and discussion
The mass spectrometer working conditions were
optimised by the direct inlet method. The peak areas
obtained for pseudomolecular ions of analytes was
chosen as the criterion of optimisation. Trazodone and
mCPP after dilution in the mobile phase of the LC-MS
system were injected into the ionisation chamber and
analysed in the full mass range (m/z 50–400 amu) at
various fragmentor voltages (30, 60 and 90 V) with the
aim of selection of the most intensive ions for SIR
analysis. Positive pseudomolecular ions of the determined substances were selected for quantitative analysis (m/z –197 for mCPP, m/z – 372 for trazodone) and
deuterated ketamine (m/z – 242). Additionally, 2 ions
each were selected for the analytes: m/z 199, 154 for
mCPP and 374, 176 for trazodone and also one, 242,
for ketamine-d4, which was used for identification together with the pseudomolecular ions. The obtained
mass spectra of trazodone, mCPP and pCPP are presented in Figure 3. Next, ESI-MS parameters were
optimised, such as: capillary voltage (CV) and
fragmentor voltage (CNV), source temperature (ST),
temperature of desolvation (DT) and desolvation gas
flow (DGF). The obtained optimum values for particular parameters are presented in Table I.
In the process of validation of the analytical procedure, calibration curves were used that were obtained
by analysis of samples of hair (50 mg) by the LC-MS
method, to which were added appropriate amounts of
working solutions of analytes of trazodone and mCPP
in the range of concentrations from 0 to 5.0 ng/mg and
also an internal standard of ketamine-d4 in an amount
corresponding to a concentration of 1 ng/mg. Calibration curves and their parameters are presented in Figure 4. Retention times, relative retention times and
monitored ions for particular compounds are presented
in Table II. Chromatograms showing the separation of
target compounds for selected ions obtained for the
Problems of Forensic Sciences 2006, LXVI, 131–139
134
R. Stanaszek, W Lechowicz
A
372.12
%
100
CV 10.1%, recovery: 70% for mCPP and 129% for
trazodone at a concentration of 2 ng/mg.
175.92
60
100
A
10.0
84.69
0
Determination coefficient R 2 =0.9916
Calibration curve equation y=2.9919*x
Response
374.14
180
140
220
300
260
340
380
420
5.0
500
460
m/z
m/z
0.0
mCPP (cone 40V)
0.00
196.96
100
0.50
1.50
B
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Concentration [ng/mg]
Determination coefficient R2 =0.9967
Calibration curve equation y=0.5420*x
Response
%
1.00
153.89
B
2.00
198.97
1.00
155.89
0
60
100
140
180
220
260
300
340
380
420
460
0.00
500
m/z
m/z
0.00
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Concentration [ng/mg]
pCPP (cone 40V)
Fig. 4. Calibration curves for A) trazodone and B) mCPP.
196.96
100
0.50
C
100
Ketamine-d 4
%
3.77
242
%
153.89
9.42
0
100
0.47
197
%
198.97
118.85
8.74
155.89
2.02
2.56
3.90
4.84
5.78
9.48
7.87
12.51
0
100
8.68
10.16
4.10
60
100
140
180
220
260
300
340
380
420
460
500
%
0
0.67
6.73
5.72
m/z
372
8.00
13.32
11.50
m/z
0
2.00
4.00
Fig. 3. Mass spectra of A) trazodone, B) mCPP and C) pCPP.
6.00
10.00
8.00
12.00
Retention time [min]
Fig. 5. LC/MS chromatograms of blank hair sample.
100
Ketamine-d4
242
%
3.83
8.88
9.75
0.67
0
%
100
0.20
0.74
4.78
mCPP
197
1.21
12.58
11.50
0
100
6.39
Trazodone
372
%
blank sample and standard sample at a concentration
of 0.5 ng/mg are presented in Figures 5 and 6. They
showed a good separation of components of the samples and lack of influence of an interfering matrix.
