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ETHANOL

ASSAY PROCEDURE

K-ETOH 02/17

(*60 Manual Assays per Kit) or

(600 Auto-Analyser Assays per Kit) or
(600 Microplate Assays per Kit)

* The number of tests per kit can be doubled if all volumes are halved

© Megazyme 2017
INTRODUCTION:
Ethanol is ubiquitous in its natural occurrence, and thus its quantitative
determination is not only important in the manufacture of intoxicating
wines, beers and spirits, but also for low-alcohol and non-alcoholic
beverages, fruit juices and a range of other foodstuffs, including
chocolates, sweets, jam, honey, vinegar and dairy products. A large
range of non-foods also contain significant quantities of ethanol, such as
cosmetics and pharmaceuticals.

PRINCIPLE:
The quantification of ethanol requires two enzyme reactions; in the first
reaction catalysed by alcohol dehydrogenase (ADH), ethanol is oxidised
to acetaldehyde by nicotinamide-adenine dinucleotide (NAD+) (1).

(ADH)
(1) Ethanol + NAD+ acetaldehyde + NADH + H+

However, since the equilibrium of reaction (1) lies in favour of ethanol

and NAD+, a further reaction is required to “trap” the products. This
is achieved by the quantitative oxidation of acetaldehyde to acetic acid in
the presence of aldehyde dehydrogenase (Al-DH) and NAD+ (2).

(Al-DH)
(2) Acetaldehyde + NAD+ + H2O acetic acid + NADH + H+

The amount of NADH formed in this reaction pathway is stoichiometric

with twice the amount of ethanol. It is the NADH which is measured by
the increase in absorbance at 340 nm (Figure 1, page 12).

SPECIFICITY, SENSITIVITY, LINEARITY AND PRECISION:

The order of addition of reagents during the assay removes any possible
interference from aldehydes and ketones. Methanol is not converted
due to the unfavourable Km-values of the enzymes used. The assay is
optimised for ethanol, however quantitative conversion of n-propanol and
n-butanol is also achieved. Higher primary alcohols react at a significantly
reduced rate and, where present, can lead to a sample-dependent creep
reaction.

The smallest differentiating absorbance for the assay is 0.005 absorbance

units. This corresponds to 0.023 mg/L of sample solution at the
maximum sample volume of 2.00 mL. The detection limit is
0.093 mg/L, which is derived from an absorbance difference of 0.020 with
the maximum sample volume of 2.00 mL.

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The assay is linear over the range of 0.25 to 12 μg of ethanol per
assay. In duplicate determinations using one sample solution, an
absorbance difference of 0.005 to 0.010 may occur. With a sample
volume of 2.00 mL, this corresponds to an ethanol concentration
of approx. 0.023 to 0.046 mg/L of sample solution. If the sample
is diluted during sample preparation, the result is multiplied by the
dilution factor, F. If, in sample preparation, the sample is weighed,
e.g. 10 g/L, a difference of 0.02 to 0.05 g/100 g can be expected.
INTERFERENCE:
Alcohols present in the distilled water and buffers used for the assay,
or in the air, can result in increased blanks or in creep reactions,
respectively. Thus, it is necessary to cover the cuvettes during assay.

If the conversion of ethanol has been completed within the time

specified in the assay (approx. 5 min), it can be generally concluded
that no interference has occurred. However, this can be further
checked by adding ethanol (~ 5 μg in 0.1 mL) to the cuvette on
completion of the reaction. A significant increase in the absorbance
should be observed.

