Determination of Methanol, Acetaldehyde, and Ethyl Acetate in Thousand Folds of Ethanol Sample by Headspace Gas Chromatography with Mass Spectrometry

Alcohol beverages have been widely consumed in several parts of the world. In this study, volatile organic compounds in alcoholic beverages including acetaldehyde, ethyl acetate, methanol, and higher ethanol were investigated and evaluated using a headspace gas chromatograph equipped with a mass spectrometer. This study evaluated the suitability of the chromatographic system, linearity, limit of detection, and limit of quantification, accuracy, and precision of the single and simultaneous determination of acetaldehyde, ethyl acetate, and methanol in thousand folds of ethanol. Results showed that the acetaldehyde concentration in local beer samples and local manual product liqueur samples ranged from 4.65 to 13.22 mg/L and from 5.55 to 75.96 mg/L, respectively, but in local industrial product liqueur samples, acetaldehyde was not detected. Methanol was only detected in a few local beer samples and locally manually produced liqueur samples within low concentrations. Ethyl acetate was only detected in all local beer samples, but it was not present in local industrial product liqueur samples.


Introduction
Ethanol level is important to consumers for the mouthfeel and favor of alcoholic beverages.Beer, wine, and liqueur have ethanol levels ranging from 3 to 6% (v/v), 7 to 21% (v/v), and 20 to 50% (v/v), respectively [1,2].Tis compound is presented in alcoholic beverages as a consequence of the carbohydrates' fermentation by yeasts.Volatile favor compounds were presented in the alcoholic beverages caused by distillation procedure [2,3].Among these compounds, acetaldehyde is one of the naturally occurring compounds that could be found in diverse beverages (such as alcoholic drinks and juices) and foods (such as dairy products and vegetables) as well as liqueurs, wines, and brandies [4].Tis compound was a favor enhancer that was added at low concentrations to provide a pleasant fruity bouquet [5,6].Acetaldehyde in the most of 18 craft beer samples was from 2.02 to 19.64 mg/L and in 8 industrial beer samples was from 2.91 to 13.28 mg/L [7].In alcoholic beverages or drinks, besides acetaldehyde, methanol is also one of the naturally occurring compounds that can be found at various levels because methanol is a byproduct that was produced due to the degradation of pectin during liqueurs' fermentation [8].Methanol in most of the 18 craft beer samples was from 4.52 to 11.13 mg/L and in 8 industrial beer samples was not detected [7].In the case of ethyl acetate, this is the most frequent ester in liqueurs and it is produced by the acetic bacteria metabolism and yeast during the liqueur fermentation.One of the symptoms of liqueur spoilage is a high level of ethyl acetate.It is well known that the high level of ethyl acetate in liqueur is a factor conditioning liqueur quality [9].Ethyl acetate in most of the 18 craft beer samples was from 13.30 to 42.33 mg/L and in 8 industrial beer samples was from 13.68 to 27.79 mg/L [7].
On the other hand, ethanol, methanol, acetaldehyde, and ethyl acetate are volatile compounds whose detection and quantifcation in alcoholic beverages can be used as a biological indicator to identify the origin of several kinds of alcoholic drinks because the volatile compounds which characterize the beverage type are diverse and originated from raw materials.And these volatile compounds are generated during production, maturation, and storage [10].
Ethanol, acetaldehyde, methanol, and ethyl acetate in alcoholic beverages were determined by several methods.Ana Catarina et al. determined acetaldehyde, ethyl acetate, and methanol in wine spirits, brandy, and grape marc spirits by the GC-FID method [11].Acetaldehyde and methanol in alcoholic beverages were determined by GC-MS [12], GC-FID, and GC-TCD [13,14].Methanol and ethanol concentrations in alcoholic drinks were detected using GC-TCD [15] and HS-GC-MS [16].Tese volatile compounds were simultaneously determined by GC-FID [17].Tere are such methods for the simultaneous determination of methanol, acetaldehyde, and ethyl acetate in a high level of ethanol in alcoholic drinks.Tis method, which is proposed in the present study, enriches the scientifc literature and has a practical, applied value.Tus, the simultaneous determination of methanol, acetaldehyde, and ethyl acetate in thousand folds of ethanol in alcoholic drinks by headspace gas chromatography with mass spectrometry is a necessary method to evaluate liqueur quality.

