A comparison of ethanol concentrations in the occipital lobe and cerebellum.

While many publications have addressed the issue of ethanol concentration in brain tissue as a better indicator of impairment than blood alcohol concentration (BAC), very few have looked at the regional distribution of ethanol in the brain and its possible significance in postmortem sampling. This paper reports on the analysis of occipital pole and cerebellar hemisphere for ethanol in 25/brain samples obtained at autopsy from the brain collection of the National Institutes of Mental Health/Stanley Foundation. When available, these concentrations were compared to BAC. The average ratio of occipital lobe alcohol concentration (OAC) to BAC for cases which also had blood samples (18/24) was 0.9, SD = 0.5, with a range of 0-1.8; the average ratio of cerebellar alcohol concentration (CAC) to BAC for these cases was 0.6, SD = 0.4, range = 0-1.2. When only those cases with a BAC > or = 0.04 g/dl (14/18 cases) were considered, the average OAC/BAC and CAC/BAC ratios were 0.8 (SD = 0.4) and 0.7 (SD = 0.4), respectively. These distribution ratios are well within the ranges reported by other authors and do not significantly differ from each other. The cortical brain region available or selected for postmortem ethanol analysis is probably not critical.

Carbon monoxide stability in stored postmortem blood samples.

Carbon monoxide (CO) poisoning remains a common cause of both suicidal and accidental deaths in the United States. As a consequence, determination of the percent carboxyhemoglobin (%COHb) level in postmortem blood is a common analysis performed in toxicology laboratories. The blood specimens analyzed are generally preserved with either EDTA or sodium fluoride. Potentially problematic scenarios that may arise in conjunction with CO analysis are a first analysis or a reanalysis requested months or years after the initial toxicology testing is completed; both raise the issue of the stability of carboxyhemoglobin in stored postmortem blood specimens. A study was conducted at the Bexar County Medical Examiner's Office to evaluate the stability of CO in blood samples collected in red-, gray-, and purple-top tubes by comparing results obtained at the time of the autopsy and after two years of storage at 3 degrees C using either an IL 282 or 682 CO-Oximeter. The results from this study suggest that carboxyhemoglobin is stable in blood specimens collected in vacutainer tubes, with or without preservative, and stored refrigerated for up to two years.

Phencyclidine blood concentrations in DRE cases.

Phencyclidine (PCP) concentration was measured in blood obtained from 259 individuals over a two-year period subsequent to Drug Recognition Expert (DRE) evaluation by the Maryland State Police. The purpose of this study was to evaluate the accuracy of the DRE in the identification of PCP-related impairment using the presence of PCP in blood to confirm drug use and to test for a correlation between PCP concentrations in blood and impairment as indicated by DRE evaluation. Of the 259 cases evaluated, 124 were identified as positive for PCP based on DRE evaluation, 130 were positive for PCP based on toxicological analysis, and 56 of the 124 were identified as positive for PCP only by DRE and subsequently confirmed to contain only PCP. The mean PCP concentration for those cases in which only PCP was identified by both DRE and toxicology was 51 ng/ml (standard deviation, 26 ng/mL) with a range of values of 12-118 ng/mL. Although no correlation was determined between PCP concentration and behavior, it is clear that, even at concentrations as low as 12 ng/mL, PCP-induced behavioral effects are measurable by DRE evaluation. This study also revealed that despite a low false-positive rate (3%) of detection of PCP use by the DRE, the false-negative rate of 8% supports the conclusion that the toxicological analysis of blood specimens for PCP provides the necessary, objective corroboration of the DRE's opinion concerning impairment.

Methylephedrine concentrations in blood and urine specimens.

Methylephedrine is a sympathomimetic amine that appears in many over-the-counter cough and cold medications throughout the world. The abuse of methylephedrine-containing medications has been reported in Japan. Although methylephedrine is not available in the United States, it was identified in 15 cases received by the Forensic Toxicology Laboratory, Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology over a two-year period; 12 of the 15 cases were collected from patients or decedents located within the confines of the continental United States. Methylephedrine was identified in each case by gas chromatography-nitrogen-phosphorus detection following an alkaline extraction and subsequently confirmed using full scan electron impact mass spectrometry. Quantitation of underivatized methylephedrine was performed using the same technique. Blood methylephedrine concentrations ranged from less than 0.05 to 0.28 mg/L (n = 14), and the mean methylephedrine concentration in urine was 1.6 mg/L (range, 0.15-6.8, n = 11 [excluding case 6]). A literature search revealed little information pertaining to the interpretation of methylephedrine concentrations in the blood. Six of the 15 cases presented here were positive for methylephedrine in the blood. Three of these cases were postmortem cases, and the other three cases were nonfatal aircraft mishaps. There is no evidence in any of these cases that methylephedrine was present at toxic concentrations; therefore, it appears from the cases reviewed in this study that blood methylephedrine concentrations less than 0.3 mg/L are not associated with significant toxicity.

