Kaplan: Clinical Chemistry, 5th Edition

Clinical References - Methods of Analysis

Alcohol
Ping Wang i

Name: Alcohol
Clinical significance: click here
Molecular formula: Ethanol Methanol Isopropanol
C2H5OH CH3OH C3H7OH
Molecular mass: 46.07 32.04 60.09
Merck Index: (11th edition) 3716, 5868, 5096
Chemical class: Alcohol
Refer to Chapter 55, Toxicology, in the 5th edition of Clinical Chemistry: Theory, Analysis,
Correlation.

Students’ Quick Hyperlink Review

• Principles of analysis and current usage
• Reference and preferred methods
• Specimen
• Interferences
• Alcohol reference intervals
• Interpretation
• Alcohol performance goals
• References
• Tables and figures
• Alcohol methods

i
Alcohol
Previous and current authors of this method:
First edition: Timothy J. Schroeder
Methods edition: Timothy J. Schroeder
Second edition: Timothy J. Schroeder
Third edition: K. Michael Parker
Fourth edition: K. Michael Parker
Fifth edition: Ping Wang

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Principles of Analysis and Current Usage
Ethanol, because of its extensive availability in such products as beverages and medicines, is the
most frequently encountered toxic substance. Detection and quantitation is often required to
determine legal impairment, evaluate patients suspected of poisoning, determine eligibility for
organ transplantation, and assess compliance with dependency treatment programs.

Alcohol analysis is frequently requested of a laboratory providing medical and/or forensic

services. The clinical laboratory must provide rapid and reliable results for patients in life-
threatening situations. The forensic laboratory, while less constrained by turnaround time
requirements, is expected to provide results that are defensible in a court of law. Today, many
laboratories performing alcohol measurements are expected to meet both medical and legal
requirements.

The term alcohol is often used to refer specifically to ethanol but can also include the other
monohydroxy alcohols that may be ingested, methanol and isopropanol. Therefore, in the clinical
setting, an alcohol or volatile testing procedure should detect all three substances. If a method is
used which measures only ethanol, results may be reported with respect to ethanol, and those
served by the laboratory must be aware of this limitation. Techniques for the determination of the
alcohols include assays that are nonspecific and semiquantitative (osmolar and diffusion
methods) and assays that are specific and quantitative (enzymatic and chromatographic
procedures) (Table 1). (In this chapter, if the method does not distinguish among ethanol,
methanol, and isopropanol, it will be identified as an alcohol procedure. If the technique is
specific for one of the alcohols, it will be identified as a method for the particular substance
detected [e.g., ethanol].)

Early chemical methods for determination of ethanol were often based on the oxidation of
ethanol by potassium dichromate or other oxidizing agents in a strongly acid medium. Reduction
of the dichromate results in a color change that can be measured to monitor the reaction. A
variation of this chemical method was introduced by Widmark. His method was based on the
simultaneous distillation and quantitative oxidation of ethanol by dichromate. A microdiffusion
method, based on essentially the same principle, has been developed for commercial use. In this
procedure, chromic acid reagent, contained within a sheet of glass fiber paper, is reduced to blue-
colored chromic oxide by ethanol. Heating the sample at 80°C to 120°C releases ethanol into the
glass fiber sheet that is placed directly above the sample according to the following reaction.

Equation 1
2K 2 Cr 2 O 2 (orange-yellow) + 10H 2 SO 4 + 3C 2 H 5 OH →
2Cr 2 (SO 4 ) 4 (blue-green) + 2K 2 SO 4 + 3CH 3 COOH + 11H 2 O + 4H+

Most of these chemical methods are considered obsolete because of their poor specificity,
tediousness, and lack of adaptation to automation [1]. Furthermore, it is important to remember
that the chemical assays are not specific for ethanol but detect volatile reducing agents. Despite
the above limitations, this type of assay does have the advantage of being amenable to a wide
variety of specimen types, including all body fluids and tissues.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

The presence of alcohol in serum leads to an increase in the osmolality of the serum when the
osmolality is measured by use of the freezing-point depression technique (Table 1, Method 2).
Osmometers that use the property of vapor-pressure depression to measure osmolality cannot be
used for the purpose of estimating alcohol concentrations, because alcohols are volatile and
contribute significantly to the vapor pressure above a solution. This phenomenon will result in a
falsely low measurement for serum osmolality.

In the presence of alcohol, there will be an increased gap between the measured (by freezing-
point depression) and the calculated osmolality values. This osmolal gap correlates reasonably
well with blood alcohol concentrations, because serum osmolality increases by 0.22 mOsm/kg
H 2 O for each 1 mg/dL of ethanol. Using the osmolal gap, Equation 2 can then be used to
estimate a serum ethanol concentration:

Ethanol (mg/dL) = osmolal gap × 4.6

Ethanol can also be estimated according to another variation of this equation as follows:

(Measured osmolality − 290 mOsm/kg) × 42.4 = ethanol (mg/L)

The factor 42.4 is the product of the molecular mass of ethanol (46.07 mg/mmol) and the
correction for the volume fraction of water in serum (0.92). As a variation on this principle, the
osmolar gap, which is the difference between the measured and calculated osmolality, can be
used to estimate the concentration of an alcohol. The difference between the measured
osmolality and the calculated osmolality is determined using the following equation:

Calculated osmolality = 2[Na+(mmol/L)] + glucose(mg/dL)/18 + BUN(mg/dL)/2.8

or
Calculated osmolality = 2[Na+ (mmol/L)] + glucose (mmol/L) + urea (mmol/L)

This difference is normally ≤ 10 mOsm/kg. However, if a low-molecular-mass toxin such as an

alcohol is present, the osmolar gap will be increased. The serum concentration of the particular
alcohol can be estimated when only one alcohol is present using the osmolar gap and the
following relationships:

Each 1 mg/dL ethanol = 0.22 osmolality increase

Each 1 mg/dL methanol = 0.34 osmolality increase
Each 1 mg/dL isopropanol = 0.17 osmolality increase

To increase the specificity of the method, Pappas et al. [2] proposed determining the ratio of the
estimated ethanol from the osmolar gap to the ethanol concentration measured by a specific
ethanol procedure. The ratio of estimated ethanol to measured ethanol was helpful in evaluating
acutely intoxicated patients for the presence of ethanol and/or another low-molecular-mass
volatile. Discrepancies in the ratio reliably predicted the presence of another volatile.

However, use of the osmolal gap to estimate ethanol concentrations may result in an
overestimation of alcohol concentrations by up to 30% [3]. This discrepancy has been attributed

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

to the atypical osmotic behavior of ethanol, which can alter the degree of dissociation of solutes
within the specimen. Regardless of these shortcomings, osmolal gap evaluations can be helpful
in the diagnosis of acutely intoxicated patients in emergency situations. In addition, the osmolal
gap is nonselective with respect to the type of alcohol detected. Thus the osmolal gap can help
reveal the presence of alcohols other than ethanol if the results of enzymatic ethanol
measurements are available.

