Determination of Aspartame Content in Liquor by High Performance Liquid Chromatography

Zhouli ZHANG Xingyu LUO Ping YONG Lan LIAO Lili YIN Yaoxin LI Min ZHANG Da YI

Abstract    [Objectives]  This study was conducted to establish a method for the determination of aspartame in liquor by high performance  liquid  chromatography.

[Methods]  The liquor samples having volumes fixed with ultrapure water were filtered by 0.22 μ m water phase， and then the content of aspartame in liquor was determined by high performance liquid chromatography using methanol （A） and ultrapure  water （B）  as mobile phase for isocratic elution.

[Results]  The results showed that a good linearity was obtained for the standard curve  （ R  2=99.98%）  in the method， and its quantitative limit was 5.34 mg/kg. Its recovery was  91.26% ， 91.92%， and 90.55%， respectively.

[Conclusions]  The method has high sensitivity， high recovery and low quantitative limit， and it was a suitable method for the determination of aspartame in liquor.

Key words   HPLC; Liquor; Aspartame

Aspartame， having a scientific name of aspartyl-phenylalanine methyl ester， is a natural functional oligosaccharide， with a sweetness 200 times that of sucrose  [1] . Long-term consumption of aspartame， an artificial sweetener， will cause certain harm to the human metabolic system and nervous system  [2-3] ， accompanied by adverse effects on the brain and the possibility of causing anxiety  [4] . Studies have shown that long-term feeding of aspartame can stimulate immune responses in the immune organs of diabetic mice， resulting in decreased immunity and the possibility of cancer  [3] . Selda and other scholars  [5]  found that long-term consumption of aspartame will accelerate the development of existing tumors in the body. Meanwhile， studies have shown that long-term use of aspartame has adverse effects on the structure of the sciatic nerve， and it cannot be completely recovered after stopping aspartame for 1 month  [6] . Liquor with moderate sweetness can give people a pleasant feeling of mellowness and sweetness. Studies have shown that the reason why the liquor produced by high-quality fermentation has a mellow and sweet feeling is that the liquor itself contains certain polysaccharide alcohols such as glycerol and glucose  [7] . The sweetness of polyols is mainly affected by the number of their own hydroxyl groups. For example， the sweetness of hexanehexol is greater than that of sucrose and butantetraol  [8] . At present， China does not allow aspartame to be added to various prepared wines and fermented liquors of various flavor  [9-13] . However， there are currently unscrupulous businesses trying to add  aspartame  to liquor to improve the aroma and taste of inferior  liquor   [14-15] ， which seriously damages the interests of consumers and producers， and brings certain difficulties to liquor supervision. The detection methods of aspartame in food focus on high performance liquid chromatography  [16-19] ， high performance liquid chromatography-tandem mass spectrometry  [20-21]  and real-time QTRAP mass spectrometry  [22] ， and there are also reports on the detection of aspartame in food by some scholars using the electrochemical sensor method  [23] . At present， there is no unified detection method for the determination of aspartame content in wine in China. In this study， combined with the characteristics that aspartame is easily soluble in polar solvents such as water， methanol and ethanol， an analytical method for the determination of aspartame in liquor by high performance liquid chromatography was designed and established， making the determination of aspartame in liquor simpler and more efficient.

Materials and Methods 

Instruments and reagents

UITIMate3000 high performance liquid chromatograph  （Thermo Fisher Scientific， USA）; XS205DU analytical balance （0.000 1 g）  （Mettler Toledo， Switzerland）.

Methanol （chromatographically pure， Fisher， USA）; aspartame standard （Dr. Ehrenstorfer GmbH， Germany）; ZORBAX XDB-C 18  column （5 mm×250 mm， 4.6 μm， Agilent， USA）;  0.22  μm aqueous filter （American Ameritech Corporation）; Milli-Q ultrapure water for laboratory use.

Liquor， commercially available.

Experimental methods 

Solution preparation

An appropriate amount of aspartame standard was accurately weighed and prepared with water into a standard working solution of 0.5 mg/ml， which was then diluted with water to aspartame with contents of 0.5， 1， 2， 3， 5， and 10 μg/ml， respectively. The aspartame standard working solutions were filtered through a 0.22 μm water-based microporous membrane for determination by liquid chromatography.

