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Recent advances in analytical techniques have enabled the detection of drugs and drug metabolites in alternative biological specimens for the purposes of workplace testing. A wide variety of specimens are available, each providing valuable information concerning prior or current drug use. The present focus is on oral fluid (saliva), hair, and sweat. An extensive evaluation by the Division of Workplace Programs of the Department of Health and Human Services is underway to determine the utility of these specimens in federally regulated programs. In future years, the testing of alternative specimens will expand our ability to understand the patterns of drug use and will become routine in all areas of forensic toxicology.

One challenge facing the laboratory forensic toxicologist today is verifying the validity of the random urine specimen submitted for workplace drugs of abuse analysis. Determining whether urine substitution has occurred is best accomplished through the inspection of the specimens appearance and the performance of specific laboratory tests, such as determining the concentration of biochemical metabolic waste products and measuring indices of urine concentration. Criteria for classifying submitted urine as substituted are postulated after an extensive review of the published scientific literature. Relevant studies that were evaluated include normal random urine reference interval studies, clinical studies involving the analysis of random urine specimens, theoretical dilutional limits, medical conditions resulting in overhydration, and water-loading studies. After compilation of the study data, derived substituted criteria of urinary creatinine ≤ 5.0 mg/dL and urinary specific gravity ≤ 1.001 are suggested. A urine specimen meeting these criteria may be considered substituted because it is not consistent with the clinical characteristics associated with normal human urine.

A gas chromatographic procedure is presented for routine detection and quantitation of methadone and its metabolites in biological fluids and tissues. The method employs an extraction of drugs with n-butyl chloride at approximately neutral pH and chromatographic identification on 3% OV-17 using SKF-525-A and n-docosane as Internal standards. A modified extraction apparatus and the recovery of methadone, 1,5-dimethyl-3,3-diphenyl-2-ethylidene-pyrrolidine (Mbl), and SKF-525-A from a variety of fluids and tissues are described. The method is sensitive to 20 ng/ml or g and is applicable to other basic drugs.

Propoxyphene (P) and its major metabolite, norpropoxyphene (N), have been determined in an increasing number of acute and drug abuse related medical examiner cases in Maryland during 1974–1976. Acute fatalities were typified by a female in her 30s with a P/N ratio in blood of 4.1, while drug abuse related fatalities were exemplified by a male in his 20s with a P/N ratio in blood of 1.9. Propoxyphene related fatalities were generally accompanied by the simultaneous detection of ethanol or other drugs. Blood propoxyphene concentrations exceeding 0.1 mg/dl appear significant in elucidating the cause of death in the absence of other evidence, while concentrations exceeding 0.2 mg/dl may reasonably be considered an independent causative factor in a fatality. The P/N ratio in brain was consistently the highest value when compared to blood, liver and kidney. Propoxyphene and norpropoxyphene blood and tissue concentrations and P/N ratios are useful in the diagnosis of propoxyphene related deaths.

A simple method for the determination of chlordecone in serum and blood employing selective separation and extraction using benzene is presented. The extracted pesticide is analyzed quantitatively by gas chromatography using an electron capture detector and a 10% DC-200 on 100/120 mesh Gas-Chrom Q column. The method is sensitive to a concentration of 10 µg/liter In a 1-mL sample and is highly reproducible showing a within-run coefficient of variation (CV) of 4.1% and an out-of-run CV of 6.9%.

Three methods (IL-182 Co-Oximeter, 11.-282 Co-Oximeter, and headspace gas chromatography) for the analysis of carbon monoxide in postmortem blood were studied and compared using a prepared reference standard, Quantra® control materials, and 62 postmortem blood specimens. The methods compared favorably with one another. The linear regression equations for the 62 postmortem blood samples (range = 1.0 to 86% saturation) were: IL-282 vs. IL-182, y = 1.11x − 3.10, r = 0.981; IL-182 vs. GC, y = 0.88x + 2.97, r = 0.973; IL-282 vs. GC, y = 1.00x − 1.24, r = 0.986.

