Deposition of 7-aminoflunitrazepam and flunitrazepam in hair after a single dose of Rohypnol.

In recent years, there has been a notable increase in the number of reports on drug-facilitated sexual assault. Benzodiazepines are the most common so-called "date-rape" drugs, with flunitrazepam (Rohypnol) being one of the most frequently mentioned. The aim of this study was to determine whether flunitrazepam and its major metabolite 7-aminoflunitrazepam could be detected in hair collected from ten healthy volunteers after receiving a single 2 mg dose of Rohypnol using solid phase extraction and NCI-GC-MS. Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault in order to reveal drug use. Ten healthy volunteers (eight women and two men, 21 to 49 years old) participated in the study. The following hair samples were collected from each volunteer: one before flunitrazepam administration, and 1, 3, 5, 14, 21, and 28 days after. In five volunteers, 7-aminoflunitrazepam was detected 24 h after flunitrazepam administration and remained in hair throughout the entire 28-day study period (0.6-8.0 pg/mg). In two cases, 7-aminoflunitrazepam appeared in hair 21 days after drug intake (0.5-2.7 pg/mg), and in two subjects 14 days later (0.5-5.4 pg/mg). In one volunteer, 7-aminoflunitrazepam was detected on day 14 and 21 but concentrations were below the quantitation limit. Flunitrazepam was detected in some samples but all concentrations were below the quantitation limit (0.5-2.3 pg/mg).

Cocaine permeability and metabolism in colonic T-84 epithelial cell line.

We studied the uptake, transport and metabolism of cocaine in human intestine using the colonic T-84 monolayers as a model. T-84 cells were grown in DMEM/Ham's F-12 medium containing 6% newborn calf serum at 37 degrees C on 1.0 microm collagen inserts. The cells develop into a polarized monolayer with the apical surface facing the upper chamber and the basolateral surface facing the lower. The monolayers develop a transepithelial resistance of > or = 600 ohms cm2 in 7 days. Varying concentrations of cocaine HCI was added in a serum free medium to the luminal side only, and after 30 min and 60 min samples from the luminal and serosal aspect were removed for analysis. Cocaine and its metabolites were measured by GC/MS. Cocaine transport across T-84 monolayers increased linearly with increasing concentration of cocaine, with no significant difference between 30 min and 60 min of exposure. Transepithelial resistance did not change even at 800 ng of cocaine suggesting no effect on monolayer viability. The metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) but not norcocaine were detected in both luminal and serosal side. The concentrations of BE and EME in the luminal side were significantly higher than in the serosal. Combined recovery of cocaine, BE and EME from luminal and serosal sides were 52 - 80% of total added cocaine. While fresh medium did not metabolize cocaine, media previously exposed to the monolayer (cell-free medium) caused a significant breakdown into BE and EME, suggesting that esterases may be released into the medium. These results indicate transfer of cocaine across this model of intestinal epithelial cell line is by simple diffusion and is concentration dependent. These studies imply that cocaine in swallowed amniotic fluid can be absorbed by the fetal gastrointestinal tract.

Cocaine and its major metabolites in plasma and urine samples from patients in an urban emergency medicine setting.

In this retrospective study, we examined the levels of cocaine and its major metabolites in plasma and urine from 29 randomly selected emergency department patients (19 males and 10 females, aged 19 to 55) whose urine screened positive for benzoylecgonine using fluorescence polarization immunoassay. Levels of cocaine along with benzoylecgonine, ecgonine methyl ester, and norcocaine were quantitated in EDTA plasma and urine from each patient using gas chromatography-mass spectrometry with selected ion monitoring. Admission diagnosis and history were also obtained for each patient. In plasma, the levels were 16-130 ng/mL for cocaine (n = 3), 27-96 ng/mL for ecgonine methyl ester (n = 9), and 18-1390 ng/mL for benzoylecgonine (n = 22). Norcocaine was not detected in any of the plasma samples. In urine, the concentration ranges were 4-40,130 ng/mL for cocaine (n = 23), 36-660,500 ng/mL for ecgonine methyl ester (n = 27), and 9-2520 ng/mL for norcocaine (n = 9). All urine samples were positive for benzoylecgonine (106-3,361,000 ng/mL), and benzoylecgonine was the only metabolite present in two urine samples (at concentrations of 407 and 435 ng/mL). Two patients had plasma and urine samples positive for all analytes (except norcocaine in plasma). The patient with the highest urinary concentrations of cocaine (40,130 ng/mL), ecgonine methyl ester (660,500 ng/mL), benzoylecgonine (3,361,000 ng/mL), and norcocaine (2520 ng/mL) had a small quantity of benzoylecgonine (465 ng/mL) in plasma. No correlation was noted with patient history, admitting diagnosis or symptomatology, or plasma/urine levels of cocaine or any of its metabolites.