Materials and Methods
2.1.Chemicals.Methanol, acetaldehyde, ethyl acetate, ethanol standard, n-butanol internal standard, acetonitrile, and water were purchased from Merck, Darmstadt, Germany.All chemical standards used for the analysis were of analytical grade.Nitrogen, hydrogen, and helium were of extrapure grade 4.5 (Air Liquide, Ho Chi Minh City, Vietnam).
Methanol, acetaldehyde, ethyl acetate, ethanol, acetonitrile standard solutions, and n-butanol internal standard solution were prepared in water solvent and stored at −18 °C.Tese solutions were sonicated for 5 min before use.

Sample Preparation and
Equipment.5 mL of samples was poured into 20 mL of glass headspace vials, n-butanol was added as internal standard, and then, the vials were sealed with caps lined with a silicon PTFE membrane.Te vial samples were incubated for 10 min at 70 °C in order to facilitate the transfer of analytes to the sample's volatile fraction.All samples were analyzed by GC-MS Termo Fisher Scientifc ISQ 72008051 gas chromatography with a mass spectrometric detector equipped split/splitless inlets, and a headspace sampling system.For separation of impurities, 1 μL of the sample's volatile fraction in the headspace was injected into the GC inlet.A TG-WAXMS 30 m × 0.25 mm × 0.5 μm (polyethylene glycol) capillary column was used to separate the volatile fractions under a constant fow of helium 1.2 mL/min.Te duration of the analysis was 8 minutes.For increased sensitivity, other method parameters were optimized.All experimental samples were analyzed in triplicate.

Validation of the Method of a Single Determination of Methanol, Acetaldehyde, Ethyl Acetate, and Ethanol in
Alcohol.Each of the acetaldehyde, methanol, and ethyl acetate standard solutions was prepared for calibration at 5 to 240 mg/L and 50 to 2400 mg/L of ethanol standard solution.Every single standard solution was determined in triplicate.All prepared standards were analyzed at optimized parameters by HS-GC-MS Termo Fisher Scientifc ISQ 72008051 gas chromatography.Te validation method of single determination was determined by the limit of detection (LOD), the limit of quantifcation (LOQ), repeatability, and recovery.
Te LOD is the smallest sample concentration at which the substance signal may be consistently recognized when compared to a blank run's baseline noise.For evaluating the detection limit, a signal-to-noise ratio of 3 : 1 is typically regarded adequate [18].
Intra-assay and inter-assay precision levels were assessed by analyzing the quality control samples.Intra-assay was assessed using a minimum of 9 determinations at 3 concentrations/6 replicates each for the procedure.Inter-assay precision levels were evaluated in three concentrations at 5, 80, and 240 mg/L for acetaldehyde, ethyl acetate, and methanol and 50, 800, and 2400 mg/L for ethanol for six consecutive days.Intra-assay and inter-assay precision was evaluated in terms of relative standard deviation (% RSD).
(2) Te validated method simultaneous determination was determined by the selectivity factor (α), the limit of detection (LOD), the limit of quantifcation (LOQ), repeatability, and recovery.

Optimization of HS-GC-MS
2.6.Application of the Optimized Method.Te optimized method was applied to 5 beer samples, 5 industrial liqueur samples, and 5 manual liqueur samples (Table 1) purchased from shops in Ho Chi Minh City, Vietnam.All samples were stored at room temperature (21 °C) and protected from light.All samples were added with internal standard prior to the analysis.

