Quantitative Analysis of Ethanol and Other Drugs
Written by KRISTI MAYO   

HANDHELD INSTRUMENTS for the determination of ethanol in breath have been used by the police to control sobriety since the early 1970s (Poon et al. 1987). Likewise, instruments suitable for evidential purposes have gone through six generations since the Breathalyzer was invented in 1954 (Wigmore & Langille 2009). The analytical principles for quantitative analysis of ethanol in breath either involve electrochemical oxidation with a fuel-cell sensor or infrared spectrometry; some devices incorporate both technologies (EC and IR) to enhance selectivity for identifying ethanol (Jones 2000). The accuracy and precision of modern breath-alcohol instruments matches that of GC analysis of ethanol in blood.

This article appeared in the July-August 2020 issue of Evidence Technology Magazine.
You can view that issue here.

This article is an excerpt from: Alcohol, Drugs, and Impaired Driving: Forensic Science and Law Enforcement Issues

The gold standard method of blood-alcohol analysis involves the use of headspace gas chromatography with flame ionization detector (HS-GC-FID) or, more recently, a mass detector (HS-GC-MS) (Tiscione et al. 2011). This type of methodology has been used for legal purposes since the 1960s and gives accurate, precise, and specific results fit for its intended purpose (Jones 1996).

Table 1 summarizes major historical developments in the analytical methods used for determination of ethanol and other drugs in biological specimens for clinical and forensic purposes.

Table 1. Historical Landmarks in the Development of Methods for Analysis of Alcohol and Other Drugs in Biological Specimens for Legal Purposes

The quantitative analysis of drugs other than ethanol is a more challenging task for analytical toxicologists for several reasons. First, as stated earlier, the concentrations of non- alcohol drugs in blood and other biological fluids (see Table 2) are 1,000–10,000 times lower than concentrations of ethanol. Second, ethanol is easily separated from the biological matrix by its volatility, whereas other drugs need to be extracted with organic solvents or solid-phase cartridges, which is more troublesome and costly (Maurer 2018).

Table 2. The Mean, Median, and Highest Concentrations of Ethanol and Other Drugs Identified in Blood Samples from Motorists Apprehended in Sweden—The Results Represent Data Accumulated Over Several Years During the Period 2000–2012

The pharmacologically active substance is first extracted from blood or tissue by adjusting the pH so that drug molecules are in their unionized form and therefore more lipophilic. The buffered mixture is then shaken with organic solvents or added to specially designed solid-phase columns. In this way, the active drug is separated from interfering substance and/or any drug metabolites prior to analysis by GC or LC using various detector systems, such as flame ionization detector, electron capture detector, nitrogen-phosphorous detector, or a mass detector (MS) with selected ion monitoring (Maurer 1999).

An important advance occurred when capillary column GC methods appeared in the 1980s, which improved sensitivity and specificity of the assay considerably. The time elapsed after injecting the sample onto the GC column to the time of appearance of a peak is known as the retention time (RT) and serves to identify the analyte (qualitative analysis) by comparing RT with known authentic substances. Alternatively, relative retention time (RRT) is another way of identification, whereby the time for elution of the GC peak is compared with RT of an internal standard added to the biological specimen before analysis (Mbughuni et al. 2016). When GC-MS or LC-MS methods of analysis are used, it is customary to make use of deuterium-labeled internal standards and both RT and mass fragmentation patterns help to identify the drugs and/or metabolites in the sample (Maurer 1992).

Today’s analytical methodology for the determination of drugs in biological specimens is highly sophisticated, fully automated, and controlled by computer systems and workstations. Separation methods based on GC or LC are first and foremost coupled with mass-selective detectors, often high-resolution instruments—so-called tandem detectors GC-MS-MS or LC-MS-MS (Maurer and Meyer 2016; Meyer et al. 2016). The positive identification of hundreds of psychoactive substances and their metabolites is no longer a difficult task for analytical toxicologists. However, the correct interpretation of the analytical results is more challenging, especially when compliance with some threshold concentration limit is an issue in criminal prosecutions. In this connection, making an allowance for uncertainty by subtracting a certain amount from the analytical result is highly recommended when concentration per se statutes are enforced (Kristoffersen et al. 2016) as is commonly done with forensic BAC determinations (Gullberg 2012). Neither should one forget pre-analytical factors, such as those associated with sampling, transport, storage, and chain-of-custody issues as well as stability of the target drug during storage (Kouri et al. 2005).

