carbamazepine and xenobiotics found in the autopsy specimen during the postmortem investigation of a body. Introduction. Carbamazepine (CBZ) (Arnizepin ...
Journal of Analytical Toxicology, Vol. 27, May/June 2003
[ TechnicalNote
PostmortemToxicologyof Carbamazepine Malgorzata Klys 1, Beata Bystrowska 2, and Beata Bujak-Gizycka 1
ICollegium Medicum]agiellonian University, Institute of ForensicMedicine, Departmentof Toxicology, 16 Grzeg6rzeckaStreet, 31-531 Krak6w, Polandand 2Collegium MedicumJagiellonian University, Departmentof Toxicology, Faculty of Pharmacy, 9 Medyczna Street,30-688 Krak6w, Poland
I Abstract ]
Materials and Methods
The study focuses on a series of 16 fatal cases in which carbamazepine and its two major metabolites (10,11-epoxide and 10,11-dihydroxycarbamazepine) were detected in body fluids and tissues collected at autopsy. The drug may be implicated in a number of deaths; however, most of these are multiple-drug intoxications with a particular contribution of ethanol. The investigations concerning toxicological findings are a source of toxicological postmortem data and show the differences in metabolism rate as depending on the concentration level of carbamazepine and xenobiotics found in the autopsy specimen during the postmortem investigation of a body.
Chemicals and reagents All chemicals and solvents were of analytical grade. Standards of CBZ; 10,11-epoxycarbamazepine(CBZ-E);and 10,11dihydroxycarbamazepine (CBZ-DHD) were obtained from Sigma (St. Louis, MO). lYifluoroacetic acid was from Fluka (Buchs, Switzerland),acetonitfile and ethyl acetate from Merck (Darmstadt, Germany), and buffer/substance IYis from Serva (Heidelberg,Germany).
Introduction
Carbamazepine (CBZ) (Arnizepin,Polpharma; Tegretol, Novartis) is structurally related to tricyclic antidepressants; however, it has different pharmacological properties. The compound was synthesized in the 1950s, approved in the U.S. for the treatment of trigeminal neuralgia in 1968, and as anticonvulsant in 1974 (1). Today, it is a well-known drug with a unique spectrum of psychotropic activity. It is used in the treatment of temporal lobe epilepsyand trigeminal neuralgia. It also shows some acute antidepressant effectsand is a useful drug in the treatment of excited states in patients suffering from schizophrenicand schizoaffectivedisorders (2,3). Though CBZ is considereda relativelysafe drug, CBZ-related deaths are occasionally encountered. This study focuses on a series of 16 deaths in which carbamazepine and its major metabolites were detected. In seven of these cases, carbamazepine was the only drug involved in four of them carbamazepine and ethanol were found. Five cases showedthe presence of additional drugs; of the latter, one also included ethanol. The results are discussedwith respect to the levelsof carbamazepine and its metabolites found and with regard to the potential for drug interactions.
Materials Postmortem autopsy specimens, samples of femoral blood, liver, and kidney were collected at autopsy during investigations of fatalities occurring in the Krak6w district between 1999 and 2000. The autopsies were performed at the Institute of Forensic Medicine (Collegium Medicum,Jagiellonian University, Krak6w,Poland) in the 24 h followingdeath. The exact time between ingestion of drug(s) and death was generally not known. In some cases, the time of life in hospital was up two days. The samples remained frozen (-22~ until the analyses were performed. Parallel with the materials taken from the authentic subjects, standards of CBZ, CBZ-E, and CBZ-DHD and "blank" samples of autopsy blood and liver taken from nonpoisoned subjects spiked with these drugs were investigated. The latter materials were used to facilitate identification, quantitation, and validation by means of liquid chromatography-mass spectrometry (LC-MS). Extraction
Samples of postmortem autopsy blood, liver, and kidney were collected for the analysis. Tissueswere homogenized in a homogenizer at 10,000 x g for 3 min and subjected to liquidliquid extraction. Quinidine at 5 pg/g was used as the internal standard (IS). All subjects of analysis were negative for quinidine.At first, samplesof 5 g were mixedwith 5 g 1Yisbuffer(hydroxymethyl-aminomethane)pH 9.0 and put in an ultrasonic
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Journal of Analytical Toxicology, Vol. 27, May/June 2003
bath for 15 rain. Afterwards, the samples were mixed with 7.5 mL of acetonitrile to carry out protein precipitation then vortex mixed and centrifuged. The supernatant was mixed with 15 mL of ethyl acetate and the solutions were separated on Whatman Phase Separators 1PS silicone-treated filter paper (Maidstone, England). The organic phase was collected, evaporated to dryness at 40~ under a stream of nitrogen, the residues were reconstituted in 1000 IJL of the high-performance liquid chromatography (HPLC) mobile phase. Ten microliters of the reconstituted extracts was injected into the LC-MS system for identification.
