Analysis of Dextromethorphan and Three Metabolites in Decomposed Skeletal Tissues by UPLC-QToF-MS: Comparison of Acute and Repeated Drug Exposures.

Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) analysis of dextromethorphan (DXM) and its metabolites-dextrorphan, 3-methoxymorphinan (3-MEM) and 3-hydroxymorphinan-in skeletal remains of rats exposed to DXM under different dosing patterns is described. Rats (n = 20) received DXM in one of four dosing patterns: acute (ACU1 or ACU2-100 or 200 mg/kg, i.p.; n = 5, respectively) or repeated (REP1 or REP2-3 doses of 25 or 50 mg/kg, i.p., 30 min apart; n = 5, respectively). Drug-free animals (n = 5) served as negative controls. Following euthanasia, the animals decomposed to skeleton outdoors. Bones were sorted by animal and skeletal element (vertebra, femur, pelvis, tibia, rib and skull), washed, air-dried and pulverized prior to dynamic methanolic drug extraction, filtration/pass-through extraction and analysis by UPLC-QToF-MS in positive electrospray ionization mode. Analyte levels (expressed as mass-normalized response ratios, RR/m) differed significantly between ACU1 and ACU2 (Mann-Whitney (MW), P < 0.05) in all skeletal elements for all analytes investigated, and between REP1 and REP2 in most skeletal elements for 3-MEM and 3-HOM, but in all skeletal elements for DXM. Between ACU1 and ACU2, and between REP1 and REP2, analyte level ratios (RRi/RRj) differed significantly (MW, P < 0.05) in 3/6 to 6/6 skeletal elements, depending on the ratios concerned, with no analyte level ratio differing significantly between both ACU1 vs ACU2 and REP1 vs REP2. Kruskal-Wallis (KW) analysis showed skeletal element to be a main effect for all analyte levels and analyte level ratios in all ACU and REP groups examined (P < 0.05). For data pooled only according to exposure pattern, KW analysis showed dose pattern to be a main effect for both analyte levels and analyte level ratios (P < 0.05). These data illustrate a dependence of these measures on dose, dose pattern and skeletal element, suggesting that some exposure patterns may be distinguished by toxicological analysis of bone.

Analysis of Dextromethorphan and Dextrorphan in Skeletal Remains Following Differential Microclimate Exposure: Comparison of Acute vs. Repeated Drug Exposure.

Analysis of dextromethorphan (DXM) and its metabolite dextrorphan (DXT) in skeletal remains of rats following acute (ACU, 75 mg/kg, IP, n = 10) or three repeated (REP, 25 mg/kg, IP, n = 10, 40-min interval) doses of DXM is described. Following dosing and euthanasia, rats decomposed outdoors to skeleton in two different microclimate environments (n = 5 ACU and n = 5 REP at each site): Site A (shaded forest microenvironment) and Site B (rocky substrate exposed to direct sunlight, 600 m from Site A). Two drug-free rats at each site served as negative controls. Skeletal elements (vertebrae, ribs, pelvic girdles, femora, tibiae, skulls and scapulae) were recovered, pulverized and underwent methanolic microwave assisted extraction (MAE). Extracts were analyzed by GC-MS following clean-up by solid-phase extraction (SPE). Drug levels, expressed as mass-normalized response ratios and the ratios of DXT and DXM levels (RRDXT/RRDXM) were compared between drug exposures, microclimate sites, and across skeletal elements. DXM levels differed significantly (P < 0.05) between corresponding bone elements across exposure groups (5/7-site A; 4/7-site B), but no significant differences in DXT levels were observed between corresponding elements. RRDXT/RRDXM differed significantly (P < 0.05) between corresponding bone elements across exposure groups (6/7-site A; 5/7-site B). No significant differences were observed in levels of DXM, DXT or RRDXT/RRDXM between corresponding elements from either group between sites. When data from all bone elements was pooled, levels of DXM and RRDXT/RRDXM differed significantly between exposure groups at each site, while those of DXT did not. For both exposure groups, comparison of pooled data between sites showed no significant differences in levels of DXM, DXT or RRDXT/RRDXM. Different decomposition microclimates did not impede the discrimination of DXM exposure patterns from the analyses of DXM, DXT and RRDXT/RRDXM in bone samples.