Identification was performed on the basis of the ratio
of intensity of ions selected for monitoring and also the
relative retention time. For mCPP the limit of detection was at the level of 0.1 ng/mg hair, and for
trazodone it was 0.02 ng/mg. Linearity for both compounds was confirmed in the range from 0 to 5 ng/mg
for a coefficient of determination (r2) > 0.99. The values obtained for other validation parameters fulfilled
criteria required for this type of analysis and were:
intra-group precision for a concentration of 0.5 ng/mg:
mCPP – CV 6.5%, trazodone – CV 14.0%, for a concentration of 2 ng/mg: mCPP – CV 5.3% trazodone –
8.74
0
2.00
4.00
6.00
8.00
10.00
12.00
Retention time [min]
Fig. 6. LC/MS chromatograms of spiked hair sample with
trazodone and mCPP 5 ng/mg and internal standard 1 ng/mg.
Problems of Forensic Sciences 2006, LXVI, 131–139
135
Determination of trazodone and its metabolite...
TABLE II. MONITORED IONS, RETENTION TIMES OF ANALYTES AND THE INTERNAL STANDARD
Analyte
Monitored
m/z
Retention time
[min]
Relative
retention time
Internal
standard
Monitored
m/z
Retention time
[min]
Trazodone
374, 372, 176
6.32
1.260
Ketamine-d4
244, 242
3.77
mCPP
199, 197, 154
4.75
1.676
4. Summary
100
3.70
%
Ketamine-d 4
242
0.67 0.87
2.89
0
100
2.89
197
2.62
%
0.13
mCPP
0.67
1.61
13.05
4.71
6.52
9.01
0
100
6.26
Trazodone
372
%
The developed method was applied to determination of trazodone and mCPP in 7 authentic samples of
hair. In Figure 7, a chromatogram obtained in the
course of analysis of the first 1 cm segment (nearest the
scalp) of a hair sample collected 1 month after the person had taken one 75 mg dose of trazodone is presented. As a result of the performed analysis of all
three segments, the following concentrations of analysed compounds were obtained: 1. segment (1 cm)
trazodone – 5.93 ng/mg and mCPP – 0.1 ng/mg; 2. segment (1 cm) trazodone – 0.04 ng/mg; mCPP was not
detected (Figure 8). Knowing the date of collection of
the sample, the average rate of growth of hair (about
1 cm/month [6, 7]) and analysing successive sequences
of hair, information can be gained – through defining
the concentrations of these substances – on the history
of their taking during particular periods of time. Detection of trazodone at the greatest concentration (5.93 g)
in the first segment (counting from the scalp) of hair of
length 1 cm and its metabolite mCPP (0.1 ng/mg) corresponds to the time of taking of this medicine and
confirms the mentioned phenomenon. Trazodone detected in successive segments may attest to the fact
that a part of the substances became incorporated into
the hair through sweat (sweat model [2]) or migrated
along the hair. mCPP, being a metabolite of trazodone,
and at the same time a more polar compound, has more
difficult conditions for migration through the cellular
membrane from blood to hair, thus its concentration
both in blood and in hair is low. Analysis of hair also
allows identification of the parent compound which
was consumed – unlike bodily fluids, in which sometimes the metabolite is present at a significantly greater
concentration than the parent substance. The six remaining hair samples collected from persons taking
narcotics from the group of amphetamine derivatives,
especially ecstasy tablets, did not reveal their presence.
0
2.00
4.00
6.00
8.00
10.00
12.00
Retention time [min]
Fig. 7. Authentic hair sample – segment 1.
6
5
4
Trazodone
mCPP
3
2
1
0
Segment 1
Segment 2
Segment 3
Fig. 8. The results of hair segmental analysis (trazodone
case).
use of the LC-ESI-MS technique was developed. Parameters of the work of the mass spectrometer were
optimised, and the analytical procedure validated. The
method was applied to analysis of real hair samples. It
enables detection of the consumption of a single dose
of trazodone (75 mg) together with its metabolite
mCPP and also differentiation between taking of the
first and the second.