Quantitation of clonazepam and its major metabolite 7-aminoclonazepam in hair.

Clonazepam (CLO) is an anticonvulsant benzodiazepine approved by the Food and Drug Administration for use in the treatment of seizures. It produces pharmacological effects (depression, amnesia) similar to other compounds from the same therapeutic class, and in combination with alcohol, its CNS-depressant action can be significantly potentiated. As with some other benzodiazepines, CLO is a drug possibly used in "date-rape" situations. A method using solid-phase extraction followed by a highly sensitive negative chemical ionization gas chromatography-mass spectrometry for the simultaneous quantitation of CLO and its major metabolite 7-aminoclonazepam (7-ACLO) in hair was developed and validated. The method has potential application to alleged drug-facilitated rape cases. To determine the feasibility of detecting 7-ACLO and CLO in hair, specimens were collected from 10 psychiatric patients treated with CLO, divided into 2-cm segments, and analyzed. Standard curves for 7-ACLO (1-1000 pg/mg) and CLO (10-400 pg/mg) had correlation coefficients of 0.998. All precision and accuracy values were within acceptable limits. 7-ACLO was present in measurable quantities (1.37-1267 pg/mg) in 9 out of 10 patient samples. CLO concentrations in hair were much lower (10.7-180 pg/mg). In 4 out of 10 cases, CLO was not detected in hair. Two patients who had never been treated with CLO before received a single 2-mg dose of the drug. Approximately three weeks later, hair samples were collected, and measurable quantities of 7-ACLO (4.8 pg/mg) were detected in the first segment (proximal) of one of those samples, and traces of the drug were present in the other sample. We concluded that the 7-ACLO is being deposited in hair in much higher quantities than the parent drug and remains there for extended periods of time. Our study also indicates that it is possible to detect 7-ACLO after a single dose of CLO as in the typical date-rape scenarios.

Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of Rohypnol.

The hypnotic benzodiazepine flunitrazepam (Rohypnol) has been identified as the drug of choice for the purposes of "drugging" unsuspecting victims and raping them while they are under the influence of this substance. The objective of this paper was to study elimination of flunitrazepam and 7-aminoflunitrazepam in urine collected from ten healthy volunteers who received a single 2 mg oral dose of Rohypnol, to determine how long after drug administration 7-aminoflunitrazepam can be detected. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of flunitrazepam (LOQ 100 pg/mL) and 7-aminoflunitrazepam (LOQ 10 pg/mL) in urine was developed. All samples were screened for benzodiazepines using optimized micro-plate enzyme immunoassay. The highest concentrations of 7-aminoflunitrazepam (70-518 ng/mL) and flunitrazepam (0.7-2.8 ng/mL) in urine were observed 6 h after drug administration in nine subjects and after 24 h in one subject. In six subjects 7-aminoflunitrazepam was detected up to 14 days after flunitrazepam administration, in one subject up to 21 days and in three subjects up to 28 days. In urine samples collected from six volunteers, flunitrazepam was detected three days after Rohypnol intake, in three subjects 24 h, and in one subject 5 days later. Benzodiazepine micro-plate enzyme immunoassay kit allowed the detection of flunitrazepam and metabolities 5 to 21 days after drug administration.