Interfering substances in the sample being analysed can be identified

by including an internal standard. Quantitative recovery of this
standard would be expected. Losses in sample handling and
extraction are identified by performing recovery experiments, i.e. by
adding ethanol to the sample in the initial extraction steps.
SAFETY:
The general safety measures that apply to all chemical substances
should be adhered to.
For more information regarding the safe usage and handling of this
product please refer to the associated SDS that is available from the
Megazyme website.
KITS:
Kits suitable for performing 60 assays in manual format (or 600
assays in auto-analyser format or 600 assays in microplate format) are
available from Megazyme. The kits contain the full assay method plus:
Bottle 1: Buffer (15 mL, pH 9.0) plus sodium azide
(0.02% w/v) as a preservative.
Stable for > 2 years at 4°C.
Bottle 2: NAD+.
Stable for > 5 years at -20°C.
Bottle 3: Aldehyde dehydrogenase solution (3.25 mL).
Stable for > 2 years at -20°C.
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Bottle 4: Alcohol dehydrogenase suspension (1.3 mL).
Stable for > 2 years at 4°C.
Bottle 5: Ethanol standard solution (5 mL, 5 mg/mL).
Stable in a well-sealed container (as supplied) for
> 2 years at 4°C.

PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:

1. Use the contents of bottle 1 as supplied.
Stable for > 2 years at 4°C.
2. Dissolve the contents of bottle 2 in 12.4 mL of distilled water.
Stable for > 1 year at 4°C or stable for > 2 years at -20°C
(to avoid repetitive freeze/thaw cycles, divide into appropriately
sized aliquots and store in polypropylene tubes).
3 & 4. Use the contents of bottles 3 and 4 as supplied. Before
opening for the first time, shake the bottles to remove any
enzyme that may have settled on the rubber stopper.
Subsequently, store the bottles in an upright position. Swirl
the bottle to mix contents before use.
Stable for > 2 years at 4°C (ADH) or -20°C (Al-DH).
5. Dilute 0.5 mL of the contents of bottle 5 to 50 mL with
distilled water. Store in a well-sealed Duran® bottle.
When diluted, this solution is stable for 2 days at 4°C.

NOTE: The ethanol standard solution is only assayed where there is

some doubt about the accuracy of the spectrophotometer being used
or where it is suspected that inhibition is being caused by substances in
the sample. The concentration of ethanol is determined directly from
the extinction coefficient of NADH (page 4).

EQUIPMENT (RECOMMENDED):
1. Volumetric flasks (50 mL and 100 mL).
2. Disposable plastic cuvettes (1 cm light path, 3.0 mL).
3. Micro-pipettors, e.g. Gilson Pipetman® (20 μL and 100 μL).
4. Positive displacement pipettor, e.g. Eppendorf Multipette®
- with 5.0 mL Combitip® [to dispense 0.2 mL aliquots of
buffer (Bottle 1) and NAD+ solution].
- with 25.0 mL Combitip® (to dispense 2.0 mL aliquots of
distilled water).
5. Analytical balance.
6. Spectrophotometer set at 340 nm.
7. Vortex mixer (e.g. IKA® Yellowline Test Tube Shaker TTS2).
8. Whatman No. 1 (9 cm) filter papers.
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A. MANUAL ASSAY PROCEDURE:
Wavelength: 340 nm
Cuvette: 1 cm light path (glass or plastic with cap)
Temperature: ~ 20-25°C
Final volume: 2.57 mL
Sample solution: 0.25-12 μg of ethanol per cuvette
(in 0.10-2.00 mL sample volume)
Read against air (without a cuvette in the light path) or against water

Pipette into cuvettes Blank Sample

distilled water (~ 25°C) 2.10 mL 2.00 mL
sample - 0.10 mL
solution 1 (buffer) 0.20 mL 0.20 mL
solution 2 (NAD+) 0.20 mL 0.20 mL
solution 3 (Aldehyde dehydrogenase) 0.05 mL 0.05 mL
Mix* and read the absorbances of the solutions (A1) after approx.
2 min and start the reactions by addition of:
suspension 4 (Alcohol dehydrogenase) 0.02 mL 0.02 mL

Mix* and read the absorbances of the solutions (A2) at the end of
the reaction (approx. 5 min). If the reaction has not stopped after
5 min, continue to read the absorbances at 1 min intervals until the
absorbances increase constantly over 1 min**.
* for example using microplate shaker, shake function on a microplate reader
or repeated aspiration (e.g. using a pipettor set at 50-100 μL volume).
** if this “creep” rate is greater for the sample than for the blank, extrapolate
the sample absorbances back to the time of addition of suspension 4.

