Journal of Analytical Toxicology, Vol. 11, July/August 1987. Distribution of Trimipramine and Its Major Metabolites in a Fatal Overdose Case. Albert D. Fraser*.
Journal of Analytical Toxicology, Vol. 11, July/August 1987
Distribution of Trimipramine and Its Major Metabolites in a Fatal Overdose Case Albert D. F r a s e r *
Department of Pathology, Dalhousie Universi~ and Toxicology Laboratory, Victoria Genera/Hospital, Halifax, Nova Scotia A r t h u r F. Isner
Toxicology Laboratory, Victoria General Hospital, Halifax, Nova Scotia Roland A. Perry
Chief Medical Examiner, Province of Nova Scotia, Halifax, Nova Scotia
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Abstract l A fatality following Ingestion of the tricycllc antidepressant trlmlpramine (SurmontlP) Is described. QuanUtation was performed by high-performance liquid chromatography. Trlmipramine and desmethyltrimipramlne concentrations were 4.8 and 2.1 rag/L, respectively, In postmortem blood. The concentration of trlmlpramine, desmethyltrlmlpramlne, and their respective 2-hydroxy metabolites were also measured in liver and urine. Analysis of gastric contents revealed a tricyclic antidepressant drug. These findings are compared to previously published reports of trimipraminerelated fatalities.
X I / R CH2 - C H CH2 - N I CH3 \CH3
X X X X
= H, R H, R = OH, R = OH, R
= = = =
CH 3 H CH3 H
Trimipramine Desmethyltrimipramine 2-Hydroxytrirniprarnine 2-Hydroxydesmethyltrirnipramine
Figure 1. Chemical structure of trimipramine and major metabolites. Introduction Trimipramine maleate (Surmontil | Rhone-Poulenc, Pharma, Inc.) has been used as an antidepressant drug in Europe since 1961. It differs chemically from its prototype imipramine by replacement of the n-propyl side chain with a 2-methyl propyl group (Figure 1). Pharmacologically, the drug is characterized by its sedative and antidepressant action (1-4). Unlike imipramine, it has been found to have a slight monoamine oxidase inhibiting activity (5). Trimipramine was noted to produce significant anxiolytic effect when 150 mg/day were ingested. Studies demonstrating its efficacy in peptic ulcer treatment have also been published (6). Few fatal overdoses involving trimipramine have been reported (7-10). To our knowledge, only one report included measurement of the active desmethyl metabolite (11). No previous reports included quantitation of 2-hydroxy trimipramine and 2-hydroxydesmethyltrimipramine.
Case R e p o r t A 25 year old male was found dead, sitting on a kitchen chair. Multiple prescription containers belonging to a friend were on the table. These included Tetracycline (250 mg), Diazepam (5 mg), Oxazepam (15 mg), and Trimipramine (50 mg). 9Reprint requests should be addressed to Dr. A.D. Fraser, Head, Toxicology Laboratory, Victoria General Hospital, 1278 Tower Road, Halifax, Nova Scotia, Canada, B3H 2Y9, (902) 428-3467.
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External examination showed no signs of injury. Internal examination revealed pulmonary congestion with scattered bilateral atelectasis. A bright yellowish fluid was found in the stomach (125 mL), and the bladder contained 300 mL of urine. The cause of death was considered drug overdose consistent with asphyxia. Specimens of femoral blood, urine, liver, and vitreous humor were submitted for toxicological analysis.
Material and M e t h o d s Standards and reagents The acetonitrile and methanol used were HPLC grade and glass-distilled (Caledon Laboratories Ltd.). Trimipramine maleate, desmethyltrimipramine maleate, 2-hydroxytrimipramine fumarate, and 2-hydroxydesmethyltrimipramine fumarate were supplied by Rhone-Poulenc Pharma, Inc. Clomipramine-HCl was obtained from Ciba Geigy Canada. Thin layer chromatography Urine and gastric contents were initially subjected to a general drug screen by thin layer chromatography (TLC). Specimens were made basic with sodium carbonate (pH 8.5) and mixed with chloroform for 10 min. After chloroform evaporation, the residue was dissolved in chloroform/methanol (1"1) and spotted on a silica gel 60 TLC plate (E. Merck and Co.). After developing in a tank saturated with ethyl acetate/methyl
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Journal of Analytical Toxicology, Vol. 11, July/August 1987
alcohol/ammonium hydroxide (170:20:10), sprayed with iodoplatinate reagent (12,13).
the plate was
Table I. Summary of Toxicological Analysis Blood (mglL)
Gas liquid chromatography Ethyl alcohol was quantitated in blood, urine, and vitreous humor by head space analysis (14) on a Carle 9500 gas chromatograph (Technical Marketing Associates). The column was packed with 5070 Hallcomid M-18 and 0.507o Carbowax 600 on 40/60 mesh Teflon 6 (Technical Marketing Associates). Peak height ratios relative to the internal standard were used for quantitation.