Enzymatic methods have become the most common means for measurement of ethanol. Alcohol
dehydrogenase (ADH), the enzyme employed in these assays, is specific for ethanol and does not
react with acetone. However, ADH shows slight cross reactivity with other alcohols. Relative to
ethanol, the cross reactivities are 6% for isopropanol, 1% for n-propanol, 3% for methanol, and
4% for ethylene glycol [4]. Although the high specificity of ADH for ethanol ensures a small
probability of interference, enzymatic assays can be the source of misleading results in cases
where intoxication results from the ingestion of other alcohols [5]. Comparison of results from
enzymatic assays with those from less specific assays (chemical or osmometric) can help in
identifying the presence of these or other alcohols. The enzymatic ethanol assay is based on the
oxidation of alcohol to acetaldehyde with concomitant reduction of NAD+ to NADH (Equation
3). The NADH that is produced may be measured directly at 340 nm, or may be coupled to
alternative detection schemes involving electrochemical, fluorometry, and colorimetric
techniques. In one such secondary reaction the NADH is coupled to a diaphorase–chromogen
system producing a red-colored solution that can be measured at 500 nm.

Equation 3:
NAD+ + alcohol alcohol dehydrogenase → NADH (340nm) + acetaldehyde + H+

Another variation on the enzymatic scheme is a fluorometric technique. The technique is termed
radiative energy attenuation, and it measures the degree of inhibition of the fluorescence of
fluorescein dye resulting from the production of a colored product [6]. In the original version of
this assay, the initial reaction of ADH with ethanol is coupled with a second reaction between
NADH and the tetrazolium salt, iodonitrotetrazolium violet dye (INT). This additional reaction,
catalyzed by diaphorase, results in the reoxidation of NADH to NAD along with the generation
of a red colored formazan-INT. This product has an absorbance peak at 492 nm, which overlaps
the excitation and emission spectral profile of fluorescein included in the reaction mixture. The
decrease in fluorescence intensity is inversely related to ethanol concentrations present in the
sample. This assay has since been reformulated to eliminate a time-consuming probe wash step
by replacing the INT with a thiazolyl blue dye (MTT). The mechanism behind the assay is
unchanged with the reduced MTT yielding a purple color with an absorbance at 565 nm [7].

Equation 4

There is good agreement between enzymatic methods and chromatographic methods [8].
However, some enzymatic methods can give falsely increased ethanol concentrations with
samples from patients with increased levels of serum lactic acid and lactate dehydrogenase [9].

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Although the enzymatic methods are not absolutely specific for ethanol, these methods usually
meet the requirements for accuracy, precision, and reliability. The need for rapid analysis in
emergency settings has led to the demand for more point-of-care instrumentation. New
instrumentation has been developed that is portable, accurate, and uses specimens such as breath
and saliva that are less invasive than traditional venipuncture. In addition, point-of-care testing is
increasingly being used in fitness-for-duty and post-accident testing and in compliance
monitoring at detoxification and correctional centers.

Saliva is an appropriate specimen for the analysis of ethanol, since the amount of ethanol
reaching the saliva should be equivalent to that in plasma or serum. This is because ethanol is of
low molecular mass, is highly water soluble, and does not bind to blood proteins and therefore
freely diffuses into saliva. A number of point-of-care saliva ethanol testing kits are commercially
available. They provide either qualitative or quantitative results, and the majority employ the
same enzymatic processes described above. With one such test system, saliva is collected on a
sterile cotton swab provided with the kit, and the swab is inserted into the test cartridge. The
saliva must then completely fill the reaction capillary tube in order for the internal quality
assurance device to indicate that a proper collection was obtained. In a process that takes
approximately 2 minutes, any ethanol in the saliva will react with the enzymes in the tube to
form a colored endpoint. The tube is fitted with a linear scale in the range of 0 to 150 mg/dL or 0
to 350 mg/dL, depending on the kit [10]. One limitation of this type of device, however, is that
20 minutes must have elapsed between the cessation of drinking and the collection of the saliva,
or an overestimation of ethanol may result. In addition, some cross reactivity and response
occurs with n-propanol and isopropanol. For the most part, these devices have been thoroughly
tested and found to be reasonably accurate, convenient, fast and free from such interferences as
methanol, ethylene glycol, acetone, and methyl ethyl ketone [10].

Breath is also an appropriate noninvasive specimen for the analysis of ethanol. A small amount
of unchanged ethanol is expired with every breath, and this amount is proportional to the amount
of ethanol in whole blood. This is based on the tenet that ethanol in alveolar capillary blood
promptly equilibrates with alveolar air in a ratio of 2100:1 (blood:breath). In other words, 2100
mL of expiratory air will contain the same amount of ethanol as 1 mL of whole blood [10].

Instrumentation that measures breath alcohol can be either qualitative or quantitative. Hand-held
devices are usually qualitative in design and are used to detect the presence of alcohol above a
certain level or give a semiquantitative estimate of the blood alcohol concentration. Hence these
devices are readily used for roadside screening of suspected drinking drivers, in emergency
medicine, and in fitness-for-duty testing. The principle behind these hand-held devices is usually
based on an electrochemical detection scheme. Ethanol from the breath is oxidized by a fuel cell
that causes the production of free electrons. The electric current generated by the free electrons is
directly proportional to the amount of ethanol oxidized by the fuel cell [4].

The most common quantitative method for detection of ethanol in breath is the use of an infrared
detection system. These devices are used in the measurement of evidential breath alcohol levels.
The results from such a test can then be used as evidence in a court of law. Measuring the
infrared energy at the beginning and end of the sample chamber quantitates the ethanol in a
breath sample. This is because the amount of ethanol present in the breath sample is proportional

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

to the amount of infrared energy lost to absorption. The wavelengths utilized are 3.4 and/or 9.5
microns, corresponding to the C–H and C–O vibrational stretching in the ethanol molecule,
respectively. Selectivity for ethanol is enhanced by the use of more than one wavelength. The
risk of misreporting an interfering substance as ethanol was further reduced by the introduction
of an instrument that makes use of five different infrared wavelengths to identify and quantitate
ethanol [4]. Gas chromatography with flame ionization detection (GC-FID) is considered to be
the reference method [11].

Chromatography is not as popular as enzymatic methods because of the need for specific
expertise for performing the analysis and the concern that the purchase and maintenance of
sophisticated instrumentation dedicated only to a single class of analytes is not cost effective.
However, gas chromatography offers the distinct advantage of simultaneous detection of other
alcohols and volatiles, such as acetone, methanol, and isopropanol.

