Journalof Analytical Toxicology,Vol. 26, January/February2002 Quantitative Determination of n-Propane, iso.Butane, and n-Butane by HeadspaceGC-MS in Intoxicationsby Inhalation of Lighter Fluid* Marie-Paule L.A. Bouche1, Willy E. Lambert1, Jan F.R Van Bocxlaer2, Michel H. Piette3, and Andr~ P. De teenheer 1,t 1Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium; 2Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium; and 3Laboratory of Forensic Medicine, Ghent University, J. Kluyskensstraat29, B-9000 Gent, Belgium I Abstract This report describes a fully elaborated and validated method for quantitation of the hydrocarbons n-propane,/so-butane, and n-butane in blood samples. The newly developed analytical procedure is suitable for both emergency cases and forensic medicine investigations. Its practical applicability is illustrated with a forensic blood sample after acute inhalative intoxication with lighter fluid; case history and toxicological findings are included. Identification and quantitation of the analytes were performed using static headspace extraction combined with gas chromatography-mass spectrometry. In order to reconcile the large gas volumes injected (0.5 mL) with the narrowbore capillary column and thus achieve preconcentration, cold trapping on a Tenax sorbent followed by flash desorption was applied. Adequate retention and separation were achieved isothermally at 35~ on a thick-film capillary column. Sample preparation was kept to a strict minimum and involved simply adding 2.5 pL of a liquid solution of 1,1,2-trichlorotrifluoroethane in t-butyl-methylether as an internal standard to aliquots of blood in a capped vial. Standards were created by volumetric dilution departing from a gravimetrically prepared calibration gas mixture composed of 0.3% of n-propane, 0.7% of iso.butane, and 0.8% of n-butane in nitrogen. In the forensic blood sample, the following concentrations were measured: 90.0 pg/t for n-propane, 246 pg/L for/so-butane, and 846 pg/L for n-butane. Introduction Charcoal lighter fluid is a consumer product for refill of cigarette lighters, consisting of a heterogeneous mixture of flammable, volatile, non-halogenated, aliphatic hydrocarbons. Its principal components, n-propane (< 6%), 2-methylpropane or iso-butane (5-25%), and n-butane (75-89%) (1) are all col* This work was presented at the 38th International Meeting of The International Association of Forensic Toxicologists (TIAFT2000)held in Helsinki, Finland, 13-17 August 2000. Author to whom correspondenceshould be addressed. E-mail: [email protected] orless, odorless, and flammable gases at room temperature. They occur as components of natural gas, from which they are refined, and are extensively used throughout the world as fuel, refrigerants, 'ozone friendly' aerosol propellants, and in organic synthesis. Volatile substance abuse (VSA), the deliberate inhalation of organic solvents and other volatile substances for euphoric purposes, is a form of drug abuse becoming increasingly popular (2). Cigarette lighter refills, solvents from contact adhesives, typewriter correction and dry cleaning fluids, halocarbon fire extinguisher and aerosol propellants, gasoline, and inhalational anesthetics are commonly abused (3,4). The products prone to abuse are cheap and quite often readily available. On top of these enticements, VSA is not illegal. Although it is very difficult to estimate the full extent of VSA, the numerous reports in the scientific literature highlighting the volatile substance-abuse-related toxicity and deaths clearly indicate that the hazardous nature of these products is not yet fully appreciated. Although there are no special reports of toxicokinetic studies on lighter refill fluid, the information published on the major components is useful for predicting its possible toxicity. The hydrocarbons are primarily resorbed by the respiratory tract and partly exhaled unchanged via the lungs. The other part is microsomally metabolized by the liver into secondary and tertiary alcohols and further oxidized to ketones, n-Propane, iso-butane, and n-butane, of which the latter demonstrates the greatest effect, are all central nervous system (CNS) depressants with a very narrow margin of safety between anesthetic and lethal concentrations. The LCs0 for the rat has been