P U B L I C A T I O N S CODON Italian Journal of Food Science, 2022; 34 (3): 91–98 ISSN 1120-1770 online, DOI 10.15586/ijfs.v34i3.2225 91 P U B L I C A T I O N S CODON Determination of aspartame and alitame in liquid dairy products and milk-containing beverages in the Chinese market Jiasi Yu1, Wei Hu2, Dan Wu1, Yun Ding1, Xin Wang1, and Ping Liu1* 1Department of Physical and Chemical Inspection, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, China; 2Shinan District Health Bureau, Qingdao, China *Corresponding Author: Ping Liu, Department of Physical and Chemical Inspection, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, 250012 Shandong, China. Email: liupingp@sdu.edu.cn Received: 4 May 2022; Accepted: 29 August 2022; Published: 6 October 2022 © 2022 Codon Publications OPEN ACCESS PAPER Abstract Aspartame and alitame are a type of food additives commonly used in recent years. However, it is difficult to con- clude that long-term use of synthetic sweeteners is completely harmless. This study aimed to establish a simple high performance liquid chromatography (HPLC) method to detect these two sweeteners, and to measure the concentrations of sweeteners in liquid dairy products and milk-containing beverages in the Chinese market. In this experiment, aspartame and alitame had a good linearity, and the average recovery values were also good with the relative standard deviations (RSDs) of 0.4–2.2%. The limits of detections (LODs) were 0.52 and 0.48 µg g–1 for aspartame and alitame, respectively. According to consumer daily purchase habits, 100 samples were purchased from supermarkets and milk tea shops in Jinan, China. The results showed that the amount of sweeteners added in all samples did not exceed the national standards, but there was a problem that the food label content was incom- plete. We hope that the relevant departments would strengthen supervision and management of food labelling, and protect the legitimate rights and interests of consumers. Keywords: alitame; aspartame; HPLC; liquid dairy products; milk-containing beverages Introduction With the development of the food industry, more and more food additives are used in food processing to improve the colour, taste, smell, nutritional value, and shelf-life of food. Food additives are synthetic or natural substances added to food, which can not only improve food quality, colour, aroma and taste but also meet the needs of anti-corrosion, preservation, and processing technology. There are more than 2000 kinds of food additives permitted by the state (GB 2760-2014), which are divided into 22 types according to their functions, mainly including sweeteners, preservatives, acidity reg- ulators, antioxidants, colorants, bleaching agents, etc. Among them, sweeteners are used in all kinds of food to increase the flavour of food. According to different sources, sweeteners can be divided into two categories, one is natural sweeteners such as licorice and stevioside, and the other is synthetic sweeteners. In the past few decades, the global prevalence of obesity has increased rapidly. The survey of lifestyle shows that excessive con- sumption of sugars such as sugary drinks may lead to obesity, diabetes, and cancer (Malik et al., 2010). The World Health Organization recommends that the intake of free sugars should not exceed 10% of the total energy intake (WHO, 2015). In order to reduce the intake of sugar, some countries have begun to levy sugar tax and have successfully reduced sugar consumption and obe- sity rate (Nakhimovsky et al., 2016). Industry is also responding to this policy by reducing sugar in food pro- cessing. Sugar is an indispensable ingredient in food processing, because it can improve the structure, colour, mailto:liupingp@sdu.edu.cn 92 Italian Journal of Food Science, 2022; 34 (3) Yu J et al. sweeteners (stevioside and mogrosides). These sweeten- ers are considered safe to eat. In the European Union, Australia, and New Zealand, sugar alcohols such as maltitol, lactose alcohol, xylitol, and erythritol are also considered safe to eat. FAO, WHO, and JECFA points out that the use of food additives within the permit- ted scope will not cause damage to the human body (Praveena et al., 2019). China’s standard for the use of food additives also refers to the international standards, which clearly stipulates the amount of sweeteners used in all kinds of food. Nevertheless, the safety of synthetic sweeteners with high sweetness is still controversial. Emerging epidemi- ological evidence shows that food additives may have non-benign biological effects and have a negative impact on human intestinal flora, leading to the destruction of intestinal mucus layer (Rinninella et al., 2019). Eating excessively processed and packaged foods is associated with increased all-cause mortality