Direct or head-space injection techniques are employed for GC analysis of volatiles. The direct
injection of blood or serum requires a thorough manual washing of the syringe after each
injection. The addition of an autosampler to the GC system improves throughput and assay
precision (CVs) while decreasing chances of carryover through the use of sophisticated washing
routines. Head-space procedures inject into the GC system a vapor sample removed from a
confined space above blood in a closed container. Head-space analysis prevents contamination of
the column and injector. Increased sensitivity of head-space analysis can be gained by the
addition of a salt, such as sodium chloride, to the specimen. The addition of salt results in an
increase in the concentration of the volatile substance in the vapor phase. Both injection
techniques require the sample to be diluted with an aqueous solution containing an internal
standard to normalize variation among specimens.

Reference and Preferred Methods

The reference method for alcohol testing is gas chromatography [11]. The choice of method for
ethanol determination depends on several factors, such as medicolegal requirements, available
instrumentation, testing personnel expertise, and turnaround-time limitations. In many states
there are specifications defining the methods that must be used when performing medicolegal
testing.

Diffusion methods have largely been replaced by more specific and quantitative procedures such
as the enzymatic or gas chromatographic techniques. Diffusion methods may still be used—only
because of their simplicity, reagent stability (more than 1 year), and low cost—in laboratories
providing very basic stat screening for volatile compounds. A test can be set up in a few minutes,
and a qualitative result can be obtained in half an hour. A true quantitative transfer of the alcohol
by diffusion requires at least 18 hours of incubation time at room temperature. This makes the
method unsuitable as a quantitative test. Since the technique detects all volatile oxidizable
substances, any of the simple alcohols, as well as compounds such as ketones, would be
detected.

Indirect methods based on osmometry should be used in very limited emergency situations when
more specific methods are not available. Any condition that changes the osmolality affects the

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

estimation of alcohol. Significant errors can occur in patients with diabetic hyperglycemia,
uremia, or hypernatremia. Substantially higher results are often obtained for the estimated
alcohol concentration based on osmometry than with those measured by a specific method [12].
The use of the estimated ethanol to measured ethanol ratio (described above) may alert one to the
possibility of ingestion of another volatile substance.

Enzymatic methods are widely used, according to proficiency testing surveys. Most laboratories
have automated chemistry instrumentation which can easily and conveniently be set up to
perform enzymatic ethanol measurements. Most analyzers are capable of directly measuring
absorbance of NADH at 340 nm, and no coupled indicator reaction is necessary. This approach,
when used on automated instruments, yields the best precision of any of the ethanol methods.
The radiative energy attenuation method provides rapid, reliable results using instrumentation
found in many laboratories and most toxicology sections. Since these enzymatic methods have
been designed to quantitate ethanol, other alcohols will not be detected. Emergency drug testing
will require additional methods to detect and quantitate methanol and isopropanol. Ingestion of
these alcohols occurs with sufficient frequency to require that toxicology laboratories have the
capacity to measure them. A quick enzymatic assay for methanol is available for quantitative
detection. This method uses alcohol oxidase to oxidize methanol to formaldehyde, which is then
further oxidized by alcohol dehydrogenase. The 340 nm absorbance change associated with
generation of NADH in the second reaction is proportional to the methanol concentration. This
assay can be adapted to automatic enzymatic platforms. Because high concentrations of ethanol
(usually > 200 mg/dL) may cause false biases in the methanol enzymatic assay, the test result
should be interpreted together with an ethanol test result and clinical presentation. Dipstick
methods using enzymatic systems with color-generating indicator reactions have been useful in
identifying ethanol at high concentrations (>100 mg/dL) [13,14].

Gas chromatography remains the reference method for alcohol testing. Proficiency testing
surveys indicate that it remains one of the commonly used methods. Although it is specific for
ethanol, the other monohydroxy alcohols can be specifically measured in the same analytical run.
Therefore, it is often the preferred method in clinical situations. With regard to sample
introduction, the direct-injection method provides results (approximately 10 min analysis time)
more rapidly than the head-space technique, and consequently it is often more suited to the
demands of emergency testing. On the other hand, when whole blood is the specimen, problems
with inlet and column fouling and syringe plugging can rapidly develop with the direct-injection
technique. Therefore, for processing a large volume of specimens, as in a forensic setting, the
head-space method is usually preferred. Combination of an enzymatic method for screening
followed by confirmation by gas chromatography provides the most accurate approach for
forensic testing.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Specimen
Specimens for the measurement of ethanol include whole blood (venous or capillary), plasma,
serum, breath, saliva, and urine. For enzymatic methodology, whole blood should be
deproteinated prior to analysis according to the manufacturer’s instructions. Anticoagulants do
not interfere with the enzymatic or gas chromatographic ethanol methods. When gas
chromatography is used, any tissue or body fluid may be used as specimen. Regardless of what
specimen is used, the samples must be well sealed to prevent loss of volatiles. Other mechanisms
of ethanol loss include metabolism by microorganisms and temperature-dependent oxidation to
acetaldehyde. The addition of sodium fluoride and refrigeration will effectively stop the loss of
ethanol by these two processes [15].

Contamination by microorganisms may also result in false apparent increases in ethanol

concentration. Urine specimens contaminated with yeast and containing modest amounts of
glucose have been shown to produce sizeable amounts of ethanol by the process of fermentation
[16]; for this reason, specimens must be kept well stoppered and preferably refrigerated to
prevent loss of ethanol. It has been shown that sealed samples of whole blood or whole blood
plus fluoride can be stored at 0°C to 3°C or at room temperature (22°C to 29°C) without
significant loss of ethanol content over a 14-day period [17]. Several microorganisms are capable
of converting glucose to ethanol—for example, Candida albicans. The presence of these
organisms along with glycosuria and prolonged storage at room temperature may lead to falsely
increased ethanol results [18]. Therefore, urine specimens must be handled in such a way to
inhibit microbial production of ethanol. A 1% solution of phenylmercuric nitrate can be added to
the urine collection container to inhibit any microorganisms which may be present in the urine.

Interferences
The use of alcohol-containing swabs to clean the venipuncture site has generally been
discouraged, but McIvor and Cosbey [19] have reported that alcohol swabs led to minimal
interference as measured by head-space GC, which was unlikely to significantly affect the
results. Specimens with increased lactate and lactate dehydrogenase concentrations can give
falsely increased ethanol results with some enzymatic assays. Anticoagulants do not interfere
with the enzymatic or GC procedures. For gas chromatography, any tissue or body fluid may be
used. Some state regulations require that ethanol analysis be performed on unclotted samples.
Senkowski and Thompson [20] reported that analysis following homogenization of the clot
produced results comparable to those obtained with unclotted specimens.