References
A method for determination of trazodone and its
active metabolite 1-(3-chlorophenyl)piperazine with
1. Baumgartner W. A., Discussion of hair analysis for drugs
of abuse, Journal of Forensic Sciences 1990, 35, 778–779.
Problems of Forensic Sciences 2006, LXVI, 131–139
136
R. Stanaszek, W Lechowicz
2. Cone E. J., Mechanism of drug incorporation into hair,
Therapeutic Drug Monitoring 1996, 18, 438–443.
3. de Boer D., Bosman I., Hidvegi E. [et al.], Piperazine-like
compounds: a new group of designer drug-of-abuse on the
European market, Forensic Science International 2001,
121, 47–56.
4. Fiorella D., Helsley S., Rabin R. A. [et al.], 5-HT2C receptor-mediated phosphoinositide turnover and stimulus
effects of m-chlorophenylpiperazine, Psychopharmacology 1995, 122, 237–243.
5. Murphy D. L., Lesch K. P., Aulach C. S. [et al.], Serotonin-selective arylpiperazines with neuroendocrine, behavioral, temperature and cardiovascular effects in humans, Pharmacological Reviews 1991, 43, 527–552.
6. Sachs H., Forensic applications of hair analysis, [in:]
Drug testing in hair, Kintz P. [ed.], CRC Press, Boca
Raton 1996.
7. Sachs H., Theoretical limits of the evaluation of drug concentrations in hair due to irregular hair growth, Forensic
Science International 1995, 70, 53–61.
8. Society of Hair Testing, Statement of the Society of Hair
Testing Concerning, the examination of drugs in human
hair, Forensic Science International 1997, 84, 3–6.
Corresponding author
Roman Stanaszek
Instytut Ekspertyz S¹dowych
ul. Westerplatte 9
PL 31-033 Kraków
e-mail: [email protected]
Problems of Forensic Sciences 2006, LXVI, 131–139
Problems of Forensic Sciences 2006, LXVI, 131–139
OZNACZANIE TRAZODONU I JEGO METABOLITU
1-(3-CHLOROFENYLO)PIPERAZYNY (mCPP) WE W£OSACH METOD¥
CHROMATOGRAFII CIECZOWEJ SPRʯONEJ ZE SPEKTROMETRI¥
MAS (HPLC-ESI-MS)
1. Wprowadzenie
Trazodon jest przeciwdepresyjnym lekiem z grupy
pochodnych triazolopirydyny (rycina 1), selektywnym
inhibitorem wychwytu zwrotnego serotoniny oraz antagonist¹ receptorów 5-HT2 wykazuj¹cym dzia³anie przeciwdepresyjne, nasenne i uspokajaj¹ce. Stosowany jest
w leczeniu depresji, a tak¿e zaburzeñ zwi¹zanych z agresywnym zachowaniem, atakami paniki, w stanach napiêcia i niepokoju, w alkoholizmie, a tak¿e w leczeniu g³odu
kokainowego. W w¹trobie lek ten jest metabolizowany
na drodze hydroksylacji, dealkilacji i N-oksydacji.