Evaluation of the Abbott ADx Amphetamine/Methamphetamine II abused drug assay: comparison to TDx, EMIT, and GC/MS methods.

Although the legitimate clinical use of amphetamine and amphetamine congeners is declining, the illicit use of these drugs remains high. There is a need for a rapid and conclusive method for detecting these compounds in routine urine drug testing, drug screening in drug rehabilitation centers, and as an aid in the diagnosis and treatment of potential overdoses. The Abbott ADx Amphetamine/Methamphetamine II assay (A/M II), a fluorescence polarization Immunoassay (FPIA), was compared to the Abbott TDx Amphetamine/Methamphetamine II assay (FPIA), the Syva enzyme-multiplied immunoassay technique (EMIT) and a gas chromatograph/mass spectrometry (GC/MS) method. Precision of the A/M II assay was evaluated on the ADx analyzer over a 14-day period in each of three modes of operation (batch, combination, and panel) and was based on within-run and between-run coefficients of variation (CVs). Within-run CVs for all three controls (low [L], medium [M], and high [H]) ranged from 0.40% to 10.60% and between run CVs ranged from 3.96% to 7.92%. Data indicated that the calibration curve was stable for 16 days. Each of the six calibrators and three controls were within 10% of their labeled concentrations when analyzed by GC/MS. Fifty routine clinical specimens from our laboratory and 74 specimens screened as positive for amphetamine or related compounds from a rehabilitation center were screened by ADx, TDx, and EMIT. Any specimen yielding a positive result by any of these three methods was confirmed by GC/MS. In-house controls, as well as clinical samples, which contained both amphetamine and methamphetamine in the same sample produced results greater than two times the expected response on the ADx and TDx.(ABSTRACT TRUNCATED AT 250 WORDS)

Citalopram distribution in postmortem cases.

This is a report of the analytical findings in 13 cases investigated by either the Office of the Chief Medical Examiner, State of Maryland or the Bexar County (San Antonio, TX) Medical Examiner's Office in which citalopram, a highly selective serotonin reuptake inhibitor used therapeutically as an antidepressant, was identified. In 8 of the 9 cases in which both blood and urine specimens were received, the urine citalopram concentration exceeded the blood concentration, indicating that urine is an appropriate specimen for screening citalopram use. The average liver to blood citalopram concentration ratio was 6.5 (range 3.1-13, n = 6). Three cases had blood concentrations less than 0.24 mg/L, which is in the reported antemortem therapeutic range of the drug. Eleven cases had blood concentrations less than 1.3 mg/L; in each of these cases, citalopram was determined to be an incidental finding to the ultimate cause of death. Quantitation of citalopram and the metabolite desmethylcitalopram in these cases yielded an average parent-to-metabolite ratio of 6.4.

Application of the Syva EMIT and Abbott TDx amphetamine immunoassays to the detection of 3,4-methylene-dioxymethamphetamine (MDMA) and 3,4-methylene-dioxyethamphetamine (MDEA) in urine.

MDMA and MDEA are hallucinogenic analogs of amphetamine. The need for laboratory monitoring of these substances has developed as a result of their increased recreational use. Since the Abbott TDx and Syva EMIT-d.a.u. immunoassays are commonly used tests for urine monitoring of drugs-of-abuse, the amphetamine assay of each manufacturer was assessed to determine the degree of cross-reactivity with MDMA and MDEA. Cross-reactivity was evaluated using a series of MDMA- and MDEA-spiked urine samples. Testing was performed over a two-day period with 3 runs/day and each sample run in duplicate. The Syva EMIT d.a.u. amphetamine assay was positive only at the highest concentration standard (10.0 micrograms/mL) for both MDMA and MDEA. Consequently, no further testing was performed with this assay. Calibration curves were generated for the TDx runs and percent cross-reactivity determinations were made. Precision for the TDx data was evaluated based on within-day and between-day coefficients of variation (CV). CVs for MDMA runs were below 6% for within-day and below 5% for between-day runs. Values of CV for MDEA were below 16% for both within-day and between runs; CVs were less than 2.5% for positive values. Cross-reactivity for MDMA ranged from 18% (10.00 micrograms/mL) to 118% (0.15 microgram/mL). Cross-reactivity for the MDEA standards ranged from 12% (10.00 micrograms/mL) to 47% (0.15 micrograms/mL). The presence of MDMA and/or MDEA in samples resulting in a negative EMIT-d.a.u. test and a positive TDx test was confirmed by GC/MS analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