Influence of dose-death interval on colchicine and metabolite distribution in decomposed skeletal tissues.

The semi-quantitative analysis of decomposed bone of rats exposed to colchicine and euthanized following different time intervals postexposure (i.e., dose-death interval, DDI) is described. Rats received colchicine (50 mg/kg, i.p.) and were euthanized 30 min (DDI1; n = 4), 60 min (DDI2; n = 4), or 180 min (DDI3; n = 4) postdose. Drug-free animals (n = 3) served as negative controls. Perimortem heart plasma was collected. Remains were decomposed to skeleton outdoors and then collected and sorted (skull, vertebrae, rib, pelvis, femur, tibia). Bones were dried, pulverized, and prepared by microwave-assisted extraction and microplate solid-phase extraction (MAE-MPSPE), followed by analysis for colchicine, 3-demethylcolchicine (3DMC), and 2-demethylcolchicine (2DMC) by ultra-high-performance liquid chromatography with photodiode array detection (UHPLC-PDA) at 350 nm. Bone type was a main effect (Kruskall-Wallis, p < 0.05) with respect to drug level (expressed as mass-normalized response ratio, RR/m) for each analyte, at each DDI. For all samples, DDI was a main effect (Kruskall-Wallis, p < 0.05) with respect to analyte level, and the ratio of analyte levels (RR3DMC/RRCOLCH, RR2DMC/RRCOLCH, and RR2DMC/RR3DMC). Bone COLCH levels varied by 19-fold, 12-fold, and 60-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Bone 3DMC levels varied by 12-fold, 11-fold and 17-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Bone 2DMC levels varied by 20-fold, 14-fold, and 14-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Values of RR3DMC/RRCOLCH varied by 16-fold, 5-fold, and 5-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Values of RR2DMC/RRCOLCH varied by 10-fold, 6-fold, and 12-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Values of RR2DMC/RR3DMC varied by 3-fold, 5-fold, and 2-fold across all bone types in the DDI1, DDI2, and DDI3 groups, respectively. Measured analyte levels in bone correlated poorly with corresponding levels in blood (r = -0.65-+0.31). Measured values of RR2DMC/RRCOLCH and RR2DMC/RR3DMC in bone also correlated poorly with corresponding values in blood. Measured values of RR3DMC/RRCOLCH were well correlated with corresponding blood levels for all bone types except skull (r = 0.91-0.97).

Analysis of dextromethorphan and dextrorphan in decomposed skeletal tissues by microwave assisted extraction, microplate solid-phase extraction and gas chromatography- mass spectrometry (MAE-MPSPE-GCMS).

Analysis of decomposed skeletal tissues for dextromethorphan (DXM) and dextrorphan (DXT) using microwave assisted extraction (MAE), microplate solid-phase extraction (MPSPE) and gas chromatography-mass spectrometry (GC-MS) is described. Rats (n = 3) received 100 mg/kg DXM (i.p.) and were euthanized by CO2 asphyxiation roughly 20 min post-dose. Remains decomposed to skeleton outdoors and vertebral bones were recovered, cleaned, and pulverized. Pulverized bone underwent MAE using methanol as an extraction solvent in a closed microwave system, followed by MPSPE and GC-MS. Analyte stability under MAE conditions was assessed and found to be stable for at least 60 min irradiation time. The majority (>90%) of each analyte was recovered after 15 min. The MPSPE-GCMS method was fit to a quadratic response (R(2) > 0.99), over the concentration range 10-10 000 ng⋅mL(-1) , with coefficients of variation <20% in triplicate analysis. The MPSPE-GCMS method displayed a limit of detection of 10 ng⋅mL(-1) for both analytes. Following MAE for 60 min (80 °C, 1200 W), MPSPE-GCMS analysis of vertebral bone of DXM-exposed rats detected both analytes in all samples (DXM: 0.9-1.5 µg⋅g(-1) ; DXT: 0.5-1.8 µg⋅g(-1) ).

Analysis of tramadol and O-desmethyltramadol in decomposed skeletal tissues following acute and repeated tramadol exposure by gas chromatography mass spectrometry.