1-(3-chlorofenylo)piperazyna, znana pod akronimem
mCPP (rycina 2), jest aktywnym metabolitem powsta³ym
w wyniku przemian w organizmie trazodonu oraz innych
leków antydepresyjnych, takich jak nefezodon, etoperidon, cloperidon, enziprazol i fefazodon, a tak¿e œrodka
przeciwbólowego mepiprazolu [3]. Aktywny metabolit
mCPP tworzy siê w wyniku N-dealkilacji azotu w pierœcieniu piperazynowym. Samo mCPP, jako substancja bêd¹c¹ pochodn¹ piperazyny, zalicza siê do tzw. narkotyków projektowanych (designer drugs) i jest wci¹¿ legaln¹ substancj¹ nie objêt¹ kontrol¹ przeciwnarkotykow¹
w wiêkszoœci pañstw, w tym równie¿ w Polsce. Mechanizm farmakologicznego dzia³ania mCPP polega na interakcji z ró¿nymi receptorami serotoniny, jak równie¿
z receptorami adrenergicznymi i dopaminergicznymi, co
prowadzi do wydzielenia serotoniny [4]. Dzia³anie to jest
analogiczne do MDMA, st¹d na rynku narkotykowym
mCPP jest zazwyczaj rozprowadzane w postaci tabletek
imituj¹cych tabletki „ecstasy”. Przyjêcie mCPP do organizmu prowadzi do szerokich neuroendokrynologicznych, fizjologicznych i psychologicznych efektów opisanych przez Murphy’ego i in. [5]. Znane s¹ przypadki
wykrycia mCPP w materiale biologicznym u osób, które
wczeœniej przyjê³y ten narkotyk.
Przyjêcie trazodonu mo¿e skutkowaæ efektami ubocznymi w postaci m.in. priapizmu u mê¿czyzn, arytmii,
bólów g³owy, zaburzonej mowy czy biegunki. Trazodon
wp³ywa tak¿e ujemnie na sprawnoœæ psychomotoryczn¹
kierowców.
W³osy stanowi¹ cenny materia³ diagnostyczny ze
wzglêdu na nieinwazyjnoœæ pobierania próbek, ich trwa³oœæ, a tak¿e fakt, ¿e analiza w³osów dostarcza informacji
na temat bliskiej i odleg³ej historii przyjmowania danej
substancji, w tym uzale¿nienia lub abstynencji [1].
Celem niniejszej pracy by³o opracowanie, optymalizacja i walidacja procedury analitycznej oznaczania popularnego leku antydepresyjnego - trazodonu - i jego
aktywnego metabolitu, 1-(3-chlorofenylo)piperazyny
(mCPP) we w³osach, pozwalaj¹cej na wykrycie pojedynczej dawki tej substancji.
2. Materia³y i metody
2.1. Próbki w³osów
Próbki w³osów u¿ywane do przygotowania „œlepej
próby” oraz próbek kalibracyjnych by³y pobrane od dzieci nienara¿onych na dzia³anie badanych zwi¹zków.
Szeœæ rzeczywistych próbek w³osów uzyskano od
osób pacjentów poddawanych leczeniu substytucyjnemu
metadonem w Poradni dla Osób Uzale¿nionych od Œrodków Psychoaktywnych Centrum Psychiatrycznego Samodzielnego Publicznego Specjalistycznego Zak³adu
Opieki Zdrowotnej „Zdroje” w Szczecinie, o których
wiadomo by³o z poprzednich badañ, ¿e przyjmowali tak¿e narkotyki z grupy pochodnych amfetaminy, w szczególnoœci tabletki „ecstasy”. Jedn¹ próbkê w³osów o d³ugoœci 3 cm pobrano od pacjenta, który miesi¹c wczeœniej
przyj¹³ jedn¹ 75 mg dawkê trazodonu. Próbki w³osów
pobierano z potylicznej czêœci g³owy wed³ug zaleceñ Towarzystwa Badania W³osów [8].
2.2. Substancje wzorcowe
Substancje wzorcowe: chlorowodorek 1-(3-chlorofenylo)piperazyny (mCPP) oraz jego izomer, 1-(4-chlorofenylo)piperazyna (pCPP), zosta³y zakupione w firmie
Sigma Aldrich (Stany Zjednoczone) w postaci substancji
sta³ych. Standard wewnêtrzny w postaci ketaminy-d4
(1 mg/ml) zakupiono w firmie Cerillian (Stany Zjednoczone).