CALCULATION:
Determine the absorbance difference (A2-A1) for both blank and
sample. Subtract the absorbance difference of the blank from the
absorbance difference of the sample, thereby obtaining ΔAethanol. The
value of ΔAethanol should as a rule be at least 0.100 absorbance units
to achieve sufficiently accurate results.

The concentration of ethanol can be calculated as follows:

c = V x MW x ΔA [g/L]
ε x d x v x 2

where:
V = final volume [mL]
MW = molecular weight of ethanol [g/mol]
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ε = extinction coefficient of NADH at 340 nm
= 6300 [l x mol-1 x cm-1]
d = light path [cm]
v = sample volume [mL]
2 = 2 moles of NADH produced for each mole of ethanol

It follows for ethanol:

c = 2.57 x 46.07 x ΔAethanol [g/L]
6300 x 1.0 x 0.10 x 2

= 0.09397 x ΔAethanol [g/L]

It follows for ethanol in (v/v) terms:

c = 2.57 x 46.07 x 0.1266 x ΔAethanol [% (v/v)]
6300 x 1.0 x 0.10 x 2

= 0.01190 x ΔAethanol [% (v/v)]

where:
0.1266 = factor to convert g/L to % (v/v), taking the density of pure
ethanol to be 0.79 g/mL.

If the sample has been diluted during preparation, the result must be
multiplied by the dilution factor, F.

When analysing solid and semi-solid samples which are weighed out
for sample preparation, the content (g/100 g) is calculated from the
amount weighed as follows:

Content of ethanol
= cethanol [g/L sample solution] x 100 [g/100 g]
weightsample [g/L sample solution]

NOTE: These calculations can be simplified by using the Megazyme

Mega-CalcTM, downloadable from where the product appears on
the Megazyme website (www.megazyme.com).

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B. AUTO-ANALYSER ASSAY PROCEDURE:

NOTES:
1. The Auto-Analyser Assay Procedure for ethanol can be
performed using either a single point standard or a full
calibration curve.
2. For each batch of samples that is applied to the determination
of ethanol either a single point standard or a calibration
curve must be performed concurrently using the same
batch of reagents.

Reagent preparation is performed as follows:

Preparation of R1:

Component Volume

distilled water 20.85 mL

solution 1 (buffer) 2.4 mL
solution 2 (NAD+) 2.4 mL (after adding 12.4 mL of H2O to bottle 2)
solution 3 (Al-DH) 0.6 mL
Total volume 26.25 mL
Preparation of R2:

Component Volume

distilled water 3.2 mL

suspension 4 (ADH) 0.25 mL
Total volume 3.45 mL

EXAMPLE METHOD:
R1: 0.200 mL
Sample: ~ 0.01 mL
R2: 0.025 mL

Reaction time: ~ 5 min at 37°C

Wavelength: 340 nm
Prepared reagent stability: > 2 days when refrigerated
Calculation: endpoint
Reaction direction: increase
Linearity: up to 110 mg/L of ethanol using
0.01 mL sample volume

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C. MICROPLATE ASSAY PROCEDURE:

NOTES:
1. The Microplate Assay Procedure for ethanol can be performed
using either a single point standard or a full calibration curve.
2. For each batch of samples that is applied to the determination
of ethanol either a single point standard or a calibration
curve must be performed concurrently using the same
batch of reagents.

