Y-STR profiling in extended interval (> or = 3 days) postcoital cervicovaginal samples

Depending upon specific situations, some victims of sexual assault provide vaginal samples more than 36-48 h after the incident. We have tested the ability of commercial and in-house Y-STR systems to provide DNA profiles from extended interval (> or =3 days) postcoital samples. The commercial Y-STR systems tested included the AmpFlSTR Yfiler (Applied Biosystems), PowerPlex Y (Promega) and Y-PLEX 12 (Reliagene) products whereas the in-house systems comprised Multiplex I (MPI) and Multiplex B (MPB). Three donor couples were recruited for the study. Postcoital cervicovaginal swabs (x2) were recovered by each of the three females at specified intervals after sexual intercourse (3-7 days). Each time point sample was collected after a separate act of sexual intercourse and was preceded by a 7-day abstention period. As a negative control, a precoital swab was also recovered prior to coitus for each sampling and only data from postcoital samples that demonstrated a lack of male DNA in the associated precoital sample was used. A number of DNA profile enhancement strategies were employed including sampling by cervical brushing, nondifferential DNA extraction methodology, and post-PCR purification. Full Y-STR profiles from cervicovaginal samples recovered 3-4 days after intercourse were routinely obtained. Profiles were also obtainable 5-6 days postcoitus although by this stage partial profiles rather than full profiles were a more likely outcome. The DNA profiles from the sperm fraction of a differential lysis were superior to that obtained when a nondifferential method was employed in that the allelic signal intensities were generally higher and more balanced and exhibited less baseline noise. The incorporation of a simple post-PCR purification process significantly increased the ability to obtain Y-STR profiles, particularly from 5- to 6-day postcoital samples. Remarkably an 8 locus Y-STR profile was obtained from a 7-day postcoital sample, which is approaching the reported time limit for sperm detection in the cervix.     

Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.     

Use of a Y chromosome probe as an aid in the forensic proof of sexual assault

Currently, the most common procedures for the forensic identification of semen that may be present due to a sexual assault include the microscopic identification of spermatozoa, acid phosphatase activity, or the detection of PSA. However, not all cases of sexual assault result in the deposit of semen. Fluorescent In Situ Hybridization (FISH) has been found to be a very sensitive and specific method for detection of the Y chromosome from male cells. This study was undertaken to demonstrate the presence of epithelial cells of male origin in the postcoital vaginal tract using a commercially available probe. Results identified Y chromosome in intact epithelial cells on postcoital Days 1 through 4, and on Day 7. Additionally, Y chromosome positive epithelial cells were identified in vaginal swabs obtained following intercourse with no ejaculation. The method developed in this study demonstrates that FISH is a sensitive method for the identification of the presence of male epithelial cells in the postcoital vagina.     

Correlation between 'on admission' blood toluene concentrations and the presence or absence of signs and symptoms in solvent abusers

For 1 year, from June 1987 to June 1988, toluene concentrations in blood samples of patients admitted to a psychiatric hospital after inhaling solvent vapor, collected on admission and 4 h later, were analyzed by gas chromatograph. Toluene levels in the first urine samples collected after admission were also analyzed and case histories were kept listing age, sex and physical and psychiatric effects. In all, 51 cases were studied--34 males and 17 females. (1) The average age of the males was 21.4 years and of the female 16.2 years. (2) The toluene concentrations in the blood collected on admission ranged from 0.3 to 22.8 micrograms/g. (3) Physical signs were observed in 9 patients with an "on admission" blood toluene concentrations of more than 3.0 micrograms/g; twice as many subjects (18), however, with blood toluene concentration greater than 3.0 micrograms/g were without physical signs. (4) The blood toluene concentrations of three cases in the condition known as twilight state were more than 10.0 micrograms/g. (5) In 24 cases with blood toluene concentrations below 3.0 micrograms/g, there were no physical signs. (6) Five subjects with blood toluene concentrations in the 0.8-5.2 micrograms/g range showed neuropsychiatric effects; however, 23 subjects in the same blood toluene concentration range did not exhibit psychiatric effects, and none of the subjects with blood toluene concentrations greater than 5.2 micrograms/g, 15 in all, had such effects.     

Ethanol distribution ratios between urine and capillary blood in controlled experiments and in apprehended drinking drivers.