Breath specimens (N = 143) from human subjects Initially analyzed for ethanol content by Infrared spectroscopy were collected onto silica gel. The specimens were retained 1.25 to 2.75 years following adsorption and subsequently analyzed by headspace gas chromatography. The results revealed good overall correlation between the direct and delayed ethanol determinations (r = 0.900). Specimens were collected by police officers following a defined protocol and the results are presented to Illustrate practical use and limitations of such a technique.

A case Involving an accidental overdose fatality resulting from an intravenous Injection of crushed Ritalin® (methylphenidate) tablets is presented. The drug was quantified by gas chromatography/nitrogen-phosphorus detection and confirmed by gas chromatography/mass spectrometry. Concentrations of drug were as follows: blood, 2.8 mg/L; bile, 5.7 mg/L; kidney, 3.0 mg/kg; liver, 2.1 mg/kg; and stomach contents, 1.6 mg total. Other drug and volatile substances were not detected.

Phencyclidine (PCP) was determined in blood, urine, and other biological specimens by gas-liquid chromatography using an OV-17 column after extraction with a n-butyl chloride:diethyl ether mixture. During the two-year-period 1976–1977, PCP was detected in 37 cases in the State of Maryland Medical Examiners population. In two of these cases, death was directly attributable to acute intoxication from the drug (blood PCP concentrations—1.5 mg/L and 25 mg/L). In the other 35 cases in which PCP was not the primary cause of death, blood concentrations showed a median value of 0.1 mg/L (range: 0.02 to 0.7 mg/L). Phencyclidine is known to be capable of eliciting psychological and physiologic changes in an individual. Such alterations in mood and physical ability appear to be an important contributing factor in the deaths of PCP-intoxicated individuals.

A fatal Intoxication Involving multiple substances Including carbamazepine (CBZ), desipramine (DI), and ethylene glycol (EG), In which the blood CBZ concentration was higher then any previously reported in the literature Is presented. Each compound, as well as the 10,11-epoxide metabolite of carbamazepine (CBZ-EP), was quantified In blood (CBZ, 79 mg/L; CBZ-EP, 11 mg/L; DI, 4.2 mg/L; EG, 3.6 g/L), bile (CBZ, 69 mg/L; CBZ-EP, 13 mg/L; DI, 23 mg/L; EG, 4.8 g/L), urine (CBZ, 6.5 mg/L; CBZ-EP, 5.7 mg/L; DI, 30 mg/L; EG, 3.3 g/L), stomach contents (CBZ, 280 mg; DI, 49 mg; EG, 2.8 g), liver (CBZ, 75 mg/kg; CBZ-EP, 11 mg/kg; DI, 49 mg/kg; EG, 2.0 g/kg), and kidney (CBZ, 41 mg/kg; CBZ-EP, 11 mg/kg; DI, 23 mg/kg; EG, 1.9 g/kg) using the following analytical techniques: high performance liquid chromotography with ultraviolet detection for CBZ and CBZ-EP, gas-liquid chromatography with mass selective detection for DI and gas-liquid chromatography with flame Ionization detection for EG.

11-Nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) was isolated using solid phase extraction and identified with high performance liquid chromatography (RPLC) with electrochemical detection (EC). HPLC/EC was compared to an established HPLC procedure using ultraviolet (UV) detection. Statistical analysis revealed good precision, linearity, and correlation between the HPLC/EC and HPLC/UV methods. Within-run and between-run precision for the HPLC/EC vs. HPLC/UV analyses yielded coefficients of variation of 4.1 vs. 6.7% and 8.3 vs. 10.9%, respectively. Standard curves over a range of 20 to 160 ng/mL of THC-COOH in fortified urine on four separate analytical runs yielded correlation coefficients of greater than 0.99 for each method. Quantitative results from 15 positive urine specimens also compared favorably between the two methods (r = 0.9923). Using an immunoassay screening technique with confirmation by HPLC/UV and high efficiency thin layer chromatography (HETLC), further evaluation of the HPLC/EC method was achieved by examining 50 cannabinoid positive and 50 cannabinoid negative clinical urine specimens and 500 sequentially collected postmortem urine specimens.

