A Fatality Related to the Veterinary Anesthetic Telazol - Journal of ...

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Journal of Analytical Toxicology, Vol. 23, October 1999 purchased from Radian (Austin, TX) as a methanolic solution at a concentration of 100 lJg/mL. SKF-525A was purchased from Sigma Chemical Co. (St. Louis, MO), and 5-ethyl-5-p-tolyl- barbituric acid was purchased from Aldrich Chemical Co. (Milwaukee, WI). The chemicals used in the extraction were analytical grade. The solid-phase extraction columns employed were United Chemical Technologies (Bristol, PA) Clean Screen ZSDAU020 extraction columns. Methanolic stock solutions were made for the tiletamine and zolazepam at a concentration of 1 mg/mL. This stock was fur- ther diluted to a combined working standard of I IJg/mL. This working standard was used to prepare calibration standards in blood of 12, 25, 50, 100, and 300 ng/mL for the tiletamine and zolazepam. An independent blood-based control was also prepared. A PCP-d5 stock solution of 3.33 IJg/mL was prepared as an internal standard from the 100-1Jg/mL Radian standard. Instrumentation A Hewlett-Packard (HP) HPLC-MS system was used for the initial screening. It consists of an HP 1050 series HPLC pump, autoinjector, and UV detector with an HP 59980B particle beam interfaced with an HP 5989B MS. It used an Altima (Deerfield, IL) C18, 5-1Jm Direct Connect Guard cartridge (#88058) Abundance #145: Tiletamine rrt=0.159 (*) 116 8000 6000 4000 II0123 2000 51 69 83 ,Jl,,..,,fl ..,,,~38,i,,,, 0 LH,J.., , .l,l.h. hd ,. 9 /z--> '"6'o"'~'o'"L66"L~6"L~6"L~ 195 Figure 1. Structure and particle beam LC-MS El mass spectrum of tiletamine. Abundance m/z--> 8000 6000 4000 2000. 0 attached to a 5-1Jm Altima C18 (20 mm x 2.1 mm) column. The HPLC was run with a gradient that began with 100% of a mobile phase A (deionized water/acetonitrile, 90:10, 0.05% trifluoroacetic acid) for 5 min and ended with 100% of a mobile phase B (deionized water/acetonitrile, 20:80, 0.05% trifluoroacetic acid) after 55 min at a flow of 0.25 mL/min with a column temperature of 40~ Total run time was 70 min which allows for re- equilibration of the column. The UV detector scanned between 205 and 290 nm. The MS was set in the electron impact (EI) mode to scan for ions in the range of 50-500 ainu. The struc- tures and LC-MS EI spectra for tiletamine and zolazepam are shown in Figures I and 2, respectively. A Hewlett-Packard (Palo Alto, CA) model 5890 series II+ gas chromatograph (GC) with a 5972 series mass selective detector was employed for the quantitation. The column was an HP- Ultra-1 crosslinked methyl siloxane column (12 m x 0.2-ram i.d., 0.3-1~m film thickness). The injector temperature was set to 250~ and the detector was set to 280~ The initial oven temperature was 135~ The temperature was then ramped to 220~ at a rate of 25~ After being held at 220~ for 1 min, the temperature was ramped to 320~ at a rate of 30~ for a total run time of 8.73 min. The MS was run in the EI mode with selected ion monitoring using a target ion and two qualifying iL I1206 223 240 268 284 319 #146: Zolazepam rrt-0.238 (*) 2~:72 5 69 145 51 i09 134k 180 199 230 i'; .... 160 1go '' '260 .... 2g, ~360' Figure 2. Structure and particle beam LC-MS El mass spectrum of zolazepam. 32~ 3s~68 394 42o '' '3~0 .... 460''' S- ~ NH~Hs OHc I ~H3 CH3 CH2 ,____~N ~ F HCl 553
ions for each compound. The target ion for tiletamine was 166 with qualifiers of 195 and 110; the target ion for zolazepam was 285 with qualifiers of 267 and 257; and the target ion for PCP-ds was 205 with 247 and 248 as its qualifying ions. Retention times for tiletamine, PCP-ds, and zolazepam were 3.74, 4.84, and 6.87 min, respectively (Figure 3). Procedure The LC-MS screening procedure in which the compounds were detected was a liquid-liquid extraction at both neutral and alkaline pHs. Buffer (1 mL, pH 7) was added to a 5-mL sample of blood or urine. Both I lag of SKF-525 A and 6 lag of 5-ethyl-5-p- tolybarbituric acid were added as internal standards. After the addition of 7 mL of hexane/toluene/isoamyl alcohol (90:5:5), the samples were vortex mixed and centrifuged. The organic layer was removed to a