Development of an analytical method for the detection of benzodiazepines, under section 5A, in blood samples of UK motorists

Mahmud, Jeshan (2020) Development of an analytical method for the detection of benzodiazepines, under section 5A, in blood samples of UK motorists. (MSc(R) thesis), Kingston University, .


A method was validated for the analysis of six benzodiazepines listed under Section 5A of the Road traffic Act. The benzodiazepines selected were diazepam, oxazepam, temazepam, clonazepam, lorazepam and flunitrazepam in line with ISO 17025 guidelines. The method can be utilised for casework analysis of blood samples in road traffic toxicology for all six benzodiazepines for assessing if a motorist’s blood sample is above the per se limits. In addition, the method was validated in line with the criteria and general guidelines stipulated in document FSR-C-133 and specifications for methods to use for Section 5A of the Road Traffic Act. The method has met all requirements detailed by FSR-C-133 including minimum number of calibrators, QC’s, along with validated parameters of linearity, accuracy, precision, percentage recovery, matrix effect and robustness. The developed method has an accuracy of ±10%, a %CV of ≤10%, linearity of ≥0.99 for all analytes. The LOQ was set at the first calibration point and the LOD was half the LOQ concentration. In all cases the LOD and LOQ displayed suitable ion ratio acceptance (≤10% of target) and accuracy (≤10% of target). The method displayed ion suppression in all analytes. The average ion suppression was -48%, this did not affect the methods accuracy or sensitivity as indicated by studies on LOD and LOQ. Further parameters tested included reproducibility with three separate extractions from three separate analyst on different days, in all three extractions the methods performance was unaffected. The analysis of all six benzodiazepines is possible, at concentrations typically detected in samples and cases found in road traffic incidents. Although a method was developed and validated for six benzodiazepines, there is scope to expand the method and add other less commonly detected benzodiazepines. Moreover, the method was validated to take into account a variety of storage conditions; room temperature, refrigerated and frozen and then analysed to ascertain whether the method was suitable in casework study where samples are not analysed on the same day. The results indicate that the blood samples are stable and all fall within ±20% of their detected concentrations.

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