Alcohol Limits and Public Safety.

On May 14, 2013, the National Transportation Safety Board (NTSB) recommended lowering the legal blood-alcohol limit to 0.05 g/dL for motor vehicle operators in the United States, in an effort to reduce the risk of injuries and deaths caused by a driver's alcohol impairment (NTSB/SR-13/01). This recommendation has prompted other organizations and agencies, including the National Safety Council, to evaluate and consider supporting this action. In order to determine the scientific and legal feasibility and advisability of lowering or establishing 0.05 per se laws, we examined 554 alcohol-related publications. Risk factors, instrument reliability, law enforcement, and adjudication issues were considered in this overview of the literature. The extensive scientific literature reviewed provides ample support for lowering the operation of motor vehicle alcohol limits to 0.05, and for supporting the NTSB recommendations. Research clearly demonstrates that impairment begins at very low concentrations, well below the recommended NTSB limit, and increases with concentration. Lowering the limit to 0.05 will save many lives and prevent injuries. Breath, blood, and saliva samples have proved to be accurate and reliable specimens for legal acceptability in a court of law.

Mass Spectra and Cross-Contribution of Ion Intensity Between Drug Analytes and Their Isotopically Labelled Analogs - Benzodiazepines and Their Derivatives.

With GC-MS as the preferred method and isotopically labeled analogs (ILAs) of the analytes as the internal standards (ISs) of choice for quantitative determination of drugs/metabolites in biological specimens, one important aspect associated with chemical derivatization (CD) is that the CD products derived from the analyte and the selected IS must generate ions suitable for designating the analyte and the IS. These ions must not have significant cross-contribution (CC), i.e., ISs' contribution to the intensities of the ions designating the analytes, and vice versa. With this in mind, the authors have reviewed literature and information provided by manufacturers, searching for suitable CD reagents, CD methods, and ILAs of the analytes related to the following 18 benzodiazepines: oxazepam, diazepam, nordiazepam, nitrazepam, temazepam, clonazepam, 7-aminoclonazepam, prazepam, lorazepam, flunitrazepam, 7-aminoflunitrazepam, N-desalkylflurazepam, N-desmethylflunitrazepam, 2-hydroxyethylflurazepam, estazolam, alprazolam, α-hydroxyalprazolam, and α-hydroxytriazolam. These analytes and ILAs were derivatized with various derivatization groups, followed by GC-MS analysis. The resulting mass spectrometric data are systematically presented in two forms: (a) full-scan mass spectra; and (b) CC data of ion-pairs with potential for designating the analytes and their respective ILAs (candidates of ISs in quantitative analytical protocols). Many of these full-scan mass spectra are not yet available in the literature and should be of reference value to laboratories engaged in the analysis of these drugs/metabolites. Full-scan MS data were further used to select ion-pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion-pairs were evaluated using data collected in selected ion monitoring mode and systematically tabulated, making the data readily available for analysts searching for this important analytical parameter.

Mass Spectra and Cross-Contribution of Ion Intensity Between Drug Analytes and Their Isotopically Labelled Analogs - Common Opioids and Their Derivatives.

For the quantitation of most drugs and their metabolites, GC-MS is currently the preferred method and isotopically labeled analogs of the analytes are the internal standards (ISs) of choice. Under this analytical setting, chemical derivatization (CD) plays a critical role in the sample preparation process. In addition to meeting the conventional objectives of CD, products derived from the selected CD method must generate ions suitable for designating the analyte and the IS; these ions cannot have significant cross-contribution (CC), i.e., contribution to the intensity of the ions designating the analyte by the IS, and vice versa. With this in mind, the authors have reviewed literature and information provided by manufacturers, searching for suitable CD reagents, CD methods, and isotopically labeled analogs of the analytes related to the following 11 opioids: heroin, 6-acetylmorphine, morphine, hydromorphone, oxymorphone, 6-acetylcodeine, codeine, hydrocodone, dihydrocodeine, oxycodone, and noroxycodone. These analytes and ISs were derivatized with various derivatization groups, followed by GCMS analysis. The resulting MS data are systematically presented in two forms: (a) full-scan mass spectra; and (b) CC data of ion-pairs with potential for designating the analytes and their respective ISs. Many (if not most) of these full-scan mass spectra are not yet available in the literature and should be of reference value to laboratories engaged in the analysis of these drugs/metabolites. Full-scan MS data were further used to select ion-pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion-pairs were evaluated using data collected in selected ion monitoring mode and systematically tabulated, readily available for analysts searching for this important analytical parameter.

