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* Inst. of Farmaceutical Chemistry and Toxicology, Univ. of Milan 20132, Italy
** Inst. of Forensic Medicine, Univ. of Macerata, Italy
*** Inst. of Forensic Medicine, Univ. of Milan, Italy
**** Inst. of Forensic Medicine, Univ. of Brescia, Italy
Even though in Italy definite rules of driving under influence of illegal substances, drugs and alcohol, have got to be approved under the traffic code, a fast screening procedure is described that allows the identification of the presence, in blood and urine, of the most common substances that normally influence the driving ability.
This procedure is based on separation from the blood and urine of acid, neutral and basic substances using solid phase extraction, with Bon Elut Certify Cartridges. A single determination with various type of solvents and at different pH is carried out for each sample. The extracts are then analyzed by linear programmed GC/MS, using capillary column.
In Italy the new highway code has enforced the prohibition of driving under the influence of drugs and psicotropic substances.
Nevertheless the regulation that will define the following topics have still to be issued:
In the meanwhile we wish to describe a simple and fast method that allows to detect the most common substances that may affect the driving ability.
The analyses are performed over the urines and preferably over the blood. In fact we think that the latter is particularly relevant as the presence of xenobiotics in the driver's blood is connected to the pharmacodynamic action that generally impair human ability. It should be noted that the law requires the proof that the driver is "in stato di alterazione fisica e psichica correlato all'uso di sostanze stupefacenti" i.e. driver physic and psychic conditions have been alterated by any drug abuse.
The analytic procedure is based on the separation from the urines and blood of acidic, neutral and basic substances by mean of just a single solid phase extraction using a "Bond Elut Certify" (BED) column.
For each sample a single column may be used with different eluents at different pH.
1 ml of urine is diluted to 4 ml with a phosphate buffer at pH 6. 1ml of blood is de-proteinized by mean of sonication (15 minutes), vortex mixing (30 sec) and added with a phosphate buffer at pH 6. 5 min. Centrifugation is following (5 min. 300 r/min) and the precipitate eliminated.
The B.E.C. column is conditioned with 2 ml of Methanol, 2 ml of water and 3 ml of a phosphate buffer solution at pH 6. Conditioning flow rate is 1.5 ml/min. The sample is then loaded onto the column at a flow rate of 1 ml/min. Before elution, the column is rinsed with 1 ml of buffer solution and dried under vacuum for 5 min. Column pH is lowered to the value of 3 by Acetic Acid teatment at a flow rate of 1.0 ml/min. Stationary phase is dried again under vacuum for 5 min and wet with 1.0 ml of hexane.
Elution is performed at a flow rate of 0.5 ml/min with the following conditions:
Fractions are collected in vials and the volume is reduced to 100µl under a nitrogen flow in a thermostatized bath at 40°C.
Analysis of the extracts are carried out on a Hewlett Packard GC-MSD system ( HP 5890 Serie II GC and HP 5970 MSD). Conditions are as follows:
|Splitless 1 min
|SE 54 15m length, 0.25mm internal diameter, 0.1-0.15µm film thickness
|80°C ---> 300°C, 15°C/min
|Helium, 1.14 ml/min constant flow
|Electron Impact Ionization, scan mode acquisition, 40:500 a.m.u.
In order to stand out substances having active hydrogens (e.g. barbiturics and benzoylegonine), all the extracts are methylated by diazomethane ether solution and analyzed again in GC-MSD at the same above conditions.
The analytic procedure has been tested on samples of urine and blood taken from volunteers who have assumed barbiturates, MDE, MDMA, amphetamine and methylamphetamine, cocaine, benzodiazepine, methadone.
Moreover in our opinion this method not only is remarkably rapid, but may also be applied to psicotropic substances that are not drugs, but nonetheless, may impair the driving ability.
The above mentioned method has not been tested for substances such as morphine and THC metabolites. In fact to achieve an acceptable sensibility of this method an hydrolisys is needed when applied to urine analyses. However for the latter substances, immunochemical screening techniques may be used to obtain preliminary indication. The accuracy thus obtained is such that the use of a different technique that requires a more complex instrumentation such as a GC-MSD system, is not justified.
Scheurer J., Moore C.M., Solid Phase Extraction of Drugs from Biological Tissues - A Review. Journal of Analytical Toxicology, 16, 264,1992.
Chen X.H., Franke J.P., Wijsbeek J., de Zeeuw R.A., Isolation of Acidic, Neutral, and Basic Drugs from Whole Blood Using. A Single Mixed-Mode Solid Phase Extraction Column. Journal of Analytical Toxicology, 16, 351,1992.
Chen X.H., Wijsbeek J., Franke J.P. de Zeeuw R.A., A single-column procedure on Bond Elute Certify for systematic toxicological analysis of drugs in plasma and urine. Journal of Forensic Science, 37, 61, 1992.
Chen X.H., Franke J.P., Wijsbeek J., de Zeeuw R.A., Determination of Basic Drugs Extracted from Biological Matrices by means of Solid-Phase Extraction and Wide-Bore Capillary Gas Chromatography with Nitrogen- Phosphorous Detection. Journal of Analytical Toxicology, 18, 150, 1994.