Healthy men drank 0.51, 0.68, and 0.85 g of ethanol per kilogram of body weight as neat whisky in the morning after an overnight fast. During 6 to 8 h after the whisky was consumed, nearly simultaneous specimens of fingertip blood and pooled bladder urine were obtained for analysis of ethanol using an enzymatic method. The mean ratios of ethanol concentration [urine alcohol concentration (UAC)/blood alcohol concentration (BAC)] were mostly less than unity during the absorption phase. The UAC exceeded the BAC in the postpeak phase. The mean UAC/BAC ratios varied between 1.4 and 1.7 when the BAC exceeded 0.50 mg/mL. When the BAC decreased below 0.40 mg/mL, the UAC/BAC ratios increased appreciably. The mean UAC/BAC ratios of ethanol were not dependent on the person's age between the ages of 20 and 60 years old, but there were large variations within the age groups. In apprehended drinking drivers (N = 654) with a mean BAC of 1.55 mg/mL, the UAC/BAC ratio of ethanol varied widely, with a mean value of 1.49. In 12 subjects (3.2%), the ratio was less than or equal to unity. In a second specimen of urine obtained approximately 60 min after an initial void (N = 135), the mean UAC/BAC ratio was 1.35 (standard deviation = 0.17). The magnitude of the UAC/BAC ratio of ethanol can help to establish whether the BAC curve was rising or falling at or near the time of voiding. The status of alcohol absorption needs to be documented if drinking drivers claim ingestion of alcohol after the offence or when back-estimation of the BAC from the time of sampling to the time of driving is required by statute.     

Determination of dimethoate in blood and hemoperfusion cartridge following ingestion of formothion: a case study

A 57-year-old male who had ingested not more than 22 g of formothion was semicomatose on admission to hospital, approximately 1.5 h after ingestion. Dimethoate, a hydrolyzed formothion, was found in blood samples collected from the patient and in the charcoal column in the direct hemoperfusion cartridge which was used 6 to 7.5 h after ingestion. It was extracted and purified by Extrelut column extraction. A gas chromatograph, equipped with a flame photometric detector and a gas chromatograph-mass spectrometer, were used to detect and confirm the presence of dimethoate. The blood dimethoate concentrations which were taken approximately 1.5 and 6 h after ingestion were 21.4 and 12.7 micrograms/g, respectively. A blood dimethoate concentration of 21.4 micrograms/g would appear to indicate a high level of formothion intoxication. The total amount of dimethoate found in the charcoal column used was 15 mg.     

The rate and kinetic order of ethanol elimination

The rate and kinetic order of ethanol elimination was evaluated in human volunteers. Part I of the study involved dosing individuals with alcoholic beverages on two separate occasions. Breathalyzer tests were performed at 15-min intervals for a period of 5 h. Attention was focused on values obtained after peak blood ethanol levels had been reached. The second part of the study included having samples drawn from alcoholics at predetermined intervals during recovery from alcoholic intoxication. Blood ethanol concentration data was analyzed for kinetic order and a comparison of ethanol elimination rates of alcoholics and non-alcoholics was made. The predicative capability of estimating a BAC from both the zero and first order theories was also investigated.It was concluded that ethanol elimination is a zero order process. For subjects classified as non-drinkers (consume less than 6 ounces of ethanol/month), the mean ethanol elimination rate as determined in the study was 12 ± 4 mg/h. For subjects classified as social drinkers (consume more than 6 ounces but less than 30 ounces of ethanol/month), the mean ethanol elimination rate was 15 ± 4 mg%/h, and for alcoholics, the mean ethanol elimination rate was 30 ± 9 mg%/h. These results indicate that the rate of ethanol elimination increases with drinking experience.     

