Journal of Food Composition and Analysis 17 (2004) 187–196

Original Article

A rapid and accurate method for determination of methanol in alcoholic beverage by direct injection capillary gas chromatography
Mei-Ling Wanga, Jih-Terng Wangb, Youk-Meng Choongb,*

Department of Food Health, Chia-Nan University of Pharmacy and Science, 60, Al-jen Road. Sect. 1, Pau-an, Jen-teh Hsiang, Tainan, Taiwan b Department of Food Science and Technology, Ta-Jen Institute of Technology, 20, Wei-shinn Rd., Yan-puu Hsiang, Pintung, Taiwan Received 16 December 2002; received in revised form 5 July 2003; accepted 28 August 2003

Abstract To directly determine methanol content in alcoholic beverages a simple and rapid method using polar megapore column (CP-Wax 58 CB, 30 m  0.53 mm) is presented. With acetonitrile as internal standard, chromatogram showed a retention time of 3.06 min for methanol and 4.21 min for internal standard. The linear range of the calibration curve for methanol was wide, ranges from 2.0 mg/mL to 20 mg/mL (r2 ¼ 0:999). Standard addition of methanol to wine and whisky lead to excellent recoveries of 101–107% and 94–103%, respectively. Examining coefficients of variation and relative errors obtained from repeated analyses in a range of concentrations (50–1000 mg/mL), this method was found to display high precision and accuracy. With this method, no pre-treatment of samples was required and it took less than 9 min for one single analysis from direct injection of original samples to obtaining data. r 2003 Elsevier Ltd. All rights reserved.
Keywords: Alcoholic beverages; Methanol; Direct injection gas chromatography

1. Introduction Methanol is an alcohol toxic to mammals and is widely used as solvent for extraction or in detergents. Accidental intake of the compound will result in severe intoxication due to accumulation of highly toxic metabolites such as formaldehyde and formic acid (Bindler et al., 1988). Acute intoxication of methanol usually causes headache, vertigo, fatigue, nausea, vomiting,
*Corresponding author. Tel.: +886-8-762-4002; fax: +886-8-762-1972. E-mail address: (Y.-M. Choong). 0889-1575/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfca.2003.08.006

