In toxicology, analysis of dried blood spots is increasingly used especially for therapeutic monitoring and pharmacokinetic studies. It offers many advantages such as low sample volume and a simplified conservation. It could also represent a new analytical approach in forensic toxicology. Blood ranks among the most usual kinds of physical evidence encountered on a crime scene. Over the two past decades, forensic toxicology followed a continuous progress, currently giving the possibility to detect various drugs in very small blood samples. This purportedly offers the opportunity to assay bloodstains for toxicological analysis, which could be of interest in some situations, e.g. determination of the victim's toxicological status even if no corpse is found at the crime scene, or of the perpetrator's if he/she bled in the surroundings of the crime scene. In this context, we investigated the stability of some xenobiotics (bromazepam, diazepam, zolpidem, methadon, tramadol and cyamemazin) in bloodstains. To reproduce realistic conditions, blood drops were deposited on different supports (glass, tile, wallpaper and cloth) over periods ranging from 24 hours to one year at room temperature (daylight and darkness).

Drug-free blood samples were spiked with an amount of 100ng/ml of each analyte. After storage, bloodstains were collected after 24H, 72H, 1 week, 1 month, 3 months, 6 months and 1 year by swabbing (glass, tile and wallpaper) and cutting (wallpaper and cloth). Samples were rehydrated with 1mL ammonium buffer (pH 9.5), sonicated 30 minutes then stored in darkness for 60 minutes. Liquid/liquid extraction was performed using methylenechloride, nheptane, isopropanol (65:25:10, v/v) with diazepam-d5 as internal standard. Then toxicological analyses were carried out by UPLC-MS/MS. Validation of the method was performed on drops of blood spiked with the same analytes and deposited directly on swabs, wallpaper and cloth.

Under these analytical conditions, the method appeared sensitive (LOQ: 2ng/mL), linear (LOQ – 5000ng/mL) and accurate (CV < 20%). Extraction recoveries were always higher than 55% and no significant matrix effects were observed. Results showed a good stability of all drugs tested even after one year of storage. Each analyte could be detected by swabbing as well as by cutting. However, more than 30% losses were observed when cutting.

This study exemplifies the stability of some xenobiotics in dried bloodstains, with no major influence from environmental conditions nor delay of exposure. This study opens the way to a new analytical approach: toxicological analysis of bloodstain samples on a crime scene may give interesting information on the influence of the drugs on a victim or perpetrator at the time the crime was committed.