Stability of 26 Sedative Hypnotics in Six Toxicological Matrices at Different Storage Conditions

Journal of Analytical Toxicology, 2016;40:663–668 doi: 10.1093/jat/bkw084 Advance Access Publication Date: 22 August 2016 Article Article Stability of 26 Sedative Hypnotics in Six Toxicological Matrices at Different Storage Conditions Dani C. Mata* Toxicology Section, Orange County Crime Laboratory, Santa Ana, CA 92703, USA *Author to whom correspondence should be addressed. Email: Abstract Forensic laboratories are challenged with backlogs that produce turnaround times that vary from days to months. Therefore, drug stability is important for interpretation in both antemortem (blood and urine) and postmortem (blood, brain, liver, stomach contents) cases. In this study, 23 benzodiazepines (2-hydroxyethylflurazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, α-hydroxyalprazolam, α-hydroxytriazolam, alprazolam, bromazepam, chlordiazepoxide, clonazepam, demoxepam, desalkylflurazepam, diazepam, estazolam, flunitrazepam, flurazepam, lorazepam, midazolam, nitrazepam, nordiazepam, oxazepam, phenazepam, temazepam and triazolam) and three sedative hypnotics (zaleplon, zopiclone and zolpidem) were spiked into the six matrices at two different concentrations for each drug. The samples were stored in either a refrigerator (4°C) or freezer (−20°C) and analyzed in triplicate at various time intervals over an 8-month period using an SWGTOX validated method. The concentrations decreased over time regardless of the initial spiked concentration, and the storage conditions had little effect on the decrease of most drugs. Conversion from drug to metabolite was difficult to determine since all 26 drugs were present in each sample. Zopiclone and phenazepam were the least stable drugs; zopiclone was the only drug that completely disappeared in any matrix (both antemortem and postmortem blood). Urine was the most stable matrix with only phenazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 2-hydroxyethylflurazepam, and zopiclone decreasing >20% over the 8 months in either storage condition. Postmortem blood, the least stable matrix, had only two drugs, zolpidem and bromazepam, decreasing <20% in the 8-month time period. Further experiments on stability of these drugs should be undertaken to remove the freeze–thaw cycle effect and more thoroughly examining drug-metabolite conversion. Introduction Sedative hypnotics, including benzodiazepines and z-drugs, are widely used for the treatment of anxiety-related conditions and sleep disorders. These are also some of the most commonly abused prescription drugs (1–4). Annual household surveys suggest that sedative hypnotics are used by 0.9–3.0% of persons over the age of 12 years, making them a common finding in driving under the influence of drugs (DUID), drug facilitated sexual assault (DFSA) and postmortem (PM) toxicology casework (1, 5) Due to backlogs in forensic laboratories, knowledge of timedependent drug concentration decrease in samples is of considerable significance. Frequently, there is a delay of weeks to months between sample collection, drug screening and quantitation. In some cases, confirmation analyses may not be performed until the case goes to court, months or years after sample collection (4). Analyte degradation during this delay is often a result of chemical or physical decomposition due to drug instability. Analyte degradation can occur due to a variety of mechanisms: binding to proteins or the sample container, physical decomposition due to storage conditions such as heat or chemical decomposition, often into metabolites, by reaction with other components of the sample, such as enzymes (4, 6). Additives and preservatives, such as potassium oxalate or sodium © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: 663 664 Mata fluoride, can delay this degradation; but they do not completely stop the decrease (2, 7, 8). Benzodiazepines and their metabolites are relatively stable compounds under short-term storage conditions. Many studies recommend specimen storage under frozen conditions, −20oC or lower, even if the sample is collected in tubes containing preservative or anticoagulant (1, 4, 7–11). Degradation has been observed in saliva and blood for the 7-amino metabolites when stored at −20oC (1, 12). In blood samples, diazepam is susceptible to hydrolysis even when stored with potassium oxalate and sodium fluoride (4, 9, 13–15). Flunitrazepam has been reported to significantly degrade in biological matrix without preservative within 1 day when exposed to sunlight (4, 9, 15, 16). PM specimens, especially blood, appear to be the most unstable matrices over time. Drummer and Robertsons found that the nitro-benzodiazepines, such as clonazepam, nitrazepam and flunitrazepam, convert to their respective 7-amino form in all bloods but more rapidly in PM blood (1, 11, 16). This conversion could be reduced by using sodium fluoride and potassium oxalate; but in many cases, the conversion has already occurred prior to specimen collection (1, 4, 14, 16). Al-Hadidi showed that temazepam was unstable after 6 months regardless of the storage temperature (8). Zaleplon, zolpidem and zopiclone, also known as z-drugs, are the three most common sedative hypnotics used to treat sleep disorders. Unlike zolpidem and zaleplon, zopiclone’s instability has been previously reported (2, 3). When stored at room temperature, zopiclone is stable in plasma for 2 days, but up to 6 months when stored at −20oC (2). At 4–8oC, zopiclone was stable for up to 6 hours, but still at a decrease of 27% of the original concentration (3). Zolpidem and zaleplon have no noted degradation in biological matrices found in literature. Delay in analysis could potentially result in reported values that are significantly lower than those in the subject at the time of sample collection. Additionally, extended time between replicate quantitation of the same sample could result in values that do not agree within parameters required for reporting or quality control. In this experiment, the stability of 23 benzodiazepines (2-hydroxyethylflurazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, α-hydroxyalprazolam, α-hydroxytriazolam, alprazolam, bromazepam, chlordiazepoxide, clonazepam, demoxepam, desalkylflurazepam, diazepam, estazolam, flunitrazepam, flurazepam, lorazepam, midazolam, nitrazepam, nordiazepam, oxazepam, phenazepam, temazepam and triazolam) and 3 z-drugs (zaleplon, zopiclone and zolpidem) was evaluated over 8 months. The drugs were spiked into antemortem (AM) blood, PM blood, urine, liver, brain and stomach contents and stored separately in a refrigerator or freezer between analyses. Experimental Matrices that were negative for all drugs present in this study were pulled from cases that had been adjudicated. AM blood samples were combined into one lot due to the limited supply. See Table I for the sample preparation and description for each matrix. The tissue dilutions chosen are common dilutions used in toxicology for each matri (...truncated)