Sample processing

In order to eliminate the interference of ethanol matrix in liquor samples， methods such as rotary evaporation  [24] ， nitrogen blowing  [25]  and water bath evaporation  [26]  are  usually  used. However， the rotary evaporation method and nitrogen blowing method take a long time， which is not conducive to the rapid detection of experimental research. Aspartame is soluble in polar solvents such as water， methanol and ethanol. Referring to the study of Zheng  et al.   [27] ， in this study， the method of adding ultrapure water to dilute samples was adopted and combined with ultrasonic treatment of the samples to reduce the interference of the ethanol matrix， decrease the influence of ethanol in the liquor samples on chromatographic peak shapes and improve the accuracy and reproducibility of the method. First， about 5 g （accurate to 0.001 g） of sample was weighed into a 25 ml volumetric flask， and diluted to constant volume with water， and the obtained mixture was transferred all to a centrifuge tube， mixed well， and ultrasonically treated for 20 min. Centrifugation was performed at 4 000 r/min for 5 min， and the supernatant was filtered through a 0.22 μm water filter and used for subsequent chromatographic analysis.

Liquid chromatography conditions

Agilent ZORBAX-XDB-C 18  column （5 mm×250 mm， 4.6 μm）; mobile phase∶ methanol（A）-water（B）=40∶ 60; flow rate： 0.8 ml/min; injection volume： 20 μl; column temperature： 30 ℃.

Recovery and precision

Three parts of blank sample were weighed， and added with 50， 75， and 150 μl of the standard working solution， respectively， and the rest followed sample processing steps in "Sample processing".

Quantitative limit solution preparation

The standard solution at the first point of the standard curve was injected into the high performance liquid chromatograph. The signal-to-noise ratio was measured， and the limit of quantification was calculated according to S/N=10∶ 1.

Sample determination

The data and images were collected and processed by Thermo Scientific Dionex Chromeleon  TM  CDS workstation. The obtained data were then calculated and processed by Excel software， and quantification was performed by the external standard method. The content of aspartame in the sample was obtained according to the following formula：

X=（C×V）/（m ×1 000）

In the formula，  X  is the content of aspartame in the sample （g/kg）;  c  is the measured concentration （μg/ml）;  V  is the constant volume （ml）; and  m  is the weighed sample volume （g）.

Results and Analysis 

Standard curve

The standard curve was injected into the high-performance liquid chromatograph， and linear regression was performed with the relative mass concentration as the X-axis and the peak area Y as the ordinate to establish a standard curve. The results are shown in Fig. 1.

The results showed that the linear relationship was good in the concentration range of 0.5-20.0 μg/ml， and the regression equation was  y =0.476 2 x -0.033 1，  R  2=0.999 8.

Reproducibility test

Six aspartame-containing liquor sample solutions were prepared according to the sample treatment method in "Sample processing". Each sample solution was injected 6 times， and determined for aspartame concentration， and the average value of the 6 determinations was taken as the content of aspartame in the sample. The relative standard deviation （ RSD ） of aspartame content was 0.02%， which indicated that the method had good repeatability， and the instrument was in a stable state for injection， and was sensitive to the determination of sample chromatographic peak area， which meets the requirements of experimental quality  control.

Precision and recovery test

The standard solution with a concentration of 1.0 μg/ml was determined 6 times in parallel. The standard deviation was 0.89% and the  RSD  was 0.99% by comparing the measured concentrations， indicating that the precision of the method was good. Then， three parts of blank sample were weighed and added with 50， 75， and 150 μl of the standard working solution respectively， and the rest followed sample processing steps in "Sample processing". Each sample solution was determined 6 times in parallel， and the average recovery and  RSD  were calculated， as shown in Table 1.

The sample recovery of aspartame was between 90.55% and 91.92%， which is close to the experimental recovery of Shao   et al.   by rotary evaporation  [24] ， indicating that the aspartame  contents  of the samples treated by rotary evaporation method and not treated by rotary evaporation method have little differences. The recovery of aspartame in liquor  （90.55%-91.92%）  is slightly lower than the average recovery （100.3%） of aspartame determined from nine beverages by Xia  et al.   [28] ， which may be due to different samples， which are affected differently by their own matrixes， resulting in different recovery. The relative standard deviation of aspartame in liquor measured by this method was between 0.68% and 1.03%， which is slightly better than the  RSD  value of aspartame in liquor （1.20%-4.52%） measured by the test method designed by Si  et al.   [29] . The value of this study is close to the  RSD  result （0.41%-1.69%） determined by the experimental method designed by Qin  et al.   [30] ， in which a  50 ml  of sample was placed in a rotary evaporation flask， rotated to dryness， and dissolved in ultrapure water to 5 ml， and the obtained solution was filtered with a 0.22 μm filter membrane， and then determined by high performance liquid chromatography. However， this study is simpler in the operation process， and  avoids  the operation that requires manual judgment and is prone to mistakes in rotary evaporation and micro-drying. It shows that the method has high precision and recovery， and can be used for the routine detection of aspartame in liquor.

