COMPLEX INTOXICATION BY TRAMADOL AND TIANEPTINE: …

1 COMPLEX INTOXICATION BY TRAMADOLAND tianeptine : A CASE REPORTW ojciechLECHOWICZI nstitute of Forensic Research, CracowABSTRACT:The appearance of a new product on the pharmaceutical market, espe-cially an antidepressant, may lead in the near future to abuse or use in suicide at-tempts. A case of a COMPLEX poisoning of a middle-aged woman with TRAMADOL andtianeptine is described. TRAMADOL and tianeptine were identified in the examined bi-ological material. tianeptine is the only drug in the dibenzothiazepine group thathas been synthesized up till now. The concentration of tianeptine in whole blood mg/l, in urine 23 mg/l (total) and mg/l (free). TRAMADOL concentrations were:in blood mg/l, and in urine 60 mg/l. The presence of both TRAMADOL and tianeptinehave been confirmed in liver and stomach WORDS: tianeptine ; TRAMADOL ; Blood concentrations; Toxicological Zagadnie Nauk S dowych, z.

2 XLIV, 2000, 130 139 Received 17 October 2000; accepted 3 November 2000 INTRODUCTIONT ianeptine (7-[(3-chloro-6,11-dihydro-6-methyldiben zo[c,f][1,2]thiaze-pin-11-yl)amino]hepta noic acid S,S-dioxide) is a tricyclic antidepressantcompound of dibenzothiazepine type (Figure 1). It is the only representativeof this subgroup. tianeptine shows serotonergic activity by enhancing thepresynaptic reuptake of serotonin [3, 4]. The half-life of tianeptine has no affinity fora1 adrenergic and H1 histaminic for these receptors is responsible for the sedative properties of otherantidepressants. The drug has no affinity for the muscarinic receptors. Itdoes not act anticholinergically either. Clinical trials lead to the conclusionthat tianeptine shows similar antidepressant efficacy to imipramine, nomi-fensine and amitryptyline in neurotic depressive syndrome treatment [2].

3 The drug is applied in the treatment of exogenic depression and anxietyin alcohol abstinence syndrome. Adverse effects which may occur duringtreatment are nausea, vomiting, abdominal pain, tremor, constipation,sleepiness, headache and dizziness. During pregnancy and breast-feedingtianeptine treatment is forbidden. The drug exhibits interaction with MAOinhibitors [3].Coaxil (Servier, France) is the only marketed pharmaceutical product con-taining tianeptine accepted by the pharmaceutical authorities in pill (30 per package) contains mg of tianeptine in sodium salt REPORTThe ambulance service was informed that a middle-aged woman hadfainted on the street. An ambulance arrived at the scene, help was given andthe woman was taken home. The next day she was found dead in her drugs were found by the deceased: Coaxil ( tianeptine ), Majamil(diclofenac), Myolastan (tetrazepam), Ketonal (ketoprofen), Cloranxen (clo-razepate) and Tramal ( TRAMADOL ).

4 This was all the information that wasgained in this case. Autopsy materials femoral blood, urine, liver and stom-ach tissues were sent for AND METHODSS tandardsA tianeptine standard was achieved by its extraction from acidic (pH = 3)suspension of Coaxil in water using diisopropyl ether (Merck, Germany).The residue gained after crystallisation was weighed and dissolved in an ap-propriate amount of methanol (Merck, Germany). The homogeneity and pu-rity of the standard solution was checked by high-pressure liquid chroma-tography with diode-array detection and gas chromatography mass spec-trometry. TRAMADOL and prazepam standards were purchased from Sigma(USA). COMPLEX INTOXICATION by TRAMADOL and tianeptine ..131 Fig. 1. Chemical structure of properties: CAS# 66981-73-5;C21H25 ClN2O4S; (acid);C21H24 ClN2O4 SNa; (sodium salt).Autopsy material preparationLiver and stomach tissues were homogenised and deproteinised usingthe sulphate-ammonium method according to Borkowski [1].

5 Both obtainedhomogenates were extracted by chloroform (POCh, Gliwice) from alkalinesolution (pH = 9) and then by diethyl ether from acidic solution (pH = 3).To free any tianeptine from its conjugates with glucuronic acid, hydroly-sis of urine samples usingb-glucuronidase and arylsulphatase (Merck, Ger-many) was performed. For this purpose 1 ml of urine was spiked with 40ml ofthe enzyme and 200ml of acetate buffer (pH = ). Hydrolysis was per-formed for 45 min at a temperature of 55 screeningDrug screening of material supplied for analysis was performed usinga gas chromatograph linked with a mass-spectrometer (GC-MS) with iontrap detector (ITD) made by Varian/Finnigan MAT (Magnum). An HP-5 MScapillary column (Hewlett-Packard) was applied for separation gradient temperature program consisting of two segments:t(0 min) 75 C,t(5 min) 75 C,Dt(5 20 min) 25 C/min.

