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A rapid method for determination of ethanol in alcoholic beverages using

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 133

Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003, Pages 133-140

A Rapid Method for Determination of Ethanol in Alcoholic

Beverages Using Capillary Gas Chromatography
MEI-LING WANG1, YOUK-MENG CHOONG2*, NAN-WEI SU3 AND MIN-HSIUNG LEE3

1. Department of Food Health, Chia-Nan University of Pharmacy and Science. 60, Al-jen Road. Sect. 1,
Pau-an, Jen-teh Hsiang, Tainan Hsien 717, Taiwan, R.O.C.
2. Department of Food Science and Technology, Tajen Institute of Technology, 20, Wei-shinn Rd., Yan-puu hsiang,
Pintung Hsien 907, Taiwan, R.O.C.
3. Department of Agricultural Chemistry, National Taiwan University, 1,sec. 4, Roosevelt Road., Taipei 106, Taiwan, R.O.C .

(Received: May 16, 2002; Accepted: September 30, 2002)

ABSTRACT
A simple and rapid method was developed to determine ethanol content in alcoholic beverages using megapore polar column (CP-
Wax 58 CB, 30 m ¥ 0.53 mm) with direct injection gas chromatography. Contrary to packed GLC method, distillation and/or stepwise
dilution of samples were not necessary by the method developed in this study. Ethanol in sample was injected directly into GC for
analysis, after adding suitable amount of internal standard, acetonitrile solution. Using this method, less than 8 min was required to
obtain result since sample preparation, and the limit of quantitation (LOQ) was about 0.5 µg/mL. Recovery studies were performed
using 0.5 mL of red wine and whisky. Each was spiked with ethanol at 50 and 100 mg, respectively. The recoveries were found in the
range of 99~104% and 99~101%, respectively. The coefficients of variation were less than 3.4%. Comparisons of the AOAC method
(AOAC 969.12 and 920.57) with current method showed no significant difference. These results suggested that precision of direct
injection GC method was higher than that of AOAC methods. Several commercial alcohol beverages, including distilled and non-
distilled spirits, were analyzed by the current method. The ethanol content of distilled and non-distilled spirit were found as: 165.2 ±
4.9~415.7 ± 17.6 and 28.2 ± 0.8~141.2 ± 4.9 mg/mL, respectively. Using this GC method, we could change the concentration in gravi-
metric percentage (%, w/v) to volumetric percentage (%, v/v) by the equation (%, w/v) = 0.814 (%, v/v) with linear coefficient R2,
higher than 0.999.

Key words: alcoholic beverages, ethanol, direct injection gas chromatography, quantitative analyses.

INTRODUCTION spectrophotometry (1,4,6), (6) enzymatic method (7-9), (7)

biosensor(10), (8) potentiometry(11), (9) gas chromatography
Ethanol content is very important for the mouth-feel (GC)(1,4,12-21), (10) capillary electrophoresis(2), (11) high
and flavor of alcoholic beverages. Ethanol contents of performance liquid chromatography (HPLC) (22-23), (12)
wine, liqueur, and beer range from 7 ~ 21%(v/v), modular Raman spectrometry(24), (13) near-infrared (NIR)
20~50%(v/v), and 3~6%(v/v), respectively(1). In general, spectroscopy (25) , (14) beer analyzer (4) , and (15) flow
ethanol contents serve as the quality index and taxation injection analysis(26-27). Due to complicated pretreatment
factor for alcoholic beverages(2). After entering WTO, procedures (e.g. sample distillation and accurate weighing
alcoholic beverages in Taiwan are taxed according to the process) and large sample volume required, the two most
ethanol contents, like the taxation system in United popular methods, picnometry and densimetric analysis, are
States(3). The higher the ethanol content in an alcoholic not applicable for samples with small amount. As for
beverage, the higher the tax. A simple, accurate, and quan- oxidation of the distillate and dichromate oxidation spec-
titative analysis method for the determination of different trophotometry, more than 5 mL of sample volume is
levels (0~50%, v/v) of ethanol content is needed as the required for analysis. Besides, the reagents used are highly
standard method of quality control for beverage manufac- toxic and classified. Low stability, low reproducibility and
turers and as a guide for government-related sanitary low accuracy are the disadvantages for enzymatic method,
agencies. biosensor, and potentiometry(9). Raman spectrometry and
Currently, ethanol determination in alcoholic capillary electrophoresis, are not popular due to the
beverages can be performed in several ways: (1) boiling expensive instruments required. Since sample distillation
point depression of the ethanol solution relative to water,(1) and accurate weighing process are still required as the pre-
(2) densimetric analysis(4), (3) refractive index method(1,4), treatment procedures, and only spectrophotometry is used
(4) oxidation of the distillate(1,4-5), (5) dichromate oxidation for analysis, HPLC method obtains a comparatively low
sensitivity(22). Recently developed NIR spectroscopy and
* Author for correspondence. Tel:886-8-7624002 ext 352;
beer analyzer are time consuming in establishing calibration
Fax:886-8-7621972; E-mail: [email protected] curves and have low accuracy as they can be interfered by
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Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

