The determination of ethanol in biological material for the forensic
purposes
The determination of ethanol (alcohol) in biological material for forensic purposes is performed in
two different methods. Primarily it is gas chromatography method with the flame ionization
detector (GC-FID) using head space, and classical volumetric method so-called Widmark method
(test). Both mentioned methods have advantages and disadvantages. Today the only one forensic
recognized method is gas chromatography using head space. Police also use the terraneous
determination of alcohol level in the exhaled air. The conversion to a blood alcohol level is
possible, but it is assumed the independent ratio of exhaled air and blood, which is variable.
Physico-chemical properties of ethanol
Ethanol is aliphatic alcohol (CH3CH2OH). It is colorless liquid with a sharp smell in an anhydrous
state. In the mixture with water the alcoholic smell is softer and sweeter. The boiling temperature of
ethanol is 78,5 °C. The density of ethanol at 25 °C is equal to 0,789 g.cm-3. Ethanol is less polar
than water and for this reason it passes more easily through cell membranes and diffuses into the
tissue. The threshold concentration of absolute ethanol, which can be captured by the human sense
of smell in the air, is 350 ppm. Ethanol is highly flammable substance and ranks among the Class 1
flammable liquids. The ethanol is freely miscible with water.
Effect of ethanol on human organism
Ethanol acts like a drug on the human organism. It has a dampening effect on the CNS (although in
the case of initial consumption causes seemingly tonic).
Ethanol is in very low concentration present in the human blood as the product of metabolic
processes. Physiological average level of ethanol for a healthy person is around 0,03 to 0,05 g.kg-1.
Above the concentration level of 0,05 g.kg-1 experiencing the mental influences, and this level is
therefore considered as a blood alcohol limit when a noticeable deterioration of motor functions is
already observed. With the further increase of blood alcohol level is gradually deepening
deterioration of motor functions and movement coordination, impaired judgment, removing
barriers, and then an impassioned speech up to aggression. The further increasing of alcohol in
blood results in the attenuation of CNS to relapse into unconsciousness. The lethal dose of ethanol
in the blood is in the range of 5-8 g kg-1 for adults and in the level of 3 g kg-1 for children. Acute
toxication of ethanol is also affected by the tolerance to alcohol, the interaction with medicals and
drugs, and also by possible hypoglycemia.
The correlation between the blood alcohol levels and acute clinical conditions in the case of acute
intoxication is reported in Table 1.
Table 1: Correlation between the blood alcohol levels and acute clinical conditions
Blood alcohol levels
(mg/dL)
Clinical conditions
Symptoms and signs
10-50
(0,1 to 0,5 g.kg-1)
sobriety
Normal behavior. The minimum behavior changes
detectable only by special tests.
30-120
(0,3 to 1,2 g,kg-1)
euphoria
Mild euphoria with increased social manifestations,
increased confidence. Reduced attention and self-control.
90-250
(0,9 to 2,5 g.kg-1)
excitation
Emotional instability, the loss of control and critical
thinking, memory lapses. The loss of motor coordination,
increased reaction time, decreased sensitivity to external
stimuli.
150-230
(1,5 to 2,3 g.kg-1)
confusion
Disorientation, mental confusion, nausea, excessive
emotional reactions, fear, sadness, anger. Visual
disturbances - double vision, perception changes of shape
and movement. Reduced response to pain, impaired
balance and coordination, slurred or incomprehensible
speech.
270-400
(2,7 to 4,0 g.kg-1)
stupor
Apathy, significantly reduced response to stimuli,
significant loss of muscle coordination, inability to stand,
incontinence, impaired consciousness (lethargy, stupor).
350-500
(3,5 to 5,0 g.kg-1)
coma
Suppression of reflexes, respiratory disorders, aspiration.
>450-500
(>4,5 to 5,0 g.kg-1)
deatht
Arrest.
Absorption, distribution and metabolism of ethanol
Absorption, distribution and metabolism of ethanol is determined by genetic and environmental
factors. The absorption of ethanol by oral administration takes place mainly in the small intestine.
