IMPORTANCE OF Q UANTITATIVE ESTIM ATIO N OF OPIATES AND C ANN ABIN OIDES FOR DETERM INATIO N OF DR IVIN G PERFORMANCE

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1 IMPORTANCE OF Q UANTITATIVE ESTIM ATIO N OF OPIATES AND C ANN ABIN OIDES FOR DETERM INATIO N OF DR IVIN G PERFORMANCE M. S taak, H. K äferstein, G. Sticht Institute of Forensic Medicine, Melatengürtel 60-62, W Köln 30, Germany Most of the experimental studies which can be applied to various aspects of traffic medicine center around cannabis. With the aid of a complex series of tests, BARNETT et al. (1982) were able to observe that there was an initial drop in the driving performance parameters under consideration followed by a gradual restoration period of some hours. The studies performed by MASON and McBAY (1985) show the dependence of the plasma concentration of THC and 9-carboxy-THC on the dose administered as well as the resulting subjective effects with good reproducibility of the pharmacokinetic and pharmacodynamic data. On the basis of an investigation performed by MOSKOWITZ (1984) it is possible to demonstrate that the dose administered and the error frequency are dosedependent: Although there are no significant differences at a dose of 50 /xg/kg body weight, there is a corresponding increase in abnormal or false reactions at doses of 100 /*g/kg and 200 /u.g/kg. These observations indicate that, in order to interpret the individual case correctly, it is not only important to know whether a narcotic has been consumed or not; it is also essential to have objective data such as the dose administered and the interval that has passed between consumption and participation in road traffic. Such data can only be provided by quantifying the active substance and, possibly, the metabolites. 1. C annabis 1.1. U rine A urine analysis concentrates exclusively on the tetrahydrocannabinol metabolite ll-nor-0-9-thc-9-carboxylic acid. The analysis can be performed under the following standardized conditions: Following the addiction of caustic soda solution, 15 ml urine are subjected to alkaline hydrolysis in the separation funnel. Then a ph value of 4 is set with acetic acid and extraction is carried out with 50 ml petroleum ether. After evaporation of the solvent and silylation of the extract, gas chromatograph and mass spectrometer (GC-MS) analyses are performed. Evaluation is carried out with external standards. The addition of internal standards is neither necessary nor sensible since 1 l-nor-a-9-thc-9- carboxylic acid concentrations of between and approx. 15 mg or even more can occur per liter urine. 490 Alcohol, Drugs and Traffic Safety - T92 Ed. by Utzelmann / Berghaus / Kroj Verlag TÜV Rheinland GmbH, Köln -1993

2 In our opinion, the following reference values are useful for interpreting the measured values (Table 1): 11-Nor- Ö -9-THC-9-carboxylic acid concentrations in urine and consumption behaviour - concentration below 0.01 mg/1: to 0.1 mg/1: cannabis consumption possible through passive smoking infrequent active consumption to 0.5 mg/1: more frequent active cannabis consumption to 1 mg/1: consumption several times per week - more than 1 mg/1: regular (daily) consumption These reference values - which must naturally take account of differences in the daily excretion pattern and diuretic effects e.g. of alcohol - are based on the experience we have collected over many years, the relationships between measured values and information supplied by cannabis consumers (as far as this is credible). In the analyses we perform for the police, 7% of the persons who have tested positive for cannabis show concentrations of more than 0.5 mg 11- Nor-0-9-THC-9-carboxylic acid per liter urine. This corresponds to the active consumption of cannabis several times per week. 36% consume cannabis infrequently and in 44% of the cases, consumption could be caused by passive smoking. It is impossible to state the time at which cannabis was last consumed in any case. Even if more than 1 mg 1 l-nor-0-9-thc-9-carboxylic acid is excreted per liter urine, which allows daily consumption to be assumed, this time point cannot be confirmed with certainty for the specific case in question but can only be stated as a probability. Although a problable daily consumption of cannabis will certainly cause relevant impairment of a person's fitness to drive, this cannot be proved by these findings alone. On the other hand, concentrations of more than 0.5 mg/1 and certainly of more than 1 mg/1 point to psychic drug dependence with the loss of driving aptitude. 491