Wavelength: 340 nm
Microplate: 96-well (e.g. clear flat-bottomed, glass or plastic)
Temperature: ~ 25°C
Final volume: 0.257 mL
Linearity: 0.1-1.2 μg of ethanol per well
(in 0.01-0.20 mL sample volume)

Pipette into wells Blank Sample Standard

distilled water 0.210 mL 0.200 mL 0.200 mL

sample solution - 0.010 mL -
standard solution - - 0.010 mL
solution 1 (buffer) 0.020 mL 0.020 mL 0.020 mL
solution 2 (NAD+) 0.020 mL 0.020 mL 0.020 mL
solution 3 (Al-DH) 0.005 mL 0.005 mL 0.005 mL

Mix* and read the absorbances of the solutions (A1) after approx.
2 min and start the reactions by addition of:
suspension 4 (ADH) 0.002 mL 0.002 mL 0.002 mL
Mix* and read the absorbances of the solutions (A2) at the end of
the reaction (approx. 5 min). If the reaction has not stopped after
5 min, continue to read the absorbances at 1 min intervals until the
absorbances increase constantly over 1 min**.

* for example using microplate shaker, shake function on a microplate reader

or repeated aspiration (e.g. using a pipettor set at 50-100 μL volume).
** if this “creep” rate is greater for the sample than for the blank, extrapolate
the sample absorbances back to the time of addition of suspension 4.
CALCULATION (Microplate Assay Procedure):
g/L = ∆Asample x g/L standard x F
∆Astandard
If the sample is diluted during preparation, the result must be
multiplied by the dilution factor, F.
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SAMPLE PREPARATION:
1. Sample dilution.
The amount of ethanol present in the cuvette (i.e. in the 0.1 mL of
sample being analysed) should range between 0.25 and 12 μg. The
sample solution must therefore be diluted sufficiently to yield a
concentration of between 0.01 and 0.12 g/L.

Dilution Table
Estimated concentration Dilution Dilution
of ethanol (g/L) with water factor (F)
< 0.12 No dilution required 1
0.12-1.2 1+ 9 10
1.20-12.0 1+ 99 100
12.0-120 1 + 999 1000
> 120 1 + 9999 10000

If the value of ΔAethanol is too low (e.g. < 0.100), weigh out more
sample or dilute less strongly. Alternatively, the sample volume to
be pipetted into the cuvette can be increased up to 2.00 mL, making
sure that the sum of the sample and distilled water components in the
reaction is 2.10 mL and using the new sample volume in the equation.

2. Sample handling.
Since ethanol is volatile, all operations should, where
possible, be performed in sealed Duran® glass bottles.

It is also necessary to show great care in pipetting, diluting and filtering

solutions. Plastic tips of dispensing pipettes should be rinsed 3 times
with the solution before taking the aliquot. Cuvettes and plastic tips
should be rinsed 3 times with ethanol-free distilled water and dried
before use.

Ensure that reagent bottles (especially the distilled water container)

are sealed immediately the required volumes are removed, to minimise
absorption of alcohol from the air. When setting up the assays, do
not employ the pipette that was used to aliquot the ethanol standard
or other concentrated ethanol solution.

3. Sample clarification.
a. Solutions:
Carrez I solution. Dissolve 3.60 g of potassium hexacyanoferrate
(II) {K4[Fe(CN)6].3H2O} (Sigma cat. no. P9387) in 100 mL of distilled
water. Store at room temperature.

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Carrez II solution. Dissolve 7.20 g of zinc sulphate (ZnSO4.7H2O)
(Sigma cat. no. Z4750) in 100 mL of distilled water. Store at room
temperature.
Sodium hydroxide (NaOH, 100 mM). Dissolve 4 g of NaOH in
1 L of distilled water. Store at room temperature.

b. Procedure:
Pipette the liquid sample into a 100 mL volumetric flask which contains
approx. 60 mL of distilled water, or weigh sufficient quantity of the
sample into a 100 mL volumetric flask and add 60 mL of distilled
water. Carefully add 5 mL of Carrez I solution, 5 mL of Carrez II
solution and 10 mL of NaOH solution (100 mM). Mix after each
addition. Fill the volumetric flask to the mark, mix and filter.