The ratio between ingested and absorbed ethanol (the absorption rate) is determined by many
factors, eg.: stomach contents, the speed of ethanol ingestion, the composition of food, leak rate of
the gastrointestinal tract. Ethanol as a small and relatively polar molecule is uniformly distributed in
dependence on the water content in individual tissues. Distributed volume of ethanol is proportional
to the total water content in the body. The factors affecting the total amount of water in the body
also affect the distribution of ethanol. The Elimination of ethanol takes place from about 90% by
metabolic processes. Only 2-10% of the dose of ethanol is excreted by breath, sweat and urine. The
metabolic elimination of ethanol depends on the enzymatic oxidation of ethanol in the liver
(alcoholdehydrogenase, aldehydedehydrogenase). The elimination rate is also dependent on genetic
variants of enzymes participating on the oxidation of ethanol.
Absorption
As already mentioned the GI tract is the main route of ethanol absorption taken orally. About 20%
of absorbed ethanol passes through the stomach by passive diffusion into the blood, the remaining
80% goes into the bloodstream through the duodenum and other parts of the small intestine.
Inhalation route of entry and the entry through the skin is not an aspect of great importance for
absorption and alcohol intoxication because of the relatively high polarity of ethanol and therefore
its large solubility in water and low solubility in fats.
The absorption of ethanol in GI takes place only by passive diffusion and thus it is controlled by
concentration gradient (according to Fick's law). The rate of ethanol absorption from the GI tract is
determined by the following factors:
a) the amount of ethanol that is in contact with the mucosal surface,
b) the time after which the ethanol remains in contact with the stomach wall,
c) the quantity of blood that flows through the point of contact of the alcohol with the gastric wall,
d) the surface area of the stomach wall, which is available for absorption of alcohol.
For healthy adult is 80 to 90% of ingested alcohol absorbed through the GI tract during 30-60
minutes.
Distribution
Ethanol is miscible with water in any ratio. For this reason in particular tissues ethanol gets the
more, the water content is higher in a given tissue. VD of ethanol is dependent upon the sex, age
and body fat (obesity). For example, women have VD of ethanol slightly lower than men because
the female body naturally contains less water and more fat per unit of mass. The distribution
volume of ethanol for adult healthy women ranging from 0.54 to 0.71 L.kg-1, while adult healthy
male VD of ethanol is in the range of 0.63 to 0.76 L.kg-1.
The binding of ethanol on protein
Ethanol does not bind on proteins of plasma or tissues. The volume of distribution is closely
correlated with the amount of water in the body. Ethanol also does not interfere with binding of
other compounds on plasma proteins.
Elimination (biotransformation) of ethanol
The main metabolic processes of ethanol are oxidative metabolic processes using enzyme
alcoholdehydrogenase and microsomal enzyme system ethanol-oxidizing (MEOS). The final
product of the enzymatic oxidation of ethanol is CO2 and H2O.
The major metabolic pathway of ethanol is based on the oxidation by alcoholdehydrogenase. The
oxidation of ethanol by alcoholdehydrogenase (ADH) takes place in the liver for the involvement of
nikotinamidadeninnukleotide cofactor (NAD+). In this case NAD+takes the role of a proton acceptor
in the oxidation of ethanol to acetaldehyde. ADH is present in the cytosol of liver cells. This
metabolic pathway of ethanol is saturated at a relatively low concentration of ethanol. Enzymes
involved in the enzymatic oxidation of ethanol exhibiting significant polymorphism (ADH,
aldehydehydrogenase (ALDH) and CYP2E1.
ALDH enzyme, which is further responsible for the oxidation of acetaldehyde, exists in several
forms. The most important are two: ALDH1 - enzyme in the cytosol of all cells including brain with
reduced catalytic activity and ALDH2 - mitochondrial enzyme present in the liver and stomach.
ALDH2 exhibits high catalytic activity for the oxidation of acetaldehyde. ALDH2 is the primary
enzyme responsible for the in vivo oxidation of acetaldehyde. The Asian population have
genetically determined deficit of the ALDH2 enzyme, which leads to increased susceptibility to
acetaldehyde formed by oxidation of ethanol. In this population this leads to increased accumulation
of acetaldehyde associated with increasing heart rate, intense facial flushing, increased sweating,
and erythrema.