A method for the determination of Δ-9-tetrahydrocannabinol (THC) in whole blood that employs hexane extraction followed by identification using gas chromatography with nitrogen/phosphorus detection has been developed. The assay is sensitive to 2 ng THC per mL blood and linear to concentrations up to 120 ng/mL (r = 0.998), Precision is demonstrated by coefficients of variation ranging from 3.6 to 9.1% (within run) and 7.1 to 8.0% (between run).

A method for the simultaneous analysis of cocaine (COC), derivatized benzoylecgonine (BE), and derivatized ecgonine methyl ester (EME) has been developed. Blood or urine containing deuterated analogs of the three analytes is extracted by pouring over a Chem-Elut column end eluting with chloroform/Isopropanol (9:1). The eluent is collected, split into two equal allquots, and evaporated to dryness. In one aliquot, dimethylformamide and dimethylformamide dipropyl acetal ere allowed to react and to derivatize BE to n-propyl COC. In the other aliquot, 4-fluorobenzoyl chloride, pyridine, and benzene are used to derivatize EME to p-fluoro-COC. COC is not affected by these reactions and may be found in either aliquot. Separation was performed on an HP 5890 GC using an HP-1 capillary column. The oven temperature was initially 100°C for 1 minute, then programmed st 30°/minute to a final temperature of 260°C. The GC/MS was operated in the SIM mode, and the masses monitored were 303, 321, and 331 respectively for COC and derivatized EME and BE, and 308, 324, end 334 for the respective deuterated internal standards. The assay showed a minimum linear range of 0.05–2.0 mg/L for COC and 0.10–4.0 mg/L for each of the metabolites.

The TDx fluorescence polarization immunoassays (FPIA) for some therapeutically monitored drugs (lidocaine, phenytoin, phenobarbital) were applied to the analysis of postmortem blood specimens. After an initial calibration using the appropriate TDx calibrators, blood bank blood fortified with known amounts of drug was analyzed by FPIA. Postmortem blood specimens containing the drugs, postmortem specimens not containing the drugs of interest (negatives), and randomly selected postmortem blood specimens fortified with known amounts of the drugs were analyzed by the FPIA and reference gas chromatographic (GC) methods. Concentrations determined in blood bank and postmortem blood specimens showed excellent correlation between the two methods and produced lines with slopes ranging from 0.98 to 1.08. In each case the correlation coefficient was greater than 0.97. Analysis of all negative specimens produced FPIA values below the limit of quantitation of the reference GC assay.

This study evaluated the capability of the Abbott ADx assays to test for phencyclidine (PCP) and barbiturates in urine specimens by comparison to TDx, EMIT, and gas chromatographic/mass spectrometric (GC/MS) methods. Within-run and between-run precision were determined using three control samples (35, 100, and 250 ng/mL PCP; 0.4, 0.6, and 1.0 mg/L secobarbital) tested over a two-week period. Controls were analyzed in both single assay (batch) and multiple assay (combination) modes. Within-run coefficients of variation (CVs) for the three PCP controls were 2.4, 2.6, and 2.2%, respectively; the between-run CVs were 4.0, 3.5, and 2.8%, respectively. The within-run CVs for the three barbiturate controls were 3.0, 2.7, and 2.7%, respectively; the between-run CVs were 5.6, 4.7, and 4.5%, respectively. Fifty specimens not containing PCP or barbiturates, 50 specimens containing PCP, and 50 specimens containing barbiturates were tested by ADx, TDx, EMIT, and GC/MS. The methods compared favorably and no false positive or false negative results were obtained by any immunoassay method for any assay when compared to GC/MS.