Single Determination of Methanol, Acetaldehyde, Ethyl
Acetate, and Ethanol in Alcohol by HS-GC-MS.Te linearity of the headspace gas chromatography mass spectrometry method was determined at seventh concentration levels ranging from 5 to 240 mg/L of acetaldehyde, methanol, and ethyl acetate standard solutions and from 50 to 2400 mg/L of ethanol standard solution.Each of these standard solutions was incubated in the headspace at 70 °C for 10 minutes, and then, the evaporative part was injected into the chromatographic system (n � 3).Te peak area and retention time of acetaldehyde, methanol, ethyl acetate standard, and internal standard were recorded, and the mean values of the peak area ratio were plotted against the concentrations to obtain the calibration curves.Linear regression of acetaldehyde, methanol, ethyl acetate, and ethanol were y � (0.0164 ± 0.0008) x + 0.0151 ± 0.0098 (mg/ L),y � (0.0025 ± 0.00004) x + 0.0008 ± 0.0003 (mg/L), y � (0.0298 ± 0.0064) x + 0.0167 ± 0.0138 (mg/L), and y � (0.0033 ± 0.0002) x + 0.0501 ± 0.0135 (mg/L), respectively (Figure 1).Te good coefcients of acetaldehyde, methanol, ethyl acetate, and ethanol were 0.9998, 0.9998, 0.9999, and 0.9999, respectively.Te results of validating the determination of acetaldehyde, methanol, ethyl acetate, and ethanol are shown in Table 2.As presented in Table 2, the limit of detection (LOD) and limit of quantifcation (LOQ) were 0.55 mg/L and 1.83 mg/L for ethanol, 0.63 mg/L and 2.09 mg/L for methanol, 0.52 mg/L and 1.72 mg/L for acetaldehyde, and 0.51 mg/L and 1.70 mg/L for ethyl acetate, respectively.Te LOD in this research was better than that in other documents.Helena et al. presented a LOD of 0.85 mg/L for acetaldehyde and 0.75 mg/L for acetone, ethanol, and methanol [18].Pontes et al. showed that the LOD was 0.87 mg/L for methanol, 0.51 mg/L for acetaldehyde, and 0.82 mg/L for ethyl acetate [19].Te method in this study has good accuracy, precision, linearity, and efciency for the quantifcation of acetaldehyde, methanol, ethyl acetate, and ethanol.

Te Optimized Parameters.
In HS-GC-MS, the samples must be held in the headspace system at high temperatures and a reproducible equilibrium must be established between a solution sample and a headspace.Te chromatographic process depends on the analytical conditions including detector temperature, inlet temperature, column temperature program, fow rate, split ratio, headspace temperature, and incubation time of the headspace.Terefore, the study of headspace conditions and GC-MS parameters in the multicomponent analysis is necessary for the HS-GC-MS method.

Optimization of Incubation Temperature of Headspace.
In the headspace technique, the sample was put into a sealed vial which was heated to an essential temperature for a period of time.More volatile compounds will tend to move into the headspace above the solution sample.Te incubation temperature of headspace was one of the factors afecting the volatile ability of compounds.In this study, after some trials of diferent headspace temperatures for 10 min, 80 °C was selected as a headspace temperature because of the great peak surface.Te results are shown in Figure 2.

Optimization of Incubation Time of the Headspace.
Solvent-vapor equilibria in the headspace play an important role in the headspace analysis.Te more volatile compounds can be evaporated into the headspace, and more volatile compounds will be injected into the column of the GC.If the sample is incubated for a too short time, less of the volatile compounds will be in the headspace, which can afect the overall peak area.In this study, the incubation time of the headspace was in the range of 5-30 minutes.Te results shown in Figure 3 show that those equilibration time periods longer than 10 min do not yield a signifcant increase in the peak area.In the case of ethanol, methanol, ethyl acetate, and acetaldehyde, the incubation time longer than 20 minutes shows a high peak area.It is of interest to note that the incubation time of the samples was 10 minutes.

Optimization of Inlet
Temperature.Te inlet was to set an optimal temperature which helped the analytes quickly vaporize.It was not only too high to cause the breakdown of these compounds but also not too low to decrease the sensitivity due to the compounds which did not vaporize.Inlet temperature was not adjusted higher or lower to optimize the performance in each diferent analysis.In our study, the inlet temperature was set in the range from 150 to 250 °C.Te results are presented in Figure 4. Figure 4 presents that the peak area of ethanol, methanol, ethyl acetate, and acetaldehyde in the chromatogram decreased with increasing temperature in the inlet.Te optimal inlet temperature was set at 150 °C.
International Journal of Analytical Chemistry  5. Table 3 shows the chromatographic system data of simultaneous analysis of acetaldehyde, ethyl acetate, methanol, ethanol, and butanol at heating rates of 3, 5, 10, 15, 20, and 25 °C/min.Te results showed that when the heating rate increased, the retention time of the analytes decreased.It was because gas chromatography relies on the evaporation temperature of the analytes to separate the analytes contained in the sample background.Terefore, when the temperature increases rapidly, the analytes will be eluted out  It is easy to completely overlap the low-intensity peaks.So, the chromatographic spectrum will only show highintensity peaks.
3.2.6.Optimization of Detector Temperature.After the volatile compounds moved the length of the GC column, they were entered into the mass spectrometer and were fragmented into an ion by an electron ionization technique.
In the electron ionization technique, an electron that was produced by a flament was accelerated and knocks an electron out of the molecule to produce a molecular ion.Tese molecular ions were detected by an electron multiplier, which essentially turned the ionized mass fragment into an electrical signal that was then detected.In this study, the detector temperature was set at 150, 170, 190, 210, 230, and 250 °C.Te efect of the detector temperature on the peak areas of ethanol, methanol, ethyl acetate, and acetaldehyde is shown in Figure 6.Te results showed that at 250 °C, the peak area of ethanol, methanol, ethyl acetate, and acetaldehyde was the highest.Te optimal detector temperature was set at 250 °C.

