Evaluation of post-mortem urine dipstick test and setting up of semi-quantification for benzodiazepines, tramadol, morphine and oxycodone on the LC-MS/MS

Abstract

Reliability of Post-mortem urine dIpstiCk test Death investigations are conducted by the police, a Public Health Service (GGD) and a forensic doctor to determine the cause of death. In case of a (suspected) unnatural death, post-mortem blood and urine samples, if available, are sent to the pharmacological laboratory of the Erasmus MC for toxicological research. Additionally, a urine dipstick test is used at the crime scene to give a quick indication of the presence of 10 or 12 drugs in the urine. The research question is: How often do discrepancies occur between the urine dipstick test strips of cocaine, MDMA and THC and the toxicological test on blood performed by LC-MS/MS? In this retrospective study, a database was created in Castor EDC in which the results of all previously performed urine dipstick tests and toxicological tests performed on the LC-MS/MS could be gathered. Due to time limitations, only 311 cases were put in this database, of which 164 cases were included for this study. Overall concordance of the cocaine dipstick results with the toxicological test results on blood by LC-MS/MS was 97%, with 3.0% false negative cocaine dipstick test strips (n=5). For MDMA, the overall concordance was 97.6%, with 1.2% false negative (n=2) and 1.2% false positive (n=2) MDMA dipstick test strips. Finally, the overall concordance for THC was 86.0%, with 0.6% false negative (n=1) and 13.4% false positive (n=22) THC dipstick test strips. There was a discrepancy between the urine dipstick test and the toxicological test on the LC-MS/MS on blood in 3.0% of the cases for cocaine, 2.4% for MDMA and 14% for THC. Possible explanations for false negative results are a low drug concentration in urine, consumption of the drug shortly before death and dilution of urine by decomposition fluids. Possible explanations for false positive dipstick results of MDMA and THC are a lower sensitivity of the LC-MS/MS, post-mortem redistribution and formation of amino acids after a high PMI. In the future, the database will be filled with more cases, which will help recognizing patterns of factors that could be responsible for the false negative and false positive dipsticks, also for the other 9 dipstick test strips.

Setting up of semi-quantification for benzodiazepines, tramadol, morphine and oxycodone on the LC-MS/MS Blood can be sent to the Erasmus MC for a toxicological screening, which can be conducted on two LC-MS/MS systems, called micro 1 and micro 2. Semi-quantification of different analytes has been set up, so that estimations can be made about these concentrations that can be interpreted as either sub-therapeutic, therapeutic, supra-therapeutic or toxic. In this project, semi-quantification for benzodiazepines was set up on the micro 1. Subsequently, semi-quantification for tramadol, morphine and oxycodone were set up on both the micro 1 and micro 2. Plasma was spiked with different concentrations of the benzodiazepines, tramadol, morphine and oxycodone. These samples were then prepared to be measured on the LC-MS/MS. All these concentrations were measured 15 times on the applicable LC-MS/MS system. Responses were calculated for each measurement and relative standard deviations (RSDs) were calculated for each chosen concentration. Calibration curves were made to obtain the equations necessary for semi-quantification. Semi-quantification can be set up for benzodiazepines, tramadol, morphine and oxycodone based on these data, except for oxazepam on the micro 1 and morphine on the micro 2 LC-MS/MS system. In the future, validation must take place periodically for all the different analytes for which semi-quantification has been set up.