A modification and validation of two urine ethanol procedures for use with the Monarch 2000 Chemistry System.

A modification of two commercially available enzymatic ethanol urine assays for use with the Monarch 2000 Chemistry System (Instrumentation Laboratory) is described. Both the Syva EMIT st Urine Ethyl Alcohol Assay and the Sigma Diagnostics Alcohol in Urine Assay, which utilize the reduction of nicotinamide adenine dinucleotide (NAD) to NADH associated with the oxidation of ethanol in the presence of alcohol dehydrogenase (ADH), were adapted to spectrophotometrically determine ethanol concentration. Precision was evaluated over a 3-day period. Within-day (n = 9) and total (n = 27) coefficients of variation (CV) were less than 7% for the controls greater than or equal to 200 mg/L (20 mg/dL). Enzymatic assay results utilizing the Monarch procedure were compared to a gas chromatographic (GC) reference method (n = 100 samples). Regression analysis of assay data with each reagent compared to the reference method resulted in correlation coefficients r = 0.972 (Syva) and 0.948 (Sigma). Both methods exhibited nonlinear results and therefore quantitative applications cannot be made. No false positive or negative results were encountered with either reagent, indicating that the assay is acceptable as a positive/negative screen for urine ethanol for a threshold less than or equal to 20 mg/dL.

MDA-MDMA concentrations in urine specimens.

Urine specimens collected from active-duty U.S. Army personnel were submitted for analysis to the Tripler Army Medical Center, Forensic Toxicology Drug Testing Laboratory as part of the random drug testing program. During an 18-month drug-screening period, 34 specimens tested positive for amphetamines with the Roche Abuscreen Radioimmunoassay for Methamphetamine (High Specificity); based on gas chromatographic-mass spectrometric (GC-MS) analysis, the presence of 3,4-methylenedioxymethamphetamine (MDMA) was suspected. These samples were subsequently submitted to the Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology for further testing. All 34 samples screened positive using both the Abbott TDx Amphetamine/ Methamphetamine II assay and the Amphetamine class assay. Confirmation and quantitation by GC-MS revealed the presence of both MDMA and 3,4-methylenedioxyamphetamine (MDA) in all samples. The MDMA concentrations ranged from 0.38 to 96.2 mg/L (mean, 13.4 mg/L) and the MDA concentrations ranged from 0.15 to 8.6 mg/L (mean, 1.6 mg/L). The mean ratio of MDA, the N-demethylation metabolite of MDMA, to MDMA was 0.15, similar to the ratio of amphetamine, the N-demethylation metabolite of methamphetamine, to methamphetamine of 0.10. The presence of MDA in urine specimens at a concentration approximately 10-15% that of the MDMA present is consistent with MDMA metabolism, which may be indicative of the use of MDMA only, as compared with the combined use of both drugs.

Vitamin B2 interference with TDx drugs-of-abuse assays.

