Analytical method for the determination of strychnine in tissues by gas chromatography/ mass spectrometry: two case reports

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1 Forensic Science Interntionl 110 (2000) locte/ forsciint Anlyticl method for the determintion of strychnine in tissues by gs chromtogrphy/ mss spectrometry: two cse reports, b E.P. Mrques *, F. Gil, P. Proenç, P. Monsnto, M.F. Oliveir, A. Cstnheir, D.N. Vieir Lbortorio de Toxicologi Forense, Instituto de Medicin Legl de Coimbr, Fculdde de Medicin d Universidde de Coimbr, Coimbr, Portugl Deprtmento de Medicin Legl y Psiquitrı, Fcultd de Medicin, Universidd de Grnd, Grnd, Spin b Received 19 Jnury 1999; received in revised form 18 Jnury 2000; ccepted 24 Jnury 2000 Abstrct This pper describes n nlyticl method for strychnine determintion in biologicl smples by gs chromtogrphy/ mss spectrometry nd their ppliction in the investigtion of two cses involving strychnine ingestion: A ftl cse nd clinicl one. The strychnine is isolted from biologicl smples using liquid liquid extrction procedure. The clen-up procedure is performed using n cid solution. Ppverine is used s internl stndrd in the quntifiction of strychnine. In the nlysed specimens, the limits of quntifiction were 0.1 mg/ ml or 0.1 mg/g. The recovery rte rnged from 75.0% to 98.7% nd the coefficients of vrition rnged from 4.8% to 10.5% Elsevier Science Irelnd Ltd. All rights reserved. Keywords: Strychnine; Rodenticide; Alkloid; GC/ MS 1. Introduction Strychnine is n lkloid which is minly extrcted from the seeds of Strychnos nux vomic, tree ntive to Indi. This compound is highly toxic to humns. It is rpidly *Corresponding uthor. Tel.: ; fx: E-mil ddress: imltoxic@.ci.uc.pt (E.P. Mrques) / 00/ $ see front mtter 2000 Elsevier Science Irelnd Ltd. All rights reserved. PII: S (00)

2 146 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) bsorbed from the gstrointestinl trct nd cts upon the centrl nervous system (CNS), cusing excittion of ll prts of the CNS. Strychnine poisoning ws quite frequent in the lst century nd in the first hlf of this century. Strychnine hs not been commercilised in Portugl since 1974, either in phrmceuticl preprtions or in rodenticide formultions. Although strychnine poisoning is now rre event, it my still occur, becuse eventhough the substnce is not vilble in our country, smll mounts of strychnine my still remin in storge, minly in rurl res, or street drugs dulterted with strychnine my be consumed [1]. The uthors describe two cses of cute poisoning by strychnine (one ftl cse nd one clinicl cse) detected by the Forensic Toxicologicl Lbortory (FTL) of the Institute of Legl Medicine of Coimbr (IMLC). In both cses, poisoning ws due to strychnine ingestion. The nlyticl methods for determining this compound in tissues nd body fluids by mens of colour rections [2], thin lyer chromtogrphy (TLC) [2,3], high performnce liquid chromtogrphy [4,5] nd gs chromtogrphy [2,3,6 8] hve been previously described. Strychnine ws detected in our Lbortory in the 1960s in experimentl studies on frogs nd by mens of colour rection, using Mndelin s regent [9]. From the 1970s up to 1995 identifiction ws performed by TLC nd gs chromtogrphy/ nitrogen phosphorus detection (GC/ NPD). In this work new nlyticl method is described. Extrction ws done ccording to the method described by Winek et l. [6], except tht we used ppverine s internl stndrd nd n cid solution in the purifiction procedure. Gs chromtogrphy/ mss spectrometry (GC/ MS), SIM mode were used for identifiction nd quntifiction. 2. Cse histories 2.1. Ftl cse In 1995, 47-yer-old womn ws found ded t home with bottle ner the body. The bottle contined smll mount of liquid. Informtion provided by the fmily indicted tht the victim suffered from depression nd hd propensity for suicide. She ws housewife. Autopsy ws performed pproximtely 12.5 h fter the discovery of the body. Body length nd weight were 145 cm nd 50 kg, respectively. No bnorml externl findings were noted during the post mortem exmintion. The internl pthology consisted on congestion nd edem of the viscer. The cuse of deth could not be determined on the bsis of these dt, becuse they correspond to those tht re normlly found in poisoning cses Clinicl cse In 1996, 45-yer-old mn ws dmitted in the Intensive Cre Unit of the University Hospitl of Coimbr (HUC) with strong convulsions nd violent contrctions. According to the HUC records, the ptient hd previously been submitted to gstric lvge in Regionl Hospitl. The tretment in the HUC consisted on prompt control of convulsions nd contrctions, with dequte mechnicl ssistnce by intubtion nd dminis-