Calibration curves and quantitation Calibration curves were constructed after the analysis of drug-free blood and liver samples (blank samples) containing known amounts of CBZ, CBZ-E, and CBZ-DHD. To prepare these standards, blood and liver samples were spiked with the studied compounds to the following concentrations: 1, 1.5, 3, 5, 10, 15, 30, 50, and 100 tJg/g. Quinidine at 5 IJg/g was used as the IS. The samples were extracted according to the described procedure. The number of quality controls per batch was six (three concentration levels in duplicate). They were assessed according to generally accepted rules (4,5). Calibration curves were constructed by plotting the peak area ratios CBZ/IS or CBZ-E (CBZ-DHD)/IS for blood and for liver. Recoveries were calculated for blood and liver. The results of the validation procedure are presented in Table I. LC-MS A Finnigan MAT LC (San Jose, CA) equipped with a pump model TSP 4000 and an autosampler model TSP AS 3000 with a 10-1~Linjection loop were used in gradient mode. The chromatographic separation was performed with a Purospher RP 18 column 125 x 3-mm i.d., 5-1~m particle size and a Lichrocard precolumn 4 x 4-mm i.d. filled with Lichrospher 60 5-~m particle size (Merck, Darmstadt, Germany). The mobile phase consisted of a gradient mixture of the phase (A) which was 1% trifluoroacetic acid (TFA) in demineralized water and mixture (B) which was 90% acetonitrile + 10% of the phase (A). The flow rate was 0.4 mDmin. The gradient was programmed as follows: 5% (A) and 95% (B) for 2 min, followed by a linear increase to 70% (A) and 30% (B) in 30 min, and then after 2 rain decreased to 5% (A) and 95% (B) for 8 rain. An LCQ ion trap mass detector (Finnigan MAT) equipped with an electrospray ionization (ESI) source was used. The ESI inlet conditions were as follows: sheath gas (nitrogen) pressure 70 p.s.i., auxiliary gas (nitrogen) 10 mL/min, heated capillary
temperature 200~ and source current 100 mA. Mass spectra of the substances involved were taken between m/z 50 and 650 at octapole offset of 10 V (positive ions). Selected ion monitoring (SIM) detection of drugs of interest in one chromatographic run was realized based on mass spectra in full scan mode and on the observed retention times. Parallel to the mass detector, a detector model TSP UV2000 (Finnigan MAT) range 200--400 nm was used.
Analysis of drugs Samples were analyzed with special emphasis on CBZ, CBZE, and CBZ-DHD. If the chromatograrns indicated a possible presence of other drugs/metabolites, an identification of the xenobiotic found was attempted by comparing its mass spectrum. UVabsorption data and retention data corresponded with those present in available libraries. When adequate matches were found, further evidence was obtained by analyzing a reference of the suspected drug, and, if possible, a calibration curve for that drug was made for quantitation.
Results The methods described above were appIied to the analysis of drugs and their metabolites in autopsy specimens of carbamazepine-related cases, which were subjects of ours investigations. The data pertaining to 16 cases are listed in Table II together with the available information on the circumstances, manner, and causes of death. Six subjects were probable suicides, five were accidents, two natural deaths, one homicide, and in two cases the manner of death could not be determined. In the 16 cases reviewed here, we evaluated blood, liver, and kidney samples (if available) in which CBZ and their two metabolites had been detected during routine postmortem toxicological examinations. An example of the identification of carbamazepine and its metabolites in autopsy blood (case 2 in Table II) is presented in Figure 1. In our series, there were four cases (Table II, cases 1-4) in which death was attributed to CBZ alone. The blood level ranged from 16 IJg/g in case 2 to almost 100 ~g/g in case 3. In the liver, the upper limit of carbamazepine found was almost twice as high (case 3). Case 3 is special because of a very high concentration level of metabolites in the blood, whereas in the liver CBZ-E reached an inexplicably low value as opposed to CBZ-DHD,which was twice as high as CBZ-DHD in relation to CBZ.
