Much of the early work on the stability of benzodiazepines in biological specimens has focused on chlordiazepoxide, which has been shown to be unstable in blood. Levine et al. found that a blood chlordiazepoxide concentration of 5 mg/L rapidly disappeared when the blood was stored at room temperature; by day 8, no chlordiazepoxide was detected. Nor-chlordiazepoxide demonstrated similar instability. When the blood was stored at 4 °C a substantial decrease was also observed, but the drug was still detectable after 2 months.

The presence of sodium fluoride and potassium oxalate did retard the degradation of chlordiazepoxide at room temperature to the extent that about 40% of the amount originally present was still detected after 2 months. Two breakdown products, demoxepam and nordiazepam, were identified and accounted for some, but not all, of the lost drug. The instability of chlordiazepoxide in blood had previously been reported by Garriott et al. In this study, a postmortem specimen containing 26 mg/L of chlordiazepoxide was stored at room temperature, and a 27% decrease was observed within 7 days. In another postmortem case involving blood and serum, the blood concentration decreased significantly but the serum concentration was unchanged over 19 days.

Other benzodiazepines have also shown similar instability. Benzodiazepines with a nitro group, such as nitrazepam, flunitazepam, and clonazepam, also decrease in concentration during storage. Kelly et al. observed no changes in blood or plasma specimens containing nitrazepam when stored at -20 °C or 4 °C over a 1-week period. However, when serum, plasma, heparinized blood, EDTA blood, or fluoride/oxalate blood was stored with exposure to light at room temperature, less than 11% of the nitrazepam originally present remained after a 3-week period. Storage of plasma and EDTA blood in the dark retarded decomposition but did not prevent it.

Addition of the anti-reductants silver nitrate and sodium metabisulfite at 2 mg/mL slowed the decomposition of nitrazepam m plasma stored either in the light or dark. Similar observations have been noted with clonazepam, a benzodiazepine used primarily as an anticonvulsant, and flunitrazepam. The instability was believed to be caused by the rapid reduction of the nitro group to the amino group. However, Kelly et al. failed to detect aminonitrazepam in their stability studies, and postulated that amino-nitrazepam undergoes dimerization to produce azo and azoxy derivatives. Robertson and Drummer performed a comprehensive study of the stability of 3 nitro-benzodiazepines, clonazepam, nitrazepam, and flunitrazepam, and their respective amino metabolizes in postmortem blood. Nitrazepam and clonazepam were stable in sterile, preserved fresh blood at 22 °C and 4 °C over 28 days.

Over the same time period, a 26% reduction in flunitrazepam was observed. In the absence of the preservative, 25 to 50% of the drugs were lost at 22 °C after 10 days. In non-sterile blood, all 3 drugs were converted to the corresponding amino compounds within 8 h at 22 °C. AII 3 drugs were stable at -20 °C up to 2 years and for 10 months at 4 °C. Surprisingly, the amino compounds also demonstrated instability, with greater instability occurring at higher temperatures. At 22 °C, a 10 to 20% decrease was observed in non-preserved blood over 45 h. At 4 °C, a 21% loss occurred after l month and at -20 °C, a 29% loss occurred after 2 months.

Flunitrazepam, nitrazepam, and clonazepam are also unstable in oral fluid. Nitrazepam and clonazepam were unstable at room temperature, being reduced to their respective amino analogs. This conversion was dependent on the composition of the oral fluid. Samyn et al. studied the stability of flunitrazepam and 7-aminoflunitrazepam in oral fluid. In the presence of 2% sodium fluoride, flunitrazepam and 7-aminoflunitrazepam concentrations in oral fluid averaged 75 to 90% of the targeted value after 48 h. Without the preservative only 23% of the flunitrazepam remained after 48 h while 7-aminoflunitrazeparn showed no significant change. Studies of other benzodiazepines indicate greater stability during storage.

Levine et al. found that diazepam was very stable when stored in blood at room temperature or at a refrigerated temperature over a Month period fly). This finding was confirmed by others. Flurazepam and N-1-desalkylflurazepam showed minimal decrease (25%) in blood concentration over a 4- to 5-month period. Al-Hadidi and Oliver studied the stability of temazepam in blood over a 1-year period at -20 °C, 5 °C, and 25 °C. Blood stored at 5 °C and 25 °C showed a decrease in temazepam concentration of less than 25% at 6 months. No change was observed under freezer storage for 6 months. At 1 year, 47 to 82% of temazepam was recovered from the blood, regardless of the temperature. El Mahjoub and Staub reported decreases in midazolam and oxazepam concentrations in blood at 4 °C and at room temperature over a 6-Month period; however, both drugs were still detectable after that time.

Benzodiazepine Summary

The literature suggests that the benzodiazepines that exhibit instability in biological specimens are those where a nitrogen atom is bonded to an oxygen atom. Chlordiazepoxide has an N-oxide structure and clonazepam, flunitrazepam, and nitrazepam have a nitro group; all are highly unstable in blood, especially in non-frozen or unpreserved conditions. AIthough other benzodiazepines demonstrate greater stability, there are some indications that these benzodiazepines have some instability as well.

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