Urinary excretion profiles of 11-nor-9-carboxy-delta9-tetrahydrocannabinol and 11-hydroxy-delta9-THC: cannabinoid metabolites to creatinine ratio study IV

The objective of this study was to compare urinary excretion patterns of two cannabinoid metabolites in subjects with a history of chronic marijuana use. The first metabolite analyzed was nor-9-carboxy-delta9-tetrahydrocannabinol (delta9-THC-COOH), the major urinary cannabinoid metabolite that is pharmacologically inactive. The second metabolite 11-OH-delta9-THC is an active cannabinoid metabolite and is not routinely measured. Urine specimens were collected from four subjects on 12-20 occasions > or = 96 h apart in an uncontrolled clinical setting. Creatinine was analyzed in each urine specimen by the colorimetric modified Jaffé reaction on a SYVA 30R biochemical analyzer. All urine specimens analyzed for 11-OH-delta9-THC had screened positive for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/mL) on a SYVA 30R analyzer and submitted for delta9-THC-COOH confirmation by GC-MS (cut-off concentration 15 ng/mL). Eleven-OH-delta9-THC was measured by GC-MS with a cut-off concentration of 3 ng/mL. Both GC-MS methods for cannabinoid metabolites used deuterated internal standards for quantitative analysis. The mean (range) of urinary delta9-THC-COOH concentration was 1153 ng/mL (78.7-2634) with a cut-off of 15 ng/mL. The mean (range) of delta9-THC-COOH/creatinine ratios (ng/mL delta9-THC-COOH/mmol/L creatinine) was 84.1 (8.1-122.1). The mean (range) urinary of 11-OH-delta9-THC concentration was 387.6 ng/mL (11.9-783) with a cut-off of 3 ng/mL, and the mean (range) of 11-OH-delta9-THC/creatinine ratio (ng/mL 11-OH-delta9-THC/mmol/L creatinine) was 29.7 (1.2-40.7). Of the 63 urine specimens submitted for delta9-THC-COOH confirmation by GC-MS, 59/63 urine specimens (94%) were positive for delta9 -THC-COOH and 51/63 (81%) were positive for 11-OH-delta9-THC. Overall, the concentrations of 11-OH-delta9-THC in urine specimens collected > or = 96 h apart were lower than delta9-THC-COOH concentrations in 50/51 of the urine specimens in this population. Further urinary cannabinoid excretion studies are needed to assess whether 11-OH-delta9-THC analyses have a role when assessing previous marijuana or hashish use in chronic users whose urine specimens remain positive for delta9-THC-COOH for an extended period of time after last drug use.     

1H NMR analysis of GHB and GBL: further findings on the interconversion and a preliminary report on the analysis of GHB in serum and urine

A 1H nuclear magnetic resonance (1H NMR) method for the determination of gamma-hydroxybutyric acid (GHB) and gamma-hydroxybutyrolactone (GBL) in human serum and urine using spiked samples has been developed. The method gives linear responses (correlation coefficients of 0.99 or greater) over the concentration range 0.01 mg/mL to 4.0 mg/mL in urine and 0.3 mg/mL to 2.0 mg/mL in serum. No sample pretreatment is required. Studies of the chemical interconversion of GBL and GHB showed hydrolysis of GBL to be rapid at pH 11.54, slower and less complete (30% hydrolysis) at pH 2.54 and slowest at pH 7.0, reaching 30% hydrolysis in about 40 days. No esterification of GHB was observed at any pH.     

Biological and DNA evidence in 1000 sexual assault cases

Using a Filemaker-based database (DNA Pro-FILES, Synchrone Infosystème Inc.), we have conducted a large-scale study on 1000 sexual assault (SA) cases where a standardized kit was submitted to our laboratory alone or with other types of exhibits. We looked at the likelihood of obtaining good quality DNA evidence, allegedly from the assailant, according to a number of parameters.The overall proportion of SA cases with DNA evidence is nearly 50%. A little more than 30% of SA kits provided DNA evidence while for 16% of cases DNA evidence could be obtained only from other exhibits.The likelihood of obtaining DNA evidence is approximately 50% in teenager and adult SA cases, but much lower for children 10 years old or younger (15%). In children cases, profiles were found mostly on clothing or skin swabs.The likelihood of obtaining DNA evidence from vaginal swabs remains good for up to 3 days after the assault (from 35% on the first day to 23% on the third day). A DNA profile was obtained from approximately 22% of anal/rectal swabs and 41% of skin swabs taken less than 1 day after the assault. Less than 10% of oral washes provided DNA evidence, all having been collected within 24 h of the assault.We found that in bodily samples, a negative result for acid phosphate (AP) is a poor predictor of the likelihood of obtaining good quality DNA evidence. Approximately 15% of vaginal swabs and 8% of anal swabs negative for AP nevertheless provided good quality DNA evidence.     

