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Letters |
Natl. Inst. of Forensic Toxicol., P. O. Box 9934 Ila, N-0132 Oslo, Norway
a Author for correspondence.
To the Editor:
Benzodiazepines are commonly abused drugs. Accordingly, urine specimens are often screened for benzodiazepines as part of drug-testing programs. However, immunoassays have different detection limits regarding the different benzodiazepines and their metabolites. Some benzodiazepines are therapeutically effective in low doses, also making detection difficult. Flunitrazepam belongs to this potent group of low-dosage benzodiazepines. The urine metabolites of flunitrazepam are reported to react weakly with most immunoassays (1). Therefore, more time-consuming chromatographic analyses are often carried out to detect flunitrazepam intake. In this letter we report the applicability of the Emit® immunoassay (Behring Diagnostics, San Jose, CA) as a preliminary screening test for flunitrazepam. The described approach has proven to be both labor- and time-saving.
Flunitrazepam is prescribed primarily as a hypnotic, but is also used in some countries as premedication in general anesthesia. Flunitrazepam is not an approved drug in the US. Recently, focus has been placed on flunitrazepam (designated as a Schedule I substance), or "Roofies," as a popular drug of abuse (2)(3). In our country, as in many other European countries, the misuse of flunitrazepam has been known for many years. The drug is taken orally in tablet form, or the tablets may be dissolved in water for drinking or intravenous injection, often in combination with heroin. According to police reports, in our country the street price for flunitrazepam is ~30 times the pharmacy price, thus reflecting its popularity among misusers.
The prevalence of flunitrazepam misuse necessitates the analysis of the drug in urine specimens. Our laboratory performs forensic and drug-abuse analyses in biological samples. Urine drug screening is mainly performed by Emit immunoassay, i.e. the monoclonal Emit II with a 200 µg/L cutoff for benzodiazepines such as diazepam and oxazepam. The CV was 15% for a calibrator at the cutoff value and 13% for a control sample at ~270 µg/L. The analyses are run on a Monarch 2000 (Instrumentation Laboratory, Lexington, MA) at 30 °C in the semiquantitative mode. Flunitrazepam is excreted as metabolites, primarily as 7-aminoflunitrazepam. The interaction of flunitrazepam metabolites with Emit immunoassay is reported as low (1) or not stated at all (Emit II Benzodiazepine Assay, instructions, 1994). Screening urine specimens with respect to flunitrazepam has thus been done in our laboratory by means of a highly sensitive gas-chromatographic (GC) method, while confirmation is performed with another analytical method (either HPLC or a different GC method). The cutoff was 60 µg/L (0.20 µmol/L) for 7-aminoflunitrazepam in the chromatographic analyses, with a CV of 11% at this concentration.
Annually we receive ~4000 urine specimens in which flunitrazepam
analysis is requested. Emit screening was performed for other drugs of
abuse, and in addition all these specimens were screened by GC. Only a
few percent were positive. This raised the question of a simpler
screening procedure, and we investigated the applicability of Emit
benzodiazepine assay in this regard. A retrospective analysis showed
that there had been a total of ~300 positive 7-aminoflunitrazepam
specimens during the last 3 years. Approximately one-half of these
samples contained another benzodiazepine drug as well and was thus
unsuitable for evaluating the Emit results with respect to
flunitrazepam metabolites. The specimens that were included in the
study (122) apparently contained 7-aminoflunitrazepam alone. In
addition, as these were authentic specimens, other flunitrazepam
metabolites were present but were not analyzed for routinely. The Emit
results of these samples are shown in Table 1
. Most samples (>80%) containing 7-aminoflunitrazepam had Emit
results above cutoff, and all samples had Emit results different from
zero.
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We now use Emit benzodiazepine immunoassay as a prescreening procedure with respect to flunitrazepam. A cutoff value at 60 µg/L has been chosen. If the Emit result of a specimen is below this value, we report a negative result, and the sample is not subjected to further analyses. With a higher Emit result, a GC screening is performed, and if positive is followed by a confirmation procedure. Also, an authentic urine control specimen containing ~60 µg/L 7-aminoflunitrazepam has been introduced. This (positive) control is analyzed together with all specimens, and the Emit result has to be higher than the negative calibrator. Otherwise, the prescreening condition is not fulfilled. Our data have shown that the risk of overlooking positive samples with an Emit cutoff at 60 µg/L is negligible. The use of Emit prescreening reduces the number of negative GC flunitrazepam analyses by as much as 80%. Some chromatographic screening analyses still must be done because of the simultaneous presence of other benzodiazepine drugs and metabolites with Emit responses. The percentage of positive flunitrazepam samples in the GC screening analyses has increased from 1.2% in 1993 to 20% with the described approach. This increment cannot be explained solely by the reduced number of analyses due to prescreening, but also probably reflects increased misuse of these drugs, as well as more adequate and aimed practice regarding the request for drug analyses.
References
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