A METHOD TO DETERMINE FUSEL ALCOHOLS IN BLOOD SAMPLES * * B. Kiihnholz, M.D. ; and W. Bonte, M.D. SYNOPSIS Fusel alcohols belong to the most important congeners of alcohol drinks. Their concentrations range between 0.01% and 1.00% of the ethanol level. Determinations of fusel alcohols in blood samples of drivers require a highly sensitive analytical method, since long-chain alcohols are partly bound to blood proteins and/or conjugated to glucuronic acid. We recommend ultrasonic disintegration, ultrafiltration, and incubation of these samples with beta-glucuronidase. In this paper, also, we discuss forensic problems which can be solved by specific blood analyses. INTRODUCTION Alcoholic beverages contain up to 600 flavor compounds (Rapp et al., 1973). These so-called congeners include aliphatic alcohols, their esters, aldehydes, ketones and other carbonyl compounds. Each drink has its own characteristic congener pattern. There are similarities between drinks of the same beverage class (e.g. between different brands of Scotch whisky), but it is also possible to discriminate 2 wines of the same vineyard and the same vintage, but of different grapes. One finds large variation in the concentration of congeners. Those of the longer aliphatic alcohols (up to C-5) range between about 1 milligram and several grams per liter; those of the higher aliphatic alcohols (the esters, etc.) are much lower. It is no problem at all to detect the lower concentrated congeners by extremely narrowed extracts of the beverages, but it is very difficult to trace them in the blood of the consumer. There are 3 limiting factors: 1) The amount of congeners to be found in the blood is proportional to the quantity consumed. It will be easier to detect them after a bottle than after a glass of brandy. * Abteilung Rechtsmedizin I der Universitat Kiel, FRG. * * Institut fur Rechtsmedizin der Universitat Gottingen, FRG. 781
2) The absorbed compounds are distributed throughout the body; this will decrease the blood levels, depending upon body weight and distribution factors. 3) One will never get more than a few milliliters of blood for analysis, which means that it is impossible to prepare narrowed extracts as when handling beverages. Because the threshold of gas chromatographic detection lies around 0.01 mg/1 (that is, 0.01 ppm), only those congeners can be detected which produce blood levels above this limit: methanol and the fusel alcohols, propanol-1, butanol-1 and -2, isobutanol, and 2- and 3-methylbutanol-l. In the beginning of our studies we wanted to show that it is likewise possible to discriminate alcoholic beverages by means of their fusel alcohol contents (Bonte 1978, 1979; Bonte et al., 1978). Analyses of about 3,000 alcoholic beverages have proven this fact. As we reported earlier in Stockholm (Bonte et al., 1981 a) we can discriminate different beverage classes (e.g. American whiskeys, Scotch whiskies, brandies, clear spirits, wines, and beers). American whiskeys contain high concentrations of methanol, isobutanol, and 2- and 3-methylbutanol-l. One can clearly distinguish them from other brandies and whiskies. Rums and liqueurs exhibit considerably lower contents of fusel alcohols and clear spirits; gin and vodka are practically free of them. As the production of alcoholic drinks is subject to legal regulations in many countries, the fusel alcohol content within one and the same beverage class very often is distributed according to the Gauss integral (Bonte & Russmeyer, 1984). After consumption of alcoholic drinks the fusel alcohols contained therein are absorbed in the intestine, similarly to ethanol, and can be traced in blood and urine samples of the consumer by means of head space technique (Bonte et al., 1981 b). The problem we faced was that the branch-chain alcohols, isobutanol and especially 2- and 3-methylbutanol-l, could not be detected in sufficient quantities; after the consumption of small amounts of distilled spirits (e.g., less than 100 ml brandy) we did not find measurable blood levels of these alcohols. The resolution of this analytical problem has required a complicated technique of preparation of the blood sample separating the alcohols in question from their biological matrix and converting the blood into an aqueous solution (Kuhnholz & Bonte, 1983). 782