Doxepin toxicity in a child following topical administration.

OBJECTIVE: To describe a case of a child with altered mental status following the topical administration of doxepin. CASE SUMMARY: A five-year-old Hispanic girl was brought to the emergency department because she was difficult to arouse at school. She had recently developed a generalized eczematous rash for which she was prescribed doxepin hydrochloride 5% cream. An entire tube (30 g) of doxepin cream was applied in the 24 hours prior to presentation. The patient was responsive only to noxious stimuli, with no focal neurologic abnormalities. She was decontaminated and observed in a pediatric intensive care unit. By 18 hours after presentation, she had fully recovered and was discharged. CONCLUSIONS: Topical doxepin, available as a 5% cream, is indicated for the treatment of pruritus secondary to eczematous dermatoses in adults. Diminished skin integrity and the application of a massive quantity of doxepin 5% cream to a large body surface area contributed to the toxicity in this child. Since the safety and efficacy of doxepin cream has not been established in children younger than 12 years, it should be used with caution in this population.

Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

Permanent cell cycle arrest in asynchronously proliferating normal human fibroblasts treated with doxorubicin or etoposide but not camptothecin.

Damage to DNA has been implicated in the induction of permanent cell cycle arrest or premature senescence in normal human fibroblasts. We tested the ability of a group of cancer chemotherapeutic agents or related compounds, which can cause DNA double-strand breaks (DSBs) directly or indirectly, to induce a permanent cell cycle arrest in normal proliferating fibroblasts. A brief treatment with etoposide, doxorubicin, cisplatin, or phleomycin D1 induced a block to S phase entry sustained through 15 days. Lower levels of these drugs did not induce appreciable levels of transient cell cycle arrest. Higher concentrations caused cell death that lacked the DNA degradation characteristic of apoptosis. Camptothecin, an agent that causes DNA single-strand breaks, which are converted to DSBs during S phase, was able to induce an efficient, but only transient, cell cycle arrest in these normal cells. The cells did not enter S phase until after removal of the camptothecin. These findings support the idea that permanent cell cycle arrest and cell death are typical reactions of these normal cells to drugs that can cause DSBs. In addition, we report data consistent with the concept that both actinomycin D and doxorubicin are sequestered by cells and slowly released in active form. This is consistent with the observation that both these drugs bind reversibly to intracellular components.

Recommendations for toxicological investigations of drug-facilitated sexual assaults.

The recent increase in reports of drug-facilitated sexual assaults has caused alarm in the general public and prompted forensic toxicologists from across North America to address the toxicological issues surrounding this matter. The authors have developed recommendations and guidelines to inform law enforcement, medical, and scientific personnel of the requirements for performing successful toxicological examinations in cases of drug-facilitated rape.

Determination of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma using solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization.

Phenethyl isothiocyanate is unstable in aqueous media and at low pH, and rapidly degrades to phenethylamine. Concentrations of phenethylamine, a phenethyl isothiocyanate marker, in dog plasma, were determined utilizing solid-phase extraction and gas chromatography-mass spectrometry with chemical ionization using acetone as the reagent gas. Deuterated d5-amphetamine was used as an internal standard. After extraction, phenethylamine and d5-amphetamine were derivatized using MBHFBA. Ions monitored for d5-amphetamine were m/z 337 and 338; and for phenethylamine were m/z 318 and 319. Precision and accuracy were studied using control solutions prepared in naive dog plasma (80 and 300 ng/ml). Intra-day variability was determined using six replicates of each control solution analyzed on a single day. The relative standard deviation for the 80 ng/ml control was 12.9% and for the 300 ng/ml it was 12.1%. Relative accuracy was 10.9% for the low control and -4.1% for the high control. Inter-day variability was determined over a 6-day period. For the 80 and 300 ng/ml control solutions, the relative standard deviations were 15.8 and 9.1%, respectively, and relative accuracy values were 10.1 and -5.2%, respectively. Standard curves were prepared in naive dog plasma and were linear over the range of phenethylamine assayed (10-500 ng/ml). The results of this study indicate that the proposed method is simple, precise, accurate and sensitive enough for analysis of large numbers of plasma samples.