Alcohol Reference Interval

Units used for reporting ethanol levels in biological specimens depend on the purpose in which
the ethanol value will be used. Clinical laboratories generally utilize SI units for reporting, which
express ethanol in terms of the number of molecular mass units of ethanol (millimoles) per unit
volume. However, if an ethanol result is to be used in court proceedings, state law usually
mandates the units used for reporting. Additionally, in the United States, most statutes require
that ethanol concentrations be expressed in terms of mass per unit volume of whole blood [21].
Law enforcement agencies in other countries may have different requirements.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

This can cause some confusion if the specimen utilized for analysis was plasma or serum,
because these specimens have a higher water content than whole blood and will therefore contain
more ethanol per unit volume than whole blood. Because of the potential for error when making
conversions of ethanol content between specimen types, some states have amended their statutes
to include per se limits for different specimen types. Ethanol, as well as other volatiles,
methanol, and n-propanol, are normally not detectable in blood or tissues. A blood ethanol
concentration of 300 mg/dL (65.1 mmol/L) or greater may be associated with coma, whereas
values greater than 400 mg/dL (86.8 mmol/L) have been reported to be fatal. Individuals who are
chronic users of ethanol can show even higher concentrations. In the postabsorptive phase,
ethanol is distributed approximately as follows compared to blood (1.0): urine (1.3), saliva
(1.12), breast milk (1.1), CSF (1.14), brain (0.8), fat (0.02), and breath (1/2100). Plasma or serum
ethanol is about 1.16 times that of whole blood, depending on the hematocrit.

Interpretation
There are both clinical and forensic applications for the analysis of ethanol. Alcohol is a primary
depressant of the central nervous system. If alcohol is taken in sufficient quantity, death can
result from irreversible depression of respiration. It is also important to distinguish altered mental
status due to ethanol intoxication from other causes, especially in cases where head trauma, low
blood glucose concentrations, or seizure are complicating the clinical diagnosis. Because of the
serious consequences of ethanol intoxication, rapid analysis is required for initiation of
appropriate therapy. Forensically, alcohol is monitored for the purpose of workplace drug
testing, post-accident investigations, driving impairment, and postmortem examinations. As the
medicolegal ramifications of ethanol intoxication are serious, the analysis must be accurate and
follow strict guidelines to ensure legal acceptance.

Medicolegal interpretation of ethanol results is usually based on state laws. Historically, 0.1 g/dL
(100 mg/dL; 21.7 mmol/L) has been used as the legal limit for blood ethanol concentration
(BEC). The trend now is toward lower BEC limits. Most states now use a BEC limit between
0.05 and 0.10 g/dL, with 0.08 g/dL being the most common limit in use. In the United States,
drivers under age 21 are held to stricter standards under zero-tolerance laws, with a BEC limit
between 0 and 0.02 g/dL. In Europe, 0.05 g/dL has been proposed as the goal for the BEC limit.
Sweden has lowered the limit further to 0.021 g/dL [22].

Endogenous ethanol production accounts for a BEC less than 0.3 mg/dL. Following acute
ingestion, BEC usually peaks at 0.5 to 2 hours (fasting) or 1 to 6 hours (nonfasting). BEC < 50
mg/dL may not be accompanied by obvious behavioral effects. As BEC increases beyond 50
mg/dL, central nervous system effects become more apparent, progressing from euphoria,
excitement, and confusion to stupor. At a BEC > 150 mg/dL, most individuals are obviously
drunk. BEC > 350 mg/dL may lead to coma and death. Chronic alcohol users may tolerate
greater BEC. Alcohol clearance is primarily by hepatic metabolism. Between concentrations of
20 and 300 mg/dL, ethanol metabolism follows zero-order kinetics at a rate of approximately 10
mL of ethanol metabolized per hour, corresponding to a decrease in blood ethanol of about 20
mg/dL/h [23]. However, alcohol metabolism varies significantly between individuals, with
alcoholics showing up to a twofold increase. Gastric alcohol dehydrogenase activity is less in
women and alcoholic men than in nonalcoholic men, leading to increased ethanol bioavailability

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

and BEC [24]. Medications such as aspirin [25] and H 2 -receptor antagonists [26,27] inhibit
gastric alcohol dehydrogenase, thereby increasing the amount of ingested ethanol reaching the
systemic circulation. Ascorbic acid has been reported to increase ethanol clearance in healthy
individuals [28].

Methanol and isopropanol are usually not detected in body fluids. Methanol concentrations
greater than 20 mg/dL are often toxic. Patients with significant methanol concentrations should
be treated with ethanol or 4-methyl pyrazole to inhibit the production of the toxic metabolites
formaldehyde and formic acid. Toxicity with isopropanol usually develops at concentrations
greater than 150 mg/dL. Acetone is produced as a consequence of isopropanol metabolism and
may be followed in addition to isopropanol concentration when monitoring the clearance of the
alcohol.

Alcohol Performance Goals

Survey data from the 2007 College of American Pathologists (CAP) Participant Summary Report
show that imprecision values (% coefficient of variation [CV]) for ethanol determinations range
from approximately 4% to 9% at a mean ethanol concentration of 40 mg/dL and CVs of 5% to
16% at a mean ethanol concentration of 24 mg/dL. Precision for laboratories measuring ethanol
by gas chromatography is similar to laboratories that employ enzymatic oxidation methods.
Acceptable performance criteria (CLIA-88) for measurement of ethanol require that laboratories
be accurate to within ±25% of the peer-group mean. For ethanol-free whole blood or serum
specimens, CAP has adopted an acceptable result range of 0 to 9 mg/dL.

References
1 Garriott JC, editor: Medicolegal aspects of alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc.
2 Pappas AA, Gadsden RH, Taylor EH. Serum osmolality in acute intoxication: A
prospective clinical study. Am J Clin Pathol 1985: 84: 74-79.
3 Bhagat CI, Beilby JP, Garcia-Webb P, Dusci LJ. Errors in estimating ethanol
concentration in plasma by using the “osmolal gap,” Clin Chem 1985; 31:647-648.
4 Jones AW, Pounder DJ. Measuring blood alcohol concentrations for clinical and forensic
purposes. In: Drug Abuse Handbook, Karch, S.B., editor, Boca Raton, FL, 1998, CRC
Press.
5 Garriott JC, editor: Medicolegal aspects of alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc. p. 221.
6 Yost DA, Boehnlein L, Shaffer M. A novel assay to determine ethanol in whole blood on
the Abbott TDX, Clin Chem 1984: 30: 1029A.
7 Garriott JC, editor: Medicolegal aspects of alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc. p. 224.
8 Jortani SA, Poklis A. Emit\R ETS\R plus ethyl alcohol assay for the determination of
ethanol in human serum and urine. J Anal Toxicol 1992: 16: 368-371.
9 Badcock NR, O’Reilly DA. False-positive EMIT\R-st\T ethanol screen with post-mortem
infant plasma, Clin Chem 1992: 38: 434.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