Mass spectrometric data characteristics of commonly abused amphetamines with sequential derivatization at two active sites.

There have been reports on improved chromatographic parameters derived from the incorporation of sequential derivatization in preparing biological specimens for the analysis of opiates. This current study was designed to characterize the mass spectrometric data resulting from sequential derivatizations of commonly abused amphetamines (along with all commercially available deuterated analogs) containing two active sites, i.e., amphetamine, methylenedioxyamphetamine, phenylpropanolamine. The first derivatization groups included in this study were trifluoroacetyl, pentafluoropropionyl, and heptafluorobutyryl, while t-butyldimethylsilyl was used as the second derivatization group. Products resulting from the first step and the two-step derivatization processes were analyzed by GC-MS. Full-scan mass spectrometric data were used to select ions with potential for designating the analytes and their respective isotopically labeled analogs in quantitative analysis protocols. Selected ion monitoring data were then collected and assessed to determine the quality of these ions when one or two different derivatization groups were incorporated in the sample preparation processes. A total of 77 full-scan mass spectra and 8 ion intensity cross-contribution tables, representing various forms of derivatization and isotopic analogs of the three amphetamines, are systematically presented for reference. Evaluations of these data concluded that many, but not all, products derived from "double derivatization" (sequential derivatization with two derivatization groups), generate ions of higher quality than those derived from "single derivatization".

Mass Spectrometric Data of Commonly Abused Amphetamines and Their Derivatives - Cross Contribution of Ion Intensity between the Analytes and Their Isotopically Labeled Analogs.

With GC-MS as the preferred method and isotopically labeled analogs of the analytes as the internal standards (ISs) of choice for the analysis of drugs/metabolites in biological specimens, one important aspect associated with chemical derivatization (CD) is that the CD products derived from the analyte and the selected IS must generate ions suitable for designating the analyte and the IS; these ions cannot have significant crosscontribution (CC), i.e., contribution to the intensity of the ion designated for the analyte by the IS, and vice versa. With this in mind, the authors have reviewed literature and commercial information on common CD reagents and methods and conducted a thorough search of isotopically labeled analogs of commonly abused amphetamine-type drugs/metabolites that are commercially available. These ISs and analytes were then derivatized with various derivatization groups. These CD products were then analyzed by GC-MS and the resulting MS data are presented here in two forms: (a) systematic presentation of full-scan spectra; and (b) tabulation of CC data for ions with potential for designating the ISs and analytes. Many (if not most) of these full-scan spectra are not yet available in the literature and should be of daily reference value to forensic and clinical laboratories that are engaged in the analysis of these drugs/metabolites. Full-scan MS data were further used to select ion-pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion-pairs were evaluated using data collected under the SIM mode and summarized in table format. These data should save enormous amounts of time and efforts for practicing laboratories in their search for this analytical parameter.

Accurate assignment of ethanol origin in postmortem urine: liquid chromatographic-mass spectrometric determination of serotonin metabolites.

Toxicological examination of fatal aviation accident victims routinely includes analysis of ethanol levels. However, distinguishing between antemortem ingestion and postmortem microbial formation complicates all positive ethanol results. Development of a single analytical approach to determine concentrations of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA), two well-known metabolites of serotonin, has provided a convenient, rapid and reliable solution to this problem. Antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio for 11-19 h after acute ingestion. The liquid-liquid extracts of postmortem urine samples were subjected to liquid chromatography-mass spectrometry (LC-MS) for the simultaneous quantitation of these two analytes, yielding detection limits of 0.1 ng/ml for each. Examination of the 5-HTOL/5-HIAA ratio was undertaken for 44 urine samples known to be antemortem ethanol-positive or antemortem ethanol-negative. Recent ethanol ingestion was conveniently and accurately separated using a 5-HTOL/5-HIAA ratio of 15 pmol/nmol, a value previously suggested using human volunteers. All 21 ethanol-negative postmortem samples were below this cutoff, while all 23 ethanol-positive postmortem samples were above this cutoff. Thus, we recommend the employment of this cutoff value, established using this straightforward LC-MS procedure, to confirm or deny recent antemortem ethanol ingestion in postmortem urine samples.

A fatality caused by accidental production of hydrogen sulfide.