METHODS Analytical Problems The short chain primary alcohols, methanol and propanol-1, and the lower secondary alcohol, butanol-2, like ethanol are highly soluble in water. Binding to protein or cellular membranes can be neglected as can conjugation with glucuronic acid. In contrast, as the length of the carbon chain increases, the water solubility decreases and the oil-water partition coefficient increases. This means that the longer-chain primary alcohols (e.g., butanol-1, pentanol-1) and their branched chain isomers are bound to the lipid double substances because of their toxicity like all oil-soluble substances they are also conjugated with glucuronic acid and, possibly, to proteins. Accordingly, concentrations of these latter alcohols in the aqueous portion of the blood are very low and nearly immeasurable without special treatment of the blood samples. Another aspect of the problem is that the fusel alcohols are usually analyzed by means of head-space techniques. It is a well-known trick to lower the limits of detection by increasing the vapor pressure with admixtures of salts, such as potassium carbonate. However, this requires aqueous solutions. Preparation Procedures First, we subject the blood sample to an ultrasonic disintegration in order to release the longer-chain alcohols from their binding to the biological matrix. The next step is ultrafiltration of the sample to produce a watery and protein-free solution. After this, beta-glucuronidase is added to the filtrate to hydrolyse the glucuron conjugates. We recommend incubation of 1 ml filtrate with 0.1 ml beta-glucuronidase at 37 C for 24 hours. Finally, we add 1 gram of potassium carbonate to 1 ml of sample; it is then ready for the head-space procedure. Reference Solutions for Calibration We take ethanol- and congener-free blood and add ethanol up 1 or 2 gms per liter and the fusel alcohols up to 1 or 2 mg per liter, each. These reference samples and that is an important condition should be taken through all steps of preparation. Unfortunately, many preparations of beta-glucuronidase are contaminated with alcohols. In our experience the least trouble will be found with a preparation from the Escherichia coli of Boehringer-Mannheim. However, it is necessary to run blank samples through all steps of the analysis. 783
Apparatus For ultrasonic disintegration we use the Ultra-Sound Generator Labsonic 1510 of Braun-Melsungen operating with a medium-size probe which can be immersed directly into the original GC vial. We recommend an operating time of not more than 1 minute and a power level of 50 W to avoid overheating of the sample. For ultrafiltration, we use an apparatus of Sartorius-GSttingen based on micro-collodion bags with a pore size of 8 nm. The cut-off molecular-weight lies about 12,400. We recommend a nitrogen pressure of 8 bars. The time necessary for filtration of 1 ml of homogenized blood is 20 to 24 hours. Our head-space gas chromatograph (GC) is a Perkin-Elmer Multifract F 45. We work with 2 glass columns and 2 FID's with 1: 1 split before the columns. The columns are packed with 15% Carbowax 1500 on Chromosorb W-NAW and 0.1% Supelco SP 1,000 on Carbopack C/Nitrogen flow-rate is about 5 ml per minute; oven temperature, 80 C. The GC is connected by interfaces to a Sigma 15 Laboratory Computer and with additional multi-range recorders. RESULTS As mentioned in Bonte and Kiihnholz (this volume), the most important utilization of congener analysis is to convict DWI-drivers who escaped after having had an accident and claimed later that the consumption of alcoholic beverage took place after the accident. Supposition is that a blood sample is drawn within 3 to 4 hours after the accident and has been stored in the laboratory for reanalysis. (Two-year storage is common in Germany.) By means of congener analysis we can specify quantitatively the alleged beverage. By means of the optimated method described above, we can detect the whole fusel alcohol pattern of brandy or whisky for example, after consumption of less than 60 ml. Furthermore, we can discriminate whether the accused has consumed beer only (in Germany, typical for drinking before driving) or beer and brandy, for example (typical allegation in court: 2 to 3 bottles of beer before driving and half a bottle of brandy after the accident). 784