Quantitative limit determination

The standard solution at the first point of the standard curve was injected into HPLC， and its content and signal-to-noise ratio were measured to determine the limit of quantification， as shown in Fig. 2.

According to S/N=10∶ 1， with the signal-to-noise ratio as the response， the calculated limit of quantification was 5.34 mg/kg. At present， China stipulates that the detection limit and quantification limit of aspartame in lactic acid beverages and liquid milk beverages are 1 and 3 mg/kg， respectively， and the detection limit and quantification limit of aspartame in solid beverages and their products are 5 and 15 mg/kg， respectively， while the detection limit and quantification limit of aspartame in liquor are not specified  [31] . The limit of quantification obtained in this study is within the relevant standard range， which meets the experimental  requirements.

Sample detection results

Ten parts of liquor were randomly selected for testing， and the samples were pretreated. Then， three groups of each sample were made for parallel analysis. The external standard method was used for quantification， and the contents of aspartame in the liquor samples were calculated， as shown in Table 2.

Among the 10 randomly selected samples， 3 were detected to contain aspartame， with the contents of 0.047 3， 0.018 8 and  0.056 3  g/kg， respectively， and the precision was between  0.06%  and 1.02%， which was in line with the national standard that the absolute value of two independent determination  results obtained  under repeated conditions shall not exceed the  requirement  of 10% of the arithmetic mean.

Conclusions and Discussion

In this study， high performance liquid chromatography was applied to detect aspartame in liquor， and the appropriate linear range， recovery and precision were selected for analysis， and actual samples were detected. The results show that the method is easy to operate， has the advantages of good precision and accuracy， high recovery， and low detection limit. The detection results are accurate and reliable， meet the analysis requirements， and meet the analysis and determination of aspartame in liquor， providing a more effective guarantee for the detection of aspartame in liquor.

References

[1]  GAO X， ZHANG F. Rapid determination of aspartame and alitame in food by reversed-phase high performance liquid chromatography with mixed standard and sample increment method[J]. Food Sci， 2020， 41（24）： 6. （in Chinese）.

[2] LIU X， MENG Y， ZHANG Y，  et al.  Characteristics of common artificial sweeteners in food and their effects on physiological metabolism[J]. J of Food Saf and Qual， 2021， 12（14）： 5734-5741. （in Chinese）.

[3] YANG B， ZHU HY， GONG C，  et al.  Study on influence of aspartame on immunity of diabetic mice[J]. J of Agri Cata， 2013， 3（7）： 2. （in Chinese）.

[4] ASHOK I， SHEELADEVI R， WANKHAR D. Effect of long-term aspartame （artificial sweetener） on anxiety， locomotor activity and emotionality behavior in Wistar Albino rats[J]. Bio & P Nut， 2014， 4（1）： 39-43.

[5] GEZGINCI-OKTAYOGLU S， ERCIN M， SANCAR S，  et al.  Aspartame induces cancer stem cell enrichment through p21， NICD and GLI1 in human PANC-1 pancreas adenocarcinoma cells[J]. Food chem toxicol， 2021（153）： 112264.

[6]  OKASHA EF. Effect of long term-administration of aspartame on the   ultrastructure  of sciatic nerve[J]. J of Micro Ultra， 2016， 4（4）： 175-183.

[7] HAN X， YAO YP， NI YP，  et al.  Analysis of the flavor composition for Ying Jiagong Baijiu’s sweet feeling （one）[J]. Liquor Making， 2019， 46（6）： 30-34. （in Chinese）.

[8] SUN BG， LI HH， HU XM，  et al.  Development trend of healthy Baijiu[J]. China Food， 2021， 16（8）： 1-6. （in Chinese）.

[9] GB 2760-2014， National standards for food safety standards for the use of  food additives[S]. Beijing： China Standards Press， 2014. （in Chinese）.

[10]  GB 2757-2012， National standards for food safety distilled spirits and their formulated liquors[S]. Beijing： China Standards Press， 2012. （in Chinese）.

[11] GB/T 10781.2-2006， Fragrant liquor[S]. Beijing： China Standards Press， 2006. （in Chinese）.

[12] GB/T 26760-2011， Sauce-flavored liquor[S]. Beijing： China Standards Press， 2011. （in Chinese）.