Table I. Validation Parameters for CBZ, CBZ-E, and CBZ-DHD
Concentration Drug CBZ
(mg/mL)
Bias (%)
RSD (%)
Blood
Liver
Blood
Liver
98.9 +- 2.91
87.0 -+ 6.42
1.4 - 18.1
1.9 - 43.4
-26.31 - 14.57
-19.08 - 16.66
CBZ-E
98.9 _+ 2.91
87.0 • 6.42
5.0 - 29.8
0.3 - 15.3
-19.66 - 23.81
-15.66 - 17.09
CBZ-DHD
42.7 + 13.82
36.8 • 0.95
1.9 - 18.5
1.6 - 35.3
-12.26 - 17.82
- 1 6 . 6 0 - 15.48
244
1-100
Mean recovery (for each level) Blood Liver n=3
Journal of AnalyticalToxicology,Vol. 27, May/June2003
Table II. Postmortem Toxicology Data of Fatal Cases Involving CBZ Alcohol
Drugsconc.
Age Gender Circumstances
Cause
Manner
(%)
(mg/L)
Blood
Liver
Kidney
1
42
M
Treatedin hospitalfor 2 days, died there, psychiatrypatient
Acute intoxication
Suicide
neg
CBZ CBZ-E CBZOH
39.40 10.40 6.52
56.48 6.26 3.70
8.29 2.38 1.25
2
50
M
Founddead in the bathroom, drug addict
Acute intoxication
Suicide
neg
CBZ CBZ-E CBZOH
16.04 3.06 3.25
130.60 20.94 50.80
NA* NA NA
3
35
F
Found dead at home, epilepsy, psychiatrypatient
Acute intoxication
Suicide
neg
CBZ CBZ-E CBZOH
99.45 274.47 199.09
218.79 8.85 428.17
40.69 85.02 73.90
4
39
F
Found dead at home
Acute intoxication
Suicide
neg
CBZ CBZ-E CBZOH
18.79 6.93 23.74
19.51 0.95 12.74
4.37 1.99 5.06
5
22
F
Death in hospital,epilepsy from childhood, pregnancy 4 months
Undetermined Natural
neg
CBZ CBZ-E CBZOH
3.14 1.60 1.21
6.35 5.02 2.02
16.62 6.87 4.87
6
7
M
Treatedbecasuseof epilepsy, murdered by strangulation
Asphyxia
Homicide
neg
CBZ CBZ-E CBZOH
4.28 2.03 0
6.50 3.18 1.63
1.24 3.98 0
7
29
M
Suddendeath in hospital, psychiatrypatient,epilepsy, mentallydeficient
Myocardial infarction
Natural
neg
CBZ CBZ-E CBZOH
10.00 0.74 0
21.27 0.33 0
8.51 0.48 0
8
54
F
Found dead nearhouse, neurologypatient,alcoholic
Acute intoxication
Accident
I3-0.5 U-2.2
CBZ CBZ-E CBZOH
32.44 11.10 0.61
14.65 0.62 0
40.34 0.81 1.05
M
No data, found on the street
Undetermined Unknown
B-0.2 U-1.6
CBZ CBZ-E CBZOH
6.85 0.37 0
112.71 19.89 12.04
NA
9
10
32
M
Founddead at home, epilepsy
Acute intoxication
Suicide
B-neg U-2.0
CBZ CBZ-E CBZOH
50.50 1.35 0.40
6.78 0.41 0.87
4.67 0.89 0.26
11
46
W
Founddead in the bathroom, epilepsy,depression
Acute multiple intoxication
Accident
B-2.6 U-3.2
CBZ CBZ-E CBZOH
20.18 0.43 0.19
362.48 0 0
58.85 1.86 0.54
12
54
M
Death in hospitalduring I day, psychiatrypatient
Acutemultiple intoxication
Unknown NA
B-2.22
CBZ CBZ-E CBZOH Estazolam
11.53 0.72 3.13 1.52
10.10 0.68 1.21 0.80
8.89 0.79 2.03 0.40
13
70
M
Death in hospitalafter2 days, drug abuser
Acute multiple intoxication
Suicide
neg
CBZ CBZ-E CBZOH Estazolam