Decomposed bone and plasma samples of rats exposed to tramadol (TRAM) under different dosing patterns were analyzed. Wistar rats received TRAM as one acute dose (n=4, 45 mg/kg, i.p.) or three doses (n=4, 15 mg/kg, i.p.), 40 min apart. Perimortem heart blood was collected, rats were euthanized and placed outdoors to decompose to skeleton. Recovered bone was ground and subjected to methanolic extraction. Bone extracts and plasma samples underwent solid phase extraction and were analyzed using gas chromatography-mass spectrometry. Levels of TRAM and the primary metabolite O-desmethyltramadol (ODMT) were expressed as mass normalized response ratios (RR/m). Levels (RR/m) for TRAM and ODMT did not differ significantly between exposure types in any of the bone types examined or for the pooled bone comparisons (Mann-Whitney, p>0.05). However, ratios of analyte levels (RRTRAM/RRODMT) differed significantly between exposure patterns for tibial and skull bone as well as for pooled bone comparisons (Mann-Whitney, p<0.05). Levels of TRAM and ODMT, as well as ratios of analyte levels (RRTRAM/RRODMT), differed significantly in plasma between exposure patterns. Bone TRAM and ODMT levels were poorly correlated to corresponding plasma levels (TRAM: r=0.33-0.57; ODMT: r=-0.35-0.23).

Discrimination between patterns of drug exposure by toxicological analysis of decomposed skeletal tissues. Part II: Amitriptyline and citalopram.

Decomposed bone and plasma samples of rats exposed to amitriptyline (AMI) and citalopram (CIT) under different dosing patterns were analyzed. Wistar rats received one acute dose (120 mg AMI/kg and 40 mg CIT/kg; n = 5) or two doses (40 mg AMI/kg and 13 mg CIT/kg, n = 5) 40 min apart. After collection of perimortem blood, the rats were euthanized and placed outdoors to decompose to skeleton. Recovered bone was ground and subjected to methanolic extraction. Bone extracts and plasma samples underwent solid-phase extraction and were analyzed using ultra-high-performance liquid chromatography. Concentrations of drugs and the primary metabolites [nortriptyline (NORT), desmethylcitalopram (DMCIT) and didesmethylcitalopram (DDMCIT)] were expressed as mass-normalized response ratios (RR/m). Concentrations (RR/m) of AMI, CIT and metabolites did not differ significantly between exposure types in plasma and all bone types examined or for the pooled bone samples (P > 0.05). However, ratios of concentrations of NORT to those of AMI differed significantly between exposure patterns for all bone types except for rib (P < 0.05). Values of DMCIT/CIT differed significantly between exposure patterns in rib, pelvi and femora (P < 0.05). Values of DDMCIT/CIT did not differ significantly between exposure types (P > 0.05), while those of DDMCIT/DMCIT were significantly different for all bones except the vertebrae and rib (P < 0.05).

Comparison of relative distribution of ketamine and norketamine in decomposed skeletal tissues following single and repeated exposures.

Bone was analyzed for ketamine and norketamine to examine whether different patterns of drug exposure could be discriminated. Rats received (intraperitoneally) one 75 mg/kg dose (Acute-1 and Acute-2 groups), three 25-mg/kg doses 1 hour apart (Repeated group), or nine single daily ketamine doses of 75 mg/kg followed by a 24-h washout period (Chronic group). Following euthanasia, all animals decomposed to skeleton outdoors. Ground samples of recovered bone underwent methanolic extraction and analysis by gas chromatography-mass spectrometry after solid-phase extraction. Drug levels (mass normalized response ratios) were compared across bone types and exposure pattern. Bone type significantly influenced drug level for the Acute-1 and Repeated dose groups, and the drug/metabolite level ratio (DMLR) for the Acute-1 group. Mean ketamine and norketamine level and DMLR varied by up to 8-fold, 7-fold and 3-fold, respectively, in the Acute-1 group, and by up to 24-fold, 5-fold and 10-fold, respectively, in the Repeated group. Drug level and DMLR differed significantly between the Acute-1 and Repeated groups for most bone types. In the Chronic group, only 1/16 and 4/16 samples were positive for ketamine and norketamine, respectively. All Acute-2 samples were positive for ketamine and norketamine. The Acute-2 and Chronic groups differed significantly in ketamine and norketamine levels, and DMLR.