this method requires long operation time (more than 4 h). Because methanol is cheap and is easily accessible. near-infrared spectroscopy (Van den Berg et al. which is a time-consuming protocol. head-space equilibrium (Davoli et al. blindness and even death (Robins et al. Cheung and Lin. low reproducibility and accuracy are their common defects. 1981. 1987). as increasing commercial exchanges of these alcoholic products between countries are expected as more and more countries join the World Trade Organization (WTO). high performance liquid chromatography (HPLC) (Chen et al... being also an AOAC qualified protocol for methanol determination (AOAC. notwithstanding the use of packed column or capillary column.. converting to capillary column (Wilson et al. it has been used in the production of imitated spirits and wine. 2001. and gas chromatography (GC) (Caggiano and Beck. They used packed column and had to distil large amount of sample (about 100 mL) to obtain enough methanol for analysis. 1990. several studies have found that the addition of pectolytic enzymes induces an increase of methanol levels in various fermentation products such as ciders (Massiot et al...ARTICLE IN PRESS 188 M. 1996). Wang et al.. membrane filtration and derivatization. Therefore. / Journal of Food Composition and Analysis 17 (2004) 187–196 blurred vision. 1963. Salvador. 1983). 1993). 1985). solvent extraction (Pollack and Kawagoa. Many methods have been developed to detect methanol at various sensitivities in beverage products. With lower resolution and less stability. As with the enzymatic. 1990). Segura et al. 1981). The AOAC qualified GC method to determine methanol suggests using packed column (AOAC. Application of GC analysis to determine methanol content was first developed by Caggiano and Beck (1963). 1997). 2002). and this has caused the death or blindness of many people in China. 1990). however. D!az et al. 1990). Among them. These pre-treatments are the most . biosensor and titrimetric methods. Pollack and Kawagoa.. many current methods for determination of methanol still suggest pre-treatment of samples with solid-phase extraction (Pereira et al. titrimetric method (AOAC. Determination of methanol by HPLC requires pre-treatment of the sample with distillation. a painstaking process to treat highly toxic reagent chromic acid. 1976. and will be interfered by carbohydrates contained in the sample (Holtzapple and Humphrey. 1991) and/or distillation (Blanch et al. Revilla and ! ! Gonzalez-SanJose. Oral lethal dose of methanol for humans ranges from 340 mg to 1 mg/kg of body weight. Servili et al. 1999). establishing the calibration curve takes a long time and the method is liable to be interfered by accompanying ethanol. enzymatic method (Mizgunova et al. 1986. 1994) or wines (Brown and Ough. 2002). Near infrared spectroscopy is a recently developed method to measure the content of methanol in alcoholic samples. biosensor detection (Sekine et al.. 1991. Wilson et al. Brazil.. the development of a quick and accurate method to determine the content of methanol in alcoholic beverages is urgently required. 1986)... However. Liu et al. Furthermore. 1998).. such as chromotropic acid colorimetric method (AOAC. 1990). Kuo et al. reproducibility of retention time obtained from packed column method is notoriously poor. 1992. The manufacturing of herbal medicine usually uses methanol as a solvent to extract natural ingredients. However.-L. ı chromotropic acid colorimetry is the most frequently used method. 1992). 1998. The working period for packed column is also known to be comparatively shorter than with capillary column. 1991) and using more sensitive detectors (Davoli et al. a fact that will also concern us about the residual level of methanol in these products (Kuo et al. AOAC. for consumer protection.. Subsequent GC method to determine methanol was modified to reduce the needed sample volume and to increase sensitivity using combinations of various packing materials (Sims. 2003).. 1991. India and Taiwan...

In order to avoid cross contamination between samples. we present a rapid GC method. respectively.10:1. 17 samples of wine and beer. The whisky. tert-butanol. The temperatures at injector port and detector were set at 210 C and 280 C. 2.06 for Trace GC.5%). and splitless injection (about 0. which was initially set at 38 C for 3 min. The remaining samples were the products of Taiwan Tobacco and Liquor Co. 1:50 (v/v) etc. 1:2. amyl alcohol and isoamyl alcohol were purchased from ALPS Chemical Co. Standard solutions of the above authentic chemicals were made up with 100 mg in 100 mL distilled water (0. the syringe for injection was completely dried out by heating with a lighter for several seconds after rinsing the syringe with distilled water. 2003).ARTICLE IN PRESS M. red wine and white wine samples were imported from France. 1-butanol. But different ı sample susceptibilities to the method has not been explored.. respectively. Italy) equipped with a computer integrator software (Chrom-Card version 1.1 mL for each injection) was used. 1:1. The feasibility remained valid even for samples with color or complex components like those in medicinal liqueur. TermoQuest.. First.2. Wang et al. ethanol. and white liquor samples were imported from China. w/v). Determination of methanol content Prior to developing the equation for calculation of methanol content. BRUNEL XO.5 mm.1. RRF value was derived from the slope of a regression curve that regressed peak area ratios of methanol to internal standard on their concentration ratios. Taiwan.3. requiring only 9 min. such as methanol. 2:1. Samples and reagents Alcoholic beverages including 12 samples of spirit. to determine the content of methanol in various alcoholic beverages by direct injection of the sample into a megapore capillary GC column. a 30 m CP-Wax 58 CB megapore capillary column (0. 1:10. 2. acetonitrile.1:5. 2. standard methanol and acetonitrile solution were mixed in the following combinations: 20:1.-L. 1-propanol. Netherlands) and a flame ionizing detector (H2: 30 mL/min and air: 300 mL/min). GC analysis The analysis of methanol was conducted in a Trace 2000 GC (TermoQuest. film thickness: 1. and then subjected to GC analysis.53 mm id. / Journal of Food Composition and Analysis 17 (2004) 187–196 189 time-consuming steps and sources of frequent errors. Milan. Milan. elevated to 250 C at the rate of 50 C/min and maintained for 1 min. Materials and methods 2. Subsequently. 5:1.. Italy). Liquid-chromatographic-grade solvents (purity above 99. the relative response factor (RRF) of methanol to internal standard acetonitrile was determined. 1:20. Direct injection has also been applied to determine volatile alcohols in wine using capillary GC column (D!az et al. Oven temperature was controlled with a temperature elevation program during analysis. the content of methanol for each sample . Hennessy VSOP. In this study. ChromPack. Flow rate of carrier gas nitrogen was set at 3 mL/min.1%. and 3 samples of Chinese medicinal liqueur were purchased from supermarkets located at Pintung or Tainan area.