A 55-year-old male Caucasian truck driver was dead at the scene after breathing hydrogen sulfide (H(2)S) produced by an accidental transfer of sodium hydrogen sulfide (NaHS) from a tanker truck to a tank containing 4% sulfuric acid (H(2)SO(4)) and iron(II) sulfate (FeSO(4)). Autopsy of the decedent's body revealed pulmonary edema and passive congestion in lungs, spleen, kidneys, and adrenal glands. Postmortem biological samples were analyzed for carbon monoxide, cyanide, ethanol, and drugs. Since a potential exposure to H(2)S was involved, blood was also analyzed for sulfide (S(2-)). The analysis entailed isolating S(2-) from blood as H(2)S using 0.5M H(3)PO(4), trapping the gas in 0.1M NaOH, and determining the electromotive force using a sulfide ion specific electrode. Acetaminophen at a concentration of 14.3 microg/ml was found in blood, and metoprolol was detected in the blood, liver, and kidney samples. The blood S(2-) level was determined to be 1.68 microg/ml. It is concluded that the cause of death was H(2)S poisoning associated with a hazardous material accident in an industrial situation.

Blood carbon monoxide and hydrogen cyanide concentrations in the fatalities of fire and non-fire associated civil aviation accidents, 1991-1998.

Blood samples submitted to the Civil Aeromedical Institute (CAMI) from aviation accident fatalities are analyzed for carbon monoxide (CO), as carboxyhemoglobin (COHb), and hydrogen cyanide, as cyanide (CN(-)). These analyses are performed to establish possible exposure of victims to smoke from in-flight/post-crash fires or to CO from faulty exhaust/heating systems. The presence of both gases in blood would suggest that the victim was alive and inhaled smoke. If only COHb is elevated, the accident (or a death) could be the result of CO contamination of the interior. Information pertaining to blood levels of these gases in aviation fatalities, in relation to the associated accidents, is scattered or not available, particularly with regard to toxicity. Therefore, considering that COHb> or =10% and CN(-)> or =0.25 microg/ml are sufficient to produce some degree of toxicological effects, the necessary information was extracted from the CAMI database. Samples from 3857 fatalities of 2837 aviation accidents, occurring during 1991-1998, were received; 1012 accidents, encompassing 1571 (41%) fatalities, were fire associated, whereas 1820 accidents were non-fire related. The remaining five accidents were of unknown fire status. There were fewer fire related fatalities and associated accidents in the (COHb> or =10% and CN(-)> or =0.25 microg/ml) category than that in the (COHb<10% and CN(-)<0.25 microg/ml) category. No in-flight fire was documented in the former category, but in-flight fires were reported in 14 accidents (18 fatalities) in the latter category. No non-fire accident fatality was found wherein levels of both gases were determined to be at or above the stated levels. There were 15 non-fire accidents with 17 fatalities wherein only COHb (10-69%) was elevated. The present study suggests that aviation fire accidents/fatalities were fewer than aviation non-fire accidents/fatalities and confirms that aviation accidents related to in-flight fires and CO-contaminated interiors are rare.

Abnormal glucose levels found in transportation accidents.

BACKGROUND: The Federal Aviation Administration's Office of Aerospace Medicine is responsible for the certification of pilots with diabetic conditions. The present study evaluated the use of postmortem vitreous humor and urine glucose levels in transportation accident fatalities as indicators of potentially incapacitating medical conditions or performance impairment. METHODS: Vitreous humor and/or urine from 192 accident fatalities were analyzed for glucose using a hexokinase method. Cases with values below the lower limit of detection (10 mg x dl(-1)) and above 3 standard deviations (SD) from the mean were not included in the final statistics. All cases more than 5 SD above the mean were deemed abnormal and a full case history was evaluated based on the available medical history. RESULTS: The mean vitreous humor glucose concentration was 30+/-21 mg x dl(-1) (N=98), while it was 27+/-16 mg x dl(-1) in urine (N=127). Of the 192 cases, 9 were identified as having abnormal glucose levels. Abnormal glucose levels were found in 5 of the 8 cases with a known diabetic condition. Glycosuria or low renal threshold was reported in 2 fatal pilots; 1 of these pilots had an abnormal glucose level. CONCLUSIONS: Hyperglycemia can be established from the vitreous humor and urine glucose levels. All of the abnormal glucose cases detected were previously identified during the medical certification process or had a medical reason for the abnormal level. Elevated vitreous humor and urine glucose levels have proven useful in identifying individuals with a pre-existing diabetic condition that might have been a factor in the accident.

Prevalence of drugs and alcohol in fatal civil aviation accidents between 1994 and 1998.