10 Jones AW. Measuring ethanol in saliva with the QED? enzymatic test device:
Comparison of results with blood- and breath-alcohol concentrations. J Anal Toxicol
1995: 19: 169-74.
11 Tagliaro F, Lubli G, Ghielmi S, Franchi D, Marigo M. Chromatographic methods for
blood and alcohol determination, J Chromatogr 1992: 580: 161-190.
12 Snyder H, Williams D, Zink B,Reilly K. Accuracy of blood ethanol determination using
serum osmolality, J Emerg Med 1992: 10: 129-133.
13 Christopher TA, Zeccardi JA. Evaluation of the Q.E.D. saliva alcohol test: A new, rapid,
accurate device for measuring ethanol in saliva. Ann Emerg Med 1992: 21: 1135-1137.
14 Schwartz RH, O’Donnell RM, Thorne MM, Getson PR, Hicks JM.
Evaluation of colorimetric dipstick test to detect alcohol in saliva: a pilot study. Ann.
Emerg Med 1989: 18: 1001-1003.
15 Ellenhorn MJ, Schonwald S, Ordog G, Wasserberger J, editors. Ellenhorn’s medical
toxicology: diagnosis and treatment of human poisoning. Baltimore, MD, 1997, Williams
& Wilkins, p. 793.
16 Lough PS, Fehn R. Efficacy of 1% sodium fluoride as a preservative in urine samples
containing glucose and Candida albicans. J Forensic Sci 1993: 38: 266-271.
17 Winek CL, Paul LJ. Effect of short-term storage conditions on alcohol concentrations in
blood from living human subjects, Clin Chem 1983: 29: 1959-1960.
18 Alexander WD, Wills PD, Eldred N, Gower R. Urinary ethanol levels and diabetes,
Lancet 1981: 1(2):789.
19 McIvor RA, Cosbey SH. Effect of using alcoholic and non-alcoholic skin cleansing
swabs when sampling blood for alcohol estimation using gas chromatography, Br J Clin.
Pract 1990: 44: 235-236.
20 Senkowski CM, Thompson KA. The accuracy of blood alcohol analysis using head-space
gas chromatography when performed on clotted samples, J Forensic Sci 1990: 35: 176-
180.
21 Garriott JC, editor: Medicolegal aspects of alcohol, ed. 3, Tucson, AZ, 1996, Lawyers &
Judges Publishing Co., Inc., p. 268.
22 Jones AW. Limits of detection and quantitation of ethanol in specimens of whole blood
from drinking drivers analyzed by head-space gas chromatography. J Forensic Sci 1991:
36: 1277-1279.
23 Gershman H, Steeper J. Rate of clearance of ethanol from the blood of intoxicated
patients in the emergency department, J Emerg Med 1991: 9: 307-311.
24 Frezza M, diPadova C, Pozzato G, Terpin M, Baraona E, Lieber CS. Higher blood
alcohol levels in women: the role of decreased gastric alcohol dehydrogenase activity and
first-pass metabolism. N Engl J Med 1990: 322: 95-99.
25 Roine R, Gentry RT, Hernandez-Munoz R, Baraona E, Lieber CS. Aspirin increases
blood alcohol concentrations in humans after ingestion of ethanol. JAMA 1990: 264:
2406-2408.
26 Caballeria J, Baraona E, Rodamilans M, Lieber CS. Effects of cimetidine on gastric
alcohol dehydrogenase activity and blood ethanol levels. Gastroenterol 1989: 96: 388-
392.
27 DiPadova C, Roine R, Frezza M, Gentry RT, Baraona E, Lieber CS.
Effects of ranitidine on blood alcohol levels after ethanol ingestion: comparison with
other H 2 -receptor antagonists. JAMA 1992: 267: 83-86.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

28 Chen MF, Boyce HW Jr, Hsu JM. Effect of ascorbic acid on plasma alcohol clearance. J
Am Coll Nutr 1990: 9: 185-189.
29 Watts MT, McDonald OL. The effect of sodium chloride concentration, water content,
and protein on the gas chromatographic head-space analysis of ethanol in plasma. Am J
Clin Pathol 1990: 93: 357-362.

Table 1: Alcohol Methods Summary

Methods for All Alcohols:

Method 1: Distillation-oxidation; colorimetric
Principle: Alcohol diffuses into gas phase and reacts with oxidizing agent, changing its
color:
2 K2Cr2O7 + 10 H2SO4 + 3C2H5OH →
(yellow-orange)
2 Cr2(SO4)4 + 2 K2SO4 + 3 CH3COOH +11 H2O + 4 H+
(blue-green)
Usage: Stat or routine; all body fluids; tissue
Comments: Nonspecific; gives reaction with all volatiles
Method 2: Osmometry; freezing-point depression
Principle: Alcohol in high concentration increases serum osmolality: (a) difference
between normal and measured value is proportional to alcohol levels, or (b) difference
between measured and calculated value (osmolar gap) proportional to alcohol
concentration
Usage: Stat; serum
Comments: Nonspecific; measured osmolality increased by non-alcohols

Methods for a Specific Kind of Alcohol:

Method 3: Enzymatic:
(a) Spectrophotometric
Ethanol:
Principle: NAD+ + alcohol _alcohol dehydrogenase→ NADH + H+ + acetaldehyde
Usage: Stat or routine; serum; urine
Comment: Specific for ethanol; other alcohols not readily measured
Methanol:
Principle: Methanol + O 2 alcohol oxidase→ formaldehyde + H 2 O 2
H 2 O 2 + formaldehyde + NAD+ formaldehyde dehydrogenase→ formic acid +
NADH + H+
Usage: Stat or routine; serum; urine
Comment: Specific for methanol; high ethanol concentration causes positive bias;
alcohol oxidase is very labile and needs to be prepared fresh before testing
(b) Colorimetric, dipstick
Principle:
NAD+ + alcohol alcohol dehydrogenase→ NADH + H+ + acetaldehyde
NADH + iodonitrotetrazolium dye diaphorase→

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 NAD+ + reduced iodonitrotetrazolium dye (insoluble)
(colored)
Usage: Stat or routine; serum; urine
Comment: Specific for ethanol; other alcohols not readily measured
(c) Fluorometric
Principle: NAD+ + alcohol alcohol dehydrogenase→ NADH + H+ + acetaldehyde
NADH + tetrazolium dye diaphorase→
NAD+ + reduced monotetrazolium dye (insoluble)
(fluorescein fluorescence decreased)
The reduced dye is colored, decreasing the amount of light reaching the
fluorescein dye reagent and therefore “attenuating” its fluorescence
Usage: Stat or routine; serum
Comment: Specific for ethanol; other alcohols not readily measured
Method 4: Gas chromatography; flame ionization detection and quantitation
Principle: Alcohol separates on chromatographic column; identification of specific
alcohol by retention time; quantitation by comparison of peak height to an internal
standard. Direct injection of sample fluid onto column or head-space analysis, where the
airspace above the sample is allowed to come to equilibrium, and the air sample is
chromatographed.
Usage: Stat or routine; all body fluids
Comments: Specific for all alcohols
Method 5: Infrared spectrophotometry
Principle: The amount of infrared energy lost due to absorption is proportional to the
alcohol concentration. Two wavelengths used to monitor for interference.
Usage: Forensic and compliance; breath analysis
Comments: Qualitative or quantitative analysis