3 1.2. Blood When analysing a blood or serum sample, it is necessary to quantify THC and ll-nor-0-9-thc-9-carboxylic acid. Matrix effects make the use of internal standards unavoidable here (Table 2). Determination of cannabinoides in blood or serum Internal standards: add 10 ng Ö -8-THC 30 ng ö - 8-THC-carboxylic acid to 0.5 ml serum (blood) ml H2O Extraction: a.) 5 ml hexane/ethyl acetate (90+ 10): Ö-9/Ö-8-THC b.) acidification with acetic acid to ph 4 5 ml hexane/ethvl acetate (90+ 10): 0-9 / ö - 8-THC-carboxvIic acid ChromatOL'ranhv: silylation with MSTFA capillary column ultra 1 12 m mass spectrometry We have analysed blood samples in this way for more than one year now, obtaining a positive result in 32% of the cases we examined. In addition, it was also possible to identify unchanged THC in 31.8% of the positive cases or in 10.2% of all cases. It was never possible to identify THC when the result for 1l-Nor-0-9-THC-9-carboxylic acid was negative. The median values are for THC and mg/l for the metabolite. In all cases in which tetrahydrocannabinol can be identified, an interval of only a few hours must be assumed between the last consumption and the taking of the blood sample. On account of the acute cannabis effect discussed above, it must be assumed that the person is unfit to drive, as in case 199/92. Case, N.N., CH 199/92 traffic accident Cannabis and traffic safety Medical report: gait unsteady speech clear consciousness clear remarks pupils delated Diagnosis: clearly under the influence of drugs Chemical-toxicoloeical findings in blood: THC U-Nor-ö-9-THC-9-carboxylic acid mg/l mg/l 492

4 Even if tetrahydrocannabinol cannot be detected and only the metabolite is identified, a person's fitness to drive may nevertheless be impaired. REEVE et al. (1983) have experimentally documented this discrepancy between subjective perception and objective discernibility of a cannabis effect. After only 430 minutes there is a decline in the subjective phenomenon in relation to the Ö-9-THC concentration in the serum or blood. In contrast, there is a considerable discrepancy between the objective defunctionalisation symptoms which exist for up to 150 minutes and the low THC levels. In such cases, any abnormalities or defunctionalisation symptoms noted by witnesses or the physician taking the blood sample are of vital importance for an evaluation. If such abnormalities are noted, even if other substances with a psychotropic effect cannot be detected, the identification of 11-Nor- )-9-THC-9-carboxylic acid alone can prove acute unfitness to drive due to the consumption of cannabis. 2. Opiates 2.1. Urine The chromatographic examination of a urine specimen for opiates must analyse both the free bases and the total amounts after hydrolysis. In any case, it must be possible to identify 6-acetylmorphine, morphine, codeine and dihydrocodeine. On account of the concentration of between approx and 500 mg/1 urine, an evaluation with external standards is the only sensible step - as in the case of cannabis. Since 6-acetylmorphine is instable in an alkaline milieu and morphine can only be extract«! effectively at ph 8.5 to 9.5, the following procedure should be used (Table 4): Determination of opiates in urine a.) Free bases: b.) Total bases: Chromatoeraohv: extraction: 5 ml urine 4-3 ml buffer ph 9 50 ml CHCl3/2-propanol (90+10) hydrolysis: 2 ml urine ml H2O + 2 ml HC1 (37%) 60 min. at 120 C extraction: adjustment to ph 9 50 ml CHCl3/2-propanol (90+10) silylation with MSTFA capillary column ultra 1 12 m mass spectrometry The identification of 6-acetylmorphine not only provides proof of the consumption of heroin; it also means that a short time interval can be assumed between the last consumption and urine sampling. In our opinion, a relevant impairment of the fitness to drive can generally be assumed as long as more 6-acetylmorphine is excreted than free morphine. 493