BACKGROUND: The use of drugs and alcohol in aviation is closely monitored by the FAA Office of Aviation Medicine's (OAM's) Civil Aeromedical Institute (CAMI) through the toxicological analysis of specimens from pilots who have died in aviation accidents. METHOD: Frozen specimens received from local pathologists were tested and the results entered into a computer database for future analysis. The data were sorted based on the class of drug, controlled dangerous substance schedules II, and I controlled dangerous substance schedules III-V, prescription drugs, over-the-counter drugs, and alcohol. RESULTS: Specimens from 1683 pilots were analyzed between 1994 to 1998. Controlled dangerous substances, CDS, (schedules I and II) were found in 89 of the pilots analyzed. Controlled dangerous substances (schedules III-V) were found in 49 of the pilots tested. Prescription drugs were found in 240 of the pilots analyzed. Over-the-counter drugs were found in 301 of the pilots analyzed. Alcohol at or above the legal limit of 0.04% was found in 124 pilots. No abused drugs were found in Class 1 air transport fatal pilots. CONCLUSION: This research supports the very low incidence rate of drugs found in the FAA random drug-testing program. Over-the-counter medications are the most frequently found drugs in fatal aviation accidents and many of these drugs could impair a pilot's ability to safely fly an aircraft. This data is helpful to the FAA in developing programs to reduce the usage of dangerous drugs and identify potentially incapacitating medical conditions that may cause an accident. Data collected from this research can be used to evaluate the effectiveness of the FAA drug-testing program.

Formation of an interfering substance, 3,4-dimethyl-5-phenyl-1,3-oxazolidine, during a pseudoephedrine urinalysis.

During fatal aviation accident investigations, biosamples from the victims are submitted to the FAA Civil Aeromedical Institute (CAMI) for drug analysis. In the process of one such analysis by CAMI, an unknown substance was found in a urine sample. Simultaneous screening by thin layer chromatography (TLC) and gas chromatography/FID (GC/FID) suggested the presence of pseudoephedrine. A subsequent routine confirmation analysis of a separate urine aliquot by GC Fourier transform infrared (GC/FTIR) and GC mass spectrometry (GC/MS) indicated that the retention times of the unknown substance matched with those of pseudoephedrine. However, its infrared and mass spectra were different--the -OH and -NH groups were missing, a C-O-C group was present, and the molar mass was 12 atomic mass units (amu) more than that of pseudoephedrine. A subsequent literature search suggested that ephedrine-like amines react with aldehydes to form oxazolidines. Therefore, the 12-amu increase could be accounted for by condensation of pseudoephedrine with formaldehyde. Since this aldehyde is present in various grades of methanol and ethyl acetate, and these solvents were used during the solid-phase extraction, 3,4-dimethyl-5-phenyl-1,3-oxazolidine was synthesized by using (+)-pseudoephedrine HCl and formaldehyde. The analytical findings of the synthesized compound were consistent with those of the unknown interfering substance, confirming that it was the oxazolidine. Aldehyde contaminants in solvents or specimens can transform drugs of interest and may result in misidentification of a compound originally present in specimens. Therefore, chemicals used in analyses should be of the highest available purity, and a multi-analytical approach should be adopted to maintain a high degree of quality assurance.

Prevalence of chlorpheniramine in aviation accident pilot fatalities, 1991-1996.

BACKGROUND: Chlorpheniramine is known to cause drowsiness, and this side effect has a potential to impair performance and could be a factor in accidents. METHODS: Therefore, to establish the prevalence of this drug in pilot fatalities of aviation accidents, a postmortem toxicology database--maintained at the Civil Aeromedical Institute--was examined for the presence of chlorpheniramine in the fatalities, occurred during 1991-1996. RESULTS: There were 47 (2.2%) accidents involving chlorpheniramine. Of these, 16 had only chlorpheniramine at 109 ng.ml-1 (n = 4) in blood and 1412 ng.g-1 (n = 12) in liver. Other drugs were also present in the remaining 31 cases, wherein chlorpheniramine concentrations were 93 ng.ml-1 (n = 18) in blood and 747 ng.g-1 (n = 12) in liver. Ninety-five percent of all quantitated blood values were at or above the therapeutic level (10 ng.ml-1), giving a 100 ng.ml-1 (n = 21) mean blood value. The drug's mean liver concentration from all cases was 1080 ng.g-1 (n = 24). The average blood value was approximately 10 times higher than the therapeutic value. CONCLUSIONS: The presence of other drugs did not appear to significantly alter the blood chlorpheniramine level, but no such correlation could be established with the hepatic value. The approximate 10-fold increase in the liver concentration was consistent with the general trend of drug distribution in the hepatic compartment. However, the contribution of postmortem redistribution of the drug to alter its concentration cannot be entirely ruled out. This study suggests that chlorpheniramine was present in some aviation fatalities at levels higher than therapeutic levels.