Computer-assisted interpretation in forensic toxicology: morphine-involved deaths

Case data from 200 morphine-involved deaths (Spiehler, V. and Brown, R., Journal of Forensic Sciences, Vol. 32, No. 4, July 1987, pp. 906-916) were analyzed for patterns and relationships using artificial intelligence (AI) computer software. Case parameters were blood unconjugated morphine, blood, brain, and liver total morphine, sex, age, frequency of use, time of death after injection, cause of death, and presence of other drugs. The programs used were Expert 4 (Biosoft-Cambridge), BEAGLE (Warm Boot, Ltd.), and KnowledgeMaker (Knowledge Garden Inc.). Interpretation was defined as estimating the dose, response, and time after drug dosing. The AI programs were used to advise on time and response outcomes for cases, to calculate the probability of the estimate being true, to develop rules for interpretation of morphine-involved cases, and to diagram a decision tree. On known cases the AI programs were successful 70 to 90% of the time in classifying the cases as to response and time. No data on dose were available in this database. The success rate in individual cases was proportional to the program-estimated probability. All three programs found the case parameters of most value in predicting response to be blood unconjugated morphine, blood total morphine, and liver total morphine. The case data most useful in estimating time of death since drug injection were blood unconjugated morphine, percent unconjugated morphine in blood, and brain total morphine. The rule induction programs found that morphine overdoses were characterized by blood unconjugated morphine greater than 0.24 micrograms/mL, liver morphine greater than 0.50 to 0.75 micrograms/g, brain morphine greater than 0.08 micrograms/g or greater than blood unconjugated morphine, and percent blood unconjugated morphine greater than 37%. Rapid deaths were characterized by percent unconjugated morphine greater than 44 to 50%; blood unconjugated morphine, as a function of other drugs present, greater than 0.09 to 0.21 micrograms/mL; and brain total morphine greater than 0.16 to 0.22 micrograms/g. This work demonstrates that inexpensive AI programs commercially available for personal computers can be useful in interpretation in forensic toxicology.     

The relationship of methanol and formate concentrations in fatalities where methanol is detected

An automated headspace gas chromatography method was developed for the determination of formate (formic acid) in postmortem specimens, based on the in situ sulfuric acid-methanol methylation of formic acid to methyl formate. Diisopropyl ether was used as an internal standard. The method was applied to over 150 postmortem cases where methanol was detected. Of the 153 cases presented, 107 deaths were attributed to acute methanol toxicity. In the vast majority of the remaining 46 deaths, the methanol was determined to be present as a postmortem or perimortem artifact, or was otherwise incidental to the cause of death. Of the 76 victims who were found dead and blood was collected by the medical examiner, all but one had a postmortem blood formate concentration greater than 0.50 g/L (mean 0.85 g/L; n = 74). The sole exception involved suicidal ingestion of methanol where the blood methanol concentration was 7.9 g/L (790 mg/100 mL) and blood formate 0.12 g/L. In 97% (72/74) of the cases where blood was available, the blood formate was between 0.60 and 1.40 g/L. In 31 of the 153 cases, the victim was hospitalized and blood obtained on admission or soon after was analyzed for methanol and formate during the subsequent death investigation; the vast majority (27/30) had antemortem blood formate concentrations greater than 0.50 g/L. Cases with samples taken prior to death with blood formate concentrations less than 0.5 g/L can readily be explained by active treatment such as dialysis. The blood formate method has also been useful in confirming probable perimortem or postmortem contamination of one of more fluids or tissues with methanol (e.g., windshield washer fluid or embalming fluid), where methanol ingestion was unlikely.     

Development and Validation of a Method Using Dried Oral Fluid Spot to Determine Drugs of Abuse

A liquid chromatography–mass spectrometry method using dried oral fluid spots was developed and validated for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, amphetamine, and 3,4‐methylenedioxymethamphetamine. The oral fluid was applied to a Whatman 903 grade paper and submitted to a drying time of 2.5 h. The extraction procedure was optimized by chemometric approach using simplex centroid design. Spots were extracted with a mixture of acetonitrile, buffer, and methanol. Calibration curves covered a linear concentration range of 40–500 ng/mL. Validation parameters of linearity, precision, accuracy, selectivity, carryover, matrix effects, and stability were evaluated and showed satisfactory results. Spot homogeneity was also satisfactory, with less than 15% of deviation from nominal concentration. Spot volume did not influence accuracy when less than 100 μL of the sample was applied to the spot. The validation of the proposed method suggests a potential application in different scenarios in toxicology.     