Table 2: Comparison of Assay Conditions for Ethanol

Parameter Enzyme Reaction Gas Chromatography
Temperature 37°C 75°C
pH 8.8 7.35
Final concentration of Reduced nicotinamide adenine n-propanol: 1.2 g/L
reagent components dinucleotide: 0.6 mmol/mL (20 mmol/L)
Alcohol dehydrogenase: 50 U/mL
Tetrasodium pyrophosphate: 0.075 mol/L
Sodium semicarbazide: 0.075 mol/L
Glycine: 0.022 mol/L
Fraction of sample volume Approximately 0.008 0.33
Sample volume 0.5 mL 0.2 mL: 0.5 mL injection
Linearity 0-600 mg/dL (130 mmol/L) 0-1000 mg/dL (217 mmol/L)
Precision* 4% (automated analyzers) 7% (automated analyzers)
Time for analysis 10 min (auto analyzers) 10 min (auto analyzers)
Interferences Enzyme inhibitors; some alcohols None known
Specimen Serum, plasma, urine Whole blood, serum, plasma, urine

*Coefficient of variation; obtained from College of American Pathologists toxicology survey data

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 Table 3: Units for Expressing Ethanol Concentrations and Common
Conversion Factors
% g/100 %
Units g/L (wt/vol) mL mg% mg/dL ppm µg/mL (wt/wt) mg/g mmol/L
g/L 1 0.1 0.1 100 100 1000 1000 0.0948 0.948 21.71
%
(wt/vol) 10 1 1 1000 1000 1E+4 1E+4 0.948 9.48 217.1
g/100
mL 10 1 1 1000 1000 1E+4 1E+4 0.948 9.48 217.1
mg% 0.01 1E-3 1E-3 1 1 10 10 9.48E-4 9.48E-3 0.2171
mg/dL 0.01 1E-3 1E-3 1 1 10 10 9.48E-4 9.48E-3 0.2171
ppm 1E-3 1E-4 1E-4 0.1 0.1 1 1 9.48E-05 9.48E-4 0.02171
µg/mL 1E-3 1E-4 1E-4 0.1 0.1 1 1 9.48E-05 9.48E-4 0.02171
%
(wt/wt) 10.55 1.055 1.055 1055 1055 10548 10548 1 10 229
mg/g 1.055 10.55 10.55 105.5 105.5 1055 1055 0.1 1 22.9
mmol/ 4.61E-
L 0.0461 4.61E-3 3 4.61 4.61 46.06 46.06 4.367E-3 4.367E-2 1

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Figure 1: Photograph of Conway diffusion dish showing sample well, A, and inner well, B,
containing reacted dichromate solution (blue-green).

Figure 2: Chromatogram of alcohol standards obtained by direct injection of the sample

into the gas chromatograph.
Retention time in minutes is recorded next to peak. Alcohols are eluted in the following
sequence: 1.545, methanol; 1.825, acetone; 2.195, ethanol; 2.880, isopropanol; 3.970, n-
propanol (internal standard).

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Procedure: Gas Chromatography of Alcohols
Table 2 compares the assay conditions for the two most commonly used methods. The
reactions for the enzyme method are given in Table 1. Actual parameters for the enzyme
procedure may vary, depending on the manufacturer of the reagents and the instrument used in
the analysis.

Principle
The following direct-injection gas chromatographic procedure can be used to separate
volatile compounds such as ethanol, methanol, isopropanol, and acetone. After sample injection
into the heated injection port, the volatiles are separated on a Carbowax column, detected with a
flame ionization detector, and quantitated by comparison of the peak height ratio of the sample to
internal standard, relative to the peak height ratio of the calibration standard to internal standard.

Specimen
Serum, plasma or whole blood may be analyzed.

Reagents and Materials

Equipment: Gas chromatograph equipped with a flame ionization detector and a 6 ft × 2
mm (I.D.) column packed with 60/80 Carbopack B/5% Carbowax 20M.

Reagents
1. Internal standard: Dilute 75 μL n-propanol to 50 mL with deionized water. Stable when
stored in a refrigerator for up to 6 months.
2. Calibration standard: Prepare a calibration standard of 79 mg/dL (17.1 mmol/L) by
diluting 1 mL of 100% ethanol (anhydrous from a sealed bottle) to 1 L with deionized
water. Stable when tightly sealed and stored in refrigerator for up to 1 month. Other
dilutions should be prepared to verify linearity of the method (0 to 500 mg/dL; 0 to 108.5
mmol/L).

Conditions: Chromatographic parameters: injection temperature, 225°C; column temperature,

75°C; detector temperature, 250°C; carrier gas (helium) flow rate, 30 mL/min; detector gas
(hydrogen) flow rate, 30 mL/min; (air) flow rate, 400 mL/min.

Assay
1. Combine 50 μL of calibration standard, assay control, or patient blood (serum) sample
and 100 μL of internal standard in an appropriate-size glass vial. Cap the vial and mix.
2. Sequentially inject 0.5 μL from each vial into the chromatograph. Chromatographic run
time is approximately 6 min.

Calculation
1. Identify the ethanol peak using the retention time:
Alcohol Retention Time
Methanol 1.545
Acetone 1.825
Ethanol 2.195
Isopropanol 2.880

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 n-Propanol 3.970
(internal standard)
See Alcohol: Figure 2 for sample chromatogram.

2. Determine the peak height (in millimeters) for the ethanol (E) and internal standard
(IS) peaks on each chromatogram.
3. Calculate peak height ratios, E/IS.
4. Calculate ethanol in samples as follows:
Ethanol (mg/dL) = _____E/IS (sample)____ × 79 mg/dL
E/IS (calibration standard)
5. If an electronic integrator is used, calculate ethanol in samples as follows:
Ethanol (mg/dL) = Factor × E/IS (unknown)
where Factor = _______79 mg/dL______
E/IS (calibration standard)
Notes
1. Direct injection of the sample into the gas chromatograph may result in inlet and column
contamination. As an alternative, analysis of the head-space vapor above a specimen may
be performed. The following procedure can be followed to prepare the head-space
samples.
a. Place 1.0 g of sodium chloride in a glass screw-capped tube (screw caps should
contain Teflon-lined silicone septa) for each calibration standard, assay control, or
patient blood.
b. Add 0.5 mL of calibration standard, control, or sample to each tube.
c. Add 0.5 mL of internal standard to each tube, seal, and mix well.
d. Place tubes at 38°C for 45 min.
e. Inject 0.5 mL of head-space vapor into the gas chromatograph.
The same chromatographic conditions and calculation procedure can be used for the
head-space method as with the direct-injection technique. Preparation of calibration
standard(s) in a matrix equivalent to that in the samples, particularly with respect to
protein, has been suggested to reduce partitioning differences of volatiles between
standard(s) and samples [29].
2. Isopropanol, methanol, and acetone can also be quantitated by this procedure. (refer to
retention times in Alcohol: Figure 2.) The same peak height ratio method with n-propanol
as the internal standard is used for the calculation.