Migraine is a headache condition found in significant frequency in the general population. One recent study has shown that riboflavin, Vitamin B2, is an effective prophylactic treatment for this headache condition. One subject in a recent study conducted by the Division of Forensic Toxicology, Armed Forces Institute of Pathology (AFIP) was taking 200 mg of riboflavin twice daily for the prevention of migraine headaches. When that subject's urine was tested using Abbott TDx drugs-of-abuse assays a number of tests resulted in a MX BKG error and all samples had BLK I values greater than those observed with normal urine specimens. The MX BKG error occurs when the BLK I value is greater than the upper limit determined by the manufacturer for a particular assay. High BLK I values may result if the specimen being analyzed contains a fluorophore that will compete with the fluorescein-labeled antibody used in the assay. This error serves as a notification that an interfering substance may be present and the assay is not performing according to manufacturer-specifications. Upon termination of riboflavin therapy the subject's BLK I values began to decrease within 60 h of the last 200 mg dose. A second subject began chronic riboflavin use to confirm this interferent effect. Elevated BLK I values resulted within 3 h of a single 200 mg dose and MX BKG errors occurred 1 h after a second 400 mg dose. No false negative results were noted with either subject (both subjects used butalbital and the first subject also used hydrocodone and diazepam during the study), suggesting that riboflavin is not an adulterant. Riboflavin use, however, does interfere with the TDx DAU assays and may result in quantitative values being determined which are of questionable validity in the face of an elevated BLK I value or may result in only an MX BKG error and no quantitative value reported. It is unclear if the interfering fluorophore is simply riboflavin itself or a combination of riboflavin and its metabolic products. Results obtained on urine samples collected from individuals using prophylactic riboflavin for migraine prevention and analyzed by TDx may be of questionable validity. Such samples may require analysis utilizing another immunoassay technique that does not employ a fluorescein-labeled antibody.

A mixed-drug intoxication involving venlafaxine and verapamil.

This case report describes the suicide of a 52-year-old woman whose cause of death was attributed to a mixed-drug intoxication involving venlafaxine and verapamil. Venlafaxine is prescribed for the treatment of depression and should be used with caution in patients with cardiovascular disease. Verapamil is a calcium channel blocker primarily used for treatment of cardiovascular disorders. The following drug concentrations were determined in postmortem fluids: verapamil--3.5 mg/L (femoral blood), 9.4 mg/L (subclavian blood), and 1.0 mg/L (vitreous fluid); norverapamil--1.0 mg/L (femoral blood), 2.1 mg/L (subclavian blood), and 0.20 mg/L (vitreous fluid); verapamil and norverapamil could not be detected in bile or urine due to the high levels of erythromycin present; venlafaxine--6.2 mg/L (femoral blood), 8.6 mg/L (subclavian blood), 5.3 mg/L (vitreous fluid), 54.0 mg/L (bile), and 72.3 mg/L (urine); and O-desmethylvenlafaxine--5.4 mg/L (femoral blood), 8.3 mg/L (subclavian blood), positive (vitreous fluid), 29.2 mg/L (bile), and 9.5 mg/L (urine). The cause of death was determined to be a mixed-drug intoxication resulting from an overdose of verapamil and venlafaxine. The manner of death was determined to be suicide.

Tissue distribution of ketamine in a mixed drug fatality.

While reports of ketamine abuse are increasing, reports of ketamine deaths and tissue concentrations associated with fatalities are rare. We report here a case of a mixed drug fatality involving ketamine and ethanol. Ketamine analysis was carried out by gas chromatography with a nitrogen-phosphorus detector (NPD). We found the following tissue concentrations: blood 1.8 mg/L; urine 2.0 mg/L; brain 4.3 mg/kg; spleen 6.1 mg/kg; liver 4.9 mg/kg, and kidney 3.6 mg/kg. The blood ethanol concentration was 170 mg/dL. Because an empty nalbuphine ampule was found in the possession of the deceased, the blood was assayed for this opioid compound using a gas chromatography/mass spectrometry (GC/MS) method. None was detected at a limit of detection of 0.02 mg/L.

Validation of two devices for evaluation of human psychomotor performance.

The Pursuit Meter III (PM III) and the Simultaneous Hand and Foot Tracking (SHAFT) task are microcomputer-based devices for the evaluation of human psychomotor performance. Both devices are pursuit-tracking tasks. The primary task (PM III) requires a subject superimpose a line over a computer-generated sine wave. The computer wave is black and the subject's wave is red. The vertical position of the subject's wave is determined by a joystick controller. The SHAFT adds a second simultaneous tracking task (FTT) that is operated by means of a foot control. Ten naive subjects performed either device for 5 sessions/day over a three-day period. Each session consisted of 5 sweeps of the sine wave pattern. Mean performance on both tasks generally improved over the assessment period, and differential stability was reached within 10 sweeps for each device.

Tissue distribution of ibuprofen in a fatal overdose.