other alcohols in alcoholic beverages. In conclusion, GC grade (purity > 99.5%) ethanol, acetonitrile, 1-propanol,
method is the most appropriate and rapid method for deter- isopropanol, acetone, 1-butanol and t-butanol were obtained
mination of ethanol contents in alcoholic beverages with from ALPS (Taiwan).
complicated alcohol contents and small sample amount.
Bouthilet et al. had developed a packed GC method to II. Methods
analyze ethanol contents in alcoholic beverages (14) .
However, the method required at least 100 mL of sample (I) Preparation of standard solution and internal standard
and distillation as the pretreatment process. The packed solution
GLC(4,12-13,15-16) and capillary GC(17-21), which required
only small sample amount, were then developed. The GC Ten grams of ethanol and acetonitrile were dispensed
methods currently available for determining ethanol into 1000 mL volumetric bottle and then distilled water was
contents in alcoholic beverages are US official methods, added to the 1000-mL mark. This was the 1% (w/v)
AOAC 968.09, 984.14, and 986.12(4), and Taiwan official ethanol standard solution or internal (acetonitrile) standard
method, CNS N6181(15). These official GC methods adapt solution.
different sample preparation procedures when dealing with
different sort of samples, and these samples require distilla- (II) Relative response factor (RRF) of ethanol to acetoni-
tion or dilution as the pretreatment process. Since packed trile
column is used, lower resolution and interference by other
alcohols in alcoholic beverages still exist(12,21). Besides, Ethanol, 1% (w/v), was mixed with 1% (w/v) acetoni-
durability of packed column and reproducibility of the trile in various ratios (ethanol:acetonitrile = 15:1, 10:1, 5:1,
retention time are relatively low(12). When capillary GC is 2:1, 1:1, 1:2, 1:5, 1:10, and 1:15). A linear regression line
used in determining ethanol contents in alcoholic was generated with the GC peak area-under-curve (AUC)
beverages, time-consuming pretreatment procedures, e.g. ratio of ethanol to acetonitrile (Y-axis) against the concen-
solid-phase extraction(17), head space balancing(18), solvent tration ratio of ethanol to acetonitrile (X-axis). Relative
extraction(19) and distillation(20-21), are still required. The response factor (RRF) is the slope of the regression line, as
capillary GC method is one of the most important modern in the Equation 1: RRF = (AS/WS) ÷ (AIS/WIS); in which,
analytical techniques because of its advantage of high reso- AS = ethanol AUC, AIS = acetonitrile AUC, WS = ethanol
lution and sensitivity. Owing to years of research using weight (mg), WIS = acetonitrile weight (mg).
GC, we found that insertion of a ball of glass wool into a
liner of GC injector could effectively prevent the non- (III) Quantitative ethanol determination
volatile compounds form getting into the analytical column
and thus, moderate the interference from the contaminants. (1) Direct injected GC method
We find that commercially available megapore capillary GC
column is superior in water resistance. Even when an Beverage sample solution (0.5 mL) was dispensed into
aqueous solution sample is injected directly into the an l-mL caped sample vial, and then 5 mL of 1% internal
column, the resolution and reproducibility of retention time standard solution (equivalent to 50 mg) was added. After
are almost the same as a new column(28-30). In this study, mixing, 0.1 µL of the sample solution was injected directly
we will prepare and inject the alcoholic beverages with into a GC with syringe. Ethanol content was calculated
adequate amount of internal standard solution directly into according to the Equation 2: Ethanol (mg/mL) = (AS/AIS) ¥
a GC analyzer without any pretreatment process,. Together (WIS/RRF) ¥ 1/V; in which, V = sample volume (mL).
with the application of appropriate column and GC condi-
tions, we plan to develop a simple, rapid and accurate (2) Dichromate oxidation method(1, 4)
method in determining ethanol contents in alcoholic
beverages. Meanwhile, the AOAC methods will be Beverage sample solution (1~5 mL) was steam distil-
compared and assessed in recovery and standard deviations lated to obtain alcoholic eluate (> 50 mL), and then
of intraday/ interday analysis in order to evaluate the oxidized with acidified dichromate. The excessive
accuracy and precision of the study method. potassium dichromate was then titrated with ferric oxide.
The ethanol content in beverage sample could be obtained
by calculating the volume difference of potassium dichro-
MATERIALS AND METHODS mate consumption between sample solution and control
I. Materials solution.