Quantitation of alprazolam and alpha-hydroxyalprazolam in human plasma using liquid chromatography electrospray ionization MS-MS.

A sensitive and specific electrospray ionization high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed for the quantitative determination of alprazolam (AL) and alpha-hydroxyalprazolam (OH-AL) in plasma. After the addition of deuterium labeled internal standards of AL and OH-AL, plasma samples were buffered to alkaline pH and extracted with toluene/methylene chloride (7:3). Dried extract residues were reconstituted in HPLC mobile phase and injected onto a reversed-phase C18 HPLC column. The analytes were eluted isocratically at a flow rate of 250 microL/min using a solvent composed of methanol and water (60:40) containing 0.1% formic acid. The analyses were performed using selected reaction monitoring. The assay was sensitive to 0.05 ng/mL for both the parent drug and metabolite and linear to 50 ng/mL. The intra-assay percent coefficients of variation (%CV) for AL at 2, 5, and 20 ng/mL were all < or = 5.6. At these concentrations, and all OH-AL intra-assay %CVs were < or = 8.4. The interassay variabilities for AL were 11.8%CV, 8.7%CV, and 8.7%CV at 2.0, 5.0, and 20.0 ng/mL, respectively. The OH-AL interassay variabilities were 9.6%CV, 9.2%CV, and 7.8%CV at the same concentrations, respectively. The assay accuracy was less than or equal to +/- 6.6% for both analytes at the three concentrations. The method was used to quantitate AL and OH-AL in plasma samples collected from 10 subjects who were administered a 1-mg oral dose of AL. The mean AL concentration peaked at 11.5 ng/mL 1 h after the dose and AL was detectable for 48 h. The mean OH-AL concentration peaked at 0.18 ng/mL 4 h after the dose and was undetectable by 36 h. Hydrolysis of the plasma samples had little effect on the detected AL concentrations but increased OH-AL concentrations substantially. Plasma/blood ratios for AL and OH-AL exceeded 1 in the study samples.

PCR-based identification of postmortem microbial contaminants--a preliminary study.

Investigation of postmortem blood can reveal the presence of significant ethanol levels. However, in some instances it cannot easily be determined if the source of ethanol is from ingestion or from postmortem endogenous fermentation by contaminating microbes. Described here is a robust polymerase chain reaction (PCR)-based method for detecting the presence of common ethanol producing microbial contaminants in human blood. A set of DNA primers were designed for use in PCR to amplify and detect the genomic DNA from humans and three test microorganisms Escherichia coli, Proteus vulgaris, and Candida albicans. A rapid and reproducible protocol was developed for isolating genomic DNA from mixed human blood-microorganism samples that yields a suitable template for PCR. The organism-specific primer pairs can detect the presence of the target microorganisms in human blood at concentrations as low as 10 colony forming units/mL. The PCR products readily can be detected after agarose gel electrophoresis. This method provides an additional means of rapidly identifying microbial contaminants in postmortem blood samples.

Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.

Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender, starting sample volume, or concentration method. Preservation by 0.25% sodium azide was acceptable for sample storage at 4 degrees C during a period of 30 days. For longer storage duration, freezing at -70 degrees C may be more appropriate. Thus, the applicability of the DQA1 + PM typing was clearly demonstrated for individualization of urine samples.

Preparation of carboxyhemoglobin standards and calculation of spectrophotometric quantitation constants.

A method was developed for the preparation of carboxyhemoglobin (COHB) standards, which were stable for more than four months with the prepared control remaining within acceptable limits during this time. A mathematical equation was developed to more accurately determine the constants A and B used in the equation COHB% = 100[(C - B)/(A - B)], where B = 0% COHB peak ratio at 540 nm and 579 nm; A = 100% COHB peak ratio at 540 nm and 579 nm; and C = the peak ratio at 540 nm and 579 nm for the blood being analyzed. The following equations were developed to calculate A and B: B = Pavg - (P) [(Pavg - Navg)/(P - N)]; A = B + (Pavg - Navg)/(P - N), Pavg = average peak ratio 540/579 for the positive standard run on the spectrophotometer; P = average decimal concentration measured on the CO-OXIMETER for the positive standard; Navg = average peak ratio 540/579 for the negative standard; N = average decimal concentration measured on the CO-OXIMETER for the negative standard. The new equations provided results consistent with those obtained from a CO-OXIMETER.