clean test tube. Buffer (1 mL, pH 9.5) was added to the remaining aqueous layer. The alkaline fraction was then extracted using 6 mL of hexane/isoamyl alcohol (99:1). The two extracts were combined and dried down under nitrogen. The samples were reconstituted with 250 laL of deionized water/acetonitrile (55:45), 0.05% trifluoroacetic acid. The samples were then dried down to approximately half their volume to remove the acetonitrile. The injection volume was 50 laL. The tiletamine and zolazepam confirmation was performed using the solid-phase method for phencyclidine as described by United Chemical Technologies (6). Two milliliters of 0.1M phosphate buffer pH 6.0 was added to 3 mL of sample. To all samples 75 laL of PCP-ds was added as an internal standard for an equivalent of 250 ng. Three grams of a 1:1 liver homogenate was measured into a test tube resulting in an actual weight of 1.5 g for the liver extract. The same amount of internal standard and buffer were added to the liver sample. In- Sample house controls were run at a concentration of 25 Blood ng/mL and 100 ng/mL for tiletamine and Urine zolazepam. Liver The columns were prepared with 3 mL of methanol (MeOH) followed by 3 mL of deionized Results and Discussion Journal of Analytical Toxicology, Vol. 23, October 1999 The screening panel for drugs of abuse by enzyme multiplied immunoassay technique (EMIT) was found to be negative for both urine and blood. Telazol did not show any cross-reactivity with the Behring Diagnostics Inc. (Cupertino, CA) benzodi- azepine assay. Although the phencyclidine assays were also negative, the urine showed an elevated negative value 16 units below the cutoff value of 25 ng/mL. It is not known if this cross-reactivity was due to the presence of ketamine or tiletamine or both. Headspace analysis for ethanol and other volatiles was also negative. The blood was also screened for aminopentamide by HPLC and was found to be negative. Tiletamine and ketamine were detected by the HPLC-MS screening. The EI spectra were searched by the Pfleger mass spectra library, which was supplied by Hewlett-Packard, and matched tiletamine as norketamine. Tiletarnine resembles norketamine in that it shares a 166 ion. After the addition of tiletamine to an in-house mass spectra library, the peak was properly labeled. The HPLC-MS also detected the presence of ketamine. Zolazepam was not extracted by the HPLC-MS screening method. Although no recovery studies were done for the solid-phase extraction of tiletarnine and zolazepam, there appeared to be no difficulty in combining the analyses of these two compounds into one method. The United Chemical Technologies extraction pro- cedures for phencyclidine and benzodiazepines were nearly iden- tical. In both extractions, sample preparation and column Table I. Concentrations of Tiletamine, Zolazepam, and Ketamine in Blood, Urine, and Liver Tiletamine Zolazepam Ketamine 295 ng/mL 1.71 ns/mL 37 n~mL 682 ng/mL 1.33 nglmL 381 ng/mL 196 ng/g 15.5 pg Analysis not performed water and finally I mL of 0.1M phosphate buffer pH 6.0. After vortex mixing and centrifuging, the Abundance TIC: 0701006.D samples were loaded onto the prepared solidphase extraction columns. The columns were then washed with 3 mL of deionized water 250000 Tile amine Zolazepam followed by 1 mL of 0.1M acetic acid and finally 3 mL of MeOH. The columns were dried for 5 rain 200000 under vacuum, and the samples were then eluted with 3 mL of methylene chloridelisopropyl alco- 150000 PCP D- 5 hol/ammonium hydroxide (78:20:2). The eluent was dried down under nitrogen and reconstituted 100000 with 100 IJL of ethyl acetate for injection onto the GC-MS. The injection volume was I laL. soooo The ketamine confirmation was performed separately using the same extraction method and 0 Time--> .... ~ , .... k_______._~ , .... , .... I .... , .... , .... ,,,,,,,,,,,,,,,, 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 GC-MS parameters as described for tiletamine and zolazepam. Ketamine had a retention time of 4.49 min. Figure 3. GC-MS total ion chromatogram of 300 ng/mL extracted tiletamine, PCP-ds, and zolazepam. 554
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