Interpretation of Analytical Results
After absorption into the bloodstream, drugs are transported to the brain and interact or bind with certain receptor sites and proteins causing impairment of thought processes, and altered performance and behavior, etc. The degree of impairment associated with drug use depends on the type of drug, the mechanism of action, the dose taken, and the time after intake when driving occurs. Drugs are eliminated from the body by metabolism and excretion at widely different rates, varying from a few hours to several days, depending on the drug’s elimination half-life.

When toxicology results are interpreted in DUID cases, it is important to consider the entire case scenario. This includes observations about the driving, results of field sobriety tests if any, clinical signs and symptoms, and the DRE examination results along with the toxicology report. The totality of information available allows reaching an evidence-based opinion about impairment caused by drug use and whether the concentrations in blood are consistent with therapeutic usage or overdosing with medication (Launianinen and Ojanpera 2014). The question of whether a patient was compliant (or not) with their medication can be gleaned by comparison with therapeutic drug-monitoring programs and concentrations of the same drugs in plasma or serum (Jones et al. 2007).

Based on knowledge of the main pharmacokinetic parameters of the drug (such as distribution volume, elimination-rate constant, and half-life), tentative conclusions can be drawn about the amount of drug in the body and sometimes when it was taken in relation to driving (Huestis et al 2005). The concentrations of drugs in blood, plasma, or serum are more closely related to amounts reaching the brain and the pharmacologic response, including impairment of body functions (Nedahl et al 2019).

Urine is an excellent specimen for a preliminary screening analysis and also provides a wider window of detection compared with blood or plasma; but urinary concentrations cannot be used to draw inferences about concentrations existing in blood nor any drug-related effects on impairment (Liu 1992). Positive results from the analysis of urine verifies prior usage; however, calculating the dose administered or the time of last intake is not possible with any degree of scientific certainty.

In the field of forensic toxicology, drugs are almost always determined in blood, whereas clinical laboratories (dealing with therapeutic drug monitoring) analyze the concentrations in plasma or serum. The concentrations of drugs in these biological media are not necessarily the same, depending on lipid- to water-solubility and the amount of binding to plasma proteins and other biomolecules (Jantos et al. 2011). In general, drug concentrations in plasma/serum are higher than in an equal volume of blood. These distribution ratios should be considered when analytical results from forensic laboratories are interpreted and compared with therapeutic concentration in clinical pharmacology. Some examples of serum/blood distribution ratios for drugs determined in blood of drivers are 1.7–2.0 for THC (Gronewold & Skopp 2011), 1.6–1.8 for diazepam (Jones Larsson 2004), and 1.01–1.15 for various alcohols, such as ethanol (Skopp et al. 2005).


About the Authors
Dr. AW Jones was born in Wales, UK, but has lived and worked in Sweden for over 40 years. He recently retired from his appointment as senior scientist at Sweden's National Laboratory of Forensic Medicine, Division of Forensic Genetics and Forensic Toxicology (Linköping, Sweden). Jones currently serves as a guest Professor in Forensic Toxicology at the Department of Clinical Pharmacology, University of Linköping, Sweden.

Professor Jørg G. Mørland received an M.D. degree from the University of Oslo in 1967 and a Ph.D. degree in pharmacology from the same university in 1975. Mørland is now a senior scientist at the Division of Health Data and Digitalization of the Norwegian Institute of Public Health and a professor emeritus at the University of Oslo.

Dr. Ray Liu took a degree in law from the police academy (now Central Police University) in Taipei, Taiwan before coming to Indiana University (Bloomington, Indiana) to study forensic science under the guidance of Professor Robert F. Borkenstein with internship training in Dr. Doug Lucas’s laboratory (Centre of Forensic Sciences in Toronto, Canada). He then studied towards a Ph.D. degree in the Department of Chemistry, Southern Illinois University (Carbondale, Illinois), which was awarded in 1976.


References

Gronewold, A., and G. Skopp. 2011. A preliminary investigation on the distribution of cannabinoids in man. Forensic Sci Int. 210:e7.