reported as 658 mg/L (4 h) for n-butane, 1379 mg/L (15 min) for iso-butane, and > 1465 mg/L (15 rain) for n-propane (5-7). Clinically, VSA is characterized by a rapid onset of intoxication and swift recovery. Small doses rapidly lead to euphoria and disinhibition, the desired psychedelic effects, but these are quite often accompanied by delusions, hallucinations, behavioral disturbances, ataxia, and confusion (3). Other sensory Reproduction(photocopyin8)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission, 35 Journal of Analytical Toxicology, Vol. 26, January/February 2002 and physiological symptoms include headache; nausea and vomiting; dizziness; loss of appetite and fine motor coordination; and irritation of eyes, nose, and throat (3,5-7). Higher doses may produce life-threatening convulsions and coma. The major risk of acute exposure to large concentrations of lighter fluid is sudden death. Nonhalogenated hydrocarbons cause sensitization of the myocardium to epinephrine-induced cardiac arrhythmia possibly resulting in cardiac arrest (3,5-9). Furthermore, death may ensue by simple asphyxia (10); gradual depletion of oxygen (oxygen deficiency) due to its replacement by the gaseous substance--especially in a confined space such as with the use of a plastic bag for sniffing--is followed by rapid loss of consciousness and respiratory and cardiac arrest. Direct drug effects and trauma may also contribute to lethality on the first use. Other medical sequelae indirectly following nbutane, iso-butane, or n-propane inhalation and possibly causing delayed death are aspiration of vomit and burns from a flash fire or explosion, for instance, after lighting a match in a small enclosed space where cigarette lighter sniffing has taken place (1). Up until now, analytical methodologies to quantitatively measure blood and tissue concentrations after lighter refill, n-bu; tane, or n-propane intoxication are scarcely described in the literature. Logically,all of them are based on gas chromatography, mostly combined with flame-ionization detection (GC-FID) (11-13). In several cases, packed GC columns are used (12,13), permitting the injection of large gas volumes (9). State-of-the-art capillary columns, however, allow better separation, and thus selectivity, and combined with sensitive and selective mass spectrometric detection, unambiguous identification and quantitation possibilities emerge. This manuscript reports on a novel, largely automated, and fully validated GC assay for the quantitative determination of n-butane, iso-butane, and n-propane in (postmortem) blood samples, using static headspace extraction and mass spectrometric detection. Experimental Instrumentation The GC unit was composed of a Hewlett-Packard (HP, Palo Alto, CA) 6890 series GC system, equipped with a CIS-4 cooled injection system (Gerstel GmbH, M~lheim an der Ruhr, Germany). Cooling of the injector liner was achieved using a pressurized Dewar vessel containing liquid nitrogen (l'Air Liquide, Marne-la-Vall~e, France), which was connected to the injector's jacket and operated electronically by means of a solenoid valve. The presence of a programmed and remotely controlled MPS multipurpose sampler (Gerstel GmbH, Mfilheim an der Ruhr, Germany), used in the headspace sampler mode, in conjunction with a tray accommodating a maximum of 60 5-mL glass headspace vials (Gerstel GmbH) allowed full automation of the method. An HP 5973 mass selective detector linked to an electronic data-handling system with ChemStation integration software (Hewlett-Packard) was used for data acquisition and storage. 36 Analytical reagents A gravimetrically prepared calibration gas mixture, containing n-propane (0.3001 • 0.009%), iso-butane (0.700 + 0.021%), and n-butane (0.800 • 0.024%) in nitrogen, packed in a steel cylinder, was supplied by l'Air Liquide (Marne-la-Vall~e, France). t-Butylmethylether (99.8% pure, high-performance liquid chromatography [HPLC] grade) was obtained from Aldrich (Milwaukee, WI), and 1,1,2-trichlorotrifluoroethane (99.8% pure) was from Carlo Erba (Milano, Italy). All other products and reagents used were of analytical grade and supplied by Aldrich. Standards Calibrators were produced by means of volumetric dilution departing from the described gravimetrically prepared calibration gas