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This research into zopiclone (ZOP) stability in stored whole blood originated from a forensic case related to a traffic accident. The accused claimed to have been drugged with ZOP. By the time of analysis, eight months after the incident, no detectable levels of zopiclone were found in a sample stored at 5±4°C. The absence of prior data on ZOP’s stability in blood underlined the need for this study, which aimed to evaluate its stability under various storage conditions, differences between authentic and spiked samples, and the effects of freeze-thaw cycles and sample processing.
Key Findings:
Storage Stability:
Blood samples spiked with zopiclone at low (0.2 μg/g) and high (0.5 μg/g) concentrations were monitored over 12 months at -20°C, 4°C, and 20°C.
Results showed concentration degradation over time, with storage temperature playing a significant role.
Authentic vs. Spiked Blood:
Stability differences were assessed using spiked and authentic samples stored at 4°C. Both types displayed similar trends, although matrix-specific factors like donor plasma protein levels (e.g., albumin and α-1-acid glycoprotein) influenced stability.
Freeze-Thaw Cycles:
Samples underwent up to three freeze-thaw cycles. Low (0.02 μg/g) and high (0.2 μg/g) concentrations exhibited some degradation, emphasizing the importance of minimizing such cycles.
Processed Sample Stability:
Extracted zopiclone in butyl-acetate was stable for at least two days at room temperature when reinjected.
Degradation Products:
Decomposition experiments showed a correlation between zopiclone degradation and the formation of degradation products like ACP when stored at 37°C for 24 hours.
Influence of Pre-Analytical Conditions:
A subsequent study highlighted the variability introduced by factors like donor-specific blood properties and pre-analytical storage conditions. To reduce inconsistencies, stability comparisons were made between authentic and spiked blood from the same donors under controlled short-term conditions.
This body of work provides critical insights into the forensic and clinical interpretation of zopiclone levels in blood, particularly after long storage periods or suboptimal handling.
The study involved a controlled experiment using pre-dosed pooled whole blood to create a consistent matrix for spiked samples. Zopiclone (ZOP) was added to aliquots to achieve target concentrations of 0.15 μg/g and 0.08 μg/g, reflecting levels commonly found in authentic samples. Post-dosed blood collected after Imovane® administration was also pooled and aliquoted for comparison.
Measurements began within 8±1 hours after sampling and were repeated daily for five days at 20°C, weekly for 12 weeks at 4°C, and monthly for three months at −20°C. Zopiclone stability was assessed by comparing spiked and authentic samples (reference and stability samples) under identical storage conditions.
For GC-NPD analysis, historical calibration curves were applied, and all samples were tested in triplicate. Additional measurements for plasma albumin and plasma α-1-acid glycoprotein were performed.
Hypotheses Tested:
Stability in Authentic vs. Spiked Samples:
H0: No significant stability differences between authentic and spiked samples.
H1: Significant stability differences exist.
Stability Across Storage Conditions:
H0: Zopiclone concentration remains unchanged under different storage conditions.
H1: Zopiclone concentration changes depending on storage conditions.
Quantitative Analysis of Zopiclone and Its Metabolites in Urine Using LC-MS/MS
Study Objective:
Zopiclone stability and its metabolites in urine samples had not been previously investigated. Factors such as temperature and pH, which influence enzymatic and chemical hydrolysis, were studied to understand their effect on zopiclone and its metabolites. The study aimed to develop and validate an LC-MS/MS method for quantifying zopiclone (ZOP), N-desmethylzopiclone (NDZOP), zopiclone N-oxide (ZOPNO), and 2-amino-5-chloropyridine (ACP) in urine to evaluate their degradation and formation over time under varying conditions.
By addressing stability concerns, this research provides insights into the interpretation of zopiclone and its metabolites in forensic and clinical settings.
LC-MS/MS Validation and Application
Validation of LC-MS/MS Method: The LC-MS/MS method underwent comprehensive validation to ensure its suitability for analyzing zopiclone, its metabolites, and degradation products. Parameters assessed included selectivity, matrix effects (ME), process efficiency (PE), lower limit of quantification (LLOQ), calibration models, precision, accuracy, and stability. This validation adhered to established guidelines for research methodologies.
Degradation and Formation Study: A controlled experiment was conducted using authentic urine samples collected after Imovane® administration. The effects of time, temperature, and pH on the concentrations of zopiclone (ZOP), N-desmethylzopiclone (NDZOP), zopiclone N-oxide (ZOPNO), and 2-amino-5-chloropyridine (ACP) were investigated using LC-MS/MS. Measurements commenced within two hours post-sampling and continued daily for five days at 20°C, weekly for up to 12 weeks at 4°C, and monthly for three months at −20°C. Daily calibration curves were employed for accuracy.
Application in Forensic Cases: The validated method was applied to two forensic cases of suspected drug-facilitated sexual assault:
Case 1: A woman was found disoriented and reported memory loss of three hours after attending a party. A urine sample was collected approximately 11 hours after the suspected assault. The sample was stored at 4°C for two months and then at −20°C for an additional month before analysis.
Case 2: A second case involved a rape allegation, with a urine sample collected within 24 hours of the incident. The sample was stored at 4°C for one week before analysis.
Estimation of Original Zopiclone Concentration via 2-Amino-5-Chloropyridine (ACP) Analysis
Background: The initial study revealed that zopiclone degrades into ACP in equimolar quantities. This degradation provided an opportunity to estimate the original concentration of zopiclone in samples through ACP measurements.
Study Aim and Approach: The objective was to validate an LC-MS/MS method for the simultaneous quantification of ACP, zopiclone, and NDZOP in blood. Degradation experiments evaluated ACP formation and zopiclone degradation under controlled conditions. Mathematical models were developed and tested to estimate the initial zopiclone concentration in authentic samples based on measured ACP levels. This approach facilitates reliable back-calculations of zopiclone levels in forensic investigations.