Detection of amitriptyline, citalopram, and metabolites in porcine bones following extended outdoor decomposition.

Skeletal remains of a domestic pig were assessed for relative distribution of amitriptyline, citalopram, and metabolites. Following acute exposure and outdoor decomposition for 2 years, drugs and metabolites were analyzed in 13 different bones. Bones were pulverized following a simple wash procedure, and drugs were extracted by passive incubation in methanol, followed by solid-phase extraction. Samples were analyzed by ultra-high performance liquid chromatography (UHPLC) and confirmed with gas chromatography-mass spectrometry. The Kruskall-Wallis test showed that bone type was a main effect with respect to drug level for all analytes, with levels varying from 33- to 166-fold. Ratios of levels of drug to that of the corresponding metabolite were less variable, varying roughly one- to eightfold. This suggests limitations in the interpretive value of drug measurements in bone and that relative levels of drug and metabolites should be further investigated in terms of forensic value.

Relative distribution of ketamine and norketamine in skeletal tissues following various periods of decomposition.

Skeletal tissues (rat) were analyzed for ketamine (KET) and norketamine (NKET) following acute ketamine exposure (75 mg/kg i.p.) to examine the influence of bone type and decomposition period on drug levels. Following euthanasia, drug-free (n = 6) and drug-positive (n = 20) animals decomposed outdoors in rural Ontario for 0, 1, or 2 weeks. Skeletal remains were recovered and ground samples of various bones underwent passive methanolic extraction and analysis by GC-MS after solid-phase extraction. Drug levels, expressed as mass normalized response ratios, were compared across tissue types and decomposition periods. Bone type was a main effect (p < 0.05) for drug level and drug/metabolite level ratio (DMLR) for all decomposition times, except for DMLR after 2 weeks of decomposition. Mean drug level (KET and NKET) and DMLR varied by up to 23-fold, 18-fold, and 5-fold, respectively, between tissue types. Decomposition time was significantly related to DMLR, KET level, and NKET level in 3/7, 4/7, and 1/7 tissue types, respectively. Although substantial sitedependence may exist in measured bone drug levels, ratios of drug and metabolite levels should be investigated for utility in discrimination of drug administration patterns in forensic work.

Examination of the effect of dose-death interval on detection of meperidine exposure in decomposed skeletal tissues using microwave-assisted extraction.

The effect of dose-death interval and tissue distribution on the detection of meperidine in selected skeletal tissues was examined using a rapid microwave-assisted extraction (MAE) methodology. Rats (n=14) were dosed with 0 (n=2) or 30 mg/kg (n=12) meperidine (i.p.). Drug-positive rats were sacrificed with CO(2) after 20, 30, 90 and 150 min (n=3 per group). Heart blood was collected immediately after death. Tibiae were excised and frozen for further analysis. The remaining carcasses were allowed to decompose outside in secured cages to the point of complete skeletonization in a rural Northern Ontario location during the late summer months. Vertebrae and pelvi were collected for each animal. Tibial marrow was homogenized in 3 mL PB6 (phosphate buffer, 0.1M, pH 6). Fresh tibiae, and decomposed vertebrae and pelvi were cleaned in PB8.5 (phosphate buffer, 0.1M, pH 8.5) and sonicated to remove remaining soft tissue. Samples of dried, ground bone (0.5-1g) suspended in 2 mL PB6 were then irradiated in a domestic microwave oven (1100 W) at atmospheric pressure for 15 min. Samples of vertebral bone (1g) were also extracted by passive incubation in methanol (3 mL, 50°C, 72 h). All supernatants then underwent solid-phase extraction and analysis by GC/MS, using electron impact ionization in the Selected Ion Monitoring (SIM) mode. Mean GC/MS responses for each tissue type were negatively correlated with dose-death interval, with correlation coefficients ranging from -0.32 to -0.87. Analysis of variance showed dose-death interval to be a main effect (p<0.05) with respect to GC/MS response for blood, marrow, tibial epiphyses prepared by MAE, and vertebral bone prepared by passive extraction, but not for tibial diaphyses, pelvi or vertebrae prepared by MAE. Overall, MAE is advantageous as a rapid extraction tool for screening purposes in skeletal tissues, but assignment of significance to quantitative expressions of skeletal drug concentrations is complex and should be approached with caution.