Gullberg, R.G. 2012. Estimating the measurement uncertainty in forensic blood alcohol analysis. J Anal Toxicol. 36:153.

Huestis, M. A., A. Barnes, and M. L. Smith. 2005. Estimating the time of last cannabis use from plasma Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol concentrations. Clin Chem. 51:2289.

Jantos, R., M. Schuhmacher, J. L. Veldstra, W. M. Bosker, and Skopp G. 2011. Bestimmund der Blut/Serum Verh.ltnisse verschiedener forensisch relevanter Analyten in authentischen Proben. Archiv Kriminolog. 227:188.

Jones, A. W., A. Holmgren, and F. C. Kugelberg. 2007. Concentrations of scheduled prescription drugs in blood of impaired drivers: Considerations for interpreting the results. Ther Drug Monit. 29:248.

Jones, A. W., and H. Larsson. 2004. Distribution of diazepam and nordiazepam between plasma and whole blood and the influence of hematocrit. Ther Drug Monit. 26:380.

Jones, A. W. 1996. Measuring alcohol in blood and breath for forensic purposes—A historical review. Forensic Sci Rev. 8:13.

Jones, A. W. 2000. Medicolegal alcohol determination—Blood- or breath-alcohol concentration? Forensic Sci Rev. 12:23.

Kouri, T., M. Siloaho, S. Pohjavaara, P. Koskinen, O. Malminiemi, P. Pohja-Nylander, and R. Puukka. 2005. Preanalytical factors and measurement uncertainty. Scand J Clin Lab Invest. 65:463.

Kristoffersen, L., D. H. Strand, V. H. Liane, V. Vindenes, I. F. Tvete, and M. Aldrin. 2016. Determination of safety margins for whole blood concentrations of alcohol and nineteen drugs in driving under the influence cases. Forensic Sci Int. 259:119.

Launiainen, T., and I. Ojanpera. 2014. Drug concentrations in post-mortem femoral blood compared with therapeutic concentrations in plasma. Drug Test Anal. 6:308.

Liu, R. H. 1992. Important considerations in the interpretation of forensic urine drug test results. Forensic Sci Rev. 4:51.

Maurer, H.H., and M. R. Meyer. 2016. High-resolution mass spectrometry in toxicology: Current status and future perspectives. Arch Toxicol. 90:2161.

Maurer, H. H. 2018. Mass spectrometry for research and application in therapeutic drug monitoring or clinical and forensic toxicology. Ther Drug Monit. 40:389.

Maurer, H. H. 1992. Systematic toxicological analysis of drugs and their metabolites by gas chromatography-mass spectrometry. J Chromatogr. 580:3.

Maurer, H. H. 1999. Systematic toxicological analysis procedures for acidic drugs and/or metabolites relevant to clinical and forensic toxicology and/or doping control. J Chromatogr B Biomed Sci Appl. 733:3.

Mbughuni, M. M, P. J. Jannetto, and L. J. Langman. 2016. Mass spectrometry applications for toxicology. EJIFCC. 27:272.

Meyer, G. M, H. H. Maurer, and M. R. Meyer. 2016. Multiple stage MS in analysis of plasma, serum, urine and in vitro samples relevant to clinical and forensic toxicology. Bioanalysis. 8:457.

Nedahl, M., S. S. Johansen, and K. Linnet. 2019. Postmortem brain-blood ratios of amphetamine, cocaine, ephedrine, MDMA and methylphenidate. J Anal Toxicol. 43:378–384.

Poon, R., B. T. Hodgson, I. Hindberg, and C. Rowatt. 1987. Evaluation of three pocket-size breath alcohol analyzers. Can Forensic Sci Soc J. 20:19.

Skopp, G., G. Schmitt, and L. Potsch. 2005. Plasma-to-blood ratios of congener analytes. J Anal Toxicol. 29:145.

Tiscione, N. B., I. Alford, D. T. Yeatman, and X. Shan. 2011. Ethanol analysis by headspace gas chromatography with simultaneous flame-ionization and mass spectrometry detection. J Anal Toxicol. 35:501.

Wigmore, J. G., and R. M. Langille. 2009. Six generations of breath alcohol testing instruments: Changes in the detection of breath alcohol since 1930. An historical overview. Can Soc Forensic Sci J. 42:276.

 
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