1A. 1-propanol. the temperature program for GC analysis was elevated rapidly to 250 C at 7 min after elution of methanol. 200. acetonitrile was a proper internal standard for methanol determination. the regression of the peak area ratios on the concentration of methanol fitted a linear relationship well (r2 ¼ 0:999).1% (w/v) acetonitrile as internal standard. The typical separation profile of acetonitrile and known constituents in wine and spirit is shown in Fig.673.ARTICLE IN PRESS 190 M. Recovery test and validation of the method Supplements of 100 and 500 mg methanol standard were added to 1 mL red wine and whisky. and 5.72 min. RRF value of standard methanol to internal standard acetonitrile was also determined. In order to evaluate the validation of the direct GC method. After spiking with 100 mL 0. and relative error of mean was for index of accuracy.49. 4. 4. / Journal of Food Composition and Analysis 17 (2004) 187–196 was determined according to the peak area of methanol and acetonitrile obtained from GC analysis and the RRF value. about 0. When compared with other seven authentic compounds. 2. the methanol content in a sample could be determined within 8–9 min.73. The other components in the sample could be excluded after 8 min.4. acetonitrile. Obviously. 500.-L. megapore capillary columns with high and medium polarity were compared. Standard deviations and coefficients of variation were calculated for index of precision. respectively. Every test was conducted in triplicate and certification of variance was also determined. Fig. a range of standard methanol concentrations (50. and that of ethanol.15. 3. The LOQ of methanol was determined by recovery and coefficient of variation of the data. 1A. Wang et al. 4. respectively. and linear range for detection of methanol was from 2 mg/mL to 20 mg/mL. as shown in Fig. The wide linear range of methanol detection also suggested that this method might be suitable for determination with samples with highly varied methanol content. As shown in Fig. 1-butanol. isoamyl alcohol and amyl alcohol were 3. respectively. When methanol concentration was serially diluted from . The limit of quantification (LOQ) of methanol by GC was also determined using diluted methanol standard when the settings of range and attenuation were at 1. Consequently.1 mL of the mixture was subjected to GC analysis. RRF value calculated from the slope of regression curve was 0. The LOQ of methanol by direct injection GC method was further determined by serial dilution of methanol standard. 2. 5. To shorten the operation time. Results and discussion In order to determine the optimum condition for GC analysis of methanol.17.42.06 min. tert-butanol. the retention time of methanol was 3.38. 100. 1 also shows that acetonitrile came out at the stable baseline region and performed clear separation from other constituents of samples derived from spirits and wine. 1000 mg/mL) was determined 3 times a day (intra-day validation) and the same determination was repeated on 5 successive days (inter-day validation). such as imitated spirits produced from industrial alcohol. Recovery of the methanol was determined by comparing the original content of methanol in the spirit sample and the content with standard addition. Preliminary tests suggested that the column with higher polarity (CP-Wax 58 CB) had the best capability to separate methanol from other components in alcoholic beverage. 5. Each determination was conducted in triplicate.

