by Pauline Griffeuille and Dr Sylvain Dulaurent
People driving under the influence of drugs do not only risk their own health but also endanger other road users. The results of the roadside tests performed by the police have to be double-checked in a laboratory, which is currently time consuming and costly. Here, a new testing method is described which provides results in a time- and cost-saving way using oral fluid samples.
Background and objectives
Most countries have defined legislation for driving under the influence of drugs (DUID) based on analytical limits (‘cut-off’ approach) or on impairment (‘effect of the drug’) . Usually, for a suspected driver, roadside testing is performed with an immunological kit using oral fluid (OF) or urine. If positive, this test has to be confirmed in a second sample by a gold standard method, usually by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
In some countries, including France, this confirmation is now typically performed using an OF sample. In France, the regulatory approach does not apply a law per se (i.e. the driver is sentenced if the drug is above a predefined limit) but a zero tolerance law (i.e. driving with a measurable amount constitutes an offense). Accordingly, the labs have to detect a list of illicit drugs at a minimum concentration. The cut-off value (or limit of detection) is 10 ng/mL for cocaine, benzoylecgonine (BZE), morphine, 6-monoacetyl-morphine (6-MAM), amphetamine, methamphetamine, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxyam- phetamine (MDA) and 3,4-methylenedioxy-N-methylamphetamine (MDMA) .
When coupled with a MS system, the probe electrospray ionization (PESI) method developed by Hiraoka makes a direct analysis of liquid samples and solid samples possible without the need for any pre-treatment . Briefly, a disposable solid needle is used as a sample probe and an electrospray ionization (ESI) is used as an emitter. This probe needle repeatedly moves down (i.e. ‘dives’ into the sample) and moves up (i.e. position when ionization occurs).
Hence, the compounds are periodically ionized and their resultant ions are pushed into the tandem MS system. Different applications of the PESI technique have been reported, for instance, in clinical toxicology, forensic science, food science and metabolic profiling [4–6]. Here we discuss how PESI-MS/MS can be applied for the determination of a selection of illicit drugs in OF in accordance with the French legislation.
Principles of the PESI-MS/MS method
In this method, 50 μL of OF samples were diluted in 450 μL of a 10 mM methanol/ethanol/ammonium formate (25/25/50) buffer. From these 500 μL, 180 μL were mixed with 20 μL of a solution containing eight deuterated internal standards at 50|ng/mL. Finally, a 10-μL sample of diluted OF was placed on the dedicated plastic sample plate and set in the PESI ion-source.
A scheduled multiple-reactionmonitoring approach with two transitions per compounds, acquired in the positive mode, was developed to determine concentrations of cocaine, BZE, morphine, 6-MAM, methamphetamine, MDEA, MDA and MDMA using a Shimadzu LCMS-8060 triple quadrupole mass spectrometer with PESI ion-source (Fig. 1).
The total run-time is 1.31 min for the detection of the eight compounds. The whole procedure was validated according to the European Medical Agency guidelines . Additionally, the laboratory of Pharmacology- Toxicology of the Limoges University Hospital has been accredited according to the International Standards Organization (ISO) 15189 standard (accreditation number: 8-2607). Briefly, a summary of the performances is given in Table 1.
Application to forensic samples
The method was applied to 52 real cases of DUID. These samples were also analysed by the dedicated LC-MS/MS method that the lab routinely employs (i.e. a method using a quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation and a Shimadzu LCMS-8050 triple quadrupole mass spectrometer) . In these OF samples, 78 molecules were detected: amphetamine (n=5), MDMA (n=17), MDA (n=1), cocaine (n=26), BZE (n=22), 6-MAM (n= 4) and morphine (n=3). When a compound was detected with the validated reference method, it was systematically identified with the new PESI-MS/MS method. There was excellent agreement between the two methods, as demonstrated by the regression analysis for cocaine and MDMA (Fig. 2)
Discussion and conclusions
For the determination of the presence and concentration of illicit drugs, laboratory analysis usually involves targeted analysis employing LC-MS/MS after liquid–liquid extraction, protein precipitation, off-line solid-phase extraction, online solid-phase extraction (2D chromatography) or ‘QuEChERS extraction’ (which consists of adding salts to a matrix that is previously mixed in a polar solvent). In 2016, our team reported a LC-MS/MS method for the determination of drugs of abuse, based on a QuEChERS sample preparation . The preparation of a classic batch that includes six calibration standards, two internal quality controls and 10 patient samples required approximately 45|min for an experienced operator. In 2018, we reported another method with a fully automated sample preparation using a CLAM-2000 (Shimadzu) . Coupled with a LCMS-8060 (Shimadzu), this robot was able to prepare a sample every 8|min and to give a result every 9|min. Here, we have described a PESI-MS/MS approach where no sample preparation is needed (except the dilution of the sample to allow ionization) and a result is given in 1.5|min.
These preliminary studies demonstrate the feasibility of using a PESI-MS/MS for the measurement of illicit drugs in saliva. Further analytical improvements are needed to integrate other compounds in the developed method.
Pauline Griffeuille* and Dr Sylvain Dulaurent PhD
Unit of Clinical and Forensic Toxicology, Department of Pharmacology,
Toxicology and Pharmacovigilance, Limoges University Hospital, France
*Corresponding author E-mail Griffeuille.email@example.com