DISCUSSION Our research on the congeners of alcoholic beverages has received increased attention in Germany since legislation has generally accepted the blood congener analysis as a method for estimating the consumed beverages. During the last 3 years we have given our expert opinions in more than 400 cases, especially in those in which hit-and-run-drivers claimed to have drunk after the accident. In about half of the cases, unambiguous comments were possible and they were accepted by the courts (Bonte et al., 1982; Ktihnholz et al., 1983). We think that this is an important argument for blood- and against breath-sampling. Moreover, there are different forensic problems which can be solved by congener analysis. Two examples are given to outline the meaning and possibilities of our investigations: 1) A 5-year-old boy was admitted to a hospital with signs of alcohol intoxication. The police thought that he had drunk from a bottle of whisky which was found in the home of his parents. Our analyses showed that the drink must have had an extremely high concentration of methanol which could not have come from the whisky. Further inquiries established that a neighbor had given a home-made beetroot-brandy (a so-called "fusel") to the boy. Our results indicated a specific therapy to prevent typical complications of the methanol intoxication, which resulted in the boy's recovery. 2) A woman of 32 years was found inside her motorcar in a forest remote from her flat. A hose pipe led from the exhaust to the interior, and blood analysis proved a combined carbon monoxide and alcohol intoxication. However, some circumstances aroused the suspicion that she was found drunk by her husband and that he took the opportunity to carry her to the forest and set up a suicide situation. Near the car lay an empty bottle of fruit brandy and the question was asked whether the woman had drunk the missing contents a short time before death, or more than an hour before at home. Congener analyses of blood, urine, stomach content, and the questionable beverage showed that all fusel alcohols of the beverage it was fruit brandy, indeed could be traced in the body fluids. Extremely high concentrations of the congeners in the stomach content and very low 785
concentrations in the urine proved that consumption could have taken place only short time before death; this conclusion was supported by the finding of relatively low concentrations of methanol and butanol-2 (a fruit-brandy specific alcohol) in the blood sample. In contrast, propanol- 1 and the long-chain alcohols, because of their fast absorption, were present in relatively high concentrations. Thus, the homicide hypothesis must be discarded. CONCLUSIONS 1) As the congener alcohols are partly bound to blood proteins and corpuscle membranes we recommend preparation of the blood samples by ultrasonic disintegration. 2) To lower the limits of (gas chromatographic) detection the vapor pressure of the head space should be raised by addition of potassium carbonate. This effect can be optimized when a watery ultrafiltrate is taken instead of the blood sample itself. 3) The long-chain alcohols, especially, can be found in the blood, nearly exclusively as glucuronides. To split the conjugations the ultrafiltrate should be incubated with beta-glucuronidase. 4) The complete procedure guarantees positive analytical findings even after consumption of only 40 to 60 ml of spirits. REFERENCES Bonte, W. (1978). Begleitsubstanzen in Wein und weinahnlichen Getranken. Blutalkohol, 15: 392-404. Bonte, W. (1979). Begleitsubstanzen deutscher und auslandischer Biere. Blutalkohol, 16: 108-124. Bonte, W., Decker, J., and Busse,J. (1978). Begleitsubstanzen hochprozentiger alkoholischer Getranke. Blutalkohol, JL5: 323-338. Bonte, W., and Russmeyer, P. (in press). Zur Frage der Normalverteilung von Begleitstoffen in alkoholischen Getranken. Beitrage zur Gerichtlichen Medizin. 786
Bonte, W., Riidell, E., Sprung, R., Bilzer, N., and Kiihnholz, B. (1982). Eine neue Methode zur Begutachtung von Nachtrunkbehauptungen: Die Begleitstoffanalyse. Neue Juristische Wochenschrift, 35: 2109-2120. Bonte, W., Sprung, R., Riidell, E., and Frauenrath, C. (1981 a). Forensic significance of congeners of alcoholic beverages. In Goldberg, L. (ed.), Alcohol, Drugs, and Traffic Saferty. Stockholm: Almkvist & Wiksell International. Pp. 1074-1081. Bonte, W., Stoppelmann, G., Riidell, E., and Sprung, R. (1981 b). Vollautomatischer Nachweis von Begleitstoffen alkoholischer Getranke in Korperfliissigkeiten. Blutalkohol, 181: 303-310. Ktihnholz, B., Bilzer, N., Bonte, W., Riidell, E., and Sprung, R. (1983). Blegleitstoffgutachten und Gerichtsentscheide bei Nachtrunkbehauptungen. In Barz, J., Bosche, J., Frohber, H., Joachim, H., Kiippner, R., and Mattern, R. (eds.), Fortschritte der Rechtsmedizin. Berlin - Heidelberg - New York: Springer. Pp. 217-222. Kiihnholz, B., and Bonte, W. (1983). Methodische Untersuchungen zur Verbesserung des Fuselalkoholnachweises in Blutproben. Blutalkohol, 20: 399-409. Rapp, A., HSvermann, W., Jecht, U., Franck, H., and Ullmeyer, H. (1973). Gaschromatographische Untersuchungen an Aromastoffen von Traubenmosten, Weinen und Branntweinen. Chemicker Zeitung, 97: 29-36. 787