The stability of cocaine (COC) in blood bank blood, postmortem human whole blood, and buffers was evaluated with consideration for the presence of the degradation products, benzoylecgonine (BE) and ecgonine methyl ester (EME). At COC concentrations commonly seen, the rate of COC hydrolysis was independent of concentration. COC was stable in blood for at least 150 days if the blood was adjusted to pH 5 and preserved with 2% NaF or organophosphates and maintained at 4°C or lower. Without preservation, most COC hydrolyzed to EME. The addition of a pseudocholinesterase (PChE) inhibitor without a reduction of pH caused COC to hydrolyze to BE. COC also hydrolyzed to BE in phosphate buffer. The rate of COC hydrolysis in all studies increased with increasing pH and temperature. COC was more stable in unpreserved postmortem blood than blood bank blood due to the lower pH of the former. The incubation of COC in enzyme solutions provided further evidence of the generally accepted hypothesis that COC is hydrolyzed to EME by PChE and to BE by chemical hydrolysis. In unpreserved blood, BE was more stable than EME at room temperature. There was little loss of BE or EME st refrigerated temperature over a period of 35 days and no evidence that EME or BE could be hydrolyzed enzymatically.

Many studies have been published comparing blood (B) ethanol (EtOH) and vitreous humor (VH) EtOH concentrations. We conducted a similar study of routinely collected specimens to determine (1) whether the reported average VH/B ratios are consistent, (2) the percentage of cases with VH/B ratios outside of the expected range, and (3) the magnitude of B EtOH that can be associated with negative VH EtOH (<0.01 g/dL). VH EtOH <0.01 g/dL was associated with 41 specimens; 34 specimens had B EtOH <0.03 g/dL while a B EtOH as high as 0.12 g/dL was measured. For B EtOH <0.10 g/dL, 67% had VH EtOH within 0.02 g/dL of B EtOH (N = 101). For B EtOH ≥0.10 g/dL, the VHtB EtOH ratio ranged from 0.10 to 1.91; the mean ratio was 1.17 and the median ratio was 1.18 (N = 205); and 64% had a ratio between 1.00 and 1.39. Vitreous humor can be used to facilitate understanding the significance of postmortem blood EtOH concentrations.

The Coat-A-Count solid phase 125I Fentanyl Radioimmunoassay was evaluated with respect to linearity and precision using equine urine fortified with fentanyl and then compared with a gas chromatographic/mass spectrometric method for quantification of fentanyl in urine. The RIA assay was found to be linear over the urine fentanyl concentration range of 0.25 to 7.5 ng/mL and precise with coefficients of variation (CV) ranging from 9.6 to 19.3%. The RIA calibrators, ranging in fentanyl concentrations from 0.25 to 7.5 ng/mL, and controls, at mean fentanyl concentrations of 0.46 and 1.32 ng/mL, were compared by both the RIA and GC/MS methods. The cross-reactivity with the 125I RIA test was determined for the fentanyl metabolites, norfentanyl and hydroxyfentanyl, and found to be 5% and 35%, respectively. The illicit fentanyl analogs were found to show significant cross-reactivity, ranging from 20 to 100%. The 125I RIA was compared to GC/MS quantifications of fentanyl in 35 positive and 20 negative case urine specimens.

A fatal case attributed to flecainide intoxication is presented. Quantitation was by capillary gas chromatography with nitrogen-phosphorus detection. The flecainide concentration in the blood was 13 mg/L as compared to a therapeutic range in serum of 0.2–1.0 mg/L. Flecainide concentrations in other specimens were as follows: bile, 160 mg/L; urine, 54 mg/L; vitreous humor, 7.4 mg/L; liver, 180 mg/kg; kidney, 74 mg/kg; and stomach contents, 120 mg.

Two cases are presented in which haloperidol was identified in postmortem toxicological analysis. One case was a suicidal overdose of the drug; the blood concentrations of haloperidol and reduced haloperidol were 1.9 and 1.4 mg/L, respectively. Bile, liver, and urine concentrations were 3.4 mg/L, 44 mg/Kg and 6.6 mg/L for haloperidol and 1.6 mg/L, 43 mg/Kg, and 5.7 mg/L for reduced haloperidol, respectively. The second case was believed to be a natural cardiac death with a blood haloperidol concentration of 0.6 mg/L. The distribution of haloperidol and reduced haloperidol in this case was bile, 0.4 and 0.5 mg/L; kidney, 0.7 and 2.3 mg/Kg; liver, 5.0 and 13 mg/Kg; and urine, 0.4 and 2.3 mg/L.

