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The Center for Human Toxicology has, together with other toxicology laboratories, been involved in the field evaluation of a radioimmunoassay procedure for the detection and quantitation of delta 9-THC in serum and an enzyme multiplied immunoassay procedure for the detection of the nor-carboxylic acid metabolite in urine. Each laboratory was selected on the basis of proven experience with commercially available immunoassay procedures. Initially, the study was designed to screen samples collected from accident casualties; however, at some of the study sites the data base was extended to include other samples. The radioimmunoassay procedure evaluated was developed at the Receptor Research Institute, and involves the use of a tritium-labeled delta 8-THC tracer. A total of eight laboratories participated in the evaluation of this method, which is prepared in kit form by the manufacturer. Thirteen laboratories evaluated the EMIT procedure developed at Syva Corporation. The study protocol required that each site screen serum and urine samples and forward all presumptive positive specimens to the Center for Human Toxicology. These positive specimens were then shipped to either Battelle Laboratories or Research Triangle Institute for confirmation analysis by gas chromatography-mass spectrometry. The results of these evaluations will be presented and will include: 1. Correlations of positive RIA and EMIT analysis with gas chromatography-mass spectrometry results; 2. The results of quality-control tests; 3. Criticisms and suggestions made by analysts at each site; 4. The results of evaluations performed at the Center for Human Toxicology.

A gas chromatographic-chemical ionization mass spectrometric (GC-CIMS) method is described for the determination of cocaine, benzoylecgonine, and norcocaine. The procedure uses stable isotopes as internal standards and a mixture of methane-ammonia as chemical ionization reagent gas. Run-to-run and within-run coefficients of variation (%) are less than 10% and the method has a sensitivity of less than 5 ng/mL from 1 mL or 1 gram of sample. The procedure has been applied to a number of cases involving cocaine intoxication and analytical data from these are described.

The dominant role of analytical chemistry in applied forensic toxicology is described briefly from an historical perspective. It is argued, however, that obligations imposed on forensic toxicologists by the law, medicine, and society for understanding toxicity in an increasing variety of case problems is really the major challenge and obligation facing toxicologists today. Case examples and rationale are presented in support of this argument. Needs and possible solutions to redress the current professional imbalance are suggested; such as a program to monitor prospectively adverse drug actions and interactions, forensic toxicology reference centers, graduate training programs, and collaborative research involving analytical toxicologists and biomedical researchers, including pathologists. There is a need to develop an industrial, governmental, and professional partnership. These proposals are discussed and justified.

The in vivo regulation of natural killer (NK) cell function by recombinant murine gamma interferon (rMuIFN-gamma) and recombinant human interleukin-2 (rHuIL-2) was investigated. Peritoneal exudate-derived natural killer (PE-NK) cells of mice treated with rMuIFN-gamma or rHuIL-2 exhibited significantly enhanced cytolytic activities against YAC-1 target cells. Compared with animals receiving treatment with either rMuIFN-gamma or rHuIL-2 alone, the sequential treatment of mice with both agents resulted in a significantly greater enhancement of PE-NK cell function, especially when rMuIFN-gamma was administered 24 h before treatment with rHuIL-2. The cytolytic activities were significantly diminished after pretreatment of effector cells with anti-Qa-5 antiserum and complement but not with anti-Lyt-3.2 antiserum. These data constitute the first evidence demonstrating the association between IFN-gamma and IL-2 in the regulation of NK cell function in vivo, reinforce the potential benefit of combined treatment with biological response modifiers, and show that the schedule of administration may be a critical requirement for optimal benefits of combined lymphokine treatment in vivo.

This study describes a population of deceased persons in which death was generally caused by ingestion of numerous drugs, of which diazepam was only one agent. This drug occurred with high frequency relative to the total case load at each site, but its toxicological importance was often of a low order, and its role in the fatal cases was judged as minimal.