Optimization of Mobile Phase Flow Rate.
Te mobile phase fow rate changed from 1.0 to 2.0 mL/min.Te results showed that the fow rate increased and the retention time of the analytes decreased.It was due to the fact that the carrier gas plays the role of the mobile phase eluting the analytes out of the column, the analytes move  International Journal of Analytical Chemistry out of the column as soon as possible.Te change in the mobile phase fow rate causes the change of peak areas of ethanol, methanol, ethyl acetate, and acetaldehyde (Table 3).Tis may be caused by the decrease in ionization efciency due to the nonstoichiometric ratio of air and the dilution of gas eluted from the column.Tus, the mobile phase fow rate of 1.2 mL/min was selected as the optimal value (Table 4).

Optimization of Slip Ratio.
One of the substantial parameters afecting method sensitivity was the split ratio.Increasing the split ratio leads to a decrease in the amounts of analytes introduced into the column.Terefore, the splitless injection technique will be the highest sensitivity.In the case of the headspace technique, ethanol, methanol, ethyl acetate, and acetaldehyde were evaporated into the headspace upper of the solution, so

6
International Journal of Analytical Chemistry  that the impurity compounds could be removed.In an alcohol sample, the ethanol amount was steadily higher than other compounds.Injection in the splitless mode or at low split ratios may potentially lead to problems with the methanol peak which can be overlapped by a large ethanol peak, analyte peak shape, and efciency of resolution.Split ratios were studied at 1 : 10, 1 : 20, 1 : 40, 1 : 60, and 1 : 100. Figure 7 shows the efect of the split ratio on the peak area of ethanol, methanol, ethyl acetate, and acetaldehyde.At a split ratio 1 : 10, the peak area of these analytical compounds was large and this ratio was set at the optimal parameter.
International Journal of Analytical Chemistry 3.2.9.System Suitability.Te mixture of ethanol, methanol, ethyl acetate, and acetaldehyde was injected into HS-GC-MS in six replications under the optimal conditions mentioned above.Te peak width, retention time, resolution, symmetry, and theoretical plates were recorded to assess the suitability of the analytical instrumentation conditions.Te results in Table 5 show the theoretical plates >2000, resolution >1.5, symmetry in the range 0.9-1.1, and the relative standard deviation of retention time <2%.Tese system suitability parameters were obtained as acceptance criteria from the International Conference on Harmonization (ICH) [20,21].Te method was suitable for the simultaneous analysis of ethanol, methanol, acetaldehyde, and ethyl acetate.

Optimized Parameters
Headspace injector conditions include the following: Te linearity of simultaneous determination of methanol, acetaldehyde, ethyl acetate, and ethanol in the headspace gas chromatography mass spectrometry method was determined at seven concentration levels ranging from 5 to 240 mg/L of acetaldehyde, methanol, and ethyl acetate standard solutions and from 50 to 2400 mg/L of ethanol standard solution.Each of these standard solutions was incubated in the headspace at 70 °C for 10 minutes, and then, the evaporative part was injected into the chromatographic system (n � 3).Recording the peak area of acetaldehyde, methanol, ethyl acetate, ethanol standard, and n-butanol internal standard, the mean values of the peak area ratio was plotted against the concentrations to obtain the calibration curves.Linear regression and good coefcients of acetaldehyde, methanol, ethyl acetate, and ethanol are shown in Table 6.
Table 5 shows that the concentration and peak area of acetaldehyde, ethyl acetate, and methanol in the concentration range from 5 to 240 mg/L and ethanol in the concentration range from 50 to 2400 mg/L correlated with linear relationship r 2 > 0.999.