Hair samples from 20 documented heroin users contained 6-acetylmorphine, a unique metabolite of heroin, in all samples. Heroin was identified in smaller amounts in seven of these samples. The identity of 6-acetylmorphine and heroin was established by comparison of full scan spectra of extracts to standard reference materials. The presence of 6-acetylmorphine generally predominated over heroin, morphine, and codeine. The mean concentrations of analytes were as follows: 6-acetylmorphine, 0.90 ng/mg, N = 20; heroin, 0.17 ng/mg, N = 7; morphine, 0.26 ng/mg, N = 20; codeine, 0.18 ng/mg, N = 15. Analysis of hair samples obtained from 10 drug-free control subjects were negative for 6-acetylmorphine, morphine, and codeine. However, a small interfering peak was observed at the retention time for heroin. Control samples soaked in aqueous solutions of heroin and 6-acetylmorphine were found to be contaminated, even though an initial wash step was included in the analysis. These data suggest that hair analysis for 6-acetylmorphine can be used to differentiate heroin users from other types of opiate exposure (e.g., poppy seed, licit morphine, and codeine); however, environmental contamination can potentially produce false positives during opiate testing.

The dramatic rise in maternal drug abuse and the incidence of positive drug findings during neonatal testing has increased the need for prenatal toxicological testing for drugs of abuse. Human amniotic fluid samples collected after 13–39 weeks of pregnancy were screened for cocaine metabolite (benzoylecgonlne) by fluorescence polarization immunoassay (FPIA). All positive samples, as well as any accompanying maternal serum, were confirmed by gas chromatography/mass spectrometry (GC/MS) for cocaine and its metabolites. Five samples out of 450 were positive for cocaine, benzoylecgonine, and ecgonlne methyl ester by GC/MS. In addition, one sample was also positive for cocaethylene. Two maternal serum samples were positive for benzoylecgonine end ecgonine methyl ester. The presence of cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene in the amniotic fluid suggests that the fetus is exposed to cocaine and its metabolites through maternal circulation. The impact of this exposure on the health of the newborn is unknown.

Plasma was obtained from 10 human subjects at various intervals after administration of two rapid doses of cocaine, either intravenously or by smoking, and multiple doses by smoking and intravenously. The plasma was analyzed for COC and its metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME). Plasma concentrations of COC were found to be dose-dependent. For patients receiving two successive doses of COC intravenously (IV) or by smoking (SM), the average half-life of COC was found to be between 38 and 39 minutes, regardless of the dose or route of administration. Considerable interindividual variation was observed. Multiple doses of both SM and IV COC were administered to three patients in a manner consistent with COC abuse. The maximum COC concentration observed was 1.2 mg/L following a total administration of 316 mg of COC over 90 min. Analysis of BE and EME confirmed that BE is the principle metabolite of COC in blood. All COC was accounted for by BE. EME, when present, did not exceed 5% of the BE concentration.

A study of the metabolism of in vivo cocaine (COC) and the stability of in vitro COC suggests that the presence of benzoylecgonine (BE) in unpreserved blood arises from in vivo COC metabolism and that ecgonine methyl ester (EME) in unpreserved blood arises from in vitro COC hydrolysis. Postmortem cases positive for COC were studied to determine if molar concentrations of EME in unpreserved blood could be used to estimate the blood COC concentration at the time of death when added to the molar COC concentrations. COC was analyzed in 10 postmortem blood specimens between 1 and 8 days following death and again 10 to 70 days after further storage. The COC lost was accounted for by its hydrolysis to EME. Good correlation (r = 0.9677, p < 0.001) was observed when the blood COC concentrations in postmortem cases were compared to blood COC concentrations predicted by the addition of blood COC and EME concentrations; hence, analysis for EME and estimation of perimortem COC concentrations can assist in defining deaths associated with COC use.