The effects of burial on drug detection in skeletal tissues.

Skeletal tissues have recently been investigated for use in post-mortem toxicology. Variables affecting drug concentration in these tissues, however, are still poorly characterized. In this work, the relative effects of burial on the response of enzyme-linked immunosorbent assay (ELISA) and gas chromatography-mass spectrometry (GC-MS) assays were examined. Rats were acutely exposed to ketamine or diazepam, euthanized and buried outdoors. After one month, the remains were exhumed and skeletal tissue drug levels were compared those of non-buried rats. A climate-controlled burial was also undertaken using defleshed bones to approximate an extended decomposition. Long bones (femora, tibiae) were isolated and separated into tissue type (diaphyseal bone, epiphyseal bone, and marrow), and according to treatment (i.e. buried or non-buried). Following methanolic extraction (bone) or simple homogenization (marrow), samples were analyzed with ELISA. Samples were then pooled according to treatment, extracted by solid phase extraction (SPE) and confirmed with GC-MS. Under the conditions examined, the effects of burial appear to be drug and tissue dependent. Ketamine-exposed tissues demonstrated the greatest differences, especially in bone marrow. In diazepam-exposed tissues, burial did not seem to greatly affect drug response and some gave greater assay response compared to the non-buried set. Overall, the data suggest that fresh tissue samples may not be representative of decomposed samples in terms of skeletal tissue drug levels.

Relative distribution of drugs in decomposed skeletal tissue.

Skeletal tissues from a domestic pig exposed to amitriptyline, diazepam, and pentobarbital were analyzed to determine the relative distribution of these drugs in bone. Following drug exposure and euthanasia, remains were allowed to decompose outdoors to complete skeletonization between summer 2007 and fall 2009. Remains were recovered and separated according to bone type. Twelve different bone types were pulverized and sampled in triplicate. Each bone sample underwent methanolic extraction (96 h, 50 °C), followed by solid-phase extraction and gas chromatography-mass spectrometry in the selected ion monitoring mode. Mass-normalized assay responses underwent ANOVA with post-hoc testing, revealing bone type as a main effect for all three drugs, but not for the diazepam metabolite (nordiazepam). The assay response varied with respect to bone type by factors of 27, 39, and 20 for pentobarbital, diazepam, and amitriptyline, respectively. The relative distribution between bone type was qualitatively similar for the three administered drugs analyzed for, with the largest response obtained from rib for all three drugs. This is the first study, to the authors' knowledge, of the distribution of different drugs in various decomposed skeletal tissues in a controlled experiment using an animal model of comparable physiology to humans. These data have implications for the interpretive value of forensic drug measurements in skeletal tissues.

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues part II: the effect of dose-death interval.

The effects of dose-death interval on the detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) are described. Rats (n=14) were administered fentanyl acutely at a dose of 0 (n=2) or 60 microg/kg (n=12) by intraperitoneal injection, and euthanized within 20, 45, 135, or 225 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae, pelvi and miscellaneous (humeri and scapulae) were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues at a given dose-death interval, according to the general order of marrow>decomposed bone>fresh bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae, decomposed pelvic bone, and decomposed miscellaneous bone were 67-100%, 0-33%, 0-33%, 0-67%, 0-67% and 0-33%, respectively, over all dose-death intervals. Although group mean %DA values showed a strong negative correlation with dose-death interval in marrow, fresh epiphyseal bone, decomposed vertebrae, pelvic and miscellaneous bone (r=-0.989, -0.930, -0.955, -0.903, and -0.974, respectively), the high variability in both fresh and decomposed bone precluded differentiation of the dose-death intervals based on %DA value alone. Overall, the results suggested that the type of skeletal tissue sampled may not be as important as the amount of residual marrow remaining in skeletonized remains.

Examination of some performance characteristics of breath alcohol measurements obtained with the intoxilyzer 8000C following social drinking conditions.

