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

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.

Determination of benzodiazepines in blood and in dried blood spots collected from post‐mortem samples and evaluation of the stability over a three‐month period

“…Regarding the stability of the analytes on DBSs (Figures and ), a slight increase of diazepam after three months (31% higher than the concentration measured at T0) was observed in one case, while the other cases provided a good stability over the whole monitored period. Our results are in good agreement with those ones obtained from two previously published studies on blood samples, fortified with benzodiazepines and monitored up to months/years at different temperatures …”

“…Alprazolam was stable in the two cases analyzed (Figure ). Mata observed a 20% degradation in blood from the initial concentration of alprazolam after two weeks . However, the author suggested that the relatively fast decrease is probably due to freeze–thaw cycles that can influence the stability of the compounds in blood.…”

“…Hence, the whole blood underwent several freeze–thaw cycles. Though this procedure could influence the stability of benzodiazepines, as already reported in the literature, we preferred to adhere to the normal protocol used in our laboratory. Each case was labeled and sealed with a proper code number.…”

“…However, it requires adequate freezing rooms with high maintenance costs. Moreover, consecutive freeze–thaw cycles of the same samples could influence the stability of the substances themselves …”

“…Benzodiazepines’ stability in blood has been evaluated by several studies . In general, all the molecules remained stable whether stored at −20°C or more, and whenever freeze–thaw cycles were limited.…”