[13] GB/T 10781.1-2006， Aromatic liquor[S]. Beijing： China Standards Press， 2006. （in Chinese）.

[14] Hubei reported that 3 batches of food were unqualified 1 batch of liquor was detected aspartame[EB/OL].[2017-02-24]. http：∥news.foodmate.net/2017/02/418739.html.[2021-12-31]. （in Chinese）.

[15] Announcement of the Food and Drug Administration of Jiangsu Province on the Announcement of the 24  th  Provincial Food Safety Supervision and Sampling Information in 2016[EB/OL].[2016-08-04].http：∥news.foodmate.net/2016/08/389963.html.[2021-12-31]. （in Chinese）.

[16] WANG PF， XUE XW， DENG WH. Determination of aspartame and ali sweet in tea shoots by liquid chromatography[J]. China Food， 2021（21）： 140-141. （in Chinese）.

[17] WANG Q， CAI LS. Study on the method of aspartame content determination in highland barley liquor[J]. J of Anhui Agri Sci， 2019， 47（21）： 214-215. （in Chinese）.

[18] LU Y， WANG LY， LIU YP，  et al.  Determination of 9 kinds of sweetenrs and preservatives in bakery products by ultra performance liquid chromatography[J]. J of Food Saf and Qual， 2021， 12（5）： 1725-1730. （in Chinese）.

[19] TIGHRINE A， AMIR Y， ALFARO P，  et al.  Simultaneous extraction and analysis of preservatives and artificial sweeteners in juices by salting out liquid-liquid extraction method prior to ultra-high performance liquid chromatography[J]. Food Chem， 2019（277）： 586-594.

[20] GONG Z， WU HP， WU LH，  et al.  Simultaneous detection of four sweeteners in Baijiu by LC-MS[J]. Liquor Making， 2021， 48（5）： 3. （in Chinese）.

[21] ZHANG SQ， FENG CT， LIN QF，  et al.  Simultaneous determination of 6 kinds of sweeteners in pastries by ultra performance liquid chromatography-tandem mass spectrometry[J]. J of Food Saf and Qual， 2021， 12（1）： 144-150. （in Chinese）.

[22] LI X， LI S， LI H，  et al.  Quantification of artificial sweeteners in alcoholic drinks using direct analysis in real-time QTRAP mass spectrometry[J]. Food Chem， 2021（342）： 128331.

[23] BALGOBIND K， KANCHI S， SHARMA D，  et al.  Hybrid of ZnONPs/MWCNTs for electrochemical detection of aspartame in food and beverage samples[J]. J electroanal chem， 2016（774）： 51-57.

[24] SHAO L， YAO YZ， LI Y，  et al.  Determination of low alcohol Luzhou flavor liquor by HPLC acesulfame potassium， saccharin sodium and aspartame[J]. Liquor Making， 2019（3）. （in Chinese）.

[25] LIU G， LEI J， ZHONG HX，  et al.  Determination of eight sweeteners in distilled spirits by HPLC evaporative light scattering detector[J]. Food Sci Tech， 2017， 42（7）： 300-304. （in Chinese）.

[26] LIU YH， TANG S， CHENG GH，  et al.  Study on the detection method of Edwan sweet and other sweeteners in liquor[J]. China Brewing， 2018， 37（11）： 172-175. （in Chinese）.

[27] ZHENG XL， TANG BB， LI XL，  et al.  Determination of 10 sweeterners in white spirits by ultra-performance liquid chromatography-tandem mass  spectrometry[J]. J of Anhui Agri Sci， 2021， 49（5）： 5. （in Chinese）.

[28] XIA QX， WANG Q， ZHANG DJ，  et al.  Determination of aspartame in nine drinks by HPLC[J]. J of Jiamusi University： Natural Sci Edition， 2021， 39（5）： 107-110. （in Chinese）.

[29] SI GR，ZHANG WW， SHI SJ，  et al.  Simultaneous determination of three sweeteners in liquor by high performance liquid chromatogrphy[J]. Liquor Making， 2014， 41（3）： 59-61. （in Chinese）.

[30] QIN GY， LI Z. Simultaneous determination of three sweeteners in liquor by high performance liquid chromatogrph[J]. Sci Tech Inno 2017（21）： 58-59. （in Chinese）.

[31] GB 2760-2014， National standards for food safety determination of aspartame and alitame in food[S]. Beijing： China Standards Press， 2014. （in Chinese）.

 Editor： Yingzhi GUANG   Proofreader： Xinxiu ZHU