The evidential value of peptidase A as a semen typing system

Human erythrocyte peptidase A (Pep A) displays a genetic polymorphism in blacks. Its occurrence in human semen was examined for its possible use as a semen typing system. Studies by starch gel electrophoresis, in which the Pep A was located by an improved method, were carried out on semen, semen stains, and vaginal swabs taken at known times after intercourse. In addition, a large number of vaginal swabs, negative for semen, were taken from females throughout their menstrual cycles and examined for Pep A activity. The results indicated that Pep A typing could be carried out on semen and semen stains. However, it was possible to determine the Pep A type on vaginal swabs only when they had been taken within about 3 h after intercourse.     

The zinc test as an alternative for acid phosphatase spot tests in the primary identification of seminal traces

The value of the acid phosphatase spot test in the primary visualization and identification of seminal traces is hampered by the sensitiveness of the enzyme to biodegradation. An alternative spot test is proposed, based on the high concentration of the more stable zinc metal in seminal plasma. The proposed zinc spot test is simple and suitable for on site investigation. Although the sensitivity in fresh stains is lower than that of the acid phosphatase spot test, this is largely compensated by the lower sensitiveness to biodegradation. The specificity for semen is higher than that of the acid phosphatase spot test. In vaginal swabs it was nevertheless seen, that samples should be taken within 24 h after alleged sexual assault to give reliable results.     

Morphine levels in urine subsequent to poppy seed consumption

Urine morphine levels after the consumption of poppy seeds were measured in two separate trials. Maximum levels of approximately 18 micrograms/ml were found using RIA, EMIT-ST and GC methodologies. Positive immunoassay results were seen up to 60 h post-ingestion. Several different lots of seeds from various sources were assayed for morphine and found to range from 4-200 mg/kg. Differentiation of poppy seed eaters from opiate users was not possible via the identification of minor alkaloid constituents of poppy seeds. It is, however, possible to analyse opiate urines with respect to 6-O-acetylmorphine. Below the level of approximately 5 micrograms/ml total opiates, GC/MS is the method of choice for this analysis.     

A New Approach for the Carbon Monoxide (CO) Exposure Diagnosis: Measurement of Total CO in Human Blood Versus Carboxyhemoglobin (HbCO)

The aim of the study is to present the application of a headspace–gas chromatography–mass spectrometry (HS‐GC‐MS) method for the determination of the carbon monoxide (CO) blood concentration and to compare it with carboxyhemoglobin (HbCO) saturation. In postmortem cases, the HbCO measured by spectrophotometry frequently leads to inaccurate results due to inadequate samples or analyses. The true role of CO intoxication in the death of a person could be misclassified. The estimation of HbCO from HS‐GC‐MS CO measurements provides helpful information by determining the total CO levels (CO linked to hemoglobin (HbCO) and CO dissociated from hemoglobin). The CO concentrations were converted in HbCO saturation levels to define cutoff blood CO values. CO limits were defined as less than 1 μmol/mL for living persons, less than 1.5 μmol/mL for dead persons without CO exposure, and greater than 3 μmol/mL for dead persons with clear CO poisoning.     

Laboratory investigation of deaths due to anaphylaxis.