n ¼ 3). Gas chromatograms of authentic compounds and samples. the recovery of methanol and the coefficient of variation between replicates increased to over 120% and 15%.2 and 108. 7: isoamyl alcohol. 1.7 and 9. the percent recoveries of methanol were between 98.173.D. This result suggests that the LOQ of methanol by direct injection method was about 2 mg/mL. and the coefficient of variation was between 2. 25 to 2 mg/mL.-L.6. when methanol concentration was diluted below 1 mg/mL. 3: acetonitrile. respectively. However.1% (w/v) authentic compounds. 5: isobutanol. 2: ethanol. / Journal of Food Composition and Analysis 17 (2004) 187–196 191 Fig. 6: butanol.4 (mean7S.ARTICLE IN PRESS M. (A) 0. (C) red wine (keys of numbers above peaks: 1: methanol. 8: amyl alcohol). Wang et al.. (B) whisky.7710. . 4: 1-propanol.

they all displayed a good recovery between 93.7 to 4. indicating the method also had high accuracy. Table 3 shows methanol content of 32 samples including 12 .3% and 3. suggesting the method performed with high precision.2 0.8 0.8 102. Calibration curve of methanol. respectively.0 0. This GC method was also subjected to methanol content analysis in several commercial alcoholic beverages and solvents. The relative errors obtained from the determination of methanol within a day or between days ranged from À4. 2. Table 1 Recoveries of methanol in spiked commercial alcoholic beverage Methanol (mg/mL) Sample Red wine Whisky a Blanka. the coefficient of variation of detections made within a day or between days ranged from 1.3 2.0 0. The data are mean 7S.b (A) 13574 25973 Added amount (B) 100 500 100 500 Detected amountb (C) 24174 64178 353710 772729 Mean recovery (%)c 106. c Recovery (%)=ðC À AÞ=B Â 100%: d Coefficient of variation was obtained from triplicate tests.8 1. / Journal of Food Composition and Analysis 17 (2004) 187–196 peak area ratio 14 12 10 peak area ratio 8 6 4 2 0 0 0.3 93.D. Table 1 shows that.8% to 4.ARTICLE IN PRESS 192 M.0 methanol conc (mg/mL) 4 8 12 16 20 methanol conc. b Recoveries from standard addition and variations of detection made within a day or a successive five-day period were also used to evaluate the method.4 0. As shown in Table 2.2 0.4 0.8% and 106.7% with a low coefficient of variation between 1. (n ¼ 3).7 101.8 3. In addition.7%.6 0.9%.4% for different concentrations of methanol standard.7 1. Wang et al.6 0. varied concentrations of methanol standard (from 50 to 1000 mg/mL) were determined three times in a day and for a successive 5-day period in the same manner.-L.6 CV (%)d 1.7 Original content of methanol in 1 mL sample. indiscriminately spiking whisky and red wine with 100 or 500 mg/mL. (mg/mL) Fig.