Trimipramine Desmethyltrimipramine 2-0H trimipramine 2-0H riesmethyltrimipramine Ethyl alcohol Gastric contents Vitreous humor
Urine (mglL)
4.8 2.1
0.58 0.1 0.1 0.09 200 1500 trimipramine positive ethyl alcohol 450 mg/L
Liver (mglkg) 51 16 1.5 1.1
High-performance liquid chromatography Liquid chromatography was performed on a Model 740 solvent delivery system by Spectra Physics with an SF 770 variable wavelength ultraviolet detector by Schoeffel Instruments and an Omniscribe recorder by Houston Instruments (all obtained from Technical Marketing Associates). Analysis was performed at ambient temperature using a 250 x 4.6-ram RP-8 column with 5-/~m particle size (Brownlee Labs). Detector wavelength was set at 205 nm. Quantitation was based on peak height analysis relative to the internal standard clomipramine. A buffer solutio~ consisting of 0.01 mol/L of potassium dihydrogen phosphate was mixed with acetonitrile and nnonylamine to form the mobile phase (550:450:0.6). This mixture was adjusted to pH 3.2 with phosphoric acid and pumped at a flow rate of 1.6 mL/min. Stock standard solutions at 100 /~g/mL (free bases) were prepared in methyl alcohol. An appropriate aliquot of the stock standard was added to drug-free serum to give working standards of trimipramine and desmethyltrimipramine at 0.3 mg/L. Five milliliters of hexane/isoamyl alcohol (97: 3) containing the internal standard (0.3 rag/L) and 0.1 mL of saturated sodium carbonate were added to 2.0 mL of sample (standard or unknown). The tubes were mixed for 20 rain and centrifuged. The organic extract was transferred to an 8-mL screw-cap tube using a plastic Pasteur pipette. A 0.1 mL mixture of mobile phase/ 0.1M phosphoric acid ( l : l ) was added. The mixture was vortexed and then centrifuged. The organic layer was aspirated to waste and 30 to 40/~L of the aqueous layer was injected onto the HPLC column with the range set at 0.02 absorbance units full scale. The HPLC procedure provided good resolution of trimipramine from its major metabolites.
Results Qualitative analysis of the urine gave a TLC pattern consistent with a tricyclic antidepressant ingestion and a trace of a benzodiazepine with the Bratton Marshall reagent. Trimipramine and ethyl alcohol were found in the gastric contents. A summary of the toxicological findings is found in Table I. Chromatograms of the serum standard, blood extract, and liver extract are found in Figure 2.
Discussion Only three published reports of trimipramine quantitation in plasma with respect to its therapeutic use were found. The first two reports (15,16) only measured the parent drug. Suckow
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(B)
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6
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Figure2. Chromatogramsof (A) serum spiked with 300 ng/rnL trimipramine and desmethyltrimipramine,(B) blood extract, and (C) liver extract. l=desmethyltrimipramine; 2=trimipramine; 3=clomipramine (internal standard); 4=2-0H riesmethyltrimiprarnine;5=2-0H trimipramine. and Cooper developed an HPLC assay for trimipramine and major metabolites using electrochemical detection (17). In a study of 29 patients, they found widely varying concentrations of trimipramine and metabolites. Demethylation of trimipramine does not appear to occur as readily as with imipramine or amitriptyline. In the therapeutic situation at steady state, the ratio of desmethyl metabolite to parent compound ranged from 1.0 to 1.6 for amitriptyline and was 1.0 for imipramine and 0.61 for trimipranine (17). Suckow felt that the difference in the side chain structure probably accounted for the retardation in demethylation of trimipramine relative to other tricyclic antidepressant drugs. In the therapeutic situation, the ratio of unconjugated 2-OH desmethyltrimipramine to desmethyltrimipramine was higher than for 2-OH desipramine to desipramine(17). Earlier studies of the antidepressant drugs made the assumption that only the parent drug and desmethyl metabolite were active compounds and that hydroxylated metabolites did not enter the brain. It is now clear that most tricyclic antidepressants have hydroxylated metabolites that are pharmacologically active and penetrate the brain (18-20). Concentrations of trimipramine in fatalities have ranged from 0.2 to 61 mg/L in published reports (7-11). Most of these cases also involved other medications such as clomipramine, doxepin, flurazepam, chlordiazepoxide, ethyl alcohol, and perphenazine. Desmethyltrimipramine was quantitated in only one case (11), and the hydroxylated metabolites have never been quantitated in postmortem tissue or fluids, to our knowledge.
