Journal of Pharmacy Practice

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Jan 3, 2012 - New York State Council of Health-system Pharmacists can be found .... Ashraf Mozayani, Harris County Institute of Forensic Sciences, 1885 Old.

Journal of Pharmacy Practice

An Overview of Alcohol Testing and Interpretation in the 21st Century Anna T. Kelly and Ashraf Mozayani Journal of Pharmacy Practice 2012 25: 30 originally published online 3 January 2012 DOI: 10.1177/0897190011431149 The online version of this article can be found at:

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New York State Council of Health-system Pharmacists

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An Overview of Alcohol Testing and Interpretation in the 21st Century

Journal of Pharmacy Practice 25(1) 30-36 ª The Author(s) 2012 Reprints and permission: DOI: 10.1177/0897190011431149

Anna T. Kelly, PhD1 and Ashraf Mozayani, PhD, PharmD1

Abstract Ethanol analysis is the most commonly carried out drug testing in a forensic toxicology laboratory. Determination of blood alcohol concentration (BAC) is needed in a multitude of situations, including in postmortem analysis, driving under the influence (DUI) and drug-facilitated sexual assault (DFSA) cases, workplace drug monitoring, and probation investigations. These analyses are carried out by direct measurement of ethanol concentrations as well as of metabolic by-products, such as ethyl glucuronide (EtG) and ethyl sulfate (EtS). This review article will discuss pharmacokinetics, including absorption, distribution, and elimination of ethanol, methods for the detection of ethanol, the effect of ethanol on human performance, the role of alcohol in injuries and fatalities, and information regarding the interactions that may occur between alcohol and other drugs. Finally, an explanation will be given on how to interpret alcohol levels as well as the extrapolation and calculation of blood alcohol levels at times prior to sample collection. Keywords ethanol, forensic, pharmacokinetics, extrapolation

Introduction Ethanol is the most commonly used and abused drug and thus understandably the most frequently tested in forensic toxicology laboratories. Therefore, an understanding about its physiological effects and the use of techniques to effectively determine its concentration in the body is imperative. Alcohol testing is performed in a variety of situations, including for postmortem, driving under the influence (DUI), drugfacilitated sexual assault (DFSA) cases, workplace testing, and probation monitoring.

Pharmacokinetics Absorption The major site of absorption of ethanol in the body is in the small intestine, and the rate of absorption is affected by a variety of factors, including the concentration of alcohol in the drink; weak or strong drinks will be absorbed more slowly than drinks falling between 10% and 30% alcohol/volume. Other factors influencing the rate of absorption are consuming food prior to or while drinking and conditions affecting gastrointestinal (GI) function; a disease that inhibits GI motility will lead to a reduced absorption rate. After alcohol has been ingested, a significant amount (10%-20%) of alcohol will be absorbed by the stomach; however, the majority of alcohol will be absorbed by the small intestine and enter the bloodstream.1 When analyzing samples for ethanol concentration, comparing the concentrations in different matrices can give an indication

as to what stage of the absorption process the body is in. In comparing blood and urine during the absorptive phase, the blood ethanol concentration is greater than that of urine, whereas during post-absorption, the reverse is true. The same relationship holds true between blood and vitreous alcohol.

Distribution Once in systemic circulation, ethanol is distributed into tissues or fluids according to water content; those areas having higher water content will have a higher ethanol concentration. While alcohol is still being absorbed, the blood alcohol concentrations (BACs) throughout the vascular system may vary considerably, especially between arterial and venous blood.2 During absorption, arterial blood may exhibit as much as 40% higher ethanol concentration than venous blood; little difference is seen after the absorption is complete. When the absorption process is completed, the alcohol concentration in the blood throughout the body is approximately the same, roughly correlating with the peak BAC. Ethanol concentrations in tissue, such as the liver or the brain, will be lower than in the blood because of the higher water content in blood. The ethanol concentration of 1

Harris County Institute of Forensic Sciences, Houston, TX, USA

Corresponding Author: Ashraf Mozayani, Harris County Institute of Forensic Sciences, 1885 Old Spanish Trail, Houston, TX 77054, USA Email: [email protected]

Kelly and Mozayani vitreous humor and oral fluid will be slightly higher than blood at equilibrium.1 The time it takes for equilibrium to occur varies depending on the sex, weight, and height of the individual as well as other factors, such as the strength of the drink and whether the individual has eaten recently.

Elimination Elimination of alcohol primarily takes place via metabolism in the liver; ethanol is converted to acetaldehyde by reaction with nicotinamide adenine dinucleotide (NAD), facilitated by alcohol dehydrogenase (ADH).1 The rate at which ethanol is eliminated from the body varies from one individual to the next. It is generally accepted that the average elimination rate of ethanol in the blood is 0.015 g/dL per h and 0.018 g/dL per h for men and women, respectively.1 Variations in elimination kinetics are discussed in more detail in the interpretation and extrapolation section. Another pathway for ethanol metabolism is the microsomal ethanol oxidizing system (MEOS).3 This pathway utilizes the cytochrome P450 enzyme CYP2E1 which catalyzes the oxidation of ethanol and also activates hepatotoxic agents. With chronic alcohol consumption, there is an increase in the MEOS pathway; therefore, alcoholics and heavy drinkers have an enhanced capacity to metabolize ethanol during chronic drinking periods.4 Additionally, because P450 enzymes are involved in the metabolism of other drugs, alcoholics may exhibit enhanced clearance of drugs from the body.5 As a result, individuals with a history of alcohol abuse will require doses of certain medications different than others in order to acquire therapeutic levels of drugs.3