The disposition of methadone, its metabolites, and other illicit drugs of abuse was investigated in head hair samples collected from heroin users (N = 20) enrolled in an outpatient detoxification study. Hair samples were assayed for methadone, methadone primary metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, EDDP), methadone secondary metabolite (2-ethyl-5-methyl-3,3-diphenylpyrroline, EMDP), cocaine, phencyclidine, heroin, and 6-acetylmorphine. Hair samples were cut, washed, and incubated in methanol. The methanolic hair wash and incubation fractions were purified with solid-phase extraction and assayed by gas chromatography-mass spectrometry. Methadone, methadone metabolites, cocaine, and phencyclidine were assayed quantitatively, and other drugs were measured qualitatively. The number of positive results and the corresponding concentration ranges were as follows: methadone, 0–15.0 ng/mg (N = 18); EDDP, trace (N = 13); EMDP, trace (N = 1); cocaine, 0–>40 ng/mg (N = 14); phencyclidine, 0–1.5 ng/mg (N = 2); heroin, positive (N = 3); and 6-acetylmorphine, positive (N = 4). These data suggest that testing hair for methadone, methadone metabolites, and other illicit drugs of abuse may be useful to drug-treatment specialists as a means of verifying drug-use history, monitoring compliance, and providing a broad measure of drug exposure.

A colchicine blood concentration of 250 µg/L was determined in a 39-year-old white female 2 hours following the ingestion of an oral overdose of the drug. At death, 40 hours following ingestion, no drug was detected. Colchicine was extracted from buffered blood using dichloromethane. The evaporated extract was redissolved in 0.1 N HCl and analyzed using a high performance liquid Chromatograph equipped with an octadecylsilane reversed phase column.

Amoxapine, a recently introduced dibenzoxazepine, has been found effective in clinical studies for the treatment of various types of depression. Two amoxapine related deaths, a 53-year-old white male and a 21-year-old white female, have been investigated by this office. Both had been prescribed amoxapine for depression. Quantitation of amoxapine was by gas chromatography using a 3% OV-17 column with confirmation by ultraviolet spectrophotometry and thin layer chromatography. Blood amoxapine concentrations were found to be 18 mg/L in the first subject, and 6.7 mg/L in the second subject. These concentrations are many-fold higher than the therapeutic serum concentrations of up to 0.21 mg/L reported in a clinical study. These cases illustrate the potential lethality of amoxapine overdosage and the need for caution in prescribing amoxapine to patients with suididal tendencies.

A sensitive and reliable procedure is presented for the measurement of the common tricyclic antidepressant (TCA) drugs in a variety of biological specimens at therapeutic as well as toxic concentrations. The method employs GC/NPD and a column packed with 3% OV-17 on Chromosorb W-HP. The pH of specimen extraction was optimized at 8.6, and 1-chlorobutane:ethyl ether (3:1) was found to be an efficient solvent for the primary extraction. Recoveries for all TCA drugs were at least 50% for each specimen. Linearity of extraction was observed over a range of therapeutic and toxic concentrations with a correlation coefficient greater than 0.99.

Blood, urine, heart, liver, and psoas muscle concentrations of tricyclic antidepressant (TCA) drugs and their N-desmethyl metabolites were determined in forty medical examiner cases. The cases were evaluated for the role played by the TCA drugs in the cause of death and compared to a literature survey of previously reported cases. The incidence of the various individual TCA drugs as a factor and the significance of the disposition of the drugs and their metabolites is included. Comparing TCA drug concentrations in the heart and skeletal muscle and considering the differences in perfusion to these organs, TCA drugs and metabolites showed an affinity for the cardiac over the skeletal muscle. The case results were correlated with experimental studies to suggest the relationships of blood concentrations with toxicity.

The Enzyme Multiplied Immunoassay Technique (EMIT) Urine Cannebinoid Assay (Syva) was adapted for automated analysis on a centrifugal analyzer allowing for cost-effective, rapid, high-volume urine testing. Confirmation of positive EMIT results was accomplished by a modification of a recently published high performance liquid chromatographic (HPLC) technique for 11-nor-Δ-9-tetrahydrocannebinol-9-carboxylic acid (THC-COOH). Further confirmation of the presence of THC-COOH was achieved by high efficiency thin layer chromatography (HETLC). The reported protocol was applied to urine samples obtained from an emergency toxicology population and a drug counseling population. Results indicated that specimens testing positive by all three methods suggest valid forensic evidence for the presence of THC-COOH.