Detection of doxepin and its major metabolite desmethyldoxepin in hair following drug therapy.

Doxepin is a tricyclic antidepressant that is widely prescribed for the treatment of mild depression. In this study, hair samples collected from a patient receiving 25 mg of doxepin daily were analyzed. Doxepin was administered to the patient for 4 months (June 15 to October 15, 1996). Five hair samples were collected: 1 and 3 months after doxepin therapy began and 1, 3, and 5 months after drug therapy ended. Solid-phase extraction was employed to isolate doxepin and its major metabolite desmethyldoxepin from the hair matrix, and gas chromatography-mass spectrometry (GC-MS) was used for quantitation of both drugs. Six-point standard curves (0.25-20 ng/mg) were prepared for both compounds with an internal standard (doxepin-d3). The standard curves for doxepin and desmethyldoxepin were linear over the range reported and had correlation coefficients of 0.984 and 0.985, respectively. The limit of quantitation for both analytes was 0.25 ng/mg of hair. In addition, the replicate analysis of control hair preparations was performed at two levels (2 ng/mg and 15 ng/mg) to determine intra- and interday variability. Doxepin and desmethyldoxepin were not detected in the patient's sample collected 1 month after doxepin therapy began. The samples collected 3 months after doxepin therapy began and 5 months after drug therapy was terminated had detectable amounts of doxepin and desmethyldoxepin. The highest concentrations of doxepin (mean, 0.59 ng/mg) and desmethyldoxepin (mean, 0.40 ng/mg) were found 5 months after doxepin therapy began, which was also 1 month after the patient had stopped using the drug. Five months after doxepin therapy was terminated, the drug and its metabolite were still present in the patient's hair. The concentration of doxepin in hair was always significantly higher than the concentration of desmethyldoxepin.

Determination of drugs of abuse in meconium.

Fetal exposure to drugs has many adverse effects upon the neonate including low birthweight, small head size and an increased risk of miscarriage and death. Correct diagnosis of drug use during pregnancy is essential if the child is to receive specialized treatment and care, which will aid in learning and behavioral development. Diagnosis will also help in the prevention of subsequent drug-exposed children being born to the same mother. Meconium is the first fecal material excreted by the newborn and is an excellent depository for drugs to which the fetus has been exposed. Its analysis is widely accepted in the scientific and medical communities since it has several advantages over urinalysis, including providing a longer historical record of drug exposure and easier collection. Various drugs and their metabolites have been detected in meconium, however, the metabolic profile of drugs in meconium differs from that of neonatal and/or maternal urine. This article addresses the determination of cocaines, amphetamines, opiates, cannabinoids, phencyclidine, nicotine and methadone in meconium using several analytical procedures including immunochemical and chromatographic methods.

Detection of cocaine, norcocaine, and cocaethylene in the meconium of premature neonates.

Our objective was to investigate the methodologic detection of cocaine abuse during pregnancy by determining the viability of meconium analysis for cocaine and its metabolites using chromatographic procedures as an alternative to urine testing using enzyme multiplied immunoassay technique. Our design was as follows: meconium and urine were taken from 106 very low birthweight premature babies. Meconium analysis for cocaine and its metabolites using extraction and chromatographic analysis was compared with the criterion standard immunoassay testing for urine. The work was carried out at The University of Chicago Hospital, Department of Pediatrics and the University of Illinois at Chicago, Department of Pharmacodynamics. Our patients were very low birthweight, premature babies (mean birthweight 1109 g; mean gestational age 29.1 weeks). Gender was evenly divided between male and female. The outcome measures were as follows: two active metabolites, norcocaine and cocaethylene, were detected in the meconium, but not in the urine, of some of the neonates. Determination of cocaine exposure in the newborn influenced assignment of babies in research studies as well as psychosocial evaluation and subsequent treatment of the neonate. Our results were: of the 106 meconium samples analyzed, 21 (19.8%) were positive for cocaine (n = 19, 0.24-0.78 mg/kg), norcocaine (n = 7, 0.10-0.56 mg/kg), cocaethylene (n = 1, 0.12 mg/kg) or combinations thereof. Benzoylecgonine was not detected in any of the samples. Of the urine samples analyzed by immunoassay, only 8 (7.5%) were positive for cocaine metabolites. We conclude that meconium is a better sample than urine for determining cocaine exposure in utero.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine liberation from body packets in an in vitro model.