Twenty-six alcoholic beverages, including 12 distilled (3) Distillation-hydrometric method(1, 4)

spirits (whisky, brandy, kaoliang wine, rice wine, Mizhiu
Tou, and medicine wine) and 14 non-distilled spirits (millet Alcoholic volatile compounds in beverage samples
wine, grape wine, fruit wine, Shaohsing wine, and beer), were separated by distillation, and the gravity of the distil-
were purchased from supermarkets in Tainan, Taiwan. LC late was measured by hydrometer. The ethanol content was
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Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

then converted. perature was set initially at 45ûC for 2 min, and then
increased to the final temperature of 245ûC in 1 min at the
(IV) The limit of quantitation (LOQ) of ethanol by GC-FID rate of 45ûC/min. Injection volume was limited to 0.1 µL.
Splittless injection mode was selected.
Ethanol standard solution (10 mg/mL) was initially
diluted with distilled water to make concentrations of 50,
25, 10, 5, 2, ,1, 0.5, 0.1, and 0.05 µg/mL, and then the RESULTS AND DISCUSSION
internal standard solution was added to each solution. I. Analysis of GC Conditions
Solution was injected directly into a GC, which was
equipped with an FID detector. The FID signal was set at During the selection of GC column, we had compared
range = 1, and attenuation = 1. The coefficient of variation the polar and mid-polar megapore capillary columns in the
(CV, %) of ethanol recovery was set at 15%. This was the determination of ethanol contents in alcoholic beverages.
LOQ of ethanol(28, 31). Each analysis was carried out in Because there was no sample pretreatment procedures
triplicate. required, and the sample could be injected directly into a
GC for analysis, polar CP-Wax 58 CB column was the most
(V) Recovery appropriate column for the determination of ethanol
contents in alcoholic beverages. When applying the GC
5 and 10 mL of 1% (w/v) ethanol standard solution conditions described in Materials and Methods, the
(equivalent to 50 mg, and 100 mg, respectively) was added retention time of ethanol standard solution was 2.73 min
into 0.5 mL of red wine and whisky (in 20-mL vial), (Figure 1A, 1B, and 1C). The resolution of megapore
respectively; and then 5 mL of 1% (w/v) internal standard capillary column (Rs = 5.8) was better than the packed
solution was added. After gentle mixing, 0.1 µL of sample GLC method (Rs = 1.4~1.9), as described in AOAC(4) and
solution was injected into a GC with syringe for the deter- CNS(15). The GC peaks of the other minute components in
mination of the ethanol content. Each analysis was carried alcoholic beverages could be separated effectively with
out in triplicate. Blank sample was analyzed at the same megapore capillary GC (data not shown).
time. In the selection of internal standard, distilled spirits
(e.g. whisky) and non-distilled spirits (e.g. red wine) were
(VI) Validation of the analysis method
47.4 2 3.35

Ethanol standard solution (500 mg/mL) was diluted

37.6 (A)
with distilled water to make concentrations of in 500, 250, 1 2.75
100, 50, 20, and 10 mg/mL. Each solution (0.5 mL) was 27.81
(mVolt)
dispensed into a 20-mL caped vial. After gentle mixing 18.01
with 5 mL of 1% (w/v) internal standard solution (equiva- 8.22
lent to 50 mg), 0.1 µL of the mixture solution was injected
-1.58
into a GC with syringe. Each concentration of ethanol 0.0 1.613 3.226 (min) 4.839 6.452 8.065
standard solution was measured in triplicate in 1 day 1 2.76 23.35
408.2
(intraday) or in 3 successive days (interday). Standard
(B)
deviation (SD) and coefficient of variation (CV, %) were 322.2
measured to evaluate precision of the method. The relative 236.2
(mVolt)
error of the mean (REM) was measured to evaluate 150.2
accuracy of the method. REM was calculated according to
64.1
the equation: REM (%) = [(measured value - true value) ÷ 3.87 4.56 5.6825 5.69 7.7239 7.7