3 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) trtion of muscle relxnts, to prevent respirtory nd hert stoppges. The HUC physicins strongly suspected strychnine poisoning, nd therefore collected biologicl fluids to confirm this suspicion by toxicologicl nlysis. 3. Experimentl biologicl mteril Body tissues were collected in the ftl cse nd fluids were collected in both cses. All the nlysis were performed in triplicte. Free post-mortem strychnine biologicl smples were collected from trffic ccident cdvers whose utopsies were performed in the IMLC, nd strychnine free urine smple ws tken from personnel lbortory Chemicls Anlyticl solvents were of HPLC grde nd regents were of nlyticl grde. They were obtined from Merck, Drmstd, Germny. Strychnine nd ppverine stndrds were purchsed from the Sigm Chemicl Co, St. Louis, MO, USA. Stock solutions were prepred in methnol t 1.0 mg/ml. They were diluted to 100 mg/ml to obtin the working solutions nd stored t 48C Equipment The equipment nd the GC/MS conditions re shown in Tble Extrction procedure After the ddition of 50 ml of ppverine (100 mg/ml) to blood (5 ml) or to urine (5 ml) nd 20 ml of ppverine (100 mg/ml) to ech tissue smple (2 g), cut in very smll pieces, strychnine ws extrcted using the previously mentioned method [6]. A sturted solution of sodium bicrbonte ws dded nd the tissue smples were homogenized t 48C using ultrsound 600 W for 10 min, with liver, lung, kidney nd smll intestine, nd for 20 min in other tissues (hert nd stomch). Strychnine extrction ws performed with 8 ml of toluene n-heptne isomyl lcohol (67:20:4) for tissue homogentes nd body fluids. After vortex gittion nd centrifugtion, the orgnic lyer ws removed to 15 ml glss tubes nd the extrction process ws repeted Purifiction procedure The orgnic lyer ws purified twice by extrction using 4 ml of 4 N hydrochloric cid solution. The cid solutions were mde lkline nd extrcted twice, using the sme solvents s before. The orgnic lyer ws evported to dryness t 408C under gentle strem of nitrogen. The residue ws reconstituted in methnol to be nlysed by GC/MS.

4 148 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) Tble 1 Experimentl conditions for the determintion of strychnine by gs chromtogrphy/ mss spectrometry GC/MS GC MSD Hewlett Pckrd (HP) 5890 series II GC; 5970 MSD Column: HP1 fused silic cpillry column with sttionry phse of cross-linked methyl silicone (12.5 m30.2 mm i.d., 0.33 mm film thickness) Injector: Split injection (1:10) Crrier gs: Helium (1 ml/ min) Tempertures: Injector: 2508C Interfce MS: 2808C Oven Temp.: C Time: 151 min Rmp rte: 15208C/ min Temp.: C Time: 257 min Ioniztion mode: EI (70 ev) Electron multiplier: 2000 V Select Ion Mss mode 3.5. Recovery Strychnine free biologicl smples were spiked with this compound t 0.1, 1.0, 10.0, 50.0 nd mg/ml or mg/g concentrtions (Tble 2) nd were then extrcted nd purified ccording to the bove described method. Ppverine ws dded to the residues t 1 mg/ ml or mg/ g concentrtions just prior to GC/ MS nlysis. Recovery ws Tble 2 Strychnine recovery nd precision (CV) in biologicl specimens t different concentrtions using GC/ MS method Concentrtion Recovery dded Men6stndrd devition (%) (mg/ml or mg/g) Blood Liver Kidney Hert Lung Urine Concentrtion Precision CV (%) dded (mg/ ml or mg/ g) Blood Liver Kidney Hert Lung Urine n53