2.3. Podstawowe odczynniki chemiczne
Acetonitryl (Gradient Grade for HPLC), chlorek
n-butylu (for liquid chromatography) oraz metanol (analytical grade) zakupiono w firmie Merck (Niemcy). Kwas
mrówkowy zakupiono w firmie Ubichem (Wielka Brytania).
138
R. Stanaszek, W Lechowicz
2.4. Przygotowanie próbek i analiza w³osów
Ka¿da próbka zosta³a poddana procesowi dekontaminacji, który obejmowa³ mycie w³osów przy u¿yciu sekwencji rozpuszczalników: dichlorometan, woda, metanol. W celu przeprowadzenia analizy segmentowej
(analizy poszczególnych odcinków w³osów), wczeœniej
wysuszone próbki w³osów poddano podzia³owi na jednolub dwucentymetrowe segmenty, zaczynaj¹c od miejsca
uciêcia w³osów, a¿ do koñca kosmyka. W celu wstêpnego rozdrobnienia próbki w³osów pociêto no¿yczkami na
czêœci o d³ugoœci ok. 1 mm, a nastêpnie zmielono w m³ynku
kulowym. Do analizy odwa¿ano po 50 mg zmielonych
w³osów pochodz¹cych z ka¿dego segmentu i poddano
kolejnym etapom analizy.
W celu sporz¹dzenia krzywych kalibracyjnych i przeprowadzenia walidacji metody odwa¿ano 50-miligramowe zmielone próbki w³osów nie zawieraj¹cych analitów.
Nastêpnie dodawano do nich roztwory robocze trazodonu i mCPP w odpowiednich iloœciach, a tak¿e 5 ml roztworu ketaminy-d4 o stê¿eniu 10 mg/ml. Ostateczne stê¿enie standardu wewnêtrznego w ka¿dej próbce wynosi³o 1 ng/mg.
Próbki w³osów wraz z dodanymi substancjami wzorcowymi poddawano alkalicznemu roztwarzaniu hydrolitycznemu. Do ka¿dej próbki dodawano po 0,4 ml 0,5 M
roztworu wodorotlenku sodu i inkubowano przez 1 godzinê w temperaturze 70°C. W celu wyizolowania analitów z otrzymanego roztworu zastosowano ekstrakcjê typu ciecz-ciecz z u¿yciem 1 ml chlorku n-butylu przez
5 min. Rozpuszczalnik odparowano w strumieniu azotu
po uprzednim dodaniu 100 ml 0,025 M kwasu solnego.
Analizê amfetamin we w³osach przeprowadzano przy
pomocy chromatografu cieczowego sprzê¿onego z kwadrupolowym detektorem mas LC-MS firmy Waters (Stany Zjednoczone) serii Alliance z komor¹ do jonizacji
przez elektrorozpylanie (ESI). Jako gaz rozpylaj¹cy stosowano azot wytwarzany z generatora azotu firmy Whatman (Stany Zjednoczone). Analizowano jony dodatnie
w trybie monitorowania wybranych jonów (SIR). Chromatograf cieczowy by³ wyposa¿ony w kolumnê LiChroCART 125 ´ 3 z wype³nieniem Purospher 60 RP-18e,
5 mm (Merck, Niemcy) termostatowan¹ w 35°C. Faza ruchoma sk³ada³a siê z mieszaniny wody redestylowanej
z dodatkiem kwasu mrówkowego (1000 ml/litr) oraz acetonitrylu z dodatkiem kwasu mrówkowego (1000 ml/litr).
Przep³yw fazy ruchomej wynosi³ 0,8 ml/min i odbywa³
siê w systemie gradientowym: 0 min – 10% acetonitryl
(AcCN), 8 min 40% AcCN, 9 min 10% AcCN i 14 min
10% AcCN. Wstrzykniêcia realizowa³ automatyczny podajnik próbek, a objêtoœæ nastrzyku wynosi³a 20 ml.