Limit of Detection and Limit of Quantifcation.
Te limit of detection and limit of quantifcation of the method are presented in Table 7. Table 7 presents the results obtained from the simultaneous determination of acetaldehyde, methanol, ethyl acetate, and ethanol in alcohol samples using the proposed method.International Journal of Analytical Chemistry 3.3.3.Repeatability.Intra-assay and inter-assay precision levels were assessed by analyzing the quality control samples.Intra-assay was assessed using a minimum of 9 determinations at 3 concentrations/6 replicates each for the procedure.Inter-assay precision levels were evaluated in three concentrations at 5, 80, and 240 mg/L for acetaldehyde, ethyl acetate, and methanol and 50, 800, and 2400 mg/L for ethanol for six consecutive days.Intra-assay and inter-assay precisions were evaluated in terms of relative standard deviation (% RSD).Te RSD of intra-assay and inter-assay precisions in Table 8 was less than 4% at three concentrations of acetaldehyde, methanol, ethyl acetate, and ethanol.

3.3.4.
Recovery.Acetaldehyde, methanol, ethyl acetate, and ethanol recovery were measured based on accuracy at 5, 80, and 240 mg/L for acetaldehyde, ethyl acetate, and methanol and 50, 800, and 2400 mg/L for ethanol.Recovery of acetaldehyde, methanol, ethyl acetate, and ethanol was found to be 95-102%, 97-103%, 98-105%, and 99-107%, respectively (Table 9).Te results showed that the limit of detection, the limit of quantifcation, linear range, repeatability, and recovery effciency of the simultaneous method were good, meeting the requirements for method validation of AOAC by GC-MS equipment.

Advantages and Disadvantages of Simultaneous Determination of Methanol, Acetaldehyde, Ethyl Acetate, and Ethanol in Alcohol by HS-GC-MS
(i) Saving analysis time: By only one injection, the simultaneous concentration of methanol, acetaldehyde, ethyl acetate, and ethanol could be the simultaneous determination of acetaldehyde, methanol, acetone, and ethanol.Te total time required for the chromatographic analysis was 15 min, which was 1.5 fold higher than our method [22].(ii) Cost savings: By only a single injection, the concentration of methanol, acetaldehyde, ethyl acetate, and ethanol in the samples can be determined simultaneously, so the cost of the analysis is smaller than in the case of individual substances.(iii) Minimizing errors in the analysis process: In the simultaneous analysis process, the steps of the analysis were minimized compared to the single analysis process, thereby limiting the errors arising from the manipulation process.(iv) Te analytes in the sample have diferent concentrations.So, it is difcult to process the samples, especially with trace concentration.To minimize this drawback, a standard addition method was performed to decrease the diference in concentration between the analytes.