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Journal of AnalyticalToxicology,Vol. 11, July/August 1987
In a large study of tricyclic antidepressant drug-related fatalities (21), the ratio of parent drug to desmethyl metabolite had a mean value of 4.7 with combined concentrations greater than 1.0 mg/L considered clearly toxic and generally associated with fatalities. Bailey et al. (22) reported an average ratio of 1.43 in blood for fatal overdose cases. In our experience, high ratios are generally seen only when a short time interval separated ingestion from death. In this case, the ratio of trimipramine to desmethyltrimipramine was 2.3. The concentration of 2-OH imipramine has been quantitated at 3.9 mg/L in postmortem blood (23). In this case, the low concentrations of hydroxylated metabolites in liver and urine were not considered significant in relation to the higher concentrations of trimipramine and desmethyltrimipramine. Measurement of hydroxylated metabolites may be important in the elderly and in situations where the total concentration of parent drug/desmethyl metabolite are near the toxic-fatal range (approximately 1.0 mg/L). To our knowledge, this is the first reported case involving quantitation of trimipramine and 3 major metabolites in postmortem tissue and fluid by HPLC.
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8. A.H. Stead and A.C. Moffatt. A collection of therapeutic, toxic and fatal blood drug concentrations in man. Hum. ToxicoL 3:437-64 (1983). 9. Methodology for Analytical Toxicology, I. Sunshine, Ed. CRC Press, Boca Raton, Florida, 1985, p. 252. 10. Clarke's Isolation and Identification of Drugs, A.C. Moffatt, Ed. The Pharmaceutical Press, London, 1986, pp. 1051-52. 11. R.C. Meatherall, D.R. Guag, J.M. Nokes, and J.R. Keenan. Toxicological findings in a death resulting from the ingestion of trimipramine. J. Forensic Sci. 28:1023-29 (1983). 12. B. Davidow, N.L. Petri, and B. Quame. A thin layer chromatographic procedure for detecting drugs of abuse. Am. J. Clin. Pathol. 50:714-19 (1968). 13. A.D. Fraser, A.F. Isner, and M.A. Moss. A fatality involving clomipramine. J. Forensic ScL 31:762-67 (1986). 14. G. Machata. Determination of alcohol in blood by gas chromatography-head space analysis. Clin. Chem. News 4:29-32 (1972). 15. S.H. Wong and S.L. Stolarum. Liquid chromatographic assay of trimipramine in plasma. Clin. Chem. 27:1101 (1981). 16. G. Caille, J.G. Besner,Y. Lacasse,and M. Vezina. Pharmacokinetic characteristics of two different formations of trimipramine determined by a new GLC method. Biopharm. Drug. Dispos. 1: 187-94 (1980). 17. R.F. Suckow and T.B. Cooper. Determination of trimipramine and metabolites in plasma by liquid chromatography with electrochemical detection. J. Pharm. Sci. 73:1745-48 (1984). 18. I. Kitanaka, R. Ross, N.R. Cutter, A.P. Zavadil, and W.E. Potter. Altered hydroxy desipramine concentrations in elderly depressed patients. Clin. PharmacoL Ther. 31:51-55 (1982). 19. R.F. Suckow and T.B. Cooper. Simultaneous determination of amitriptyline, nortriptyline and their respective isomeric 10-hydroxy metabolites in plasma by liquid chromatography. J. Chromatogr. 230:391-400 (1982). 20. J.L. Bock, E. Giller, S. Gray, and P. Jatlow. Steadystate plasma concentrations of cis and trans-lO-OH-amitriptyline metabolites. Clin. PharmacoL Ther. 31:609-16 (1982). 21. J.H. Hebb, Y.H. Caplan, C.R. Crooks, and W.J. Mergner. Blood and tissue concentrations of tricyclic antidepressant drugs in postmortem tissue: Literature survey and a study of forty deaths. J. Anal ToxicoL 6:209-16 (1982). 22. D.N. Bailey and R.I. Shaw. Tricyclic antidepressants: Interpretation of blood and tissue levels in fatal overdose. J. Anal. ToxicoL 3:43-46 (1979). 23. A.D. Fraser, E. Susnik, and A.F. Isner. Analysis of 2-hydroxyimipramine in an imipramine related fatality. J. Forens. Sci. 32: 543-49 (1987). Manuscript received August 26, 1986; revision received December 1, 1986.