Concentration = Peak height ratio (unknown) × Standard concentration

Peak height ratio (standard)

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Alcohols Gas Chromatography: Direct Injection
Important Procedural Note: Do not use injection syringes that have been exposed to
methanol as a washing solvent. Use deionized water when rinsing all syringes.

Principle
Most volatile compounds such as ethanol, methanol, and isopropanol will separate from
biological fluids in the heated injection port of a gas chromatograph. These compounds
will then be separated from each other by the GC column and can be identified and
quantitated by comparing their peak height ratio to a known internal standard.
Specimens of Choice
A. For nonlegal/nonforensic quantitative uses: 0.5 mL of serum collected in a red-top
tube; or plasma collected in either a heparinized or EDTA tube.
B. For nonlegal/nonforensic qualitative uses: 1 mL urine from spot aliquot collection
with no preservatives added.
C. For legal/forensic* quantitative use: 1.0 mL whole blood collected in a gray-top
NaF tube.
D. For legal/forensic* qualitative uses: 1 mL urine from spot aliquot collection with
no preservatives added.
All specimens may be transported at room temperature. For nonlegal
cases, serum or plasma should be separated from RBCs and may be stored at
room temperature until time of testing (up to 3 days) then stored frozen at −20°C.
For legal/forensic whole blood and all urine specimens, store at 2°C to 8°C prior
to testing.
*Note: For legal/forensic uses, specimen must be accompanied by a Chain of
Custody Form.
Indications
As an aid in assessing ethanol or other alcohol abuse which may relate to acute or chronic
clinical problems (i.e., coma, hepatic damage, drug/alcohol interactions, psychiatric
disorders, acidosis); to assess ethanol abuse for forensic purposes, including employee
monitoring and postmortem studies.
Reagents and Materials
A. n-Propanol internal standard. Using a 1.0 mL serological pipet, deliver 0.4 mL of
n-propanol to a 100 mL volumetric flask. Dilute to mark with distilled H2O. Store
in 100-mL reagent bottle at room temp. Stable for 2 months.
B. Alcohol working standards.
Note: For routine instrument calibration, a “working mixed standard” will be
prepared from individual standards.
1. Ethanol standard (150 mg/dL)—aqueous “Setpoint” standard (Stephens
Scientific Co.). Store refrigerated at 2°C to 8°C. Stability as indicated by
manufacturer.
2. Isopropanol calibration standard (157 mg/dL)—Using a 0.2-mL
serological pipet, deliver 0.2 mL of 100% chromatoquality isopropanol (2-
propanol) to a 100-mL volumetric flask. Dilute to mark with distilled
water. Store at room temperature. Stable for 2 months.
3. Methanol calibration standard (158 mg/dL)—Same as #2 above, using
chromatoquality methanol.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 4. Alcohol Working Mixed Standard: Combine 100 µL each of ethanol,
isopropanol, and methanol standards with 100 µL of internal standard.
Prepare fresh as needed. (Total volume: 400 µL.)
5. Ethanol linearity check standards—To respective 100 mL volumetric
flasks, add 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mL of 100%
chromatoquality ethanol, representing concentrations of 79, 158, 237, 316,
395, 474, 553, and 632 mg/dL of ethanol, respectively. Dilute to mark
with distilled water. Store at room temperature. Stable for 2 months. To be
used for monthly instrument linearity check.
C. 12 × 75 mm glass culture tubes.
D. 100 µL Eppendorf pipet.
E. Utak Volatiles Control Serum.
F. Hamilton 7002 NWG syringe.
Instruments and Parameters
A. The parameters given here are for a Shimadzu Mini-2 gas chromatograph.
Detector: Flame ionization
Column: 5% Carbowax 20M on 60/80 Carbopack B (Supleco); 6 ft, 2 mm ID
Temperatures: Col. 90°C; Det. 240°C; Inj. 240°C.
Attenuation: 32
Range: 10
Nitrogen flow: 50 mL/min
Gas pressures at tank: air, 20; nitrogen, 50; hydrogen, 30 mm Hg
B. Model 3390 Hewlett Packard GC Terminal

Procedure
A. Nonforensic analytical runs will consist of an internal control, a calibration
standard if required, and patient specimens.
1. Assess standardization by running a single control at the beginning of each
shift or after any instrument changes (i.e., septum change, gas tank
change, etc.).
2. If control is within acceptable range, proceed to step 4.
3. If control is out of acceptable range, repeat the control. If it is still
unacceptable, perform a recalibration.
4. Run the patient sample. If patient sample is positive for any alcohol,
prepare a second internal standard and sample mixture, and reinject for a
duplicate analysis. For quantitative analyses (i.e., serum or blood) average
the duplicate values for a reportable concentration. If patient
chromatogram is negative, a single analysis is all that is necessary.
5. Run any other appropriate standard (i.e., methanol or isopropanol) if
patient specimen indicates need.
B. Forensic analytical runs will consist of a calibration standard, an internal control,
and the patient specimens. Note that a standard and control must be analyzed with
each specimen or batch of specimens.
1. Assess standardization by running the 150 mg/dL “setpoint” calibrator. If
the calibrator result is between 142 and 158 mg/dL, proceed by running a
control. If the calibrator is outside these limits, perform a recalibration.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 2. Run the single internal control. Assess its acceptability (will have been
determined previously).
3. Run the patient/subject specimen as with nonforensic runs, analyzing all
positive samples in duplicate.
4. Indicate type of specimen (i.e., whole blood, urine) in the report by
appending computer comments “Test performed on whole blood,” or
“Test performed on urine.”
C. Sample Preparation
1. Add 100 µL (with a 100-µL Eppendorf pipet) of internal standard solution
to a glass culture tube.
2. Add 100 µL (with a 100-µL Eppendorf pipet) of patient specimen, control,
or appropriate standard to the tube and mix by agitation. Analyze
immediately (within 1 min of preparation).
3. Inject 1.0 µL of the mix into the GC. (Note: Injection amounts may vary
with time.) Peak height of internal standard should be a minimum of ½
full-scale response. Use syringe designated for alcohols; do not rinse with
methanol.
4. Immediately press [START] on the integrator.
5. Proceed to “Calculations.”