A rapid and simple method is described for the separation of quinidine (Q), its metabolites 3-hydroxyquinidine (30HQ) and 2'oxoquinidinone (QD) as well as the quinidine salt contaminant, dihydroquinidine (DHQ) in plasma by high pressure liquid chromatography (HPLC). A silica column was used with methanol: 1 N ammonium nitrate: 2 N ammonium hydroxide (28:1:1 v/v) as the mobile phase to separate the compounds and a fluorescence spectrophotometer for detection. Application of this method is demonstrated by the analysis of human plasma samples collected from a volunteer during a phenytoin-quinidine drug interaction study.

One or more drugs were detected in 81 percent of 440 male drivers, aged 15-34, killed in motor vehicle crashes in California; two or more drugs were detected in 43 percent. Alcohol, the most frequently found drug, was detected in 70 percent of the drivers, marijuana in 37 percent, and cocaine in 11 percent. Each of 24 other drugs was detected in fewer than 5 percent. Except for alcohol, drugs were infrequently found alone; typically, they were found in combination with high blood alcohol concentrations. The causal role of drugs in crashes was assessed by comparing drivers with and without drugs in terms of their responsibility for the crash. Alcohol was associated with increased crash responsibility; the role of other drugs could not be adequately determined.

Analytical techniques are available to reliably detect and, if necessary, quantify drugs of abuse and their metabolites in urine. A variety of different immunoassays are likely to remain at the heart of initial screening methods but with an increasing emphasis on confirmation of positive findings by GC-MS. The quality of work in laboratories (which may make considerable financial gain through increased volume of samples) must be guarded by both internal quality assurance and external proficiency testing, but most of all by ensuring that staff are truly forensic toxicologists with adequate credentials and experience. Laboratories must be credible to the courts and to their clients, which is simply not the case today. For the past 20 years, the toxicologist has played an increasingly important role as an ombudsman of public-health issues; this new dimension of professional responsibility has almost frightening proportions. The alarm that is rightly felt in the profession can only be assuaged and brought into perspective by increasing the knowledge base from which toxicological opinions are rendered, and by recognizing that, however critical, the analytical laboratory report is only a part, and never the whole case. Just a few areas of immediate needs have been mentioned, but they are critical, they cannot be avoided, and they must receive appropriate attention very soon.

Levo-alpha-acetylmethadol (LAAM, 0.2 or 2.0 mg/kg/day) was orally administered to female Sprague-Dawley rats for one month prior to and throughout pregnancy. The rats were killed on the 18th day of gestation along with a group of 18-day pregnant females given a single oral 2.0 mg/kg dose of LAAM 24 hours earlier. Although cumulation of LAAM or its active metabolites was not seen in plasma or brain of pregnant rats given drug chronically, significant cumulation was observed in whole fetus and in fetal brain. In addition, a 2-3 fold elevation in the concentrations, and an even greater elevation of total content, was noted in the newborn pup. These data suggest that opiate intoxication soon after birth may be a factor responsible for the increased morbidity and mortality of rat pups prenatally exposed to LAAM.

A simple, isocratic, reversed-phase, high-pressure liquid chromatographic procedure was developed for the determination of chlordiazepoxide and its major metabolites in plasma and urine. The within-run coefficient of variation was 3.4-8.0%, and the day-to-day variation was 4.0-8.0%. Recoveries of 80-91% with sensitivity limits of 50 ng/ml were obtained for the parent drug and its metabolites. Plasma and urine samples collected after single intravenous and single oral doses were analyzed using this procedure.

A versatile method for the quantitation of emetine and cephaeline in biological samples is described. Two milliliters of samples containing N-propylprocainamide as the internal standard are buffered to pH 9 and extracted with n-butyl chloride. After subsequent back extraction into 0.01 M hydrochloric acid, a portion of the acid layer is analyzed by reversed-phase high performance liquid chromatography with fluorescence detection. Routinely, the minimum level of detection for both drugs is 5 ng/mL and linearity is demonstrated from 5 to 2500 ng/mL.