To establish a useful laboratory protocol to investigate possible cases of fatal anaphylaxis, we measured mast-cell-derived tryptase levels and allergen-specific immunoglobulin E (IgE) antibody levels in sera obtained prior to or within 24 h after death from 19 anaphylaxis victims. Elevated serum tryptase levels (range = 12 ng/mL to 150 micrograms/mL) were found in nine of nine Hymenoptera sting fatalities, six of eight food-induced fatalities, and two of two reactions to diagnostic therapeutic agents. Tryptase levels were normal (less than 10 ng/mL) in 57 sequential sera obtained postmortem from six control patients. Tryptase could not be measured in pleural or pericardial fluids for technical reasons. Serum IgE antibodies were elevated in five of the nine Hymenoptera sting fatalities and in eight of the eight fatal food reactions; assays were unavailable for the two diagnostic/therapeutic agents. If elevated, the victim's serum IgE antibodies to food could be used to identify allergens in uneaten portions of foods consumed shortly before the anaphylactic event. IgE antibodies were moderately stable during storage in a variety of anticoagulants at room temperature for up to 11 weeks. Elevated mast-cell-derived tryptase levels in postmortem sera reflect antemortem mast cell activation and may be used as a marker for fatal anaphylaxis. If assays are available for IgE antibodies to relevant allergens, such assays provide evidence for antemortem sensitization; these assays may be modified to identify allergens in foods consumed by victims of food-induced anaphylaxis.     

Detection and disposition of JWH-018 and JWH-073 in mice after exposure to "Magic Gold" smoke

The disposition in mice of the cannabimimetics JWH-018 and JWH-073 in blood and brain following inhalation of the smoke from the herbal incense product (HIP) "Magic Gold" containing 3.6% JWH-018, 5.7% JWH-073 and less than 0.1% JWH-398 (w/w) is presented. Specimens were analyzed by HPLC/MS/MS. The validation of the method is also presented. Five C57BL6 mice were sacrificed 20 min after exposure to the smoke of 200 mg of "Magic Gold" and a second set of five exposed mice were sacrificed after 20 h. Twenty minutes after exposure to "Magic Gold" smoke, blood concentrations of JWH-018 ranged from 42 to 160 ng/mL (mean: 88 ng/mL ± 42) and those of JWH-073 ranged from 67 to 244 ng/mL (mean: 134 ng/mL ± 62). Brain concentrations 20 min after exposure to "Magic Gold" smoke for JWH-018 ranged from 225 to 453 ng/g (mean: 317 ng/g ± 81) and those of JWH-073 ranged from 412 to 873 ng/g (mean: 584 ng/g ± 163). Twenty hours after exposure to "Magic Gold" smoke, JWH-018 was detected and quantified in only two of the five blood samples. Blood concentrations of JWH-018 were 3.4 ng/mL and 9.4 ng/mL. JWH-073 was detected in only one blood specimen 20 h after exposure at 4.3 ng/mL. Brain concentrations 20 h post exposure for JWH-018 ranged from 7 to 32 ng/g (mean: 19 ng/g ± 9). JWH-073 was not detected in 20 h post exposure brain specimens. JWH-398 was not detected in any of the blood or brain samples. The disposition data presented with the limited data available from human experience provide reasonable expectations for forensic toxicologists in JWH-018 or JWH-073 cases. As with THC after smoking marijuana, blood and brain concentrations of JWH-018 and JWH-073 after HIP smoking can be expected to rise initially to readily detected values, and then drop dramatically over the next few hours to several ng/mL or ng/g, and finally to be at extremely low or undetectable concentrations by 24h apparently due to extensive biotransformation, and redistribution to body fat.     

Postmortem redistribution of fentanyl in the rabbit blood

Postmortem redistribution of fentanyl in the rabbit was investigated after application of the 50-μg/h Durogesic pain patch. Patches were applied for 48 hours. Two cycles of patch administration were used before characterization of the postmortem redistribution. Fentanyl showed marked redistribution into the femoral and pulmonary veins of the rabbit through 48 hours after the animals were humanely killed and the pain patches removed. The plasma concentration of 2.34 ng/mL in the femoral blood before killing the animals increased 5.6-fold by 48 hours after patch removal to 13.2 ng/mL. This postmortem concentration is approximately 3-fold the C(max) determined during antemortem pharmacokinetic analysis, 4 ng/mL, which was achieved 24 hours after the application of the second 50-μg/h Durogesic pain patch. After blood sampling for 48 hours after animal termination with patch removal compared with sampling for 48 hours from animals not terminated and with patch removal, the exposure ratios in the terminated animals were approximately 30-fold, indicating that between the postmortem redistribution of fentanyl and the cessation of hepatic clearance of fentanyl in the rabbit, the postmortem redistribution of fentanyl leads to an elevated measures of postmortem blood concentrations relative to antemortem blood concentrations.