Forensic drug testing for opiates. VII. Urinary excretion profile of intranasal (snorted) heroin.

The urinary excretion profile of free and conjugated morphine and 6-acetylmorphine was determined by gas chromatography-mass spectrometry (GC-MS) and immunoassay for six healthy male subjects after intranasal administration of 6 and 12 mg of heroin HCI. Results were compared with heroin administration (6 mg) by the intramuscular route. Heroin metabolites were rapidly excreted with peak concentrations appearing in the first or second specimen collection after drug administration. Concentrations of total morphine and 6-acetylmorphine after intranasal heroin were similar to those after intramuscular administration, but free morphine concentrations after the lower intranasal dose were significantly lower than the same dose given intramuscularly. Detection times for total morphine by GC-MS and immunoassay (300-ng/mL cutoff concentration) were generally 24-36 h, but were reduced to less than 12 h at the higher cutoff concentration of 2000 ng/mL. 6-Acetylmorphine concentrations were highly variable and short-lived; detection times (10 ng/mL) were approximately 2-3 h for most subjects, but some failed to produce positive specimens. Of 14 specimens with 6-acetylmorphine concentrations of 10 ng/mL or more, ten were associated with total morphine concentrations greater than 2000 ng/mL, and four specimens had total morphine concentrations less than 2000 ng/mL. Overall, intranasal administration of heroin produced a similar profile of excretion of heroin metabolites to intramuscular administration.

Methamphetamine and amphetamine derived from the metabolism of selegiline.

Routine methamphetamine testing identified a urine specimen with inconsistent screening and confirmation results. The methamphetamine RIA screening test (Diagnostic Products Corporation) indicated a borderline positive specimen, while the achiral confirmatory GC/MS result showed 4690 ng/mL of methamphetamine and 1895 ng/mL of amphetamine. Analysis of the specimen after derivatization with S(-)-N-trifluoroacetylprolyl chloride showed only the presence of 1-amphetamine and 1-methamphetamine. It was later learned that the individual providing the specimen had been taking Selegiline. Selegiline, (-) propynylmethamphetamine, is a monoamine oxidase inhibitor used for the treatment of Parkinson's disease. It is sold under the trade name Eldepryl. Its major metabolites are 1-methamphetamine, 1-amphetamine and N-desmethylselegiline. Urine specimens from other Selegiline users were obtained and analyzed. A characteristic metabolic pattern was noted, exemplified by a ratio of 1-methamphetamine to 1-amphetamine of about 2.8. This is in contrast to what is observed in the urine of individuals who ingest pure 1-methamphetamine, such as with Vicks Inhaler, where the 1-methamphetamine to 1-amphetamine ratio in the urine is usually greater than 8. Caution is advised when interpreting methamphetamine results without using a chiral identification technique.

Identification of parent benzodiazepines by gas chromotography/mass spectroscopy (GC/MS) from urinary extracts treated with B-glucuronidase.

Urinary glucuronide metabolites of the benzodiazepines were converted back to the parent molecules after treatment with B-glucuronidase. The benzodiazepines were extracted by a one-step liquid/liquid extraction from urine or by a liquid/solid phase extraction. For the limit of detection (LOD), a standard solution of diazepam and oxazepam was serially diluted and analyzed to the point at which a reproducible analytical result was no longer obtained. Using a temperature program and a splitless mode of injection, excellent quantitation was achieved within an 8-min run time. Based upon specimens obtained from patients under a physician's care, we have determined that urinary concentrations of the benzodiazepines > 200 ng/ml are most likely due to abuse rather than to a prescribed ingestion under strict medical surveillance. Therefore, the calibration standard and cutoff concentration for a positive result was set at 200 ng/ml.

Simultaneous analysis of codeine, morphine, and heroin after B-glucuronidase hydrolysis.