Widmark method
Widmark method is still relatively extended very accurate and reliable analytical method. Its
advantages are high sensitivity and relative simplicity, which makes it one of routine laboratory
tasks. It is also used as a principle in a breathalyzer test in detection tubes, which determine an
indicative consumption of alcohol on the basis of color change of detection tubes.
To perform the Widmark test, about 5-8 ml of blood is collected. No alcohol or other volatile
substances can be used for disinfection to distorting the results. The principle of Widmark method
consists in distilling off the ethanol in the blood and its oxidation of known excess of potassium
dichromate in sulfuric acid. The excess dichromate is determined by iodometric titration.
A disadvantage of this test is its non-specificity because other reducing agents behave like the
ethanol and other volatile reducing substances, such as e.g. acetaldehyde, isopropanol etc.
Gas chromatography with flame ionization detector (GC-FID)
Ethanol (as well as other volatile substances) is evaporated to the space above the liquid from
examined blood in a closed flask at a temperature of about 60 °C. The resulting gas sample is
injected into the gas chromatograph column - this technique is called "head space". On the resulting
chromatogram is the retention distance (retention time) the quality level of individual split
components of a mixture, and the peak area is a measure of quantity. The determination is carried
out using the internal standard. Under constant conditions, the retention time of alcohol as well as
other volatile substances that may be contained in the examined sample is always the same.
Fig. 1: Schematic illustration of static head space analysis
The determination of ethanol in biological material by Widmark
method
Task: Determine the ethanol content in the biological material (blood or urine)
Tools: Widmark flask, volumetric flask of 500 mL, chaff bowls, measuring cylinder, burette,
beakers, dryer, analytical balance, pipettes and tips, centrifuge.
Fig. 2: Widmark titration flask
Solutions and reagents:
1) solution of 5% KI,
2) solution of 0,25% potassium dichromate in concentrated sulfuric acid (equal to 0,01 M
solution),
3) solution of 1% starch,
4) 10% solution of hydrochloric acid,
5) 50 mM sodium thiosulfate in water,
6) 5 mM sodium thiosulfate in water.
These solutions and reagents are pre-prepared.
Workflow:
1. The determination of the exact concentration of the standard solution of 0.25% potassium
dichromate
10 mL of the stock solution of potassium dichromate is pipette into titration flask and 30 ml of 5%
potassium iodide solution and 10 mL of 10% hydrochloric acid are added. The mixture is
thoroughly mixed and titrated with 50 mM sodium thiosulfate solution to yellow color. Then 1 mL
of 1% solution of starch wax is added and solution is further titrated to permanently slightly blue
color.
From the consumption of 5% potassium iodide solution is calculated the exact concentration of
potassium dichromate solution.
2. The preparation of the biological material to the analysis
a) Blood - the tube with the submitted blood is centrifuged for 5 minutes at 3000 rpm. After
centrifugation, 100 ul of serum is removed for one titration.
b) The urine is pipette directly without prior centrifugation.
3. The determination of ethanol
a) 1 ml of 0.25% solution of potassium dichromate in concentrated sulfuric acid is exactly metered
into 2 Widmark flasks.
b) 2 chaff bowls are weighed on an analytical balance and their weight is record. 100 uL of
examined biological material and the blank (100 uL solution of potassium dichromate in
concentrated sulfuric acid) are pipette into 2 already weight chaff bowls. Bowls are
differentially weighed and the portion is subtracted.
c) Bowls containing biological material are inserted into the mounts of Widmark flasks.
d) Flasks are placed into the dryer for 2 hours at 60 °C. After two hours the flasks are pulled out
and allowed to cool down at room temperature.
e) Chaff bowls are carefully removed from the Widmark flask and the content is diluted with 25
mL of deionized water and mixed. 1 ml of 5% potassium iodide is added and the solution turns
yellow. Then 3 drops of starch wax solution is added and the solution turns blue and is mixed.
f) The solutions in Widmark flasks are titrated with 5 mM sodium thiosulfate into a permanent
bleached. The exact consumption in mL is subtracted.