2.33 0.79 0.59 1.15
4.19 0.09 0.94 1.56
2.07 0.36 1.45 0.75
14
33
M
Founddead in hostel
Acute multiple intoxication
Accident
neg
CBZ CBZ-E CBZOH Phenobarbital
7.08 1.68 3.14 23.5
1.57 0.29 1.27 -
NA
CBZ CBZ-E CBZOH Diazepam Nordiazepam
3.66 1.82 0.62 0.2 I .I
14.95 1.51 6.14 0.6 10.9
6.89 3.84 4.22 0.5 6.9
CBZ CBZ-E CBZOH Morphine (total)
3.48 0.05 0 1.13
5.86 0.03 0.02 2.10
15
16
26
M
Founddead at home, alcoholic, psychiatrypatient
Probably multiple intoxication
Accident
neg
F
Found dead at the park, no data
Acute multiple intoxication
Probably accident
13-2.9 U-3.5
-
NA
* NA, notavailable.
245
Journal of Analytical Toxicology, Vo[. 27, May/June 2003
Cases 5-7 show therapeutic concentrations of CBZ plus low levels of its metabolites, which is in line with the ruling that, in these cases, death was because of other causes. Nine cases (cases 8-16) presented here with lower concentration levels of CBZ, in the range from therapeutic to fatal, also had certain concentrations of other xenobiotics including ethanol, benzodiazepines, and morphine, which emphasizes the importance of taking into consideration potential interactions with other drugs or preexisting disease (e.g., epilepsy). As to the possibility of interactions between the mentioned xenobiotics, CBZ was considered by the pathologists to be a factor contributing to toxicity in these deaths. In this group of nine cases, four (cases 8-11) were considered combined CBZ/ ethanol intoxications. The concentration of ethanol in blood was low, whereas in urine it was significantly higher indicating death in the late phase of elimination. The remaining five cases (cases 12-16) were mixed-drug intoxications, in which CBZ and its metabolites were found together with other drugs. We calculated the parent drug to metabolites (P/M) concentration ratio in our series of CBZ poisonings (Table II) involving CBZ/CBZ-Eand CBZ/CBZ-DHD.The results are shown in Figure 2 for blood (Figure 2A), liver (Figure 2B), and kidney (Figure 2C) samples. The lowest P/M values can be noted in cases involving only CBZ, whereas the highest values are found in cases of combined CBZ/ethanol intoxications (cases 8-11). An intermediate position is taken by cases involving CBZ and other drugs such as estazolam, phenobarbital, and diazepam. These relationships are especially clear in the blood and kidney. Because the concentrations of metabolites in the liver in mixed cases were low (including negative values), or even not measurable in mixed intoxications of CBZ and other drugs, the P/M ratios for this tissue show high values.