Detection of Ethanol The analysis of samples for alcohol in a forensic laboratory has been carried out using different methods. One of the older approaches is an enzymatic approach, in which the action of ADH to oxidize ethanol to acetaldehyde is measured spectrophotometrically; the intensity of the measured signal is proportional to the concentration of ethanol present in the specimen.2 The problem with this method is the potential for interference by other alcohols, such as isopropanol and butanol; an example of this was reported by Vasiliades et al, which demonstrated how nonselectivity of ADH led to erroneous blood ethanol levels in a case of ethanol and isopropanol ingestion.6 The use of gas chromatography–flame ionization detection (GC-FID) circumvents this issue, because various volatile compounds including ethanol, methanol, acetone, and isopropanol can be separated and their concentrations determined.7 GC techniques use either direct or headspace injection, with headspace analysis being the most efficient because there is little column contamination as compared with direct injection, in which the specimen is injected into the GC in liquid form. Headspace analysis leads to improved performance and extended column lifetime.1 The headspace technique uses the principle of Henry’s Law, which states that, at a given

31 temperature, the amount of a volatile in the airspace above the sample is proportional to the concentration of the volatile in the sample.2 By warming the specimen to 37 C in a sealed vial, the volatile compounds are separated from the sample matrices and diffuse into the headspace until equilibrium is reached.7 A small amount of the headspace is then injected into the GC. This approach is sensitive, precise, and capable of quantifying ethanol levels as low as 0.01 g/dL.1 This analysis can be performed on a number of different matrices, including blood, urine, hair, saliva, vitreous humor, bile, liver, or spleen. In forensic situations, it is common to adjust the determined ethanol concentration in matrices other than blood in order to obtain a value that can be compared to BAC; the specimen-to-blood ethanol concentrations vary depending on the water content of the matrix.1 One example is the analysis of serum as opposed to whole blood; the measured ethanol concentration will be higher for serum because it has a 12% to 20% higher water content than whole blood.1 A study comparing ethanol levels in plasma, red blood cells, and whole blood gave serum:whole blood ratios of 1.12 to 1.17 and plasma:whole blood ratios of 1.10 to 1.35.8 If the hematocrit, the proportion of whole blood volume that is occupied by red blood cells, is known, this can be used to convert a serum ethanol concentration to a blood ethanol concentration.1 In addition to analyzing for ethanol concentration, other metabolic by-products can be detected in order to determine whether an individual has recently consumed alcohol. Less than 0.1% of ingested ethanol is conjugated with glucuronic acid in the body, forming ethyl glucuronide (EtG), which is excreted in the urine.9,10 Ethyl sulfate (EtS), another metabolite of ethanol, is formed by conjugation with sulfate; this conjugation is catalyzed by sulfotransferase.11 EtG and EtS offer an extended time period over which testing can take place because they are not cleared from the body as rapidly as ethanol; detection in urine is possible from 24 hours up to 5 days after ingestion of large repeated doses of ethanol ingestion.12-14 The levels of EtG in blood, urine, and oral fluid have been found to demonstrate a dose-dependent relationship.15 In addition to blood, urine, and oral fluid, there have been several studies regarding the detection of EtG in hair samples as an indication of alcohol consumption.16,17 In a study in which alcohol was administered to rats, as was reported for the analysis of other matrices, it was found that EtG was incorporated into the hair in a dose-dependent manner.16 Further studies are needed to gain a better understanding of the mechanism behind the incorporation of EtG into hair, whether through blood flow, sweat, or some other pathway.

Fetal Alcohol Toxicity and Testing in Utero Exposure of Alcohol Another area in which alcohol testing has been performed is in neonates to determine whether there was any recent intrauterine exposure to ethanol. Chronic ethanol exposure in utero can lead to birth defects, classified as fetal alcohol spectrum disorder (also known as fetal alcohol syndrome). Several effects can

32 result from exposure, including growth deficiency, craniofacial abnormalities, and central nervous system damage (structural, neurological, or functional).18 Meconium, the earliest stool of a baby, is composed of materials ingested during the time the infant spends in the uterus. Analysis of meconium for the presence of EtG has been conducted, and this matrix has been found to be the most reliable for determining recent intrauterine exposure to ethanol.19 EtG and EtS testing are useful in a variety of situations, including DUIs, DFSAs, and workplace monitoring of alcohol intake. However, in the case of EtG analysis, due to the high sensitivity of this test, the Substance Abuse and Mental Health Services Administration (SAMHSA) advises that it be used in combination with other analyses and not alone because it is not able to distinguish between alcohol consumption and exposure.20 A study of different analytical approaches for the quantitation of EtG and EtS in urine using liquid chromatography/ mass spectrometry (LC/MS) found that the limit of quantitation (LOQ) for EtG in urine21 could be