mixture. After thorough ventilation of the decompressor, a 1.2-L Teflon gas sampling bag (Alltech, Deerfield, IL), provided with a barbed on-off valve and a septum port, was extensively flushed and allowed to fill completely. Subsequently, the bag was sealed off and carefully brought to atmospheric pressure. In the next step, different aliquots of this stock gas mixture, depending on the calibrator concentration, were sampled through the septum port and brought into separate calibrated and evacuated 125-mLgas sampling bulbs (Alltech)using a 2.5mL gas-tight sample lock syringe (1002SLHamilton, Bonaduz, Switzerland). After adjustment to atmospheric pressure and equilibration for at least 10 min, which promoted adequate and homogenous diffusion of the gases, 0.5 mL of gas was sampled from each calibrator gas bulb with a gas-tight sample lock syringe and carefully transferred into a sealed 5-mL glass headspace vial containing 0.5 mL of blood free of volatile poisons. An overdraw-and-compress technique whereby an excess of gas was drawn into the syringe (approximately 1 mL) was applied, the syringe was locked, and the plunger was pushed down to precisely 0.5 mL. The syringe then was very briefly unlocked, ~eleasing the excess gas. This technique facilitated the adjustment to atmospheric pressure. Standards were then shaken vigorously and left to equilibrate overnight. To each vial 2.5 DL of a liquid solution containing 1,1,2trichlorotrifluoroethane (158.6 rag/L) in t-butylmethylether was added as an internal standard (IS) by means of a very precise Hamilton CR-700 constant rate syringe (Bonaduz, Switzerland) and then mixed thoroughly. Sample collection and preparation The procedure developed aims the detection of VSAby blood analysis. A real intoxication case was assayed in order to substantiate the usefulness of our approach. This sample concerns a nearly 16-year-oldmale adolescent who was found dead at the bottom of a staircase. At about midnight, his father heard some stifled giggling, but no particular noise indicating a fall. A gas cylinder (used for refilling cigarette lighters) was found about 6 m from the body. Paraphernalia present indicated possible drug experimentation, and an external examination was performed on the same day. Because a gas intoxication by sniffing was suspected, and in order to perform a standard toxicological investigation, 20 mL Journal of Analytical Toxicology,Vol. 26, January/February2002 of subclavian blood and a small amount of urine were sampled by syringe. The syringes were immediately sealed off and frozen at -20~ Analysis was performed no longer than 10 days after collection. Just before analysis, the sample was allowed to thaw at 4~ and swirled gently. Approximately 0.5 mL of blood was directly injected, in fivefold, into clean, sealed headspace vials. Subsequently, the correct sample volume was calculated based on the gravimetrically assessed density of the postmortem blood. By analogy with the standards, 0.5 mL of blank air and 2.5 laL of liquid solution of 1,1,2-trichlorotrifluoroethane (158.6 mg/L) in t-butylmethylether were added to each vial. Headspace extraction and injection Prior to injection, every vial was preheated for 20 min at 60~ comparable with a standard toxicologic analysis by headspace GC for volatile poisons (e.g., ethanol) at our laboratory, in order to minimize influence of environmental temperature fluctuations and maximize vapor phase enrichment. After pressurization with carrier gas and equilibration (1 min), 0.5 mL of the headspace was introduced in the injector liner by means of a 1-mL gas-tight syringe (Gerstel GmbH) at a speed of 5 mL/min. In order to enhance sensitivity, cryofocusing in the injector liner was applied. To this end, the deactivated liner was packed with Tenax| TA (20/35 mesh) obtained from Alltech. The CIS4 injector was operated under the following conditions in all runs: during a precool time (5 min prior to the injection of the analyte), the liner cold trap was cooled down to -80~ by liquid N2 departing from a Dewar vessel. The liner was maintained at that temperature until 0.30 min after the injection was completed, thus ensuring adequate analyte adsorption onto the Tanax and concentration. Subsequently, the cold trap was flash heated up to 200~ (heating rate: 12~ and held at