Discussion
CBZ has a relatively small volume of distribution of 0.8-2.0 L/kg, which implies that the drug and possibly its metabolites do not accumulate extensively in tissues. CBZ apparently is not subject to postmortem redistribution; in a series of deaths, the heart/femoral blood concentration ratio averaged 0.9 (6). Although a total of 33 metabolites of CBZ have been described, the major pathway of its biotransformation is 10,11-epoxide formation by hepatic monooxygenase, followed by subsequent hydrolysis to 10,11-dihydroxycarbamazepine by microsomal epoxide hydrolase. A minor pathway results in iminostilbene formation, and, finally, glucuronic acid conjugation also occurs. The quantitative importance of the epoxide-diol pathway is affected by the presence of other xenobiotics (e.g., ethanol or anti-epileptic drugs). A half-life of between 5 and 13 h was determined in polytherapy, which is usually higher under single and/or chronic use. The distribution characteristics of 10,11epoxide, with an activity similar to that of the parent drug, have been studied because of its potential contribution to the therapeutic and toxic effects of the parent drug (1,2,7-9). Although the biochemical properties of 10,11-epoxide have been quite well established, 10,11-dihydroxycarbamazepine has not been so thoroughly investigated. From our studies, the detection of CBZ~ may characterize more fully the course of carbamazepine metabolism, dependant on time of ingestion. Our observations indicate that in cases of intoxication with CBZ alone, the formation of both metabolites may occur in roughly the same amounts, with some slight deviations in favor of one or the other. In some mixed cases of CBZ/ethanol intoxications, the amount of CBZ-DHD formed may be lower than the amount of CBZ-E, whereas in other instances of mixed cases CBZ/drugs poisoning the NL:2.57E7 RT:0.00-39.99 generation of CBZ-DHD may be more ef11.95 Jig-Quinidine ] m/z =324,5-315,5 325.26 fective than the formation of CBZ-E. ,.~,( MSLech09 These studies show that biotransformation of CBZ is a complex process, probably depending on various factors such as the o 20,70 dose and concentration of CBZ, but the 2 3 i ~ NL:S.13E7 m/z =236.5-237,5 process itself may be also modified by MSLech09 other xenobiotics or dependent on genetic predispositions. According to various authors (1,2, 14. 3 6 10-12), the therapeutic range of serum 2g.23 NL:1.03E6 CBZ is 4-12 lag/g. At levels above 12 lag/g, the frequency and severity of side effects increase dramatically, and massive overdoses result in serum concentration 18.3321 higher than 20 lag/g with initial agitation ~~f~ 271. NL:I.S2E7 m/z = 270.5-271.5 followed by stupor, and in severe cases M S Lech09 coma (1,2,13). CBZ overdose is a serious situation because the symptoms are diffiJ , , , ~ cult to control. Fatalities have occurred 5 o 10 15 20 25 30 35 after the ingestion of as little as 5 g of Time(min) CBZ by an adult (7). Therapeutic and Figure 1. Total ion current used for CBZ, CBZ-E, and CBZ-DHD in autopsy blood examined (case2 in toxic ranges for other drugs present in Table II). the autopsy specimens were reviewed for interpretation of the obtained results
246
Journal of Analytical Toxicology, Vol. 27, May/June 2003
(1,2,10-12). In our series, there were four cases (Table II, cases 1--4) in which death was attributed to carbamazepine alone, and the concentration levels in autopsy blood and other specimens were considered toxic and fatal. CBZ metabolites were found in relatively high concentrations, especially in the liver and in particular cases, the P/M ratio occurred at different values to the precursor. In cases 8--11 (mixed intoxications with ethanol), the presence of CBZ at high levels with relatively low levels of its metabolites renders the probability of drug toxicity greater. Regardless of this, ethanol seems to be a serious contributor to a final toxic effect in mixed cases of intoxications. In association with CBZ-ethanol intoxication cases, Fraser (14) postulated that the most significant effect of alcohol might be the induction of cytochrome P450 2E1 to produce more toxic drug metabolites (e.g., creative free radicals). Alcohol may also affect other pharmacokinetic aspects (e.g., the rate of gastric emptying). Some authors (2) assert that when a patient is taking more than one anti-epileptic drug, one cannot identify the drug causing the toxicity solely on the basis of clinical findings. Anti-epileptic drugs may interact widely with each other, and toxicity may easily occur because the magnitude of these interactions is unpredictable. The latter appears to be confirmed by our observations in the remaining cases in Table II (cases 12-16) and constituting complex intoxications with drugs. It should be stressed that, in the mentioned cases, the concentrations of CBZ in the specimens tested were within the therapeutic range and that there were significant concentrations of metabolites. In cases 12 and 13 (Table II), apart from CBZ,another anti-epileptic drug, estazolam, was found at concentrations close to toxic for estazolam (> 0.1 l~g/g).In case 14, an interaction between CBZ and phenobarbital at a therapeutic concentration (for phenobarbital 20--40 ug/g) may have occurred. Similarly, in case 15, a possible cause of death may be attributed to the result of a CBZdiazepam interaction, the latter also at a therapeutic level (for diazeparn 0.125-0.75 I~g/g).In the cited cases, mixed intoxications with drugs seemed the only cause of death, because other factors were excluded during extensive postmortem investigations. (1,2,10-12). One case of our series (case 16, Table II) involved a mixed intoxication with ethanol, opiates, and CBZ.However,CBZ and its metabolites were found at low therapeutic concentrations, whereas morphine (1.13 pg/g) and ethanol (2.9 %) were found at toxic levels. It may be noted that morphine levels > 0.2 ug/g in blood are being considered toxic (12). CBZ intoxications have been a subject of investigation of a number of authors. The majority of the cases involved nonfatal intoxications, which are seen with an increasing tendency (13,15-20) in the context of clinical observations. Moreover, these observations generally indicate that the pharmacokinetics of various drugs (including CBZ) in overdoses differ significantly from those in normal therapy (21,22). Fatal poisonings with carbamazepine alone occur rarely (1,11,23). Interestingly, May et al. (23) reveal that the last premortem serum levels of CBZ and CE were highly correlated with the
postmortem serum concentration values. This is an important observation for clinical and forensic toxicologybecause the evaluation of postmortem drug concentrations in body fluids or organs requires distinct knowledge on any changes that may occur after death (e.g., redistribution).