A 26-year-old white man was found dead near his home. The decedent had had a history of ibuprofen overdose and had recently received a physician's order for 800 mg ibuprofen every 4 h for back pain. Postmortem examination was performed and was unrevealing except for heavy lungs (1,140 g combined weight) and a brownish-white granular residue in the stomach. Samples of heart blood, femoral blood, liver, brain, and gastric contents were submitted for toxicological analysis. Qualitative screening detected only the presence of ibuprofen. Quantitation of ibuprofen was performed using reverse-phase high-performance liquid chromatography (HPLC) with ultraviolet detection. The analytical column was an Econosphere C-8 column (150 mm, 4.6 mm I.D.) with 5 microns particle size preceded by a C-8 5 microns guard column. The mobile phase was 46% methanol and 54% 0.2 M acetate buffer at a flow rate of 2 ml/min. Fenoprofen was used as an internal standard at 200 mcg/ml. A linear response (r = 0.99) was achieved over a concentration range of 25-600 mcg/ml. Ibuprofen was identified and quantitated in the following tissues: heart blood (518.0 mcg/ml), femoral blood (348.3 mcg/ml), liver (942.1 mcg/g), brain (283.9 mcg/g), and gastric contents (131 mg total).

Fluvoxamine distribution in postmortem cases.

Four postmortem cases are reported in which the selective serotonin re-uptake inhibitor fluvoxamine was identified. Fluvoxamine was detectable using a standard alkaline drug screen, chromatographed well using a HP-1 column, and did not require derivitization for quantitation. Two of the cases reported were drug intoxications; fluvoxamine was only an incidental finding in the other 2 cases. Central and peripheral blood values are reported, as well as antemortem blood, bile, vitreous fluid, and urine values. No solid organs were obtained in any of the cases. Quantitations were performed using both an analytical standard and a fluvoxamine tablet for the preparation of calibrators. A comparison of quantitative values was made to evaluate the feasibility of using a tablet as the drug source for the preparation of calibrators when a pure reference material is unavailable. Postmortem peripheral blood concentrations ranged from approximately 0.5 mg/L in a case of suicidal shooting to approximately 6 mg/L in a case of drug overdose. Evidence of postmortem redistribution was noted in the only case for which both central and peripheral blood were obtained. Quantitations using an extracted drug tablet for the preparation of calibrators correlated well with quantitations using a pure reference material.

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography.

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with beta-glucuronidase. Separation is achieved using a C8 reversed-phase column with a methanol-water-phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 micrograms/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.

The use of microcomputer-based psychomotor tests for the evaluation of benzodiazepine effects on human performance: a review with emphasis on temazepam.

1. The literature relating to the effects of benzodiazepines in general, and temazepam in particular, on human psychomotor performance as assessed using microcomputer-based testing batteries is surveyed. 2. The adverse effects of central nervous system depressants on performance is an important mediocolegal issue and frequently comes into question in on-the-road and on-the-job accidents. The use of microcomputer-based testing batteries allows for performance evaluation both in the laboratory and at-the-scene, as well as providing the opportunity to model a large number of different behaviours required in routine yet complex psychomotor tasks. 3. The conclusions in general are: (1) The benzodiazepines as a class of drugs impair both cognitive and motor performance. These effects are often subtle when low doses are involved or when testing occurs the morning following evening administration of the medication. (2) No single psychomotor task adequately simulates complex daily tasks such as automobile driving. A battery of tests that evaluates a number of the components of such tasks is necessary to determine adequately the full range of effects of these medications.

The effects of temazepam and ethanol on human psychomotor performance.

We have studied the effects of temazepam, alone and in combination with ethanol, on psychomotor performance in six healthy men and women using a battery of five microcomputer-based tasks before and 30, 90, and 150 min after treatment. The tests were pursuit tracking, divided attention, two four-choice reaction time tests and tapping rate. The entire battery required 25 min. The subjects also reported their mood at each testing time using a computerized bipolar mood scales test. Temazepam (15 mg) plus ethanol (peak blood concentration of 11 mmol.l-1) significantly impaired divided attention, tracking, and reaction time over a 3 h period. There was significant impairment versus placebo for each drug alone on some of the tests. Plasma and urine concentrations of temazepam and temazepam glucuronide were measured, but there was no significant temporal correlation between impairment and drug or metabolite concentration in either plasma or urine. The subjects knew when they had taken ethanol, but could not discriminate temazepam from ethanol whether alone or in combination. The subjects rated their performance similarly after each of the four treatment conditions. The performance on the tracking, divided attention, and PAB reaction time tasks used in this study was impaired by a combination of temazepam and ethanol in doses which may not cause impairment when each is given alone.