Analysis of the opiates, morphine and codeine, often proceeds by way of acid hydrolysis for release of the parent morphine from its glucuronide formed during metabolism. Following use, heroin is rapidly deacetylated to 6-monoacetylmorphine (6-MAM), which can be detected in the urine for a short time following injection of heroin. Only a small amount of 6-MAM may be further metabolized to morphine glucuronide. Thus, in general, the urine specimen has not been hydrolyzed prior to analysis for heroin, using a separate procedure from morphine and codeine. Simultaneous analysis of morphine, codeine, 6-MAM and heroin would be complicated by loss of identity between morphine and heroin when heroin converts to morphine following acid hydrolysis for removal of the glucuronide moiety from morphine glucuronide. Another significant problem in simultaneous analysis is the relative disparity in concentration between morphine/codeine and 6-MAM/heroin (which might be present in the urine specimen). In the proposed method of analysis, free morphine resulting from B-glucuronidase rather than acid hydrolysis of morphine glucuronide is derivatized with propionic anhydride to form dipropionylmorphine. Heroin that does not react with B-glucuronidase remains unhydrolyzed as the diacetylmorphine derivative. Some of the more exacting steps for the acid procedure are eliminated altogether making overall costs for the enzyme procedure comparable to those of the acid hydrolysis method. The enzyme reaction mixture is purified through a solid phase column system. The optimal conditions for concentration of enzyme, temperature of hydrolysis and pH are individually characterized for B-glucuronidase hydrolysis and the ions which identify the propionyl derivatives are characterized for the simultaneous analysis of morphine, codeine, 6-MAM and heroin.

Effect of pre-enlistment testing on the confirmed drug-positive rate for Navy recruits.

In June 1988, the armed forces of the United States began testing prospective candidates for military service for alcohol, marijuana metabolite (THC-COOH), and cocaine metabolite (benzoylecgonine) through the Military Entrance Processing Station (MEPS) program. Comparison of the confirmed positive drug rates for pre-inductees into the Navy during the MEPS program testing period with the confirmed drug-positive rates for 5 years before and 1 year after MEPS testing showed a decrease in the confirmed positive rate for THC-COOH and an apparent initial decrease for cocaine metabolite as a result of MEPS testing. The overall results for cocaine seem to be overshadowed by other demographic factors operating in the population tested.

Creatinine analysis in single collection urine specimens.

Controlled and uncontrolled fluid intake studies were conducted on series of volunteers over the 6 or 12 h of the study periods. Urine specimens were obtained from each subject randomly or at specified times relative to fluid ingestion. Creatinine analysis performed by a modification of the Abbott TDx procedure demonstrates that the values obtained from single collection specimens fall almost in the same range as the values from 24 h pooled collection specimens. The creatinine concentration can be used to indicate possible adulteration of urine specimens by dilution as a means of avoiding detection of use of drugs of abuse. Between 4 and 7 h are required for a decrease in creatinine concentration to about 100 mg/dL from an initial mean of about 170 mg/dL. A minimum of 6 h is needed for any creatinine value to fall to 50 mg/dL or less. Thus, it appears that creatinine output is sensitive to the amount of fluid ingested, but the relationship is neither linear nor immediate. The absence of a significant creatinine concentration in a specimen can be used as an indication of direct or indirect adulteration of the urine specimen by dilution or replacement with water. At NDSL-Great Lakes, a decline of the creatinine concentration to 30 mg/dL is used as a cutoff for differentiating between urine specimens that might have been tampered with to avoid detection of drug use and those specimens that are dilute for other reasons. Values at 10 mg/dL or less are suggestive of replacement by water. The information is provided to local commands for investigation prior to initiation of punitive action by the command.

Factors influencing confirmed drug positives for Navy and Marine Corps recruits.

Confirmed use of marijuana and cocaine in Navy and Marine Corps recruit populations was examined in terms of age, month of enlistment, recruit class size, education level, and Armed Forces Qualifications Test (AFQT) scores. Between 1982 and 1989, confirmed use of the drugs showed a seasonal variation, with the highest levels in the fall and winter. The rate for marijuana and cocaine was inversely related to the size of the recruit class (r = -0.80) and completion of high school (r = -0.82). The recruit class is larger and younger in the summer. AFQT scores were directly related to age. Education level appears to be one major determinant in addressing drug use.

Liquid-liquid extraction systems for THC-COOH and benzoylecgonine.

Because DuPont Instruments has discontinued commercial availability of the PREP automated sample processor, an alternate method for the general extraction of THC-COOH and benzoylecgonine from a urine matrix in the military drug screening laboratories' standard procedure is needed. The liquid-liquid extraction method presented for isolation of THC-COOH uses extraction with isobutanol-hexane followed by back extraction into an aqueous alkaline medium and results in a percent recovery of 104.24 +/- 9.71%. The isolation of benzoylecgonine involves extraction into ethanol-methylene chloride followed by evaporation and derivatization with 1-iodopropane; recovery is 102.43 +/- 3.13%.

Limits of linearity and detection for some drugs of abuse.