4. General considerations.
(a) Liquid samples: clear, slightly coloured and approximately
neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used
undiluted (such as wine or fruit juice), the pH of the solution should be
increased to approx. 9.0 using 2 M NaOH, and the solution incubated
at room temperature for 30 min.
(c) Carbon dioxide: samples containing a significant amount of
carbon dioxide, such as beer, should be degassed by increasing the pH
to approx. 9.0 with 2 M NaOH and gentle stirring, or by stirring with
a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with
no ADH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly
coloured samples should be treated by the addition of 0.2 g of
polyvinylpolypyrrolidone (PVPP)/10 mL of sample. Shake the tube
vigorously for 5 min and then filter through Whatman No. 1 filter
paper.
(f) Solid samples: homogenise or crush solid samples in distilled
water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water
at a temperature above the melting point of the fat, e.g. in a 100 mL
volumetric flask at 60°C. Adjust to room temperature and fill the
volumetric flask to the mark with distilled water. Store on ice or in a
refrigerator for 15-30 min and then filter. Discard the first few mL of
filtrate and use the clear supernatant (which may be slightly opalescent)
for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing
protein with perchloric acid; alternatively, clarify with Carrez reagents.
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(i) Samples containing micro-organisms: filter samples through a
0.2 micron filter using a syringe apparatus.

SAMPLE PREPARATION EXAMPLES:

(a) Determination of ethanol in wine.
The ethanol concentration of white and red wine can generally be
determined without any sample treatment (except dilution according to
the dilution table). Typically, for wines with 10-15% (v/v) ethanol, a dilution
of 1:1,000 and sample volume of 0.1 mL are satisfactory.
(b) Determination of ethanol in beer, cider and alcoholic fruit
juices.
After removal of carbon dioxide by increasing the pH of the solution
to approx. 9 with 2 M NaOH and gentle stirring, dilute the sample
according to the dilution table and analyse. Typically, for beverages of
3-8% (v/v) ethanol, a dilution of 1:500 and sample volume of 0.1 mL are
satisfactory.
(c) Determination of ethanol in “non-alcoholic” and “low
alcohol” beers and other beverages.
After removal of carbon dioxide by increasing the pH of the solution
to approx. 9.0 with 2 M NaOH and gentle stirring, dilute the sample
according to the dilution table and analyse. Typically, a dilution of 1:50
and sample volume of 0.1 mL are satisfactory.
(d) Determination of ethanol in spirits (whisky, brandy, etc.).
The ethanol concentration of spirits can generally be determined
without any sample treatment (except dilution according to the
dilution table). However, care should be taken as two dilution steps
will generally be required. Typically, for spirits of 30-60% (v/v) ethanol, a
dilution of 1:10,000 and sample volume of 0.1 mL are satisfactory.
(e) Determination of ethanol in fruit juice, concentrates and
related beverages.
The ethanol concentration of clear, neutral solutions can generally be
determined without any sample treatment (except dilution according
to the dilution table). When used undiluted, the pH of acidic solutions
should be increased to approx. 9.0 with 2 M NaOH. Turbid liquids
generally only require filtering before the dilution step. Coloured
solutions are usually suitable for analysis after dilution to an appropriate
ethanol concentration. However, if coloured solutions require analysis
undiluted, they may need decolourising as follows: treat the solution
with polyvinylpolypyrrolidone (PVPP) or activated charcoal at
2 g/100 mL and filter through Whatman GF/A glass fibre filter paper.
Typically, no dilution is necessary and sample volumes up to 0.5 mL will be
required.

