Stability of Nitrobenzodiazepines in Postmortem Blood

Abstract

Studies were undertaken to determine the stability of nitrobenzodiazepines and their 7-amino metabolites in water and blood. At 22°C nitrazepam and clonazepam were stable in sterile fresh blood containing preservative over 28 days, whereas 25% of flunitrazepam was degraded. At 37°C all three drugs were substantially lost over 9 h (29–51%). There was only a small loss observed for the 7-amino metabolites and no substantial amounts of parent drug and 7-amino metabolite were degraded in water under these conditions. In the absence of preservative substantial amounts (25–50%) of parent drugs were lost in fresh blood over 10 days at 22°C. In bacterially-contaminated postmortem blood all three drugs were completely degraded over 8 h at 22°C with almost all drug completely converted to the respective 7-amino metabolite. These metabolites were also partially degraded (10–20%) over 45 h at 22°C. All 3 nitrobenzodiazepines were stable in blood stored for up to 24 months at −20°C, or 4°C over 10 months. Their respective 7-amino metabolites were, however, relatively unstable at −20°C with a significant loss (29%) after 2 months. At 4°C a 21% loss occurred after 1 month. Freeze/thawing was found not to affect the concentration of nitrobenzodiazepine and 7-amino metabolites. These results show that the nitrobenzodiazepines and their metabolites are unstable chemically and metabolically in blood. We advise that blood collected for the purpose of nitrobenzodiazepine determinations should be preserved with sodium fluoride, stored at −20°C and assayed as soon as practicable, preferably within a week of collection.

Changes in Androgenic Steroid Profile Due to Urine Contamination by Microorganisms: A Prospective Study in the Context of Doping Control

“…Metabolic activity of bacteria may lead to misinterpretation of analytical results (1)(2)(3). In the field of sports medicine, androgenic anabolic steroids are included in the list of forbidden substances by the International Olympic Committee.…”

Flunitrazepam Product Detected in the Stomach of an Autopsy Case Buried Under the Ground for Two Years

“…When FNZ was added to blood under sterile conditions, 75% of FNZ remained on the 28th day of incubation. In contrast to FNZ, 90% of 7-amino FNZ remained after incubation in bacterially contaminated post mortem blood for 45 h and 88% of 7-amino FNZ remained after 28 days' incubation under sterile conditions [10]. Samples incubated with Bacteroides fragilis, Clostridium perfringens and mixed culture resulted in nearly complete conversion of FNZ [11].…”

“…The change in the content of FNZ and its metabolite were studied after the FNZ addition to blood [10] or medium [11]. When FNZ was incubated for 8 h in bacterially-contaminated post mortem blood at 22°C [10], only 4% of FNZ remained, and the remaining 96% was converted rapidly to 7-amino FNZ.…”

“…When FNZ was incubated for 8 h in bacterially-contaminated post mortem blood at 22°C [10], only 4% of FNZ remained, and the remaining 96% was converted rapidly to 7-amino FNZ. No other metabolites were detected in the incubation.…”

Development of an Aseptic Urine Collection Kit and its Evaluation in Preventing the Bacterial Decomposition of Nitrazepam

“…It is well established that nitrobenzodiazepienes, such as NZ, are readily converted to the corresponding 7-amino-metabolites by bacteria. [14][15][16] Most of bacteria contain an oxygensensitive enzyme capable of reducing nitroaromatic compounds into the corresponding amines, and such bacteria in the gastrointestinal tract is the most important site of the reduction of nitrobenzene in humans. 17,18) Robertson and Drummer demonstrated that NZ was completely converted to 7- aminonitrazepam within 240 min at 22°C in the postmortem blood specimen from a cadaver 48 hr after death, where various kinds of enteric bacteria were detected.…”