(true value) ¥ 100%]. -21.9

0.0 1.613 3.226 (min) 4.839 6.452 8.065
1 2.73 2
(VII) GC conditions 575.2 3.31

455.1 (C)
This study used Trace GC 2000 (TermoQuest, Milan,
335.0
Italy), which was equipped with computer integrator (mVolt)
software (Chrom-Card version 1.06 for Trace GC, 214.9
TermoQuest, Milan, Italy) and an FID detector. The flow 94.8 3.463.83 4.54 5.56 5.886 6.26 6.74 6.94 7.7
rates of H2 and air were set at 30 and 300 mL/min, respec-
-25.3
tively. The temperature of the FID detector and the 0.0 1.613 3.226 (min) 4.839 6.452 8.065
injection port was set at 285ûC, and 225ûC, respectively. Figure 1. Gas chromatograms of (A) ethanol and acetonitrile
Nitrogen (N2) in the flow rate of 2 mL/min was used as the authentic compound; (B) ethanol in non-distilled spirit (red wine) and
carrier gas. The CP-Wax 58 CB separation column (30 m ¥ (C) ethanol in distilled spirit (whisky) by splittless injection method.
0.53 mm, Chrompack, Netherlands) was used. Oven tem- Peaks: 1 = ethanol, 2 = acetonitrile (IS).
136

Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

added with minute quantity of 1-propanol, 2-propanol, ace- sample amount and simple pretreatment procedures, is
tonitrile, acetone, 1-butanol, and t-butanol. The ethanol developed and can be adapted in different sort of alcoholic
contents were then analyzed according to the above- beverages with ethanol contents between 0~500 mg/mL.
mentioned method. The results had shown that retention Ethanol standard solutions in the concentrations of 5,
times of the 6 standard solutions were 4.43, 4.37, 3.32, 10, 20, 30, 40, and 50% (w/v) were prepared, and then 0.5
4.06, 5.96, and 5.72 min, respectively (data not showed). mL of each solution was dispensed into a 20-mL caped
When GC chromatograms of distilled spirits were vial. After adding and gentle mixing with 0.5, 1.0, 2.0, 2.5,
compared with non-distilled spirits, no overlapping of ace- 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mL of 1% (w/v) internal
tonitrile GC peaks was observed. Meanwhile, the GC standard solutions, respectively 0.1 µL of each solution was
peaks of acetonitrile and ethanol were closer to each other then injected directly into a GC for analysis. In order to
than the other compounds. Acetonitrile was then selected achieve better accuracy, samples with high contents of
as the internal standard. Due to the possibility of its ethanol needed larger amount of 1% (w/v) internal standard
existence in some sort of alcoholic beverages (1,12) , 1- solution (Figure 2). For different beverage samples with
propanol, acetone, t-butanol, or 1-butanol, which acted as ethanol contents of 5, 10, 20, 30, 40, and 50% (w/v), the
the internal standards GLC methods (AOAC(4), CNS(15), ratios of 1% (w/v) internal standard solution required were
and other researches (12, 16)), are not appropriate as the 2, 4, 6, 8, 10, and 12, respectively. In order to obtain
internal standard for the determination of ethanol contents results with high consistency, 10x amount of the internal
in alcoholic beverages. Acetonitrile is the most appropriate standard solution was added when ethanol contents in
internal standard for the determination of ethanol contents alcoholic beverages were determined in this study.
in alcoholic beverages, because no acetonitrile could In conclusion, we have developed a method which
possibly exist in alcoholic beverages. requires only 0.5 mL, even 0.1 mL (data not shown), of
For selection of the GC conditions, we initially beverage sample amount mixed with adequate amount
selected low temperature, 45ûC for 2 min, and then (10x) of 1% internal standard solution. This sample
increased rapidly (at the rate of 45ûC/min) to 245ûC in 6 solution is suitable for injecting directly into a GC for
min. Ethanol and acetonitrile were eluted at 80ûC and determination of ethanol content. No pretreatment
100ûC, respectively. Through this process, sample compo- procedure is required. This study method, is more simpli-
nents will be eluted very rapidly and it takes 7-8 min to fied than official methods AOAC(4) and CNS(15), which
complete a sample analysis. require dilution or distillation as the pretreatment proce-
dures. This study method is also easier in operation
II. Relative Response Factor of Ethanol to Iinternal compared with methods developed by Collins et al.(2) (CE
Standard method) and by Antonelli(12) (packed GLC method), which
require ultra-filtration (0.45 µm filter) and dilution as pre-
In this study, we selected acetonitrile as the internal
standard for quantitative determination of ethanol contents
in alcoholic beverages. In order to obtain an accurate quan- 100
tification, the RRF value of ethanol against acetonitrile
needed to be specified, then the ethanol contents could be 80
calculated according to Equation 2. When the AUC ratio of
Recovery (%)

ethanol to acetonitrile (Y-axis) was plotted against the con-

60
centration ratio of ethanol to acetonitrile (X-axis), a linear
regression equation (Y = 0.952 X) was generated (R2