Application of Simultaneous Determination of Methanol, Acetaldehyde, Ethyl Acetate, and Ethanol in Alcohol by HS-GC-MS.
Because the ethanol concentration in the alcohol samples was at 5-40%, the samples should be diluted to analyze at the appropriate ratio.Meanwhile, methanol, acetaldehyde, and ethyl acetate concentrations were lower than ethanol concentrations.Terefore, the dilution samples have done a standard addition at 5 mg/L for each methanol, acetaldehyde, and ethyl acetate, and an internal standard was added into the dilution sample.Table 10 presents the actual sample analysis results.
Ethanol concentration in liqueur product samples varies for each product group.Ethanol concentration in beer was in a range from 4.5% to 5.9%; meanwhile, the ethanol concentration in local liqueur samples (industrial products and manual products) was in a range from 25.7% to 39.6%.Tese results were in accordance with the declaration of the manufacturers.Most of the beer samples did not contain methanol or contained a little methanol level.In contrast, the content of ethyl acetate in beer samples was high, in the range of 30-40 mg/L.Meanwhile, the content of acetaldehyde ranged from 4.6 to 13.2 mg/L.Beer was produced through fermentation and then fltration without distillation.So, beer samples have a lot of impurities in them, such as acetaldehyde and ethyl acetate.
Industrial liqueur products have no or contain very little methanol, acetaldehyde, and ethyl acetate (except for RNUL products which contain above 40 mg/L of acetaldehyde and ethyl acetate).Most industrial liqueur products were produced using multistage distillation towers to distillate and help separate and decrease impurities in the liqueur.In the group of manual liqueur products, the ethanol concentration is from 25.6 to 37.8%, which depends on the needs of each product.Most of the manual liqueur product samples contained acetaldehyde, but its signifcant diference lies in the levels of diferent production facilities.In contrast, ethyl acetate and methanol concentrations did not present or were found very little in manual liqueur products.Te reason is that all local artisanal distilleries use simple or self-designed distillation equipment.So, the process of separating ethanol from impurities does not completely depend on the system and conditions of the distillation process.Terefore, the results in the group of manual liqueur products are signifcantly diferent in impurity concentration between production facilities.Te methanol, acetaldehyde, and ethyl acetate concentrations in these liqueur products were less than these concentrations in several documented previous studies.According to Kokkinakis et al., the concentrations of acetaldehyde, ethyl acetate, and methanol in bottled spirits and in-bulk spirits were 297.58 mg/L, 429.16 mg/L, and 698.02 mg/L International Journal of Analytical Chemistry and 199.75 mg/L, 1067.66 mg/L, and 781.20 mg/L, respectively [23].Te methanol and acetaldehyde contents of fermented plant beverages in Tailand were less than 29 mg/L and 45 mg/L, respectively [24].

Conclusions
Tis study was taken to analyze the simultaneous concentration of acetaldehyde, methanol, ethyl acetate, and ethanol in alcohol, which are most commonly consumed by the habitants of Ho Chi Minh City, Vietnam.Te HS-GC-MS parameters for simultaneous determination of these volatile compounds were optimized.Te simultaneous method was validated.In beer samples, there were impurities such as acetaldehyde and ethyl acetate because the production process did not go through distillation.In industrial liqueur products, there is very little methanol, acetaldehyde, and ethyl acetate because multistage distillation towers were used to distillate to help separate and decrease impurities in the liqueur.For manual liqueur products, there are still impurities such as methanol and acetaldehyde because simple or self-designed distillation equipment was used to separate impurities during the production process.12 International Journal of Analytical Chemistry

Figure 2 :Figure 3 :
Figure 2: Efect of headspace temperature on the peak area of ethanol, methanol, ethyl acetate, and acetaldehyde.

Figure 5 :
Figure 5: Chromatograms of ethanol, methanol, ethyl acetate, and acetaldehyde in the GC oven temperature set from 38 °C and increased to 50 °C at 3 °C/min, where it was held for 1 min and then 50 °C and increased to 170 °C at 35 °C/min.Te retention time periods t R of acetaldehyde, ethyl acetate, methanol, ethanol, and butanol were 1.81 min, 3.41 min, 3.55 min, 4.24 min, and 8.01 min, respectively.

Figure 4 :
Figure 4: Efect of inlet temperature on the peak area of ethanol, methanol, ethyl acetate, and acetaldehyde.

Figure 6 :
Figure 6: Efect of detector temperature on peak areas of ethanol, methanol, ethyl acetate, and acetaldehyde.

Figure 7 :
Figure 7: Efect of split ratio on the peak area of ethanol, methanol, ethyl acetate, and acetaldehyde.

Table 1 :
Sample name, number of samples, and number of individuals for each sample.

Table 2 :
Te results of validating the determination of acetaldehyde, methanol, ethyl acetate, and ethanol.

Table 3 :
Chromatographic system data: retention times, selectivity factor, peak area, resolution, symmetry, and theoretical plates at several heating rates.

Table 4 :
Chromatographic system data: retention times, selectivity factor, peak area, resolution, symmetry, and theoretical plates at several mobile phase fow rates.

Table 7 :
Limit of detection and limit of quantifcation of the HS-GC-MS method.

Table 8 :
Te repeatability of the HS-GC-MS method.

Table 9 :
Recovery of the HS-GC-MS method.

Table 10 :
Concentrations of methanol, acetaldehyde, ethyl acetate, and ethanol in alcohol samples in Ho Chi Minh City.
Note. n/d: not detected (below detection limit).