A gas liquid chromatographic procedure for the determination of methaqualone and metabolite l (2-methyl-3(2-hydroxymethylphenyl)-4-(4H)-quinazoline) in plasma and saliva is described. The method requires 1.0 mL of sample and involves internal standard addition, extraction with butyl chloride, chromatography on a 1% SP-1,000 column packing using nitrogen phosphorous detectors. Within-run and day-to-day coefficient variations were less than 8% and the procedure has a sensitivity limit of 20 ng/mL. The method has been used to monitor both parent drug and metabolite in plasma and samples collected after a single oral dose.

1-alpha-acetylmethadol (LAAM) is a new drug under development for the treatment of heroin dependence. A new analytical method applicable to the accurate biodispositional study of the drug and its metabolities is described and critically discussed in this report. The procedure involves sample preparation and direct organic solvent extraction using eta-butyl chloride, amide derivatization by molecular rearrangement, and gas chromatography-chemical ionization mass spectrometry-selected ion monitoring, with methane as the carrier and ammonia as reagent gases. Deuterated (d3 stable isotopes of LAAM and its metabolites are used as internal standards. The method is free from qualitative interferences and has quantitative sensitivity to 5 ng/ml for 2.0 ml samples with 10-15% accuracy and precision in the range 5-100 ng/ml; and 2-5% at concentrations up to 750 ng/ml. Specimens of plasma, whole blood, urine, bile, brain, liver, and other visceral samples have been successfully analyzed, as well as in vitro preparations such as hepatic microsomes. By appropriate data processing, the method lends itself to routine analysis and high volume work; even manually the method is capable of at least 50 samples per week. A simplified procedure for the analysis of LAAM and its metabolites in urine only is also presented and discuet up and use the methods.

The disposition of 1-alpha-acetylmethadol (LAAM) in plasma and urine was monitored by GC/CIMS following oral administration of 10 doses (0.73-1.5 mg/kg) over 42 days, to twelve human subjects. Plasma concentration-time course profiles fitted a two-compartment, first order kinetic model. Mean plasma t1/2 alpha for LAAM was 2.4 hours; t1/2 beta was 37.5 hours for the first dose and 46.8 hours for the last dose. The mean terminal half-life for nor-LAAM was 38.2 hours for first and 64.6 for last dose; for dinor-LAAM t1/2 beta was 168 hours, last dose. Drug accumulation occurred in some subjects, but within the study range, dosage was not related to maximum plasma levels nor to accumulation. In urine, the sum of LAAM, nor-LAAM, and dinor-LAAM represented 25% of the dose, and unconjugated methadol metabolites, 1.6-1.7%.

Syrup of ipecac contains the nauseant alkaloids emetine and cephaeline. Although thousands of doses are given yearly, no data exist on the absorption of these alkaloids in man. We gave 30 mL of USP Syrup to ten adult patients. Blood and urine samples were obtained at approximately one-half and two hours after administration, and the entire volume of vomitus was saved. The samples were then analyzed for cephaeline and emetine by a high-performance liquid chromatographic (HPLC) assay developed in our laboratory. All patients vomited within 30 minutes, but the amounts of alkaloid regurgitated varied from 22 +/- 14% in six patients to 80 +/- 16% in the remaining four. Only six patients had emetine or cephaeline in their blood by two hours (range, 5 to 73 ng/mL), although ten patients had detectable concentrations of the alkaloids in their urine. Measured over two hours, no patient eliminated more than 0.5% of the dose by the urinary route. In our study ipecac was absorbed by all who received it; the extent of absorption varied widely, and elimination by the renal route was small.

A quantitative "high-pressure" liquid-chromatographic assay for tricyclic antidepressant drugs in plasma or serum is described in which amitriptyline, nortriptyline, imipramine, desipramine, doxepin, and desmethyldoxepin are separated with a 10-micrometer particle size silica column and a methanol/NH4OH/NH4NO3 solvent system. The drugs and two internal standards are extracted with hexane/isoamyl alcohol, the solvent layer is evaporated at 40 degrees C, and the drugs are detected at 254 nm. Drug concentrations are linear with absorbance from 25 to 1000 micrograms/L; within-assay and between-assay CVs are less than or equal to 10%.