T.,o --: ................. I-1
II
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
FFl
Ii!
~-~
~.
is .~
.
-.
, 1 3 I
.-.
!. "_~-'~-'..
~
--,c,..mi[m,i
me-
..
, .?.~9
Figure 2. Concentration ratios of CBZ to CBZ-E and to CBZ-DHD in autopsy specimens of all cases tested: cases 1-7 CBZ, cases 8-11 CBZ + ethanol, and cases 12-16 CBZ + other drugs in blood (A), in liver (B), and in kidney (C).
247
Journal of Analytical Toxicology,Vol. 27, May/June2003
The interaction between CBZ and other drugs/ethanol may be manifold and complex. On the one hand, there may be kinetic factors (e.g., in the metabolism), and on the other there may be dynamic implications (3,8,18,19). These interactions mostly take place in the rnicrosomal enzyme system in the liver. They may result in multiple pathologies, reduced homeostatic responses, reduced renal and possibly hepatic function, and altered pharmacodynamic responses. The P/M ratio may be a very useful factor for the interpretation of toxicological results including the manner of ingestion and the metabolic activities and perhaps also for the evaluation of possible interactions between the xenobiotics involved. Apple (24) suggested this for tricyclic antidepressants. Other authors (11) have stated that for different drugs, high P/M ratios may indicate an acute overdose and low ratios a high chronic dosage. However, a high P/M ratio can also be because of a reduced metabolism of the parent drug, either because of an interaction with other drugs or a genetically low metabolic capacity [i.e., in the so-called poor metabolizers who lack a particular metabolic enzyme (25)]. However, a number of possible CBZ/ethanol/drug interactions have been found, which may explain some of the high metabolic ratios for certain substances because the "poor metabolizer pitfall" is relatively rare. Because the literature on postmortem concentration levels of xenobiotics involved in fatal intoxications is mainly available in the form of case reports, we hope that this study will contribute to the database extending the field of forensic toxicology.
References 1. R.C. Baselt and R.H. Cravey. Disposition of Toxic Drugs and Chemicals in Man, 4th ed. Chemical Toxicology Institute, Foster City, CA, 1995. 2. R.H. Levy, A.J. Wilensky, and G.D. Anderson. Carbamazepine, valproic acid, phenobarbital, and ethisuximide. In Applied Pharmacokinetics Principles of Therapeutic Drug Monitoring, W.E. Evans, J.J. Schentag, and W.J. Jusko, Eds. Lippincott, Williams & Wilkins, Philadelphia, PA, 1992. 3. W. Daniel, L. Janczar, L. Danek, W. Legrum, and K.J. Netter. Pharmacokinetic interaction between carbamazepine and neuroleptics after combined prolonged treatment in rats. NaunynSchmiedeberg's Arch Pharmacol. 145:598-605 (1992). 4. R. Causon. Validation of chromatographic methods in biomedical analysis. Viewpoint and discussion. J. Chromatogr. B 689: 175-180 (1997). 5. F. 8ressolle, M. Bromet-Petit, and M. Audran. Validation of liquid chromatographic and gas chromatographic methods. Application to pharmacokinetics. J. Chromatogr. B 686:3-10 (1996). 6. M. Dalpe-Scott, M. Degouffe, D. Garbutt, and M. Drost. A comparison of drug concentrations in in postmortem cardiac and peripheral blood in 320 cases. Can. Soc. Forensic Sci. J. 28: 113-121 (1995).