. Even though we could not determine whether the high levels methanol resulted from the use of pectolytic enzyme in fermentation process or faked spirits produced by diluting medicinal or industrial alcohol. using this direct injection GC method.8 À2.1) 52279(1. it is difficult to comment on them.3) Repeat injection for three times in the same day (n ¼ 3). 4. (n ¼ 3). spirits. Therefore. this method displayed a capability to rule out dangerous alcoholic samples in a very short time. c Precision is indicated with recovery of methanol and coefficient of variance (%) in parentheses.4 À1.4) 9873(2. our data did not confirm this general view. this method will be very useful for monitoring the methanol content in the fermentation process at brewer factories and for routine examination of methanol content in commercial products performed in governmental laboratories. showed a low methanol levels (roughly 30–100 mg/mL). Conclusions From a pilot screening of several alcoholic beverages. and the level in other wine samples were comparatively high but were within the safe limits (roughly 120–200 mg/mL).1 2. white wine contained less methanol than red wine (Cabanis and Cabanis.5 4872(4.D. we found two spirits containing methanol amounts beyond the legal limits. which was about 5 times higher than that of fruit wine 2 in the same category.2) 10272(2.7) 511717(3. 17 wines and beers and 3 Chinese medicinal liqueur.1 4. As shown in Table 3.9 4. Samples of wine and beer showed various levels of methanol content.-L. d Accuracy is indicated with the results of relative error (%) of individual detection to the mean value of detection.. Repeat injection for three times each day and continuously for five successive days (n ¼ 15). though the methanol content of white wine 1(12879 mg/mL) was still in the normal range described. most samples contained methanol between 160713 and 340726 mg/mL (mean7S. The highest methanol level was found in fruit wine 3 (320717 mg/mL). 857727 and 985743 mg/mL (mean7S. 1999).6) 21076(3. Generally. Due to the lack of information on the production process of these samples.2) 19875(2. respectively.3) 1015729(2. Two spirit samples belonging to whisky and Gao-liang displayed a remarkably high content of methanol.2 2.ARTICLE IN PRESS M. In spirit samples where ethanol content ranged from 40% to 54%. . For the other samples tested. n ¼ 3).D. The Chinese medicinal liqueur samples. n ¼ 3). Taiwan beer contained consistently low methanol content (roughly 22–34 mg/mL).D. which were prepared for Gao-liang by Taiwan Tobacco and Liquor Co..2 Precisionc 5272(2.1 1. the data of recovery are mean 7S. / Journal of Food Composition and Analysis 17 (2004) 187–196 193 Table 2 Precision and accuracy analysis of methanol detection by direct injection GC method at the concentration from 50 to 1000 mg/mL Concentration (mg/mL) Within a daya Precisionc 50 100 200 500 1000 a b Between daysb Accuracyd À4.7) 988723(2. Wang et al.2 À1.8) Accuracyd 4.

5 10.-L. Data are mean7S. Wang et al.5 10.ARTICLE IN PRESS 194 M. but also accurate and reliable when examined in the light of the relative errors and coefficients of variation between repeated analyses and recoveries from standard addition tests.5 16 16 15 15 12 12 4 4 35 35 35 7672 12573 12879 167714 196711 202710 164715 6373 320717 16578 199714 15774 18479 12373 12075 2271 3471 6873 10278 3175 Note: The number following each item is given arbitrarily to indicate that the samples were obtained from different companies. it has wide detection range (2 mg/mL– 20 mg/mL) and this fact makes this method very useful for quantifying various concentrations of . / Journal of Food Composition and Analysis 17 (2004) 187–196 Table 3 Methanol content detected in some alcoholic beverage Sample Spirits Whisky 1 Whisky 2 Whisky 3 Gao-liang 1 Gao-liang 2 Gao-liang 3 XO 1 XO 2 VSOP 1 VSOP 2 White liquor 1 White liquor 2 Wine and beer Red wine 1 Red wine 2 White wine 1 White wine 2 Sweet red wine 1 Sweet red wine 2 Fruit wine 1 Fruit wine 2 Fruit wine 3 Yellow wine 1 Yellow wine 2 Shao-Hsing wine 1 Shao-Hsing wine 2 Millet wine 1 Millet wine 2 Taiwan Beer 1 Taiwan Beer 2 Chinese medicinal liqueur 1 Chinese medicinal liqueur 2 Chinese medicinal liqueur 3 Ethanol content (%) 45 45 45 54 54 54 40 40 40 40 40 40 Methanol content (mg/mL) 857727 260713 214723 322723 186711 985743 294728 190711 160713 292721 213725 340726 14 14 14 14 10. Direct injection of spirit or wine samples into a 30 m CP-Wax 58 CB megapore capillary column with a flame ionizing detector has proven useful to determine methanol content in a very short time (o9 min). The present method is not only quick.D. Furthermore.5 10. (n ¼ 3).5 10.

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