that temperature for 6 min, providing flash desorption and adequate transfer of the analyte onto the capillary column. During this desorption phase, the injector split ratio was 0.7:1, thereby eliminating pressure effects in the injector liner, albeit losing minimal sample mass. At 0.75 min after the injection was completed, the split ratio was increased to 40:1 in order to flush the liner and avoid peak tailing and carry-over. Chromatographic conditions Isothermal chromatographic separation was achieved on a WCOT fused-silica CP-Select 624, custom-made capillary column (41 m x 0.25-mm i.d., 2.1-1am film thickness, Chrompack, Middelburg, The Netherlands). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. Oven temperature was 35~ for a runtime of 14.50 min, and the MS transferline temperature was maintained at 100~ The mass selective detector was operated in the full-scan mode (mass range, rn/z 15 to 300). The compounds were identified by comparing retention times and mass spectra in the samples with those obtained by injection of standards. Quantitation and method evaluation For quantitation purposes, mass fragmentograms of mlz 29 (n-propane), mlz 43 (/so-butane and n-butane), and mlz 101 (IS) were constructed and integrated. Linearity. Standard curves were prepared in bank blood as previously described over a concentration range from 0 to 487.2 IJ~L for n-propane, to 1498 IJg/L for/so-butane, and to 1712 pg/L for n-butane. For each curve, six different concentration levels, including a zero-calibrator, were analyzed. For calibration purposes, peak-arearatio (ana]yte/]S) was regressed against accurate, recalculated (depending, for example, on calibrated gas bulb volume) concentration of the standards. In an effort to account for data heteroscedasticity, weighed linear regression (weighing factor: 11y)was applied to calculate the calibration curve. Precision. Precision of the method was evaluated by analyzing standards at three different concentration levels for each compound (seeTable I for detailed concentrations) on the same day (five replicates, within-day reproducibility) and over sepa- Table I. Method Validation Data n-Propane Within-day reproducibility (nt = 5) Total reproducibility (n = 11) Linearity Intercept Slope r* LOD (pg/L) LOQ (pglL) n-Butane iso-Butane spiked conc. spiked conc. (pg/L) CV%* 96.5 292.3 487.2 96.5 292.3 487.2 6.0 4.4 4.7 5.9 3.4 4.5 value 0.00075 0.00053 0.99931 1.4 5.5 spiked conc. (pg/L) CV% 297 899 1497 297 899 1497 5.6 3.9 4.8 6.0 4.1 4.5 value 0.00244 0.00121 0.99924 2.6 10.5 (pg/L) CV% 339 1027 1711 339 1027 1711 9.7 4.8 5.2 9.3 4.3 4.1 value 0.00384 0.00099 0.99859 3.0 12.0 * CV%,coefficientof variation. n, numberof determinations. * r, correlationcoefficient. 37 Journal of Analytical Toxicology, Vol. 26, January/February 2002 rate days (11 replicates, total reproducibility). Peak-area ratios were transformed to concentrations using calibration curves prepared and analyzed simultaneously. Limits of detection and of quantitation. The limit of detection (LOD) was determined by injecting decreasing concentrations of the analytes in blood. The LOD was defined as the lowest concentration that produces a response three times the noise level of a blank blood sample and could still be assayed with acceptable reproducibility. The limit of quantitation (LOQ) was es- A tablished at a signal-to-noise ratio of 10 (14), conditional to an effortless quantitation at that level with an error of less than 15%. Selectivity. In order to monitor the selectivity of the method, several volatile organic compounds (VOC), including popular laboratory solvents and some inhalation anesthetics were chromatographed at a concentration in large excess to the highest calibration point. To that end, solutions of these compounds were prepared in t-butylmethylether and then 2.5 IlL was spiked into a glass headspace vial containing 0.5 mL blank blood. Retention and mass spectral behavior were examined and compared with that of the analytes and the internal standard. General drug screening A comprehensive drug screening was also performed on the postmortem blood sample using the enzyme-multiplied immunoassay technique (EMIT), radioimmunoassay, and chromatographic techniques (HPLC following alkaline extraction and GC-MS) (15,16). Time (mln) B 5 4 ~ 8 ~ 1'0 