Calculations and Quality Control

A. Integrated calculations
Note: For all positive patient samples, duplicate analyses are performed.
Average these duplicate results for the reportable quantitative result for
serum and/or blood assays.
1. The integrator prints direct alcohol concentrations in mg/dL, as well as
retention times for the alcohols of interest.
2. If only qualitative analysis is required (i.e., urine), compare retention times
of patient sample peaks to those of the known control. Urine
concentrations at or greater than 10 mg/dL for any of the alcohols will be
reported as “Positive.” Concentrations below 10 mg/dL will be reported as
“None detected.” Urine results will not be reported quantitatively.
B. Manual calculations (to be used whenever direct instrument integration is
unavailable).
1. Ethanol
(a) Measure the peak height of the n-propanol I.S. and of the ethanol
in millimeters.
(b) Compute the peak height ratio (PHR) for the 150 mg/dL standard,
control, and patient:
peak height of ethanol
PHR = peak height of I.S.
Compute the concentration of ethanol in the patient and control:
PHR of patient or control × 150
Concentration (mg/dL) = PHR of 150 ethanol std
2. Methanol

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 a. Measure peak heights as above in “a” for patient, control, and 158
mg/dL methanol standard.
b. Calculate PHR as above in “a.” Compute concentration of
methanol in the patient and control:
PHR of patient or control × 158
Concentration (mg/dL) = PHR of 158 methanol std
3. Isopropanol
a. Measure peak heights as above in “a” for patient, control, and 157
mg/dL isopropanol standard.
b. Calculate PHR as above in “a.” Compute concentration of
isopropanol in patient and control:
PHR of patient or control × 157
Concentration (mg/dL) = PHR of 157 isopropanol std
C. Acetone—report only internally as being positive. Do not quantitate or officially
report.
D. Concentration conversions (For use when converting to other concentration
units):
1. To convert from mg/dL to µg/mL, multiply by 10.
2. To convert from mg/dL to grams %, divide by 1000.
E. An acceptable run includes control values within acceptable limits and patient
duplicate analyses within 10% of the duplicate mean. If outside of 10%, analyze a
third sample preparation, and recalculate from the two closest values.

Interpretation
A. For ethanol, 100 mg/dL denotes “legally intoxicated.” Greater than 400 mg/dL is
considered critical. Critical values for methanol and isopropanol are 30 mg/dL
and 200 mg/dL, respectively. Reportable concentrations are up to 1000 mg/dL for
each alcohol. Call all critical values to the caregiver.
B. For alcohol concentrations above 632 mg/dL, dilute the patient specimen 1:1 (50
µL plus 50 µL) with distilled H2O, add 100 µL internal standard, and analyze.
Multiply calculated mg/dL result by 2.
C. Run a complete ethanol curve to establish linearity monthly, when changing GC
columns, or when other changes dictate.
D. If methanol is detected in the patient sample, run a blank (distilled water) taken
through the procedure, then rerun the patient. This is to rule out false positives
due to methanol from wash bottle. If methanol appears in the blank, change water
rinse.
E. Acetone will most frequently be found in patients who are acidotic or who have
ingested isopropanol where acetone is produced as a metabolite. Acetone
detection is noted but not officially reported.
F. False-positive isopropanol levels may be a result of improper blood drawing
technique (i.e., using an alcohol swab). A good indication that a serum
isopropanol is due to swab alcohol is that there will not be any acetone in the
sample. Also, if available, confirm the presence of isopropanol by analyzing the
patient’s urine. A negative corresponding urine is also indicative of a false-
positive serum result.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

 G. The current method sensitivity for each alcohol is 10 mg/dL. Negative results
should be reported as < 10 mg/dL (<10).
H. Because of the relatively high threshold concentrations for reporting alcohols (10
mg/dL), other volatiles, although they may appear in the chromatographic field,
will generally not be detected. Even so, if clinical histories indicate specific
nonalcohol volatiles as being possible poisons, the area supervisor should be
consulted. This would include compounds such as acetonitrile, ethyl acetate,
chlorinated hydrocarbons, and formaldehyde.

Alcohols, Serum, Plasma, Whole Blood, Quantitative

1. Process a Utak control with a known ethanol concentration at the beginning of
each shift for each staff member who will be performing ethanol concentration
measurements on that shift.
2. To a 12 × 75 mm tube, add 100 µL of serum, plasma, or whole blood.
3. Add 100 µL of n-propanol internal standard.
4. Inject 1 µL into gas chromatograph.
5. Integrator will directly report ethanol concentrations in mg/dL.

Alcohol Spot Test (Qualitative)

Principle
In a Conway microdiffusion dish, alcohols will diffuse out of an aqueous specimen, be
trapped, and form a color complex with potassium dichromate.
Specimen
Serum (1 mL) drawn in a red-top tube with or without separator gel, whole blood drawn
in a heparinized or EDTA tube, or urine (1 mL) spot aliquot.
Indications
To be used as a general adjunct alcohol screening procedure. To qualitatively assess the
presence of ethanol, methanol, or isopropanol.
Reagents and Materials
All chemicals used should be analytical reagent grade.
A. Conway microdiffusion dish.
B. Potassium dichromate.
Into a 1-L flask, dissolve 1.0 g of potassium dichromate in 500 mL of water. Add
0.1 g of silver nitrate to the solution. Carefully, add 500 mL of concentrated
sulfuric acid. Stir to mix, transfer to an amber reagent bottle, and store at room
temperature. Stable for 1 year.
C. Negative control.
Distilled water blank.
D. Positive control (790 µg/mL).
To a 100-mL volumetric flask, add 0.1 mL of absolute 100% ethanol. Fill to mark
with distilled water. Transfer to a reagent bottle. Store at room temperature.
Stable for 3 months.
E. 500-µL Eppendorf pipet.
F. 1-mL serological pipet.
Instrument and Parameters

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.

Clinical References - Methods of Analysis 6-23

None
Procedure
Note: With reach run analyze a positive and negative control.
A. Cover the bottom of the middle ring of the microdiffusion dish with blood, serum,
or urine by adding 0.5 mL of sample with an Eppendorf pipet.
B. Add 0.5 mL of potassium dichromate to the center well with a 1 mL serological
pipet.
C. Place the cover on the dish, and allow it to stand at room temperature for 2 h.
Read promptly.
D. Observe the color produced.
Calculations (Observations) and Quality Control
A. The dichromate reagent will change color in 15 min to 2 h if ethanol or other
volatile reducing substances are present, with the concentration of substance
being proportional to time of color change. Color change is from yellow, to green,
to blue.
B. A yellow “nonreactive” color indicates no alcohols above the procedure threshold
cutoff of 20 mg/dL.
C. A green to blue color is indicative of the presence of:
Ethanol
Isopropanol
Methanol
Other reducing substances
D. For all positive samples, proceed with confirmatory testing using gas
chromatography.
E. An acceptable run includes a green-to-blue color formation with the positive
control and no green-to-blue color with the negative control.
Interpretation and Notes
A. A positive finding indicates the presence of a reducing substance, such as alcohol,
but may not be indicative of toxicity or impairment.
B. Sensitivity: 20 mg/dL; report negatives as “None detected.”
C. Specificity: Nonspecific method; unable to identify specific alcohol present; will
react with other reducing substances.

Methods of Analysis © 2010 by Lawrence A. Kaplan and Amadeo J. Pesce.