0.999). The slope of the linear regression line, 0.952, is the 40
RRF of ethanol to the internal standard. The linearity of
the adjusted regression line is in the range of 0-500 mg/mL. 20

III. The Effect of Internal Standard Volume to the Accuracy

0
of Ethanol Content Quantification for Various Alcoholic 0 2 4 6 8 10 12 14 16
Beverages Volume ratio of IS to ethanol

5%(w/v) 10%(w/v) 20%(w/v)

Ethanol contents in commercial alcoholic beverages 30%(w/v) 40%(w/v) 50%(w/v)
vary a lot, even for the same sort of beverages, e.g. 3~6%
Figure 2. Effect of the volume ratio of 1% internal standard (IS)
(w/v) for beers, 7~21% (w/v) for fermented wines, and
solution to various concentration of standard ethanol solution(1) on
20~50% (w/v) for distilled spirits.(1) Therefore, different the quantitative determination(2) of ethanol content.
pretreatment procedures are required when using the (1) The volume ratio of IS solution to standard ethanol solution, v/v,
current official packed GLC methods for the determination 0.5 mL of standard ethanol solution was used in each analysis.
of ethanol contents in various alcoholic beverages. In this (2) Recovery(%) = (level of ethanol detected)/( ethanol level of the
study, a rapid capillary GC method, which requires smaller standard solution) 100%.
 137

Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

treatment procedures. spirit (whisky), were found in Table 2. The results show
that the recoveries of ethanol with 50 and 100 mg fortifica-
IV. The Limit of Quantitation (LOQ) of Ethanol by GC-FID tion level into 0.5 mL of red wine and whisky were in the
range of 99~104%, and 99~101%, respectively, with coeffi-
In this study, 1% (w/v) ethanol standard solution was cient of variable (CV) less than 3.4%. When 6 ethanol
successively diluted, and then mixed with adequate amount samples with known concentrations (10~500 mg/mL) were
of internal standard. The mixture solutions were then analyzed using the current method, the coefficient of
injected directly into a GC, equipped with an FID detector. variant (CV, %) for intraday (1 day) and interday (succes-
The FID signal was set at range = 1, and attenuation = 1. sive 3-day) analyses was 1.0~4.7%, and 1.5~3.6%, respec-
The coefficient of variation (CV, %) of ethanol recovery tively. These results indicate that the precision of the study
was set at 15%. This was the LOQ of ethanol.(28, 31) The method is very high. In addition, the REM of intraday and
results showed that the LOQ of ethanol was around 0.5 interday analyses was -3.5~4.2% and -1.0~3.0%, respec-
µg/mL (Table 1). tively. It indicates that the study method possesses very
good accuracy (Table 3). Using the study method, after
V. Validation of the Analysis Method beverage samples are mixed with adequate amount of
internal standard solution, they are ready for injecting
The recoveries of ethanol standard solutions, which directly into a GC for the determination of ethanol contents.
spiked into a non-distilled spirit (red wine) and a distilled It takes 7~8 min to complete an analysis. The GC chro-
matograms were illustrated in Figure 1A, 1B, and 1C. This
Table 1. Limit of quantitation, LOQ, of ethanol using gas chromato- study method is one of the most simple, rapid, and accurate
graphy with FID detector by direct injection method methods published for quantitative determination of ethanol
Ethanol content (mg/mL)a Recovery (%)b CV (%)c contents in alcoholic beverages. It also can be proposed as
50.0 105.4 ± 0 2.4 2.3 the reference method for routine analysis.
25.0 97.5 ± 0 4.4 4.5
10.0 98.4 ± 0 6.6 6.7 VI. Comparison of Study Method with Dichromate
05.0 104.7 ± 0 9.2 8.8 Oxidation Method and Distillation-Hydrometric Method
02.0 106.7 ± 0 5.4 5.1
01.0 095.7 ± 0 8.9 9.3
00.5 105.3 ± 13.3 12.6
The study method was compared with dichromate
00.1 107.2 ± 22.3 20.8 oxidation method (AOAC 969.12) and distillation-hydro-
00.05 138.4 ± 41.1 29.7 metric method (AOAC 920.57) for the determination of
a: FID range = 1, Attenuation = 1. ethanol contents in red wine, rice wine, and whisky (Table
b: Average of triplicate analyses. 4). No statistically significant difference between these 3
c: Coefficient of variation (cv %). methods (p < 0.05, n = 3) was observed, which indicates