5 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) performed compring the smple results with methnol stndrd solutions t different concentrtions, with ppverine, lwys t the sme concentrtion (1 mg/ml or 1 mg/g) Precision Strychnine free biologicl smples were spiked with this compound t 10.0 mg/ml or 10.0 mg/g concentrtion nd with ppverine t 1.0 mg/ml or 1.0 mg/g concentrtion (Tble 2), nd were nlysed by the method previously described. 4. Results nd discussion In the ftl cse, the presence of strychnine ws confirmed in the vestigil liquid contined in the bottle by GC/ MS, mode SCAN. The strychnine concentrtions found in post-mortem specimens re presented in Tble 3. The quntity determined in the stomch is higher thn lethl dose of strychnine through ingestion. In the tissue smples, the liver hd the highest concentrtion, being more thn twice tht in blood. Similr toxicologicl findings were found in some of the erlier reports [2,3,6 8,10 12]. Tble 4 shows the strychnine distribution in biologicl smples from published investigtions of ftlities. In the ftl cse described in this pper there ws no fmily or clinicl informtion concerning to the cuse of deth. The utopsy result ws not specific, lthough the pthologicl findings gree with those in published ftlities from cute poisonings [2,3,5 7,9,10] which do not mention specific pthology. Strychnine poisoning ws only dignosed from the results of the toxicologicl nlysis performed in the FTL. In the clinicl cse, the ptient showed typicl symptoms of strychnine poisoning. The clinicl picture of strychnine poisoning cn be confused with epilepsy, tetnus nd hysteri, but the clinicins strongly suspected strychnine poisoning. The strychnine concentrtions re shown in Tble 3. The strychnine level ws low in the gstric contents, becuse the ptient hd previously hd gstric lvge. The blood concentrtion found (0.35 mg/ ml) ws lower thn the blood concentrtion (0.8 mg/ ml) detected in clinicl cse of strychnine poisoning cused by phrmceuticl preprtion reported by Creusot et l. [13]. It ws lso lower thn the concentrtion limit (0.8 mg/ml) reported by Dumont et l. [9] for ptients tht do not show ny tonic contrctions or respirtory rrest. The concentrtion Tble 3 Strychnine concentrtions in humn tissues nd body fluids tken from clinicl nd ftl cse (mg/ml or mg/g) Blood Liver Kidney Hert Lung Urine Smll Gstric intestine contents One clinicl cse One ftl cse cse n53

6 150 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) Tble 4 Published dt on strychnine tissue distribution from ftl cses (mg/ ml or mg/g) Blood Liver Kidney Lung Urine Smll Gstric intestine contents Xor n Xor n Xor n Xor n Xor n Xor n Xor n rnge rnge rnge rnge rnge rnge rnge 1979 [2] [10] [3] [11] [6] [12] [7] [8] Totl contents (mg). found could be explined once tht the smple ws collected for nlysis some time fter the ingestion of the compound. The urine concentrtion (15.0 mg/ ml) further illustrtes this fct. Clinicl tretment in this cse ws imed t stopping the convulsions. Respirtory support ws efficient. Tble 5 gives the retention times nd the selected ion msses for strychnine determintion. Fig. 1 shows totl ion chromtogrm (TIC) of blood smple spiked with strychnine nd internl stndrd t 1.0 mg/ ml, the selected ions nd their bundnce in SIM mode. The strychnine presents two isomers, the mjor one ws used in for the quntifiction. The interfering peks on the chromtogrms of the different specimens were nonexistent. The limit of quntifiction by this method ws 0.1 mg/g or 0.1 mg/ml for the nlysed specimens. The clculted recoveries vry from 75.0% to 98.7% (Tble 2). The recovery vlues were the lowest for lung smples nd highest for the liver. The coefficients of vrition (CV) rnged from 4.8% to 10.5% (Tble 2), with the lung smple being the one which showed greter vrition. 5. Conclusion This study emphsizes the importnce of toxicologicl nlysis in detecting cute strychnine poisonings. In the ftl cse the cuse of deth ws only determined from the Tble 5 Retention time nd selected ion mss for determintion of strychnine Compounds Retention Ions (m/z) time (min) Identifiction Quntifiction Strychnine isomers 10.03; ; Ppverine ;