1'~ Safety considerations In order to minimize laboratory environment pollution and thus protect the operator, a charcoal trap was attached to the split vent of the GC. All experiments were carried out in a wellventilated room and, whenever possible or necessary, under a fume hood. The gas cylinder containing the calibration mixture was placed in a special safety closet overnight. Halogenated volatiles were meticulously collected in separate disposal canisters. Furthermore, the method demands no additional safety precautions. General guidelines for work with organic gases and volatile solvents have to be respected. Time (mln) Figure 1. Total ion current chromatogram of blank blood (A) and of a calibrator containing approximately 400 pg/L of n-propane, 1250 pg!L of/so-butane, and 1430 iJg/L of n-butane (B). Peak identification: 1, n-propane; 2,/so-butane; 3, water; 4, n-butane; and 5, internal standard (1,1,2-trichlorotrifluoroethane). Results and Discussion As an analytical toxicologist, one sometimes comes across intoxication cases that can not be entirely elucidated using standard toxicologic techniques such as routine screening by immunoassay techniques and standard confirmatory techniques (e.g., HPLC). In case of the described forensic sample, standard toxicological analysis only disclosed the presence of toxicologically irrelevant levels of ethanol (0.08 g/L blood and 0,12 g/L urine), cannabinoids (69 IJg/Lin urine only), caffeine (0.53 mg/L in urine only), and cotinine (0.44 mg/L in blood; 3.79 mg/L in urine). The analyses using general purpose HPLC--diode-array detection (DAD), GC-FID, GC-nitrogen-phosphorus detection (NPD), and GC-MS confirmed these preliminary results. However, the headspace GC-FID chro4 w 7 8 9 10 11 Time (min) matogram of the blood revealed three very small peaks at the start of the run, but these Figure 2. Total ion current chromatogram of the postmortem blood sample. Peak identification were useless from a quantitative point of view and massspectra (seeinsets): 1, n-propane; 2, iso-butane; 3, water; 4, n-butane; and 5, internal because of the inappropriateness of the anastandard (1,1,2-trichlorotrifluoroethane). lytical conditions. In an effort to provide sound 38 Journal of Analytical Toxicology, Vol. 26, January/February2002 quantitative data concerning this intoxication case, a novel assay for determination of the toxic gases in blood was developed. Headspace GC-MS assay Besides being eminently suitable for the components of interest because of their volatile nature, the use of headspace GC combined with MS detection fulfills numerous analytical requirements. Headspace extraction is indeed very popular on account of its rapid sample processing capacity and suitability for direct quantitative determination of VOC in a great number of different matrices (9,11-13,17-19). Thick-film capillary GC provides a simple, versatile, fairly rapid, and extremely powerful technique for the separation and quantitation of multiple compound mixtures, and, in combination with sensitive and selective MS detection, unambiguous identification and quantitation possibilities undeniably emerge. Figure I depicts the total ion current chromatograms of the headspace GC-MS analysis of a blank blood sample and of a standard containing approximately 400 IJg/Ln-propane, 1250 1Jg/L iso-butane, and 1430 lJg/L n-butane. As can be seen, the three analyte peaks are well separated from each other and characterized by an excellent peak shape. The n-propane and nbutane peak are devoid of any interference. Only the iso-butane peak is not baseline separated from the peak representing water, also present in the headspace of every blood specimen, but the latter causes no interference whatsoever in regard to the quantitation of iso-butane. The retention times of n-propane, isobutane, n-butane, and the internal standard, 1,1,2-trichlorotrifluoroethane, are 3.72, 4.28, 4.80, and 9.33 min, respectively. In our assay, oven temperature was isothermal at 35~ This temperature ensured adequate retention and could still be kept stable, regardless of environmental conditions. The total run time was 14.50 min, allowing elution of a large peak oft-butylmethylether, the solvent used in the liquid internal standard solution, after the GC run but during the in-between run time