Table 2. Recoveries of the ethanol in spiked commercial alcoholic beverage by direct injection gas chromatographic method
Sample Blanka (mg) (A) Amount added (mg)(B) Amount foundb (mg) (C) Recovery(%)c CV(%)d
Red wine 90.5 ± 2.7 50 142.7 ± 4.0 104.0 2.8
100 189.1 ± 3.9 98.6 2.1
Whisky 317.9 ± 11.6 50 367.3 ± 10.3 99.0 2.8
100 418.5 ± 14.4 100.6 3.4
a: Ethanol in 0.5 mL alcoholic beverage.
b: Average of triplicate analyses.
c: Recovery(%) = (C–A) / B ¥ 100%.
d: Coefficient of variation (cv %).

Table 3. Precision and accuracy of intraday and interday validation for concentration range from 10 to 500 mg/mL
Concentration Intraday (n = 3)a Interday (n = 9)b
(mg/mL Precision Accuracy Precision Accuracy
Mean ± SD (CV %) REM(%)c Mean ± SD (CV %) REM(%)
10 9.9 ± 0.1 (1.0) -1.0 10.2 ± 0.2 (2.0) 2.0
20 19.7 ± 0.4 (2.0) -1.5 19.8 ± 0.3 (1.5) -1.0
50 52.1 ± 1.9 (3.6) 4.2 50.7 ± 0.8 (1.6) 1.4
100 96.5 ± 3.1 (3.2) -3.5 102.4 ± 2.7 (2.6) 2.4
250 259.3 ± 8.5 (3.3) 3.7 255.6 ± 7.1 (2.8) 2.2
500 487.8 ± 23.1 (4.7) -2.4 515.2 ± 18.7 (3.6) 3.0
a: n = 3, Repeated injection three times on the same day.
b: n = 9, Repeated injection three times each day and a successive three-day.
c: REM = relative error of the mean.
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Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

Table 4. Comparison of AOAC methods and proposed method for the determining of ethanol content in alcoholic beverages.
Sample Proposed methoda Dichromate oxidation methodb Distisllation hydrometric methodc
Ethanol (mg/mL) CV (%) d Ethanol (mg/mL) CV (%) d Ethanol (mg/mL) CV (%)d
Red wine 107.4 ± 2.9 2.7 110.6 ± 15.7 14.2 114.1 ± 13.8 12.1
Rice wine 158.2 ± 6.0 3.8 151.4 ± 16.8 11.1 153.2 ± 14.7 9.6
Whisky 326.3 ± 7.8 2.4 311.3 ± 37.7 12.1 307.6 ± 40.5 13.2
a: Direct injection GC method in this study.
b: AOAC 969.12 method.
c: AOAC 920.57 method.
d: Average of triplicate analyses, coefficient of variation (cv%).