Several problems associated with the gas chromatographic determination of DPX and NDPX are discussed and an improved method for their extraction and quantification is described. The method involves two separate extractions, one for DPX with SKF 525A as the internal standard and one for NPDX with dinor-LAAM as the internal standard. The use of two internal standards improved quantitative reproducibility by almost 50%. It was also found that routine DPX and NDPX assays were better determined on packed columns than on capillary columns because the quality of the samples and of the columns was critical. The use of two IS and the double extraction procedure is recommended for general toxicological analyses.

This survey continues and expands earlier studies (Finkle et al., 1976a, b). A total of 27 medical examiner or coroner offices across the USA and Canada were visited. The combined jurisdictional population of these 27 sites is 64.8 million people: 56.5 million people in the USA (27% of the US population) and 8.3 million in Canada (Ontario). Since 1969 through mid-1983, a total of 4412 cases provided information sufficient for inclusion in this study. In each of the cases the presence of propoxyphene, and often its major metabolite, in the blood or tissues of the deceased was confirmed by toxicological analysis at the survey site. The following is a summary of the survey findings: The incidence of propoxyphene-associated deaths reached a peak in 1977, then declined by 22.2% from 1977 to 1978 and by 33.3% from 1978 to 1979, and by a further 10-18% since then. This continuing decline has occurred despite increased interest in propoxyphene misuse and the existence of improved analytical methods for the detection of propoxyphene and its metabolites. The decline is greater than can be accounted for by the decline in propoxyphene prescribing. The most common manner of death was suicide, accounting for about 45% of the cases; it can be safely assumed that the suicides were under-reported. A large majority of the suicides involving propoxyphene were multiple-drug intoxications, including alcohol. Propoxyphene alone was noted in about one-sixth of the suicides. These findings confirm those of the earlier studies, i.e., that a high proportion of the deaths associated with propoxyphene are suicides, and, in most cases, the deceased were victims of multiple-drug toxicity. More than 90% of cases involved persons between the ages of 20 and 40 years. There were very few instances of paediatric, adolescent or older adult deaths associated with propoxyphene. There is no evidence to support the view that the deceased were part of the street drug-abuse population. A history of heroin abuse appeared in less than 5% of the cases. Even fewer people had been known to have abused propoxyphene before their deaths, but 18% of the population had been known to 'self-medicate', using multiple drugs without appropriate medical supervision. The distribution of males to females approximated that of the US population.(ABSTRACT TRUNCATED AT 400 WORDS)

Human gamma interferon given for up to 5 days by subcutaneous infusion or intraperitoneal injection did not significantly alter mouse hepatic microsomal oxidative drug-metabolizing enzyme activities. In contrast, murine gamma interferon and human alpha interferon given for 5 days at the same dose (10(7) units/kg) caused 25 and 50% decreases, respectively, in hepatic microsomal cytochrome P-450 concentrations. The human alpha interferon-induced decline in cytochrome P-450 was accompanied by a significant drop in p-nitroanisole demethylase activity and significant elevations in serum alanine aminotransferase and cytosolic glutathione S-transferase activities. An elevation in glutathione-S-transferase was the only significant change found following human gamma interferon administration. Microsomal UDP-glucuronosyltransferase activity was unaffected by any interferon.

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.

Murine IFN(gamma) and human IFN(alpha)-AD:Bgl were compared over a limited dose range and after single and multiple dosing for their effect on male mouse liver oxidative and conjugative drug metabolizing enzymes. Both IFNs depressed the microsomal cytochrome P-450 concentration but did not alter cytosolic glutathione S-transferase nor microsomal UDP-glucuronosyltransferase activity. Both IFNs showed some slight hepatotoxicity (elevated serum ALT), alpha AD:Bgl more than gamma, especially after multiple dosing. While the IFNs did not produce significant increases in liver weight, they did increase the yield of microsomal protein. The increased endoplasmic reticulum may compensate for the decreased cytochrome P-450 concentration and so account for the lack of observed effect of the IFNs on hexobarbital sleep times in vivo. Overall, the minimal effects of murine gamma-IFN on the mouse liver were no different than those of human alpha AD:Bgl.