Analiza by³a prowadzona w trybie monitorowania
wybranych jonów. Wartoœci parametrów pracy spektrometru mas, które by³y stosowane w trakcie przeprowa-
dzanych badañ, umieszczone s¹ w tabeli I. Ca³kowity
czas akwizycji wynosi³ 17 min.
3. Wyniki i dyskusja
Optymalizacja pracy spektrometru mas zosta³a przeprowadzona metod¹ bezpoœredniego wprowadzenia
próbki do urz¹dzenia (direct inlet). Jako kryterium optymalizacji wybrano pole powierzchni uzyskane dla pseudomolekularnych jonów analitów. Trazodon i mCPP po
rozpuszczeniu w fazie ruchomej systemu LC-MS by³y
wstrzykiwane do komory jonizacyjnej i analizowane
w ca³ym zakresie mas (m/z 50–400 amu) przy ró¿nym napiêciu fragmentora (30, 60 i 90 V) w celu wyboru najbardziej intensywnych jonów do analizy SIR. Do analizy
iloœciowej wybrano dodatnie jony pseudomolekularne
oznaczanych substancji (m/z - 197 dla mCPP, m/z – 372
dla trazodonu) i deuterowanej ketaminy (m/z – 242). Dodatkowo wybrano po 2 jony dla analitów: m/z 199, 154
dla mCPP i 374, 176 dla trazodonu oraz jeden 242 dla ketaminy-d4, które wykorzystywano do identyfikacji wraz
jonami pseudomolekularnymi. Uzyskane widma masowe
trazodonu, mCPP i pCPP przedstawione s¹ na rycinie 3.
W dalszej kolejnoœci przeprowadzono optymalizacjê parametrów ESI-MS, takich jak: napiêcie kapilary (CV)
i fragmentora (CNV), temperatury Ÿród³a (ST), temperatury desolwacji (DT) i przep³ywu gazu desolwacyjnego
(DGF). Uzyskane optymalne wartoœci dla poszczególnych parametrów przedstawione zosta³y w tabeli I.
W procesie walidacji procedury analitycznej wykorzystane zosta³y krzywe kalibracyjne uzyskane poprzez
analizê metod¹ LC-MS próbek w³osów (50 mg), do których dodawano odpowiednie iloœci roboczych roztworów analitów trazodonu i mCPP w zakresie stê¿eñ od 0 do
5,0 ng/mg oraz standardu wewnêtrznego ketaminy-d4
w iloœci odpowiadaj¹cej stê¿eniu 1 ng/mg. Krzywe kalibracyjne oraz ich parametry zosta³y przedstawione na rycinie 4. Czasy retencji, wzglêdne czasy retencji i monitorowane jony dla poszczególnych zwi¹zków przedstawione s¹ w tabeli II. Chromatogramy obrazuj¹ce rozdzia³
badanych zwi¹zków dla wybranych jonów uzyskane dla
œlepej próby oraz próbki wzorcowej o stê¿eniu 0,5 ng/mg
przedstawione s¹ na rysunku 5 i 6. Œwiadcz¹ one o dobrym rozdziale sk³adników próbek i braku wp³ywu interferuj¹cej matrycy. Identyfikacji dokonywano na podstawie stosunku intensywnoœci wybranych do monitorowania jonów oraz wzglêdnego czasu retencji. Dla mCPP
wyznaczono granicê detekcji na poziomie 0,1 ng/mg w³osów, a dla trazodonu wynosi³a ona 0,02 ng/mg. Liniowoœæ dla obu zwi¹zków zosta³a potwierdzona w zakresie
od 0 do 5 ng/mg przy wspó³czynniku determinacji (r2) > 0,99.