Table 5. Ethanol levels determined in alcoholic beverages. (e.g. acetic acid or sulfur dioxide) in distillation process(1).
Sample Ethanol Content Also, highly toxic reagents used in dichromate oxidation
Distilled spirit mg/mL %, v/v method make the operation and waste disposition trouble-
Whisky 1 327.6 ± 11.8 40.3 ± 1.4 some and dangerous.
Whisky 2 317.8 ± 12.4 39.0 ± 1.5
In the application point of view, the simple, rapid, and
Whisky 3 331.3 ± 13.2 40.7 ± 1.6
White liquor 1 328.9 ± 09.5 40.4 ± 1.2 direct-injected capillary megapore column GC method
White liquor 2 413.6 ± 19.7 50.8 ± 1.8 developed in this study has shown more advantages and
Kaoliang1 327.5 ± 12.4 40.2 ± 1.5 values compared with the AOAC (4) and CNS official
Kaoliang2 415.7 ± 17.6 51.1 ± 1.7 methods(15). Currently, different beverage samples require
XO 313.0 ± 13.6 38.5 ± 1.7 unique sample preparation procedures when the official
VSOP 289.7 ± 08.2 35.6 ± 1.0 AOAC and CNS packed GLC methods are applied, and dis-
Rice wine 165.2 ± 04.9 20.3 ± 0.6
tillation or dilution is required as the pretreatment
Mizhiu Tou 324.3 ± 10.3 39.8 ± 1.3
Medecine wine 197.7 ±05.3 24.3 ± 0.7
procedure. Since the resolution is low for packed GLC
Non-distilled spirit methods (12), the accuracy for quantitative analysis and
Millet wine1 77.2 ± 02.9 9.5 ± 0.4 reproducibility of ethanol contents will be interfered for
Millet wine2 81.3 ± 01.4 9.9 ± 0.5 those beverages with high and complicated contents of
Red wine 1 90.1 ± 03.7 11.1 ± 0.5 volatile compounds (e.g. fruit and medicine wines)(21). In
Red wine 2 106.5 ± 04.5 13.1 ± 0.5 addition, packed GLC column has shorter shelf life and
Red rose 1 128.4 ± 05.5 15.8 ± 0.7 lower durability. Besides, currently adapted capillary GC
Red rose 2 117.1 ± 03.7 14.4 ± 0.5
methods require time-consuming sample pretreatment pro-
Fruit wine1 91.2 ± 02.6 11.2 ± 0.3
Fruit wine2 112.5 ± 03.4 13.8 ± 0.5 cedures, such as solid-phase extraction(17) , headspace
White wine 1 100.4 ± 03.1 12.3 ± 0.4 balancing(18), solvent extraction(19), and distillation(20-21).
White wine 2 103.9 ± 03.7 12.8 ± 0.5 The study method we developed is a simple and rapid
Shao-Hsing wine 1 141.2 ± 04.9 17.3 ± 0.6 method, without any sample pretreatment procedure
Shao-Hsing wine 2 138.6 ± 05.5 17.0 ± 0.7 required. After beverage samples mixed with adequate
Beer 1 28.2 ± 00.8 3.5 ± 0.1 amount of the internal standard solution, the samples are
Beer 2 29.8 ± 00.6 3.7 ± 0.1
ready to be injected into a GC for determination of ethanol
a: Average of triplicate analyses.
contents.

the study method we proposed obtains very high accuracy. VII. The Ethanol Contents of Commercial Alcoholic
The ethanol contents determined by the study method were: Beverages
107.4 ± 2.9 mg/mL in red wine, 158.2 ± 6.0 mg/mL in rice
wine, and 326.3 ± 7.8 mg/mL in whisky (CV = 2.4~3.8%). The ethanol contents of commercial alcoholic
The ethanol contents determined by dichromate oxidation beverages are usually labeled in volume percent (%, v/v).
method were: 110.6 ± 14.2 mg/mL in red wine, 151.4 ± Sometimes, it is convenient to be labeled in weight-volume
16.8 mg/mL in rice wine, and 311.3 ± 37.7 mg/mL in percent (%, w/v). In this study, the ethanol standard
whisky (CV = 11.1~14.2%). The ethanol contents deter- solutions, 10%, 20%, 30%, and 40%, were prepared both in
mined by distillation-hydrometric method were: 114.1 ± weight-volume percent (%, w/v), and in volume percent (%,
13.8 mg/mL in red wine, 153.2 ± 14.7 mg/mL in rice wine, v/v). After beverage sample were mixed with adequate
and 307.6 ± 40.5 mg/mL in whisky (CV = 9.6~13.2%). amount of internal standard and injected into a GC for the
These results also indicate that the study method has better determination of ethanol content, the relative response
precision than the AOAC methods. Both AOAC methods factor (RRF) was then calculated according to the above
require dilution and distillation as the pretreatment proce- mentioned equation. The linear regression line of ethanol
dures. High deviation may be found if the connection tubes weight-volume percent (%, w/v, Y-axis) against ethanol
are not tightly secured, condensation efficiency is low, or volume percent (%, v/v, X-axis) was: Y = 0.814 X, with R2
the sample contains high contents of volatile compounds = 0.999 (Figure 5).
 139

Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

The ethanol contents of the 26 commercial alcoholic 4. AOAC Official Method of Analysis. 1990. Wines. In
beverages (12 distilled spirits and 14 non-distilled spirits) Official Methods of Analysis of AOAC International.
were analyzed with the study method. As shown in Table 15th edition, pp. 739-750.
5, the ethanol contents of 12 distilled spirits were: 5. Caputi, A. Jr. and Wright, D. 1969. Collaborative study
317.8~331.3 mg/mL (39.0~40.7%, v/v) in whisky, of the determination of ethanol in wines by chemical
328.9~413.6 mg/mL (40.4~50.8%, v/v) in white liquor, oxidation. J. Assoc. Off. Anal. Chem. 52: 85-88.
327.5~415.7 mg/mL (40.2~51.1%, v/v) in kaoliang wine, 6. Caputi, A. Jr., Ueda, M. and Brown, T. 1968.
313.0 mg/mL (38.5%, v/v) in XO, 289.7 mg/mL (35.6%, Spectrophotometric determination of ethanol in wines.
v/v) in VSOP, 165.2 mg/mL (20.3%, v/v) in rice wine, Am. J. Enol. Vitic. 19: 160-165.
324.3 mg/mL (39.8%, v/v) in Mizhiu Tou, and 197.7 7. Jones, A. W. 1995. Measuring ethanol in saliva with the
mg/mL (24.3%, v/v) in medicine wine. The ethanol QED enzymatic test device: comparison of results with
contents of the 14 non-distilled spirits were: 77.2~81.3 blood and breath alcohol concentration. J. Anal.
mg/mL (9.5~9.9%, v/v) in millet wine, 90.1~128.4 mg/mL Toxicol. 19: 169-174.
(11.7~15.8%, v/v) in grape wine, 91.2~112.5 mg/mL 8. McCloskey, L. P., and Replogle, L. L. 1974. Evaluation
(11.2~13.8%, v/v) in fruit wine, 100.4~103.9 mg/mL of an enzymatic method for estimating ethanol in wines
(12.3~12.8%, v/v) in white wine, 138.6~141.2 mg/mL using an enzyme kit. Am. J. Enol. Vitic. 25: 194-197.
(17.0~17.3%, v/v) in Shaohsing wine, and 28.2~29.8 9. Mason, M. 1983. Report of the technical subcommittee
mg/mL (3.5~3.7%, v/v) in beer. The ethanol contents of on ethanol determination via immobilized enzyme. J.
these alcoholic beverages were consistent with their labels, Am. Soc. Brew. Chem. 41: 89-90.
without any fraud found. 10. Mason, M. 1983. Ethanol determination in wine with an
immobilized enzyme electrode. Am. J. Enol. Vitic. 34:
173-175.
CONCLUSIONS 11. Kokovkin, V. V. and Smolyakov, B. S. 1995. Ethanol
determination in aqueous solutions and wine stocks by
In this study, it takes only 7~8 min to complete a potentiometry with ion selective electrodes. J. Anal.
sample analysis for the determination of ethanol content in Chem. 50: 569-573.
a beverage sample. A sample solution (0.5 mL) is mixed 12. Antonelli, A. 1994. Ethanol determination by packed
with adequate amount (5 mL) of 1% (w/v) internal standard GLC: a quick method with small sample amount and
solution (acetonitrile, equivalent to 50 mg), and injected high sensitivity. Vitic. Enol. Sci. 49: 165-167.
into a capillary GC. The study method we developed can 13. Caputi, A. Jr. and Mooney, D. P. 1983. Gas chromatog-
be applied to alcoholic beverages with different alcoholic raphy determination of ethanol in wine: Collaborative
contents, and with the advantages of simple sample pre- study. J. Assoc. Off. Anal. Chem. 66: 1152-1157.
treatment procedures, rapidity and accuracy, and maybe a 14. Bouthilet, R. J., Caputi, A. Jr. and Ueda, M. 1961.
routine analysis method in substitution of current AOAC Analysis of ethanol in wine by gas liquid partition chro-
and CNS methods. matography. J. Assoc. Off. Anal. Chem. 44: 410-414.
15. CNS, 1983, Method of test for beverage-Determination
of alcohol content. Chinese National Standard, Taiwan.
ACKNOWLEDGEMENT Code No. 10292 (N6181).
16. Stackler, R. and Christensen, E. N. 1974. Quantitative
This research was supported by Project 90-FH-10 from determination of ethanol in wine by gas chromatogra-
Chia-Nan University of Pharmacy and Science. The authors phy. Am. J. Enol. Vitic. 25: 202-207.
would like to thank Dr. Hui-Cheng Chen for his translation 17. Kumazawa, T., Seno, H., Lee X. P., Ishii, A., Suzuki,
work. O., and Sato, K. 1996. Detection of ethanol in human
body fluids by headspace solid phase micro extraction
(SPME) capillary gas chromatography. Chromatogr. 43:
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Journal of Food and Drug Analysis, Vol. 11, No. 2, 2003

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