interval for vial transport and injector cooling. When conducting capillary GC analysis of gases, there are two major factors to be considered. First of all, samples usually hold the key analyte at very low concentration. This implies the injection of a large volume of gas, in a reproducible way, in order to place enough analyte mass onto the capillary column and thus reach the intended sensitivity. Secondly, chromatographic or instrumental restrictions (e.g.,very small column internal diameter) preventing direct loading of such gas volumes, have to be taken into account. Cryofocusing on an adsorbent, located in the injector liner, proved pivotal in order to reconcile the large injected gas volumes with the small internal diameter of the capillary column. Consequently,sample preconcentration and enrichment and a minimization of injection bandwidth were successfully achieved. During the injection, the analyte is introduced into the pre-cooled CIS-4 (-80~ and adsorbed onto the Tenax and most of the air is purged out of the system at a split ratio of 7:1. Flash heating of the injector liner is consequently followed by immediate desorption and transfer of the volatiles onto the capillary column. During this phase, split ratio was kept minimal to increase sensitivity as much as possible. Prior to, as well as after every injection, the injection syringe was thoroughly flushed with carrier gas in order to exclude memory effects and carry-over. MS detection was our first choice on account of its high sensitivity and selectivity. Furthermore, as data are still collected in full scan mode, this holds the opportunity to effortlessly enhance sensitivity by switching to SIM mode. On top of the chromatographic retention data, the full-scan MS detection offered unambiguous identity confirmation by means of the mass spectrum. The EI mass spectra of n-butane, iso-butane, and n-propane are represented in the insets of Figure 2. The ions at m/z 29 (n-propane), m/z 43 (iso-butane and n-butane), and rn/z 101 (IS) were selected as the target ions for quantitative purposes using reconstructed mass chromatograms. Calibration Aliquots of a calibration gas mixture were brought into sealed and evacuated gas sampling bulbs of exactly known volume and allowed to evaporate completely. Subsequently, after the bulbs were brought to atmospheric pressure and were again left to equilibrate for at least 10 min, 0.5 mL of gas was sampled and transferred into a headspace 2-1 vial containing 0.5 mL of blank blood. In order o to allow establishing of the subtle equilibrium n n-Propane between the gas phase and the liquid phase, 1.5 -I y = 0.00057x calibrators were vigorously shaken and kept at 0 r = 0.99961 room temperature overnight. As of this point, o iso-Butane standards and samples were handled identi"~ 1 -I ~= o.oo1~ 0.99944 cally. The gas sampling bulbs used for preparaA n-Butane .= y = 0.0096x + 0.00648 tion of the standards were labeled 125 mL, but r = 0.9995 0.51 their true internal volume varied up to 15%. So, in order to construct a correct calibration curve, the exact concentration of each standard in micrograms per liter was calculated ac0 500 10'00 15'00 2000 cordingly, taking into consideration the Concentration (pg/L) calibrated volume of the used gas bulb and the exact amount of analyte present in that Figure 3. Typical calibration curves for n-propane, iso-butane, and n-butane in whole blood. particular gas bulb, the latter depending on 39 Journal of Analytical Toxicology, Vol. 26, January/February 2002 Table II. GC-MS Retention Data for Compounds Evaluated as Possible Interferants Compoundname Retention time (min) methane < 3.5 ethene < 3.5 ethane < 3.5 n-propane 3.72 2-methylpropane (iso-butane) 4.28 water 4.51 n-butane 4.80 2,2-dimethylpropane 5.00 difluoromethyl-l,2,2,2-tetrafluoroethylether (desflurane) 5.26 methanol 5.62 2-methylbutane 6.62 n-pentane 7.61 ethanol 8.08 fluoromethyl-l,1,1,3,3,3-hexafluoro-2-propylether (sevoflurane) 8.44 di-ethylether 8.50 2-chloropropane 8.99 1,1,2-trichlorotrifluoroethane (IS) 9.33 2-propanone (acetone) 9.72 dimethoxymethane 9.87 carbondisulfide 10.43 2-propanol 10.57 1-chloro-2,2,2-trifluoroethyl-difluoromethylether (isoflurane) 10.88 acetonitrile 11.24 2-chloro-l,l,2-trifluoroethyl-difluoromethylether (enflurane) 11.32 methylacetate 11.46 2*methylpentane 11.74 2-chloro-2-methylpropane 11.82 cyclopentane 11.96 dichloromethane 12.07 1-chloropropane 12.26 2-methyl-2-propanol 12.57 t-butylmethylether 13.06 3-methylpentane 13.07 n-hexane 14.80 1,1,I -trifluoro-2-iodoethane 15.41 1,1-dichloroethane 16.11 2-bromo-2-chloro-1,1,1 -trifluoroethane (halothane) 16.21 2,2,2-trifluoroethanol 16.57 di-isopropylether 16.58 3-chloro-1 -butene 16.68 1-propanol 17.16 1-bromo-2-fluoroethane 18.12 methylcyclopentane 18.87 2-chlorobutane 19.02 1-chloro-2-methylpropane, propionitrile, cis-l,2-dichloroethylene, > 20 trichloromethane, 2-butanol, tetrahydrofuran, 1-bromopropane, n-heptane, 1,2-dichloroethane, tetrachloromethane, 1-chlorobutane, 2,2-dimethoxypropane, cyclohexane, 1,1,1-trichloroethane, ethylacetate, 3-methyl-2-butanone, 1-pentanol, 2,3-dichloro-l-propene, 1-butanol, cyclopropylmethylketone, 2-pentanol, 1,4-dioxane, methoxyflurane (pentrane), 1,3-dichlorotetrafluoro-2-propanone, dibromomethane, tetrachloroethylene, n-propylacetate 1-pentanol, 2,3-dichloro-l-propene, 40 the exact volume of calibration mixture transferred from the Teflon sampling bag to the gas bulb. One very important factor to be considered during preparation and manipulation of gas mixtures and liquid dilutions (IS solution) is the environmental temperature at which the operator is working. For practical reasons, all handling was performed at room temperature (approximately 20~ and, naturally, hand manipulation of the glass recipients containing the gases or solutions was avoided as much as possible. Internal standard To enhance the precision of the analysis, an appropriate internal standard was needed. Hence, we could compensate for unexpected losses and variability in vial or injection volume. After screening of several compounds, based on their chemical structure, volatility, and retention profile, 1,1,2-trichlorotrifluoroethane was chosen because of its perfect retention time and typical mass spectrum (see the insets in Figure 2). Fur* thermore, 1,1,2-trichlorotrifluoroethane is halogenated, and thus xenobiotic, available in a high degree of purity, and elutes at an interference free spot in the chromatogram close to the analytes. For preparation of the IS solution, t-butylmethylether was used as a solvent. Compared with the hydrocarbons, it is characterized by a higher boiling point, and therefore it elutes later (tn= 13.0 rain), causing no interference and offering a large time window for elution of the IS and, in view of the speed of analysis, retention is still acceptable. Sample preparation In view of the highly volatile nature of the compounds of interest, both sample collection and storage are pivotal (20): the blood was sampled by syringe and immediately frozen at-20~ In our assay, sample handling was kept to a strict minimum and simply involved injecting the postmortem blood, after thawing at 4~ and gentle swirling, directly into a clean, sealed vial. Only 2.5 I~L of a liquid internal standard solution was added afterwards. Method validation As has become requisite in bioanalytical applications, a thorough validation of our method was performed. The method validation data are presented in Table I. A good linearity over a broad range (from 0 to 487.2 IJg/L for n-propane, to 1498 I~g/L for iso-butane and to 1712 lJg/L for n-butane) was obtained for the relationship between the peak-area ratio (analyte/IS) and the corresponding calibration concentrations: the average correlation coefficient (r, n = 11) was 0.9993 for n-propane, 0.9992 for iso-butane, and 0.9986 for n-butane (see Figure 3). Within-day (n = 5) and total (n = 11) reproducibility were investigated at three different concentration levels. Coefficients of variation for n-propane, iso-butane, and n-butane varied between 3.4 and 9.7%, undoubtedly indicating a good reproducibility. The LOD in blood, using the S/N = 3 criterion and acceptable qualifier ion ratios, were 1.37, 2.63, and 3.01 lJg/L for the three compounds, respectively. The respective LOQs (S/N 10 ) were established at 5.50, 10.50, and 12.00 IJg/L. It must be stressed that, in assessing both the LOD and the LOQ not only the signal-to- Journal of Analytical Toxicology, Vol. 26, January/February 2002 noise definition (for LOD S/N 3, for LOQ S/N 10 ) was applied but also more stringent spectral demands. In