STUDY OBJECTIVE: To quantitatively assess cocaine liberation from various body packet materials. DESIGN: 100-milligram cocaine packets (plastic bags with various wrapping techniques, paper, and condoms) were placed in a simulated gastric medium. Samples were also tested in an alkalinized gastric medium, with determination of both cocaine and benzoylecogonine concentrations using high-performance liquid chromatography with ultraviolet detection. RESULTS: Cocaine liberation was greatest in acid medium, with increasing liberation from condom packets to cellophane bags (three wrapping techniques used) to paper packets. The same trend was noted in alkaline medium but with a far lower maximum cocaine concentration accompanied by rapid hydrolysis to its inactive metabolite, benzoylecgonine. CONCLUSION: Cocaine liberation of a known quantity of drug is dependent on the wrapping method and material used; thus, a good history from the "body-stuffer" is essential to predict potential cocaine liberation and toxicity. Rapid hydrolysis of cocaine to its inactive metabolite in an alkaline medium implies a role for gastric alkalinization in the acute management of these patients.

Determination of cocaine and benzoylecgonine in human amniotic fluid using high flow solid-phase extraction columns and HPLC.

A new solid-phase extraction procedure for the determination of cocaine and benzoylecgonine in amniotic fluid, using high flow co-polymeric sorbents is reported. The recoveries of cocaine and benzoylecgonine within the range 0.1-1 mg/l were 95.7% and 50.3%, respectively. The use of high-flow sorbents allowed the easy extraction of amniotic fluid regardless of sample viscosity or physical nature. The use of these solid-phase columns provided many advantages over the more commonly used solvent extraction, including an increase in extraction speed and efficiency, reduced operator time, reduced solvent use and disposal volumes and exceptional extract quality. Further, the determination of amniotic fluid obtained from pregnant cocaine users may provide important information about handling of cocaine by the fetus at various gestational ages. The procedure was successfully applied to amniotic fluid from suspected cocaine abusers.

Determination of cocaine and its metabolites in brain tissue using high-flow solid-phase extraction columns and high-performance liquid chromatography.

A new solid-phase extraction procedure for the determination of cocaine and some of its metabolites in brain tissue, using high-flow co-polymeric sorbents is reported as a substantial improvement on our recently reported procedure. The recovery of cocaine, norcocaine and cocaethylene was excellent as was the reproducibility of the extraction. The use of high-flow sorbents allowed the easy extraction of tissue without the need for a time-consuming lipase digestion, regardless of sample viscosity or physical nature. The use of these solid-phase columns provided many advantages over the more commonly used solvent extraction, including an increase in extraction speed and efficiency, reduced operator time, reduced solvent use and disposal volumes and exceptional extract quality. The procedure was successfully applied to rabbit brains spiked with cocaine, benzoylecgonine, norcocaine and cocaethylene.

The effect of some microsomal enzyme inducers on amitriptyline distribution in rats.

The influence of different types of enzyme inducers: phenobarbital, Aroclor 1254 and benzo(a)pyrene, on distribution of amitriptyline and its metabolite nortriptyline in rats was investigated. The level of amitriptyline and nortriptyline in serum, brain, heart and liver was determined by gas chromatography. The maximum concentration values of amitriptyline and nortriptyline as well as areas under concentration-time curves (AUC0-6) in serum and organs were statistically compared using Student t test and AUCCOMP computer program. The results suggest that studied xenobiotics significantly influence the distribution of amitriptyline and nortriptyline in rats. The changes of these drugs concentration in target organs may be very important from the toxicological point of view.