Uzyskane wartoœci innych parametrów walidacyjnych
spe³nia³y kryteria wymagane dla tego typu analiz i wynosi³y: precyzja wewn¹trzgrupowa dla stê¿enia 0,5 ng/mg:
Problems of Forensic Sciences 2006, LXVI, 131–139
Oznaczanie trazodonu i jego metabolitu...
mCPP - CV 6,5%, trazodon – CV 14,0%, dla stê¿enia
2 ng/mg: mCPP - CV 5,3% trazadon - CV 10,1%, odzysk: 70% dla mCPP i 129% dla trazodonu przy stê¿eniu
2 ng/mg).
Opracowana metoda zosta³a zastosowana do oznaczenia trazodonu i mCPP w 7 autentycznych próbkach
w³osów. Na rycinie 7 przedstawiono chromatogram
uzyskany w trakcie analizy pierwszego (od strony skóry
g³owy) 1 cm segmentu próbki w³osów pobranych w terminie 1 miesi¹ca po przyjêciu przez osobê jednej 75 mg
dawki trazodonu. W wyniku przeprowadzonej analizy
wszystkich trzech segmentów uzyskano nastêpuj¹ce stê¿enia analizowanych zwi¹zków: 1. segment (1 cm) trazodon – 5,93 ng/mg i mCPP - 0,1 ng/mg; 2. segment
(1 cm) trazodon – 0,04 ng/mg; mCPP nie zosta³o wykryte
(ryscina 8). Znaj¹c datê pobrania próbki, œredni¹ szybkoœæ wzrostu w³osów (ok. 1 cm/miesi¹c) [6, 7]) i analizuj¹c kolejne sekwencje w³osów, mo¿na - poprzez okreœlenie stê¿enia tych substancji - uzyskaæ informacje na temat historii ich przyjmowania w poszczególnych odcinkach czasu. Wykrycie trazodonu w najwiêkszym stê¿eniu
(5,93 g) w pierwszym (licz¹c od skóry g³owy) odcinku
w³osa o d³ugoœci 1 cm oraz metabolitu mCPP (0,1 ng/mg)
odpowiada czasowi przyjêcia tego leku i potwierdza
wspomniane zjawisko. Wykryty trazodon w nastêpnych
odcinkach mo¿e œwiadczyæ o tym, ¿e czêœæ substancji
wbudowa³a siê do w³osa poprzez pot (model potowy [2])
lub uleg³a migracji wzd³u¿ w³osa. mCPP, bêd¹ca metabolitem trazodonu, a tym samym zwi¹zkiem bardziej polarnym, ma utrudnione warunki do migracji poprzez
b³onê komórkow¹ z krwi do w³osa, st¹d jego stê¿enie zarówno we krwi, ale tak¿e i we w³osach, jest niskie. Analiza w³osów pozwala tym samym zidentyfikowaæ macierzyst¹ substancjê, która zosta³a przyjêta, w przeciwieñstwie do p³ynów ustrojowych, w których niejednokrotnie
metabolit jest obecny w znacznie wiêkszym stê¿eniu ni¿
macierzysta substancja. Szeœæ pozosta³ych próbek
w³osów pobranych od osób przyjmuj¹cych narkotyki
z grupy pochodnych amfetaminy, w szczególnoœci tabletki
„ecstasy”, nie wykaza³o ich obecnoœci.
4. Podsumowanie
Opracowano metodê oznaczania trazodonu oraz jego
aktywnego metabolitu 1-(3-chlorofenylo)piperazyny z wykorzystaniem techniki LC-ESI-MS. Parametry pracy
spektrometru mas zosta³y zoptymalizowane, a procedura
analityczna zwalidowana. Metoda zosta³a zastosowana
do analizy rzeczywistych próbek w³osów. Pozwala ona
wykryæ przyjêcie pojedynczej dawki trazodonu (75 mg)
wraz z jego metabolitem mCPP oraz rozró¿niæ przyjêcie
tego pierwszego od drugiego.
Problems of Forensic Sciences 2006, LXVI, 131–139
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