Akademik Veri Yönetim Sistemi
61st annual meeting of the International Association of Forensic Toxicologists (TIAFT 2024), Sankt Gallen, İsviçre, 2 - 06 Eylül 2024, ss.178-179
Background & Aims: The anxiolytic benzodiazepine diazepam (DZP) was first introduced and licensed in the United States in 1963. It is categorized as a benzodiazepine with both long-acting and fast-acting properties. It is frequently recommended to treat a range of diseases including acute recurring seizures, severe muscle spasms, stiffness related to neurological illnesses, anxiety disorders, and alcohol withdrawal. DZP is highly efficient in relieving symptoms associated with acute alcohol withdrawal, such as restlessness, shaking, drunk hallucinations, and severe delirium tremens. The therapeutic drug monitoring (TDM) of DZP is crucial for observing patients’ adherence to treatment. Furthermore, the detection of diazepam and its major metabolite (NDZP) holds significant importance in forensic toxicology due to its potential for misuse. There are several reasons why NDZP was chosen for this study. These; It is a major metabolite, has a longer half-life compared to other metabolites, and TDM studies are mostly carried out based on the ratio of DZP and NDZP. In a chronic dosing study, the steady-state metabolite and parent drug plasma concentrations ratio was found 1.26 (Greenblatt et al., 1981). Similarly, Hiemke (2016) stated that the NDZP/DZP ratio between 0.9 and 1.9 is the normal level. However, Hiemke et al. (2018) reported that the detection of DZP plus NDZP concentration above 3000 ng.mL-1 is the laboratory alert level. Our aim with this method we have developed is to produce a method that gives fast, reliable, and accurate results within the specified limits. The developed method can be utilized for both forensic toxicology and TDM purposes.
Methods: The analysis of DZP and nordiazepam NDZP was conducted using an Agilent HPLC system with a UV detector. A Kinetex F5 column was used for separation. The mobile phase consisted of acetonitrile and phosphate buffer in a 30:70 ratio, set in isocratic mode with a flow rate of 1.0 mL/min. The column thermostat was set to 40°C, the injection volume was adjusted to 5 µL, and the detection wavelength was set at 238 nm. Solutions were filtered and degassed before analysis. A protein precipitation method was used to remove proteins from plasma samples. The validation experiments ensured selectivity, linearity, recovery, accuracy, precision, LOD, and LOQ. Specificity was evaluated with plasma samples from six individuals. Matrix-matched QC samples and calibration samples were prepared at four concentrations including LLOQ and six concentrations respectively. Intra- and inter-day repeatability studies were conducted using QC samples and at the end of the study, accuracy and precision parameters were evaluated. Quantitation was performed accounting for relative peak areas.
Results & Discussion: The linearity of the calibration curves drawn at six different points within the desired plasma drug concentration range is demonstrated by R2 values of 0.9989 and 0.9993, respectively. The LOD and LLOQ values were calculated as 0.066 and 0.2 µg.mL-1 for DZP, and 0.052 and 0.2 µg.mL-1 for NDZP, respectively. Upon examination of the results, it is observed that the accuracy values range from 98.62% to 113.25% for NDZP and from 93.20% to 114.24% for DZP. These values meet the ±15% deviation criterion specified in the ICH and FDA bioanalytical method validation guidelines. The RSD% values obtained in the precision study were calculated as 0.42% to 4.06% for NDZP and 0.97% to 5.39% for DZP. As a result of our analysis of real samples (n=84), NDZP was detected in all samples, while DZP remained below the detection limit in fifteen samples.
Conclusion: As a result of our study, a rapid, accurate, and precise sample preparation and analysis method has
been developed. The applicability of the method was proven by applying the developed and validated method to
real samples.
Acknowledgments
This study was supported by the Scientific and Technical Research Council of Turkey (TUBITAK, grant number
121C441, 221) and awarded to Selin OZKAN-KOTILOGLU.