7 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) Fig. 1. TIC of blood smple spiked with strychnine nd ppverine t 1.0 mg/ml nd its selected ions. results of the toxicologicl findings, nd in the clinicl cse the dignosis ws confirmed by the nlyticl dt. Pthologicl dt nd post-mortem concentrtions of strychnine re consistent with the published dt. The nlyticl method described is simple, specific nd sensible, nd provides recovery nd precision for the identifiction nd quntifiction of strychnine in forensic nd clinicl investigtions. Acknowledgements We thnk Isbel J. McDonld for her ssistnce in reviewing lnguge. References [1] A.R.L. Wijeseker, H.D. Henry, P. Rnsinghe, The detection nd estimtion of (A) rsenic in opium, nd (B) strychnine in opium nd heroin, s mens of identifiction of their respective sources, Forens. Sci. Int. 36 (1988) [2] J.S. Oliver, H. Smith, A.A. Wtson, Poisoning by Strychnine, Med. Sci. Lw. 19 (1979) [3] M. Vn den Heede, A. Wuters, J. Cordonnier, A. Heyndrickx, J. Timpermn, The toxicologicl investigtion of two unexpected deths due to poisoning by strychnine, in: H. Brndenberger, R. Brndenberger (Eds.), Proceedings of the interntionl meeting of TIAFT, Switzerlnd, 1985, pp [4] A. Alliot, G. Brynt, P.S. Guth, Mesurement of strychnine by high performnce liquid chromtogrphy, J. Chromtogr. 232 (1982)

8 152 E.P. Mrques et l. / Forensic Science Interntionl 110 (2000) [5] P. De Squi-Snnes, P. Nups, P. Le Brs, V. Burgt, Evlution of n HPTLC Method for the Determintion of Strychnine nd Crimidine in Biologicl Smples, J. Anl. Toxicol. 20 (1986) [6] C.L. Winek, W.W. Wgdy, F.M. Esposito, W.D. Collom, Ftl strychinine ingestion, Anl. Toxicol. 10 (1986) [7] F.A. Benomrn, K.D. Henry, Homicide by strychnine poisoning ( cse report), in: R. Klus Mueller (Ed.), Contributions to Forensic Toxicology, MOLINA press, Leipzig, 1994, pp [8] C. Giroud, A. Sutter, M. Augsburger, I. Rivier, Ftl strychnine self-poisoning in hospitl, in: B. Jcob, W. Bonte (Eds.), Advnces in Forensic Sciences, Forensic Chemistry, Vol. 5, Verlg Dr. Koster, Berlin, 1995, pp [9] Strychnine, in: E.G.C. Clrke (Ed.), Clrke s Isoltion nd Identifiction of Drugs, The Phrmceuticl Press, London, 1986, p [10] R.B. Bselt, in: Disposition of Toxic Drugs nd Chemicls in Mn, 2nd Edition, Biomedicl Publictions, Dvis, CA, 1982, pp [11] J.A. Perper, Ftl strychnine poisoning A cse report nd review of the literture, J. Forens. Sci. 30 (1985) [12] D. Dumont, C. Piv, D. Mthe, H. Gstinne, Intoxiction igue pr l strychnine (` propos de 60 cs), L strychnine ne tue ps que les tnpes, J. Medecine Legle-Droit Medicl, T 32 (1989) , na2 bis numero supplement. [13] G. Creusot, S. Moulin, P. Hllereu, Intoxiction pr l strychnine ` propos d une observtion, J. Medecine Legle-Droit Medicl, T 32 (1989) 57 61, na2 bis numero supplement.