248
7. Body fluids postmortem material. In Clark's Isolation and Identification of Drugs and Pharmaceuticals, A.C. Moffat and E.C.G. Clarke, Eds. The Pharmaceutical Press, London, England, 1986, pp 428-429. 8. W. Daniel. Metabolism of psychotropic drugs: pharmacological and clinical relevance. Pol. ]. PharmacoL 47:367-379 (1995). 9. J.L. Maggs, N.R. Pirmohamed, N.R. Kitteringam, and B.K. Park. Characterization of the metabolites of carbamazepine in patient urine by liquid chromatography/mass spectrometry. Drug Metab. Dispos. 25:275-280 (1997). 10. A.H. Stead and A.C. Moffat. A collection of therapeutic, toxic and fatal blood drug concentrations in man. Hum. Toxicol. 3: 437-464 (1983). 11. H. Druid and P. Holmgren. A compilation of fatal and control concentrations of drugs in postmortem femoral blood. J. Forensic ScL 4:79-87 (1997).
12. D.R.A. Uges. Therapeutic and toxic drug concentrations. The Bulletin of the International Association of Forensic Toxicologists, Supplement 26/1. Quo's Printing, Fountain Valley, CA, p 8. 13. D.E Weaver. Massive carbamazepine overdose: clinical and pharmacologic observations in five episodes. Neurology38:755-759 (1998). 14. A.G. Fraser. Pharmacokinetic interactions between alcohol and other drugs. Clin. Pharmacokinet. 33:79-90 (1997). 15. H. Thabet, N. Brahmi, J. Zagdoudi, M. Amamou, A. Hedhili, N. Ben Salah, and M. Yacoub. Acute self-induced poisoning with carbamazepine (in French). PresseMed. 28:955-958 (1999). 16. T. Rosel, J. Schneider, J.T. Fischer, and K.F. Druschky. Carbamazepine poisonings caused by confusing the drugs (in German). Dtsch. Med. Wochenschr. 125:352-356 (2000). 17. T. Tateishi, M. Asoh, H. Nakura, M. Watanabe, M. Tanaka, 1. Kumai, and S. Kobayashi. Carbamazepine induces multiple cytochrome P450 subfamilies in rats. Chem. Biol. Interact. 117: 257-268 (1999). 18. W.W. Shen. Cytochrome P450 monooxygenases and interactions of psychotropic drugs: a five-year update. Int. J. Psychiatry Med. 25:286-290 (1995). 19. W.W. Shen. The metabolism of psychoactive drugs: a review of enzymatic biotransformation and inhibition. Soc. Biol. Psychiatry 41:814-826 (1997). 20. M. Bertram, C.W. Fabien, S. Schwarz, and S. Achwab. Massive carbamazepine overdose: clinical and neurophysiological findings. J. Neurol. 245:745-747 (1998). 21. J.J.Sue and M. Shannon. Pharmacokinetics of drugs in overdose. Clin. Pharmacokinet. 23:93-105 (1992). 22. J. Rosenberg, N.L. Benowitz, and S. Pond. Pharmacokinetics of drug overdose. Clin Pharmacokinet. 6:161-192 (1981). 23. T. May, U. Jurgens, B. Ranbeck, and R. Schnabel. Comparison between premortem and postmortem serum concentrations of phenobarbital, phenytoin, carbamazepine and its 10,11-epoxide metabolite in institutionalized patients with epilepsy. Epilepsy Res. 33:57-65 (1999). 24. F.S.Apple. Postmortem tricyclic concentrations: assesingcause of death using parent drug to metabolite ratio. J. Anal. Toxicol. 13: 197-198 (1989). 25. H. Druid, P. Holmgren, B. Carlsson, and J. Ahlner. Cytochrome P450 2D6 (CYP2D6) genotyping on postmortem blood as a supplementary tool for interpretation of forensic toxicological results. Forensic Sci. Int. 99:25-34 (1999). Manuscript received September 24, 2001 ; revision received June 14, 2002.