6 Acquisition was carried out in ionscanning mode (TIC) in a mass-to-charge range of 45 and stomach tissue extracts were examined using thin layer chro-matography (TLC) on G-60 (Merck) silica plates. Separation of componentswas accomplished using the following organic solvent systems: benzene : di-ethyl ether (1:1), chloroform : acetone (9:1), methanol : 25% ammonia (98:2),chloroform : methanol : 25% ammonia (90:10:1), cyclohexane : benzene :diethylamine (67:2:4).TABLE I. THE MAIN PARAMETERS OF THE MASS SPECTROMETER IN THE LC-MSTECHNIQUEP arameterValueFragmentor voltage70 VCapillary voltage4000 VVaporizer temperature300 CDrying gas temperature300 CDrying gas flow10 l/minDrying gas pressure50 psiCorona current4mATianeptine and TRAMADOL quantitation was performed using an HP-1100liquid chromatograph coupled with a LC-MS (Hewlett-Packard) mass-spec-132W. Lechowicztrometer.

7 Ionisation was carried out in an atmospheric-pressure chemicalionisation chamber (APCI). The fundamental parameters of the mass-spec-trometer are displayed in the table below (Table I).A LiChroCART 125x4 Purospher RP-18e (Merck) column was applied fordrug separation. The mobile phase consisted of acetonitryl and water withaddition of ml/l acetic the identification of tianeptine and TRAMADOL , the best results wereachieved using methanol : 25% ammonia (98:2) developing solution. The fol-lowing reagents sequence coloured drugs to yellow-orange, and then brown(Dragendorff reagent, ferrum chloride, potassium iodoplatinate).The retention index,Rt, of tianeptine in the developing system was of TRAMADOL by mass spectrum was not a great problem. How-ever, identification of tianeptine was difficult, as the Mass spectra library(NIST, USA), with more than 65 thousand compounds, does not containtianeptine s mass spectrum.

8 Therefore search results only indicated an-other compound with similar fragmentation to tianeptine , but not tianep-tine itself. An appropriate match result was only obtained after introducingthe mass spectrum of tianeptine into the library (Figure 2).Both tianeptine and TRAMADOL quantitation by the LC-MS technique wasnot difficult thanks to selectivity and relatively high concentrations (Fig-ure 3). Concentrations of tianeptine and TRAMADOL are shown in Table INTOXICATION by TRAMADOL and tianeptine ..133 Fig. 2. Spectrophotometric and mass spectra of tianeptine . UV maximum at 205,270 nm; MS (70 eV) 228, 293, 194, 213, 165 II. CONCENTRATIONS OF tianeptine AND TRAMADOL IN ANALYSEDAUTOPSY SAMPLESC ompoundWholebloodUrine(non-hydrolysed)Ur ine(enzymatichydrolysis) ++ +++(+) positive analysis, not concentrations of TRAMADOL and tianeptine in whole blood ac-cording to available data are and TRAMADOL concentration in the whole blood of the dead woman was5mg/ml, so it significantly exceeded the highest therapeutic levels for thedrug.

9 It should be noted that TRAMADOL plasma levels in reported fatal casesare about 2mg/ml. A very high concentration of TRAMADOL in urine confirmsingestion of a lethal dose. A simultaneous high concentration of tianeptine ,exceeding the highest therapeutic levels by more than ten times, may, how-ever, be difficult to interpret, for two reasons: firstly, there are no availablepublications concerning fatal tianeptine poisonings, and, secondly, clinicaltrials have shown that very high doses of tianeptine are tolerated( mg = 27 pills).The lack of papers concerning fatal tianeptine cases may indicate thatuse of this drug is safe. Nevertheless, tianeptine overdoses do occur and sopublicising of such cases seems to be INTOXICATION by TRAMADOL and tianeptine ..134 Fig. 3. Extracted ions chromatogramof tianeptine and TRAMADOL obtainedduring blood analysis of a tianeptine identification in autopsy material and interpretation ofthe obtained results may cause some problems, due to a lack of physico-chemical data and unknown toxic and lethal (if any) concentrations in casesof analyses of autopsy material showed that about 15% of alltianeptine occurs in urine in free form (unconjugated with glucuronic acid).

10 Therefore screening analysis may require the hydrolysis of glucuronic andsulphate conjugates of tianeptine . A very high concentration of TRAMADOL inwhole blood and urine may indicate a fatal poisoning. The situation is simi-lar for tianeptine , but in the described case results obtained in clinical trialssuggest non-fatal :1. B o r k o w s k i T ., Metody wyosabniania trucizn organicznych z materia u biolo-gicznego,Archiwum Medycyny S dowej i Kryminologii1968, t. XVIII, s. 95 Martindale the complete drug reference, edition 32, Parfitt K. [ed.], Pharmaceu-tical Press, London P o d l e w s k i J . K . , C h w a l i b o g o w s k a - P o d l e w s k a A ., Leki wsp czesnejterapii, PZWL, Warszawa W i l d e M . I . , B e n f i e l d P ., tianeptine . A review of its pharmacodynamic andpharmacokinetic properties, and therapeutic efficacy in depression and coexist-ing anxiety and depression, INTOXICATION by TRAMADOL and tianeptine .