DNA typing as a strategy for resolving issues relevant to forensic toxicology.

To investigate aircraft accidents, multiple postmortem biological samples of victims are submitted to the Civil Aeromedical Institute for toxicological evaluation. However, depending upon the nature of a particular accident, their body components are often scattered, disintegrated, commingled, contaminated, and/or putrefied. These factors impose difficulties with victim identification, tissue matching, and consequently authentic sample analysis and result interpretation. Nevertheless, these limitations can be overpowered by DNA typing. In this regard, three situations are hereby exemplified where DNA analysis was instrumental in resolving a tissue mismatching/commingling issue, pinpointing an accessioning/analytical error, and interpreting an unusual analytical result. Biological samples from these cases were examined for six independently inherited genetic loci using polymerase chain reaction (PCR) suitable for analyzing degraded DNA generally encountered in putrefied/contaminated samples. In the first situation, three of five specimen bags from one accident were labeled with two different names. DNA analysis revealed that one of these bags actually had commingled specimens, originating from two different individuals. Therefore, the sample was excluded from the final toxicological evaluation. In the second situation, an unacceptable blind control result was reported in a cyanide batch analysis. By comparing DNA profiles of the batch samples with those of the known positive and negative blind controls, it was concluded that the error had occurred during the analysis instead of accessioning. Accordingly, preventive measures were taken at the analytical level. The third situation was related to the presence of atropine at toxic concentrations in the blood (318 ng/mL) and lung (727 ng/g) with its absence in the liver, spleen, and brain. DNA analysis of the blood and liver samples exhibited their common identity, ensuring that the submitted samples had indeed originated from one individual. The selective presence of atropine was attributed to its possible administration into the thoracic cavity by the emergency medical personnel at the accident site for resuscitation, but circulatory failure prevented its further distribution. These examples clearly demonstrate the applicability of the PCR-based DNA typing to enhance the effectiveness of forensic toxicology operation.

Role of metabolites in aviation forensic toxicology.

In aviation accident investigations, specimens from fatal aircraft victims are analyzed for drugs. The presence of drugs suggests possible associated medical conditions for which they might have been taken. As drugs are mostly present in therapeutic to subtherapeutic levels in aviation forensic toxicology cases, determination of parent drugs and their metabolites in multispecimens is of significance. Although chemically reactive metabolites are difficult to detect, physiologically active and inactive metabolites can be analyzed. Selective and sensitive techniques are available, but unavailability of metabolite reference standards, endogenous substance interference, and low tissue metabolite levels limit the analyses. However, the majority of primary metabolites can be effectively characterized/quantitated. Demonstrating the presence of drug (e.g., terfenadine, cocaine, THC) metabolites provides a compelling evidence for exposure to the parent drug and facilitates interpretation of results, particularly when the metabolites are active. Such analyses are not as helpful if the metabolites are also available as drugs (e.g., diazepam, temazepam, oxazepam).

Determination of alprazolam and alpha-hydroxyalprazolam in human plasma by gas chromatography/negative-ion chemical ionization mass spectrometry.

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma. After the addition of deuterium-labeled internal standards, plasma samples were buffered to pH 9 with 1 ml of saturated sodium borate buffer, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane and analyzed on a Finnigan-MAT mass spectrometer operated in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was linear from 0.25 to 50 ng ml-1 for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml-1 and 4.6% at 50 ng ml-1 and that for alpha-hydroxyalprazolam was 15.8% at 0.5 ng ml-1 and 4.2% at 50 ng ml-1. The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plasma samples collected after a single 2 mg oral does of alprazolam. A peak concentration of 32.9 ng ml-1 of alprazolam was detected at 1 h following the dose.

The identification and quantitation of triamterene in blood and urine from a fatal aircraft accident victim.

Triamterene, a diuretic drug used in combination with other drugs for the treatment of hypertension, was found in the blood and urine of a fatal aircraft accident victim. The extraction and identification of triamterene is difficult. It exhibits poor extraction efficiency using some standard base-extraction procedures, and the parent drug is unsuitable for analysis using gas chromatography. In this case, a thin-layer chromatography solvent system and high-performance liquid chromatography were used to identify and quantitate triamterene in blood and urine. Triamterene is a strong absorber in the ultraviolet region and has an unusual UV spectrum, which simplifies the identification and quantitation of this substance by high-performance liquid chromatography.