The limits of linearity (LOL) and detection (LOD) are important factors in establishing the reliability of an analytical procedure for accurately assaying drug concentrations in urine specimens. Multiple analyses of analyte over an extended range of concentrations provide a measure of the ability of the analytical procedure to correctly identify known quantities of drug in a biofluid matrix. Each of the seven drugs of abuse gives linear analytical responses from concentrations at or near their LOD to concentrations several-fold higher than those generally encountered in the drug screening laboratory. The upper LOL exceeds the Department of Navy (DON) cutoff values by factors of approximately 2 to 160. The LOD varies from 0.4 to 5.0% of the DON cutoff value for each drug. The limit of quantitation (LOQ) is calculated as the LOD + 7 SD. The range for LOL is greater for drugs analyzed with deuterated internal standards compared with those using conventional internal standards. For THC acid, cocaine, PCP, and morphine, LOLs are 8 to 160-fold greater than the defined cutoff concentrations. For the other drugs, the LOL's are only 2 to 4-fold greater than the defined cutoff concentrations.

Comparison of drug abuse in different military populations.

Quantitative analytical data, generated at the Navy Drug Screening Laboratory, Great Lakes, Illinois, expressed as percent confirmed positives for four drugs of abuse (marijuana metabolite, cocaine metabolite, amphetamines, and opiates) are summarized and compared according to their population of origin. The four populations of interest included U.S. Navy and Marine Corps recruits and service school members. Conformed positive urines for marijuana showed a small but significant decline (p less than 0.001) from about 1.2% confirmed positive among U.S. Navy recruits entering service school commands in 1984 to 0.9% among Navy service school members in 1988 and from 2.0% among U.S. Marine Corps recruits entering service schools in 1984 to 0.8% among Marine Corps service school members in 1988. Navy and Marine Corps recruits showed a significantly higher (p less than 0.001) confirmed positive use rate (6.1 and 3.3%, respectively) compared to service school members, perhaps reflecting their recent civilian use pattern. The relatively high confirmed positive cocaine rate among all groups may have reflected an increasing trend in all populations, confirming a similar trend in high school and other civilian populations. Generally, the frequency of confirmed positive urines with amphetamines and opiates, based upon the findings at the Navy Drug Screening Laboratory at Great Lakes, has been static except for an apparent recent increase in amphetamine use in 1988. The decline in confirmed positive drug urinalyses among service school members from both the Navy and Marine Corps indicated that perhaps education and maturity had a positive effect upon their behavior.

Clinical evaluation of a single antibody RIA assay for alpha-fetoprotein.

Open neural tube defects have been associated with elevated levels of alpha-fetoprotein in maternal serum and amniotic fluid. In the present study, specimens were collected during 14 to 20 weeks of gestation in five laboratories. Dates were confirmed by ultrasound. Among 1,049 subjects included in total serum alpha-fetoprotein determinations, 25 infants were born with neural tube defects, one patient had intrauterine fetal demise, and 1,023 delivered normal infants. The false-negative rate for serum alpha-fetoprotein was 0.38%, and the false-positive rate was 0.29%. For the total 1,211 amniotic fluid specimens, 23 infants were born with neural tube defects. The false-negative rate for amniotic fluid alpha-fetoprotein was 0.25%. All were closed lesions. There were no false-positive values. This study reaffirms the value of screening for neural tube defects by means of serum alpha-fetoprotein RIA. It also negates the argument that serum screening would promote excessive amniocentesis.

Radioimmunoassay for detection of antibody to hepatitis A virus. Results of clinical evaluation.

The performance of a solid-phase radioimmunoassay (HAVAB) for the detection of antibody to hepatitis A virus (HAV) was evaluated in clinical studies. The procedure was reproducible by eight investigators, and laboratory-to-laboratory variations were minimal. The sensitivity of the test was about equal to or slightly greater than that of immune adherence hemagglutination (IAHA) for detecting antibody in serum, but IAHA gives somewhat higher titer values than HAVAB. A survey of the incidence of anti-HAV in selected populations revealed an overall frequency of 48% and a correlation with age and lower socioeconomic status. The specificity of the test was demonstrated with specimens from patients who had clinical hepatitis A. Seroconversion to anti-HAV positivity was demonstrated with HAVAB to coincide with onset of illness, about two to three weeks earlier than it was detected by IAHA. HAVAB provided a convenient test for demonstrating the immune status of subjects, and was useful as an aid in diagnosing hepatitis A.

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd