Linggo, Mayo 11, 2014

Ethanol Assay

Sadie, James Karl
Estacio, Katrina
Pineda, Anna Criselda

What is Alcohol?

“Alcohol" has been synonymous with "spirituous" liquids for the past years. The history of its consumption, along with codes or laws restraining its consumption was way back in 1700 B.C. There are four known types of alcohol: ethyl alcohol, methyl alcohol, isopropyl alcohol and butyl alcohol or butanol. Ethyl Alcohol, or ethanol (C2H5OH), is commonly used in making alcoholic beverages or liquor. The remaining types, if ingested can result in blindness and death, even in relatively small doses.

Ethyl alcohol or Ethanol is the intoxicating agent used as an ingredient in making beer, wine and liquor. It is created by fermentation of yeast, sugars, and starches. Grapes and grains like barley and wheat are most commonly used in making the alcoholic beverages.

            Ethyl Alcohol simply called as alcohol comes second to caffeine as the most widely utilized drug in the society. Alcohol, being a legal substance many employers do not often test for it as part of their urine drug testing program. Typically, a urine alcohol assay is done using a biochemical method where in alcohol, oxidizes acetaldehyde in the presence of alcohol dehydrogenase if it is present in the specimen. Simultaneously after oxidation, Nicotinamide adenine dinucleotide (NAD) is reduced to NADH. Upon spectro-photometric analysis of NADH the concentration of alcohol can now be obtained. Gas chromatography with flame ionization detection is the most common confirmatory method used. The screening and confirmation cut-off values for Alcohol are 0.02 or 0.04 g/dL.

How do we measure the concentration of Ethyl Alcohol in the body?

Just a “nice to know” facts, alcohol is metabolized in the body by our liver. It is first converted to an aldehyde, specifically ethanol to acetaldehyde, by the enzyme Lactate Dehydrogenase isoenzyme-6 or also known as the Alcohol Dehydrogenase. Then it is furthermore converted into an acid, acetaldehyde to acetic acid, by the enzyme Aldehyde Dehydrogenase present in our liver. Acetic acid is soluble in water and eliminated in the body as a waste via urine, perspiration, and even in our breath.

Ethanol concentration can be measured using blood, urine, saliva or breathe tests. Toxic concentration merely depends on the individual’s tolerance and usage although levels greater than 300-400 mg/dL can be fatal due to respiratory depression. Conversion unit: one millimole of ethanol per liter of blood is equal to 4.61 milligrams of ethanol per 100 milliliters of blood.

There are three methods known to measure the level of Ethyl alcohol in the body. It could be measured using Blood tests, Urine tests and Breath tests.

Below are specific methods used in the determination of Ethyl alcohol levels:



Blood alcohol (also known as blood alcohol content) is the concentration of ethanol on a person’s blood stream and is usually measured as weight per volume and is converted to percentage. This blood alcohol content is used to determine if the person is legally drunk with alcohol for law enforcement or medical purposes. Because alcohol is absorbed in the stomach and the small intestine, it will go then into the bloodstream and will travel throughout the body and brain which can affect the cognitive functioning of a person that can lead to injuries and unnecessary act.
Analysis of urine and blood samples for ethyl alcohol levels is one of the most frequently performed tests for drugs-of-abuse. The results of these tests are utilized for medico-legal purposes that include identifying impairment or legal intoxication, and diagnosis and treatment of alcohol abuse and poisoning.


There are several methods in determining the concentration of ethanol in the BLOOD, these includes:
  • Headspace Gas Chromatography-Flame Ionization Detector (HSGC-FID)
This method provides a procedure for the quantitation of ethyl alcohol in blood by FID with simultaneous confirmation by Mass Spectroscopy and can also be utilized as an identification method for inhalants such as 1,1-difluoroethane.
The mechanism of FID is based on the detection of electrically charged particles (ions) produced during combustion of organic compounds in a hydrogen flame. The generation of these electrically charged particles or ions  is equal to the amount of organic species in the sample gas stream.
  • Direct Blood-Injection Method for Gas Chromatographic Determination of Alcohols
This method is a simple and rapid method that is described for determining different alcohol levels in the blood. This method uses less than 1 microliter of blood, mixed with an internal standard, and is injected directly into a gas chromatograph equipped with flame-ionization detector. This method is sensitive to less than 10 micrograms per ml.
This method has the same principle with Headspacer gas chromatography FID but has one notable difference.  It uses less than 1 µl of blood, mixed with an internal standard, and is injected directly into a gas chromatograph equipped with a flame-ionization detector. 

·        High Performance Liquid Chromatography


·         High-performance liquid chromatography (HPLC) is a method based on chromatography is widely used in forensic science. The “HP” acronym is sometimes referred to the words high pressure (versus high performance), but it refers to the same analytical system. HPLC is useful in drug analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics to name a few forensic applications.
·         A solvent that flows under high pressure over coated beads (mobile phase) with the solid stationary phase. While flowing through the column, molecules contained in the sample separate selectively between the stationary and the mobile phase. Those that interact more with the stationary phase will wrap behind those molecules that separate with the mobile phase. As a result, the sample established at the front part of the column and will emerge in separate bands (called peaks), bands emerging first being the components least interacted with the stationary phase and as a result flows quicker through the column. The components that appear last will be the ones with most interaction with the stationary phase and thus moved the slowest through the column. A detector is placed at the end of the column to identify the elute components.

Ethanol was quantified by measurement of the succeeding raise in acetaldehyde level after reaction with alcohol dehydrogenase and nicotinamide adenine dinucleotide (ADH-NAD) using the fluorimetric HPLC method. Ethanol standards ranging 0.3 to 200 mg/dl were examined and the boundary of quantification of the fluorimetric HPLC method was found to be 6 mg/dl. Assessment of the HPLC method accuracy was evaluated by testing blood samples containing 6-200 mg/dl of ethanol and comparing its results to those of the ADH-NAD enzymatic method (r2 = 0.993). The blood ethanol concentrations of a volunteer after a pulse of 0.3 g/kg of ethanol was determined with the described HPLC method and were compared to the results from the ADH-NAD enzymatic method (r2 = 0.986). Thus, the fluorimetric HPLC method for the measurement of ethanol is of potential clinical utility.

(The following concentration of BAC may vary depending on the method or test that is used)
0.02-0.03 BAC
Euphoria, loss of shyness,
0.04-0.06 BAC
Warmth sensation, Euphoria, exaggeration of behavior and emotional intensified
0.07-0.09 BAC
Impairment of balance, speech, vision and hearing. Euphoria, reason and memory are impaired
0.16-0.19 BAC
Dysphoria, nausea
0.25 BAC
Severely impaired mental, physical and sensory functions,
0.30 BAC
May pass out and difficult to awaken
0.35 BAC
Possibility of coma. This is the level of surgical anesthesia
0.40 BAC and up
Coma, possible death due to respiratory arrest


Enzymatic Method


This is a method utilized for the quantitative determination of chemical substances in solution using enzymes. Enzymatic methods quantify substances that have the capability of taking part in chemical reactions catalyzed by enzymes, as well as substances that activate or inhibit enzymes. This method is highly sensitive and specific, since enzymes catalyze the substance conversion with rapidly and selectively.

In the determination of the substrate in an enzyme-catalyzed reaction, addition of an enzyme and other components to the sample is essential. Upon completion of the reaction, the reaction product quantity in the solution is to be measured. For example, the quantification of ethanol in a specimen using alcohol dehydrogenase is carried out in the presence of the enzyme’s coenzyme, nicotinamide adenine dinucleotide (NAD). Through the enzyme-catalyzed reaction it is converted quantitatively into reduced NAD. Establishing the concentration of the reduced NAD and calculate the concentration of ethanol is possible if absorption is measured. This method allows determinations of 1 microgram (xg) of alcohol in 1 milliliter of solution.

EMIT® Ethyl Alcohol Assays

EMIT® Ethyl Alcohol Assays have been intended to accurately quantify ethyl alcohol in urine, serum, and plasma. In clinical studies, EMIT tests have been proven to be accurate, and they also correlate well with other assays of ethyl alcohol measurement such as, gas chromatography.
Due to the evaporative property of ethyl alcohol, testing for its level needs particular precautions and procedures during collection of sample, handling, and analysis, to ensure the accuracy of the results.


The Emit® II Plus Ethyl Alcohol Assay is based on an enzymatic reaction. Reagent 1 contains the buffering system. Reagent 2 contains alcohol dehydrogenase (ADH), the coenzyme nicotinamide adenine dinucleotide (NAD), buffer, preservatives, and stabilizers. The ADH catalyzes the oxidation of ethyl alcohol to acetaldehyde. During this reaction, NAD is reduced to NADH. The increase in absorbance at 340 nm is proportional to the concentration of alcohol in the specimen.

• For in vitro diagnostic use.
• Do not leave alcohol-containing solutions exposed longer than necessary. Tightly cap upon storage.
• Do not use expired calibrators.
Preparation of Reagents
The Emit® II Plus Ethyl Alcohol Assay reagents are ready to use.
Storage of Assay Components
• Store reagents upright at 2–8°C and tightly capped when they are not to be used.
• Unopened reagents are stable until the expiration, if stored at 2–8°C.
• Do not freeze reagents nor store them to temperatures above 32°C.


• The assay requires serum, plasma, or urine.
• Avoid use of alcohol as disinfectant in collecting or storing blood specimens.
(Use aqueous zephiran (benzalkonium chloride), iodine, or other suitable aqueous
• Make sure the sample tube is tightly closed to avoid evaporation of alcohol.
• Fluoride/oxalate are the preferred for storing blood prior to analysis of plasma specimens. The anticoagulants citrate, EDTA, fluoride/oxalate, and heparin may be used.
• Urine samples should be stored refrigerated with the sample container. Before opening, invert it gently several times.
• If not tested  immediately, specimens may be stored refrigerated at 2–8°C for up to 3 days. After 3 days, specimens should be stored frozen.  Avoid repeated thawing.
• Frozen specimens must be thawed and thoroughly mixed before analysis.
• High turbidity specimens must be centrifuged prior to analysis.
• Urine samples with the pH 3.0–11.0 do not require adjustment of pH.
• Specimens should be treated and handled as potentially infectious.


• In diluting specimens having high alcohol levels, the following factors may affect the results: diluting with the appropriate fluid (Emit® Ethyl Alcohol Negative Calibrator or deionized, distilled water), and precision of the dilution.
• Other substances or factors (eg, technical or procedural errors) may as well interfere with the test and cause inaccurate results.

The Emit® II Plus Ethyl Alcohol Assay quantifies alcohol levels in human urine, serum, or plasma having levels 10–600 mg/dL (0.01–0.60%, 0.1–6.0 g/L) alcohol.
Note: To convert mg/dL to g/L ethyl alcohol, multiply by 0.01.
Toxic dosage for children is at 3 g/kg body weight, but a more little amount can be fatal in the presence of induced hypoglycemia or drug intake. Alcohol-tolerant adults have been observed to endure blood levels of 1500 mg/dL (1.50%, 15 g/L) provided with treatment.
Another Enzymatic Method similar to EMIT is DrugCheck Alcohol rapid test.

Gas Chromatography


A highly rapid and sensitive gas chromatographic method for ethanol determination in various biological specimens (human whole blood, serum, urine, and fecal supernatants) was developed. This method uses direct injection of the specimen into the gas chromatograph, without pretreatment. Injection was done in between the glass beads. 50 or more injections of the specimens could be done before replacement of the liner by a new one. This injection technique permits direct injection of large sample volumes of up to 10 μL without troubling the gas chromatographic separation. Sensitivity of the method was made by the injection of these samples. The detection boundary for ethanol is 0.1 mg/L (2 μmol/L) when an injection volume of 5 μL was used.


Gas chromatography (GC) is a method widely used for the identification, quantitation and separation of mixture components. In this technique, a sample is converted to the vapor state and a flowing stream of carrier gas (often helium or nitrogen) sweeps the sample into a thermally-controlled column.
 In the case of gas-liquid chromatography, the column is filled with solid particles coated with a non-volatile liquid, called as the stationary phase. As the sample mixture travels through the column, the sample components which act together with the stationary phase use up more time in the stationary phase vs. the moving gas phase and thus it requires more time to flow through the column.

The main goal of GC is the detection and separation of components of a mixture and it is thus necessary to be able measure the efficiency of a chromatographic column. One way to express column efficiency is through calculating the height, equivalent to a theoretical plate (HETP) which is then the column length divided by the number of theoretical plates.
Ethyl Glucuronide (EtG) is a direct metabolite of beverage alcohol (ethanol).  Its presence in the urine may aid in detection of alcohol consumption, even it is no longer quantifiable.  EtG in the urine may indicate that alcohol was ingested.

Conventional laboratory methods identify the actual alcohol in the system, which reflects current consumption within the past few hours. The presence of EtG in urine means that ethanol was ingested within the past 3 to 4 days, or about 80 hours after ethanol is eliminated from the body. 
Through ETG alcohol testing, it can be detected in the urine for up to 3-5 days following consumption of alcohol. ETG assays are extremely helpful for the measurement of younger people’s alcohol consumption as well as people under supervision of the criminal justice system when they tend prohibit alcohol consumption. 
Studies show that alcoholics who abstain will show undetectable levels of EtG in their urine after more or less 80 hours of detoxification.Studies state that EtG ‘s stability is more than 4 days at room temperature.  Recent experiments also show that upon heating urine at 100 degrees C stability of ETG increases.Thus, heat does not yield to the breakdown of EtG. 

Methods used in detection of EtG include immunoassay (EIA or ELISA), gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS).  Immunoassay have limited specificity and sensitivity with a limit of detection of approximately 1000 ng/mL.  GC/MS and LC/MS methods are known to be more specific than immunoassay, and offer detection limits of approximately 500 ng/mL.

DRI Ethyl Glucoronide Assay

DRI Ethyl Glucoronide Assay is an enzyme immunoassay intended for the semi-quantitative and qualitative determination of ETG in human urine at 500 – 1000 ng/ml.
DRI ETG Assay is a liquid ready-to-use homogenous enzyme immunoassay. This assay uses specific antibodies that can detect ETG without any significant cross reactivity to other glucoronide compounds. It is also based on competition between a free drug from the urine with fixed amount of antibody binding sites and drug labeled with Glucose-6-Phosphate Dehydrogenase (G6PDH). In the absence of free drug from the sample, the specific antibody binds the drug labeled with G6PDH and causes a decline in the enzyme activity. A Direct relationship is created by this phenomenon in urine and enzyme activity. This active enzyme has the capability to convert NAD to NADH yielding to change in absorbance that can be measured spectrophotometrically at 340 nm.

Precautions and Warnings:

1. This test is for in vitro diagnostic use only. Reagents are harmful if ingested.
2. Reagents used in the assay contain sodium azide which may react to lead or copper plumbing. When disposing reagents always flush a large volume of water to prevent azide build up.
3. Do not use expired reagents.


            As stated earlier, ethanol levels in the body can be expelled in the air that we breathe out. Scientists and researchers made another alcohol assay which makes use of breath as specimen in testing alcohol levels in the body. Breath alcohol analysis started in the year 1950’s wherein there is only limited knowledge in pulmonary physiology. Only little amount of alcohol is present in the breath of a person and the estimated ratio of breath and blood alcohol content is 2100:1. Therefore, breath analyzers are usually used for screening tests especially used by police in screening “drunk driving” or drivers who are under the influence of alcohol. Still the best specimen of choice in testing alcohol concentration of the body is blood.


·      Analyzers Based on Color Change


These types of breath analyzers are the oldest types. They are based on the principle of color change which uses chemicals that alter in color in the presence of alcohol. Chemicals present in the analyzer include Silver nitrate, Potassium dichromate, and Sulfuric acid. Alcohol from breath is absorbed by the Sulfuric acid, which also provides acidic medium in order to facilitate the process, turning to orange in color and then converts Potassium dichromate into Chromium sulfate which is green in color. The Silver nitrate catalyzes the reaction. Reaction of color change is measured by photocells connected to a meter.
Breathalyzer was the first machine invented by Smith and Wesson.

·         Analyzers Based on Infrared Spectroscopy


These types of breath analyzers follow the Beer-Lambert’s Law but uses Infrared light. Assay is done wherein a continuous spectrum of IR light is passed through the specimen allowing light to be absorbed and transmitted. Beer-Lambert’s Law states that the concentration of a substance is directly proportional to the absorbed light and inversely proportional to the transmitted light. Therefore, the greater the absorbed light, the greater the amount of alcohol present in the breath and vice versa; the greater the transmitted light, the lesser the alcohol present.
The Intoxilyzer was a brand of breath analyzer functioning based on Infrared Spectroscopy developed by Omicron in Palo Alto, California.

·         Analyzers Based on Fuel Cell Technology


These types of analyzers contains fuel cell which is a porous disk coated with platinum oxide on both sides. Acidic solutions with various salts are contained within the porous layer allowing charged particles to travel along the medium. It also contains platinum wires which connects the 2 sides of the disk containing platinum oxide. If alcohol is present on the person’s breath, alcohol is converted by the platinum oxide, into acetic acid, hydrogen ions, and electrolytes on the top surface. Hydrogen, which is a charged particle, combines with oxygen in the air converting to water and removing electrons from the platinum oxide. This results to excess of electron on top while deficit of electron on the bottom, allowing electrons to flow through the platinum wire creating an electric current. This electric current is proportional to the amount of alcohol present from the air exhaled. The current is converted to equivalent blood alcohol by the microprocessor of the machine.

Alcotest and Alco-Sensor are example of machine which utilizes Fuel Cell Technology.


·         High amounts of substances which are structurally similar with alcohol metabolites being tested

·         High amount of acetone in the air

·         Acetone is converted to isopropyl alcohol by the Alcohol dehydrogenase
·         Methanol poisoning
·         Exposure to toluene or xylene

Negative effects of Alcohol Abuse

Long-term and frequent alcohol consumption is associated with diseases of the liver and heart, damage to the nervous system, psychiatric problems like depression and anxiety and even cancer.
Furthermore, Alcohol consumption can cause sudden changes in behavior. Even at low doses may impair judgment and coordination. It can also induce feelings of relaxation, suppression of anxiety, and in some, it may even increase self-confidence. Nevertheless, as the dose is increased, normally beyond six ounces of 100 proof alcohols. These euphoric feelings it causes begin to pave way to feelings of depression. Intoxication occurs when the liver is loses its ability to metabolize more than one ounce of alcohol every hour. Thus, if a person consumes alcohol exceeding the body’s ability to metabolize, intoxication occurs. Intoxication may last from one to 12 hours, and its after-effects (“hang-over”) for more or less 24 hours.

Repeated consumption of alcohol may lead to tolerance that in turn leads to greater doses required to attain its desired effects. If a person develops dependence to alcohol, a sudden cessation of its intake will most likely produce withdrawal symptoms. 

 Alcohol can be toxic if its amount reaches a conc1entration above 460 milligrams of alcohol per 100 milliliters of blood (0.46 g/dL). Respiratory depression can cause death upon severe alcohol intoxication, and this can be accelerated if alcohol is to be taken with CNS depressant medications.

Positive effect of Drinking Alcohol

            Looking at the brighter side, drinking alcohol still has a benefit in an individual’s body. Some say that it is good for the heart. Well, a lot of studies were already conducted and showed that it reduces one’s risk of death from Coronary Heart Disease or CHD, but according to the researchers that only those individuals that drink moderately are qualified in this good news. Defining “moderate drinking,” in some studies, refers to less than one drink per day. And defining “one drink,” in the US, considers approximately 12 ounces of beer, 5 ounces of wine, or 1½ ounces of spirits (hard liquor such as gin or whiskey). Each sums up at approximately 12 to 14 grams of alcohol. Due to these studies, researchers came up to a hypothesis that alcohol may prevent the risk from a condition of having an impaired blood flow to the extremities resulting from narrowed blood vessels or Peripheral Vascular Disease. But according from the data from Framingham Heart Study, there is no significant relationship between alcohol and PVD, but further studies from Physicians’ Health Study showed that there is a 26% risk reduction from PVD among “moderate drinkers.” But still, comparing these positive effects of drinking alcohol from its negative effects, in our opinion, it is not beneficial to drink alcohol since a lot of negative effects arise in consuming such drinks.


Michael L. Bishop: Clinical Chemistry Techniques, Principles, Correlations 6th edition copyright 2010 by
Lippincott Williams & Wilkins, Wolters Kluwer business, pp. 624-627
Porter WF, Moyer TP: Clinical toxicology. In Tietz Textbook of Clinical Chemistry. 4th edition. Edited
by CA Burtis, ER Ashwood. Philadelphia, WB Saunders Company, 1993, pp 1155-1235
The Role of the Laboratory in the Investigation of Solvent Abuse Hum Exp Toxicology 1982; v. 1, p.299-
The Science of Drinking: How Alcohol Affects Your Body and Mind By Amitava Dasgupta “Alcohol: Balancing Risks and
Benefits” “Tenth Special Report to the United States
Congress on Alcohol and Health: Health Risks and Benefits of Alcohol Consumption”
“DRI Ethyl Glucoronide Assay”

“Liquid chromatographic determination of ethyl alcohol in body fluids”

“Quantifying ethanol by high performance liquid chromatography with precolumn enzymatic conversion and derivatization with fluorimetric detection”

“High-Performance Liquid Chromatography”

“Highly sensitive gas chromatographic analysis of ethanol in whole blood, serum, urine, and fecal supernatants by the direct injection method”
“Enzymatic Methods of Analysis
“Drug Check Alcohol Rapid Test”
“Ethanol Analysis by Headspace Gas Chromatography with Simultaneous Flame-Ionization and Mass Spectrometry Detection”
“EMIT Ethyl Alcohol Assays:  Answers to Frequently Asked Questions”
“Ethyl Glucoronide (ETG) Alcohol Testing”

“Alcohol ETG Urine Testing Resources”

“Emit II Plus Ethyl Alcohol Assay”

“Gas Chromatography: Principles and Determination of Percent Alcohol”