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(f) Determination of ethanol in solid foodstuffs (such as
liqueur chocolates).
Homogenise solid foodstuffs (~ 10 g) using a mortar or homogeniser
if necessary. Add 2 g of representative material to 50 mL of ethanol-
free water and stir for 30 min in a sealed Duran® bottle (heat at
60°C if necessary). Cool the extract (if necessary) and quantitatively
transfer to a 100 mL volumetric flask. Dilute to the mark with
ethanol-free water. Filter the turbid solution, dilute if necessary
(according to the dilution table) and analyse. Typically, a dilution of
1:10 and sample volume of 0.1 mL are satisfactory.
(g) Determination of ethanol in vinegar.
Generally, the analysis of vinegar only requires filtration and dilution
before assay. However, the pH of samples to be analysed undiluted
should be increased to approx. 9.0 using 2 M NaOH before filtration.
Typically, a dilution of 1:20 and sample volume of 0.1 mL are satisfactory.
(h) Determination of ethanol in jam.
Accurately weigh approx. 5 g of representative material into a
100 mL Duran® bottle and extract with 50 mL of ethanol-free
water with agitation for 30 min at 60°C (with the bottle sealed).
Cool the extract and, if acidic, adjust the pH to 9.0 using 2 M NaOH.
Quantitatively transfer the solution to a 100 mL volumetric flask
and adjust the volume to the mark with ethanol-free water. Filter
turbid solutions and dilute if necessary according to the dilution table.
Typically, no dilution is necessary and sample volumes up to 0.5 mL will be
required.
(i) Determination of ethanol in honey.
Accurately weigh approx. 10 g of representative material into a
100 mL volumetric flask containing 40 mL of ethanol-free water and
stopper the flask immediately. Dissolve the honey by slight agitation
at 60°C for 10 min. After cooling, adjust the volume to the mark with
ethanol-free water. Filter the turbid solution and dilute if necessary
according to the dilution table. Typically, no dilution is necessary and
sample volumes up to 0.5 mL will be required.
(j) Determination of ethanol in dairy products.
Accurately weigh approx. 10 g of representative material into a
100 mL Duran® bottle and extract with 50 mL of ethanol-free water
with agitation for 30 min at 60°C (with the bottle sealed). Cool the
extract and quantitatively transfer the solution to a 100 mL volumetric
flask and adjust the volume to the mark with ethanol-free water. Filter
turbid solutions and dilute if necessary according to the dilution table.
Typically, no dilution is necessary and sample volumes up to 0.5 mL will be
required.

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(k) Determination of ethanol in raw unpasteurised kombucha.
Remove micro-organisms by filtering through a 0.2 micron filter using a
syringe apparatus. Remove residual gas from the sample by mixing on a
vortex mixer for approx. 30 s. Typically, a dilution of 1:200 and a sample
volume of 0.1 mL are satisfactory.

REFERENCE:
Beutler, H. O. (1988). Ethanol. “Methods of Enzymatic Analysis”
(Bergmeyer, H. U., ed.), 3rd ed., Vol. VI, pp. 598-606, VCH
Publishers (UK) Ltd., Cambridge, UK.
Absorbance, 340 nm

Blank (M)
Sample (M)
Blank (C)
Sample (C)

Incubationtime,
Incubation time,min
min

Figure 1. Increase in absorbance at 340 nm on incubation of 5 μg of

ethanol with alcohol dehydrogenase and aldehyde dehydrogenase in
the presence of NAD+. (M) Megazyme kit; (C) competitor kit.

NOTES:

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NOTES:

13
NOTES:

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 Bray Business Park, Bray,
Co. Wicklow,
A98 YV29,
IRELAND.
Telephone: (353.1) 286 1220
Facsimile: (353.1) 286 1264
Internet: www.megazyme.com
E-Mail: [email protected]

WITHOUT GUARANTEE
The information contained in this booklet is, to the best of our knowledge, true and accurate, but
since the conditions of use are beyond our control, no warranty is given or is implied in respect of
any recommendation or suggestions which may be made or that any use will not infringe any patents.

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