Calculation of the amount of ethanol in biological material
c = 100.1,13 . (Vb-Va)/m . x
c…the concentration of ethanol in g/kg
Vb…the consumption of sodium thiosulfate in the blank
Va…the consumption of sodium thiosulfate in the titrated sample
m…sample weight in mg
x…factor reflecting the type of biological material
In the case of the determination of alcohol in the blood serum is necessary to divide the entire result
by number 1.2. The resulting alcohol concentration then corresponds to the content of ethanol in
whole blood.
The preparation of solutions:
1. The solution of 5% potassium iodide
25 g of KI is diluted in 475 mL of deionized water. The solution should be stored in dark
bottle.
2. The solution of 0.25% potassium dichromate in concentrated sulfuric acid
1.25 g of potassium dichromate is diluted in slightly heated deionized water of about 15
mL and quantitatively transferred to a volumetric flask of 500 mL. The solution is up to
the mark with concentrated sulfuric acid. Potassium dichromate may not crystallize. The
solution must be retained in a dark bottle and prepared up to 2 days prior to exercise.
3. The solution of 1% starch wax
1 g of starch is mixed in a beaker with a few mL of deionized water to form slurry. The
slurry is poured by 120 ml of boiling deionized water and boiled for 15 min. Then the
solution is allowed to evaporate to a final volume of 100 mL. The solution should be
stored in a dark bottle.
4. The solution of 10% hydrochloric acid
100 mL of concentrated hydrochloric acid (37%) is mixed with 324 mL of deionized
water.
5. The solution of 50 mM potassium thiosulfate
26 g of potassium thiosulfate pentahydrate is diluted in 100 mL of deionized water and
conserve with the addition of 1 g of mercuric cyanide. It is necessary to store the
prepared solution in the fridge.
6. The solution of 5 mM potassium thiosulfate
25 mL of 50 mM potassium thiosulfate is diluted in 250 mL of deionized water. The
solution must be prepared prior to each exercise.
The determination of ethanol by gas chromatography
Task: Determine the ethanol content in the biological material (blood or urine).
Tools: glass vials, rubber stoppers, crimp cap, crimp pliers, pipettes and tips, centrifuge.
1. The preparation of the biological material to the analysis
a) Blood - the tube with the submitted blood is centrifuged for 5 minutes at 3000 rpm. After
centrifugation, 100 µl of serum is removed for one titration.
b) The urine is pipette directly without prior centrifugation.
2. The analysis of urine
a) 0.5 ml of the biological material (serum, urine) and 0.5 ml of internal standard - 0.4 ‰ (v/v)
solution of tertiary butanol are pippeted into the standard glass vials.
b) The vial is sealed with a rubber stopper and crimped cap under the teacher supervision. Caped
vial is placed on a thermostat gas chromatograph.
c) The series of calibration solutions containing a known concentration of ethanol of 0.05 to 4.0 ‰
always with the same concentration of the internal standard are prepared by the same way.
Caped vials are placed on a thermostat gas chromatograph.
d) The setting of the parameters of GC-FID analysis and the analysis start is done together with
the teacher.
2. Evaluation
The subtracted area of the peaks corresponds to the internal standard peak and ethanol in the
calibration standards and biological samples. Construct the calibration curve and calculate the
concentration of ethanol in the analyzed biological sample.
The obtained result is multiplied by a conversion factor reflecting the type of biological material:
blood = 0,7937
urine = 0,9852
Questions:
1. Write the equation expressing the reactions of metabolism of ethanol and ethylene glycol.
2. Describe the principle of the flame ionization detector (FID), which is used in gas
chromatography for the detection of organic substances.
3. What is the principle of detection devices for approximate determination of ethanol in breath test
drivers for alcohol?
Literature:
1. B. Levine, Principles of Forensic Toxicology, AACC Press; Fourth edition (2013).
2. A. Negrusz, G. Cooper, Clarke's Analytical Forensic Toxicology, Pharmaceutical Press; 2
edition (2013).