doing so, the double identification potential (retention time and mass spectrum), even in the lowest range was completely preserved. Because volatile organic compounds (VOC) are omnipresent, not only in the global atmosphere, but also, in greater amount, in the typical laboratory environment, selectivity considerations are paramount. Table II shows the retention times of a list of VOCs and other compounds that were considered as potential interferences. For obvious reasons, this is a volatility-limited list that includes other gases, some inhalational anesthetics, frequently used solvents, and products that were screened as possible internal standard. Furthermore, selectivity is guaranteed not only from a chromatographic viewpoint but additionally through the selectivity of MS ion extraction, the mass fragmentograms allowing sound quantitation of the compound of interest. Applicationof the method A substantiation of the usefulness of our method was achieved by analyzing the above mentioned forensic blood sample. An intoxication with lighter fluid was, in view of the circumstantial evidence, obviously suspected and clearly corroborated by the data resulting from our analytical approach. On external examination of the deceased, a normally built boy measuring 1.58 m in height was seen. The rigor mortis was extreme. The livores were extremely pronounced and violet. The face was congested and cyanotic; petechiae were discernable on both upper eyelids. Vomit was seen at the mouth, and a hemorrhagic edema was flowing out of nose and mouth during manipulation of the body. Only a recent small ecchymosis was just noted beneath the left eyebrow, which is consistent with a forward fall. The following concentrations were measured: 90.0 I~g/Lfor npropane, 246.3 IJg/L for/so-butane and 846.4 IJg/L for n-butane. These levels appear to be several-fold lower then the scarce values (after lighter fluid or n-butane inhalative intoxication) supplied by the current scientific literature (15). Indeed, mostly semiquantitative data are presented (9) or only a superficial qualitative investigation of the available matrices is performed (1,17). Moreover, of the available data the report of Graefe et al. (11) concerns a suicidal death involving the use of a gas-filled plastic bag over the head of the deceased, logically resulting in sky-high blood and tissue concentrations. Hence, in consideration of our analytical findings and the circumstances surrounding this case, on top of the absence of any anatomical or alternative toxicological evidence as to the cause of death, the boy's death could now be certified as due to toxic effects of n-butane inhalation and considered accidental in nature. Conclusions This manuscript reports on a sensitive, automated, and fully validated GC assay for the quantitative determination of npropane, iso-butane, and n-butane in postmortem blood using static headspace extraction followed by capillary GC separation and MS detection. Cryofocusing was successfully applied to reconcile the large gas volumes injected with the small internal column diameter at the same time enhancing sensitivity of the method. The intricacies of gas analysis with respect to sampling, calibration, and chromatographic separation were successfully overcome. In addition, quantitative data from a fatal intoxication with lighter fluid are generated and prove the hidden danger in volatile substance abuse. Acknowledgments M.P. Bouche acknowledges her position with the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen). This work was supported in part by Grant BOF 011BO697 (Ghent University, headspace GC-MS). References 1. B.M. Huston and K.R. Lamm. Complications following butane inhalation and flash fire. Am. J. Forensic Med. Pathol. 18:140-143 (1997). 2. J.M. Bland and J. Taylor. Deaths from accidental poisonings in teenagers. Deaths due to volatile substance misuse are greatly underestimated. Br. Med. J. 316:146 (1998). 3. R.J. Flanagan, M. Ruprah, T.J. Meredith, and J.D. Ramsey. An introduction to the clinical toxicology of volatile substances. Drug Saf. 5:359-383 (1990). 4. R.J.Flanagan and R.J.Ires. 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Manuscript received February 19, 2001 ; revision received June 4, 2001.
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