The Intoxilyzer 8000C was used to measure breath alcohol concentration (BrAC) in 10 healthy subjects under social drinking conditions. Measurements commenced within 5 min of the end of drinking (EOD). For 14 blood-breath pairs, measured BrACs were compared to corresponding venous blood alcohol concentrations (vBAC) of samples drawn at least 30 min after EOD and within 5 min of the corresponding breath test. BAC was analyzed using an enzymatic method. Concentration differences between breath and blood (BrAC - vBAC) ranged from -32 to +3 mg/dL (untruncated BrAC) and from -32 to -4 mg/dL (truncated BrAC). The Invalid Sample message was actuated in five out of 23 BrAC profiles. In the remaining 18 samples, residual mouth alcohol was evaluated by comparing the maximum difference between successive (5 min apart) measurements (MID5) over 20-30 min after EOD with the precision of replicate BrAC values taken 30-40 min after EOD (5 mg/dL or less; precision unaffected by breath sample volume over the range of 2-3 L). MID5 values occurred within the first three measurements in 16/18 cases, indicative of a significant mouth alcohol effect. Thus, mandatory delays should be used with the Intoxilyzer 8000C prior to testing to minimize the probability of overestimation of BrAC due to mouth alcohol.

Detection of acute diazepam exposure in bone and marrow: influence of tissue type and the dose-death interval on sensitivity of detection by ELISA with liquid chromatography tandem mass spectrometry confirmation.

Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC/MS/MS) were used to detect diazepam exposure in skeletal tissues of rats (n = 15) given diazepam acutely (20 mg/kg, i.p.), and killed at various times postdose. Marrow, epiphyseal, and diaphyseal bone were isolated from extracted femora. Bone was cleaned, ground, and incubated in methanol. Marrow underwent ultrasonic homogenization. Extracts and homogenates were diluted in phosphate buffer, and then underwent solid-phase extraction and ELISA. Relative sensitivity of detection was examined in terms of relative decrease in absorbance (ELISA) and binary classification sensitivity (ELISA and LC/MS/MS). Overall, the data showed differences in relative sensitivity of detection of diazepam exposure in different tissue types (marrow > epiphyseal bone > diaphyseal bone), which is suggestive of heterogenous distribution in these tissues, and a decreasing sensitivity with increasing dose-death interval. Thus, the tissue type sampled and dose-death interval may contribute to the probability of detection of diazepam exposure in skeletal tissues.

Microwave-assisted extraction in toxicological screening of skeletal tissues.

The use of microwave-assisted extraction (MAE) in screening of decomposed bone tissue for model drugs of abuse is described. Rats received 50 mg/kg (i.p.) pentobarbital (n=2), 75 mg/kg (i.p.) ketamine (n=2) or 16 mg/kg (i.p.) diazepam (n=1), or remained drug-free (control). Drug-positive animals were euthanized within 20 min of drug administration. Animal remains were allowed to decompose in a secure outdoor environment to the point of complete skeletonization. Bones (tibiae, femora, vertebrae, ribs, pelvi, humeri and scapulae) were collected and pooled (according to drug) in order to minimize effects due to inter-bone differences in drug distribution. Bones were crushed and cleaned of marrow and residual soft tissue in alkaline solution or phosphate buffer with ultrasonication. Cleaned bones were then ground and underwent MAE in phosphate buffer (pH 6), methanol or a methanol:water mixture (1:1, v/v) at atmospheric pressure in a domestic microwave oven, or passive extraction in methanol. Bone extracts (control and drug-exposed) containing methanol were evaporated to dryness before reconstitution in phosphate buffer (pH 6) and subsequent analysis by ELISA, while bone extracts containing only phosphate buffer were assayed directly by the same ELISA protocol. Measured absorbance values were expressed as the decrease in absorbance, measured as a percentage, relative to the corresponding drug-free control bone extract. The semi-quantitative nature of the ELISA assay allowed examination of the effects of extraction solvent and bone sample mass on the assay response for each drug examined, and subsequent comparison to assays of extracts obtained through passive methanolic extraction of various bone tissues. Overall, the time required for maximal extraction varied with extraction solvent and bone mass for each drug investigated, with significant extraction occurring with all solvent systems examined. MAE may represent a substantially faster extraction system than passive extraction, with significant extraction recovery observed within 1 min of exposure for all samples examined. The implications of these results in the context of the available literature on drug analysis in skeletal tissues are discussed.

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues.

The detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) is described. Rats (n=15) were administered fentanyl acutely at a dose of 0 (n=3), 15 (n=3), 30 (n=3) or 60 microg/kg (n=6) by intraperitoneal injection, and euthanized within 20 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae and pelvi were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues under a given dose condition, according to the general order of marrow>vertebrae approximately pelvi>epiphyseal bone approximately diaphyseal bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae and decomposed pelvic bone were 100%, 16-33%, 0-16%, 0-33% and 66-100%, respectively, at the 60 microg/kg dose level. While mean %DA values showed a strong positive correlation with those in marrow and blood measurements and the administered dose (r=0.997 and 0.986), such a correlation was not observed in assays of decomposed tissues (r=-0.157 and -0.315). These results suggest that the type of skeletal tissue sampled and position within a given bone may be important considerations in the choice of substrate for fentanyl screening in skeletal tissues, but that quantitative analysis of drugs in decomposed bones may be of limited interpretive value.

Assessment of response of the Intoxilyzer 8000C to volatiles of forensic relevance in vitro, part I: acetone, isopropanol, and methanol.

The response of the Intoxilyzer 8000C (version approved for evidentiary breath alcohol testing in Canada) to volatile solvents in vitro is described. Acetone, isopropanol, and methanol were prepared as aqueous solutions or dilutions of standard alcohol solution (SAS; 1.21 mg ethanol/mL) to generate apparent blood ethanol concentrations (aBEC) of 50 or 80 mg/dL. Solvent concentrations examined were relevant to clinical or impaired driving scenarios. Replicates of 20 aBEC measurements were made for each mixture and the actuation of the "INTERFERANT DETECT" message (IDM) was noted. Measurements of aqueous acetone (0-40 mg acetone/dL), isopropanol (0-100 mg isopropanol/dL), and methanol (0-100 mg methanol/dL) yielded aBECs of 0, 0-43, and 0-55 mg/dL, respectively. The minimum concentration examined at which the IDM was actuated in 100% of replicates was 25, 30, and 100 mg/dL for acetone, isopropanol, and methanol, respectively. The maximum concentration examined at which the IDM was actuated in none of the replicates was 5, 10, and 50 mg/dL for acetone, isopropanol, and methanol, respectively. In examinations of acetone/isopropanol mixtures in diluted SAS where the IDM was not always actuated, the maximum BEC overestimation was 10 mg/dL. Overall, the potential for significant undetected BEC overestimation is low and may be further reduced through truncation of test results and subject observation.

Effects of tissue type and the dose-death interval on the detection of acute ketamine exposure in bone and marrow with solid-phase extraction and ELISA with liquid chromatography-tandem mass spectrometry confirmation.

Ketamine exposure was detected in skeletal tissues by ELISA and liquid chromatography-tandem mass spectrometry (LC-MS-MS). Rats (n = 9) received ketamine hydrochloride acutely (75 mg/kg, i.p.) and were euthanized within 15, 30, or 90 min. Drug-free control animals (n = 3) were also euthanized. Extracted femora were separated into epiphyseal and diaphyseal fragments, with marrow isolated from the medullary cavity. Bone was ground and incubated in methanol. Extracts were dried and reconstituted in phosphate buffer (0.1 M, pH 7.3), and marrow was homogenized in alkaline solution. Both then underwent solid-phase extraction. Extracts were assayed by ELISA, with data expressed in terms of relative decrease in absorbance (%DA, drug-positive tissues vs. matrix-matched drug-free controls) and binary classification test sensitivity (S). Generally, %DA decreased in the order of marrow > epiphyseal bone > diaphyseal bone, and was negatively correlated with dose-death interval (DDI). Measured S values were 100% in ELISA analysis of extracts of all tissue types. Sensitivity values were computed from LC-MS-MS data using a 5 ng/mL cutoff. Sensitivity values for ketamine detection were 100%, 0-100% and 0%, at the 15, 30, and 90 min DDI, respectively, and sensitivity values for norketamine detection were 0-66%, 0-66%, and 0% at the 15, 30, and 90 min DDI, respectively. These results suggest that the tissue type sampled and DDI may influence the sensitivity of detection of ketamine exposure in skeletal tissues.

Utility of immunoassay in drug screening in skeletal tissues: sampling considerations in detection of ketamine exposure in femoral bone and bone marrow following acute administration using ELISA.

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.