5 2.2. Blood Reference must be made to internal standards for the quantitative determination of opiates in blood or serum. The following procedure has proved satisfactory (Table 5): Extraction procedure for determination of opiates 1.0 ml serum (blood) add 3.0 ml H2O, 300 ng dj-morphine, 150 ng dß-codeine precipitate with 5.0 ml HCIO4 (4%) (TURNER, 1964) a.) Free bases: b.) Total bases: 3.5 ml supernatant extract at ph 9 with 5.0 ml CHCl3/2-propanol 3.5 ml supernatant add 3.9 ml H20 and 2.0 ml HCI (37%) hydrolyse at 120 C for 60 min. extract at ph 9 with 5.0 ml CHC13 /2-propanol In all cases we examined the free bases and the total amounts of opiates in blood or serum. The results are shown in Table 6. Results of opiates analysis in blood median mg/1 positive free total morphine 53.5% from heroin 31.0% from codeine 12.7% from heroin or codeine 9,8% dihydrocodeine 16.9% We identified morphine in 53.5% of all cases. The median concentration of the free base is mg/1. This is much lower than the concentration of whole morphine amounting to 0.25 mg/1. The case presented in Table 7 shows how important chemical and toxicological analyses can be for clarification of the clinical picture and the actual facts of the matter. 494

6 Narcotics and traffic safety Case: N.N., 26 years, CH 1431/92 stopped by police because of abnormal driving performance Medical report: constitution gait finger-finger-test nose-finger-test speech consciousness thought process behaviour mood remarks haggart, gaunt unsteady uncertain unsicher clear clear persevering aggressive irritated pupils are dilated and reacting to light Diagnosis: clearly under the influence of drugs Chemical-toxicological analyses OPIATES urine mg/1 blood mg/1 free bases: acetylmorphine 0.38 morphine 15.2 codeine 1.4 total opiates: morphine 279 codein Here we are dealing with the acute consumption of heroin by a chronic narcotics addict dependent on intravenous drug administration. An objective clinical diagnosis can be made on the basis of the driving performance, medical findings and the results of chemical/toxicological analyses which can also be used to substantiate the consequences: The abnormalities observed by the physician taking the blood sample point to acute unfitness to drive. The dilated pupils could even point to starting withdrawal symptoms. In any case, they are not the result of an acute cannabis or cocaine effect. A concentration of 0.03 mg free morphine per liter blood would also indicate withdrawal symptoms, especially in a person who is addicted to opiates. In addition to dertermining whether the person was fit to drive at the time in question, his driving aptitude also had to be tested. We must be dealing with a chronic opiate addict here since this alone can explain the unusually high whole morphine concentration with the loss of driving aptitude. 495

7 Conclusions Epidemiological, experimental and pharmacological and clinical experience must be supported to the greatest extent possible by a differentiated chemical/toxicological analysis in order to evaluate driving safety and driving aptitude in an individual and realistic manner using the principles of traffic medicine and hence to make an important contribution to traffic safety by preventing the occurrence of accidents. Purely qualitative verifications, which are only suitable for verifying the consumption of heroin or cannabis do not suffice for this purpose. Since matrix effects in urine are low, adequate reproducibility of the results can be attained with standardized methods. If only blood or serum is available, tetrahydrocannabinol and ll-n or-0-9- THC-9-carboxylic acid must be quantified. Internal standards are essential. We recommend the addition of Ö -8-tetrahydrocannabinol and ll-n or-0-8- THC-9-carboxylic acid. If Ö-9-THC can be identified, it can be assumed that the person in question is unfit to drive. But even if THC cannot be identified, acute unfitness to drive can be proved in the individual case as a result of the discrepancy between a low or no longer detectable THC concentration and objective defunctionalisation symptoms. In the case of opiates, we assume an acute effect to be present for at least as long as more 6-acetylmorphine can be detected in the urine than morphine. When analysing blood or serum, reference must be made to internal standards for quantification purposes. Here we recommend the use of deuterium-labeled morphine and deuterium-labeled codeine and, as in the case of urine analyses, the determination of the free bases and the total amounts after hydrolysis. L iterature 1. Barnett G et al (1985) Behavioral pharmacokinetics of marijuana. Psychopharmacol 85: Mason AP, McBay AJ (1954) Cannabis: Pharmacology and interpretation of effects. J Forens Sei 30: Moskowitz H (1984) Marihuana and driving. Accid Anal Prev 17: Reeve VC, Robertson WB, Grant J, Soares JR, Zimmermann EG, Gillespie HK, Hollister LE (1983) Hemolyzed blood and serum levels of Ö9-THC: Effects on the performance of roadside sobriety tests. J Forens Sei 28: Turner LK (1964) A study of barbiturate estimation. In Decomposing samples of post-mortem whole blood. J Forens Med 11: