High resolution, high accuracy non-targeted LC-HR-MS/MS dried urine spot screening for drug of abuse testing

Abstract

Screening for drugs of abuse in urine samples collected during roadside traffic testing is common practice in Switzerland. The use of an onsite rapid test for the detection of drugs and their metabolites is generally limited by a restricted panel of detectable drugs or substance classes (e.g. buprenorphine, benzodiazepines, cocaine, EDDP, morphine or opiates) with limited sensitivity and specificity. To improve the screening for legal and illegal drugs, the already collected urine sample typically undergoes an additional screening routine in a forensic laboratory. By using dried urine spots (DUS), sample handling, shipment, and storage could be substantially simplified, when compared to the sample handling of liquid urine. To prepare a DUS, the driver's urine is pipetted on a prepared paper card. The subsequent drying of the specimen, starting immediately after its collection, can prevent the post-sampling formation or degradation of substances (e.g. caused by bacteria). Furthermore, the DUS can be stored and shipped at ambient temperature and need no cooling or freezing. To evaluate the potential of DUS screening, 17 authentic urine samples containing multiple substances, were pipetted (10 μL) onto Autocollect TFN filter paper cards (Ahlstrom, France). After drying, the DUS cards were extracted (6 mm Ø extraction head) into glass vials with an insert (100 μL, methanol/water, 70/30, v/v), using a fully automated CAMAG DBS-MS 500 HCT autosampler in combination with a PAL RSI 3 robot. Subsequently, the prepared extracts were analyzed on a Sciex 5600 TOF instrument using a non-targeted screening and library searching approach. Thereby, 1 μL of the extract was injected onto a Kinetex C8 column and a ballistic gradient was used (mobile phase A: H2O+ 0.1% HCOOH, mobile phase b: MeCN + 0.1% HCOOH). A run time of 15 minutes was applied. To evaluate the performance of DUS, the screening results were compared to the analysis of the same urine sample in liquid form, using the same method on the same LC-MS instrument (injection of 1 μL of a 1/10 diluted urine). More than 50 different legal and illegal drugs were successfully identified within the investigated 17 urine samples using the DUS screening approach: 6-MAM, alprazolam, amoxicillin, amphetamine + metabolite, bisoprolol, bupropion + metabolite, caffeine + metabolites, carbamazepine + metabolites, citalopram + metabolite, cocaethylene, cocaine + BE + EME, codeine + metabolite, fentanyl, gabapentin, ketamine + metabolites, lacosamide, lamotrigine, levetiracetam, lidocaine, lorazepam + metabolite, losartan, metamizole-metabolites, metformin, methadone + EDDP, methylphendiate, metoclopramide, metoprolol, midazolam + metabolite, morphine + metabolite, nicotine + cotinine, ondansetron, oxazepam, paracetamol + metabolites, phenytoin, pregabalin + metabolite, promazine, propranolol, quetiapine + metabolites, sildenafil + metabolite, THC-COOH, torasemide, tramadol + metabolite, tranexamic acid, trazodone-metabolite, trimipramine + metabolites, venlafaxine-metabolite, zolpidem + metabolite. When compared to the analysis of liquid urine, the following compounds could not be identified: 2× nicotine, 1× promazine, 1× THC-COOH. Overall, 96% of the target substances that have been detected in liquid urine were identified correctly using the DUS approach. The DUS contain 10 μL of urine, therefore the signal intensity compared to liquid urine analysis, where we used 30 μL, is on average 68% lower. In most cases all substances were identified correctly using the DUS screening approach. Because of lower signal intensities for the DUS in one case of nicotine, promazine and THC-COOH were not properly identified. DUS Screening offers a simple, cost-effective and easier sample handling alternative to the traditional use of liquid urine. Based on the obtained results, a pilot study has been initiated (during roadside traffic control by the local police) in order to verify the usability in the forensic workflow.