and increased global incidence of obesity, metabolic syndrome, and inflam- matory bowel disease (IBD) (Kim et al., 2015; Marion- Letellier et al., 2019; Pinget et al., 2019; Rico-Campà et  al., 2019). For example, with the increase in the sales of processed food, the incidence of IBD in China has increased significantly (Ng et al., 2013). Recent stud- ies have shown that the metabolic and inflammatory effects of food additives may be induced by changes in intestinal flora. Artificial sweeteners in food can change the number of intestinal bacteria, increase the patho- genicity of intestinal bacteria, and interfere with the normal environment of human and animal intestinal mucosa (Bian et al., 2017; Swidsinski et al., 2009; Zuo et al., 2018). Studies suggest artificial sweeteners such as saccharin, sucralose and aspartame can induce glucose intolerance in rats. Artificial sweeteners have metabolic effects and may lead to type 2 diabetes, obesity, and other diseases (Suez et al., 2014). Consumption of synthetic sweeteners during pregnancy and lactation has adverse effects on the metabolism of infants, which may lead to metabolic disorders in later life (Olivier-Van Stichelen et al., 2019). Plows et al. find that the intake of synthetic sweeteners during pregnancy can lead to glucose intolerance, hyperglycaemia, short- ened pregnancy time, male fetal growth restriction, and female fetal hypoglycaemia (Plows et al., 2020). In addi- tion, studies have shown that consumption of aspartame during pregnancy can increase the obesity rate and make offspring more prone to anxiety behavior (Palatnik et al., 2020). Long-term consumption of synthetic sweeteners is closely related to memory loss, Alzheimer’s disease, and depression (Burke and Small, 2015; Gao et al., 2018), in addition to carcinogenicity and genotoxicity (Mao and Song, 2018; Purohit and Mishra, 2018; Zhao and Wang, 2018). and taste of food. Hence, reducing the sugar content in food may have a negative impact on the taste of food. To ensure sweet taste while reducing sugar, synthetic sweet- eners are usually used instead of sugar. The human body does not digest and absorb sweeteners (such as sugar alcohols). The content of sweetener is high and provides little energy, so its dosage is very small. In order to pre- vent obesity, cardiovascular disease, diabetes, and other chronic diseases, consumers tend to choose foods con- taining lower calorie sweeteners. As a result, “sugar free” or “no added sugar” products with synthetic sweeteners instead of sugar have become more and more popular. At present, the artificial synthetic sweeteners approved for use in China mainly include aspartame, alitame, sac- charin sodium, acesulfame, sodium cyclamate, sucralose, etc. Aspartame, the chemical name of L-aspartyl-L phenyl- alanine methyl ester, also known as proteoglycan, sweet- ener, aspartame essence, aspartame mother, aspartame, etc., belongs to dipeptide derivatives. At room tempera- ture, it is white crystalline powder. The taste is very sim- ilar to sucrose, and the sweetness is 100–200 times of sucrose. In China, aspartame can be used as sweetener and flavour enhancer in dairy products, pastries, season- ings, drinks, jellies, puffed foods, etc. After people con- sume aspartame, it completely decomposes into aspartic acid, phenylalanine, and methanol in the stomach and intestines, and then is absorbed in the blood. Patients with phenylketonuria lack phenylalanine hydroxylase, and their excessive intake of aspartame causes accumula- tion of phenylpyruvate in the body and damages the ner- vous system. Thus, foods containing aspartame should be marked as follows: aspartame (containing phenylalanine). Alitame, the chemical name of L-α-aspartyl-n-(2,2,4,4- tetramethyl-3-sulfotrimethyl)-d-alanamide, also known as aspartame, is a dipeptide derivative, which is generally a white crystalline powder without odour and hygroscop- icity. The sweetness of alitame is 2000 times higher than that of sucrose, and its properties are relatively stable, especially for heat and acid. Alitame is easily soluble in water or hydroxyl containing solvents, but it is difficult to dissolve in lipophilic organic solvents. Alitame is often used with other sweeteners and has a good sweetening synergistic effect. China approved the use of alitame in 1994. It can be added to dairy products, frozen bever- ages, preserves, beverages, gum-based candies, jellies, and other foods as a sweetener (Wang and Shen, 2019). At present, with the increasing demand for “sugar free” or “low sugar” products, synthetic sweeteners can be an ideal and calorie-free sugar substitute in the food industry. The FDA has approved six synthetic sweet- eners (edvantame, aspartame, acesulfame, neotame, saccharin, and sucralose) and two natural non-nutritive Italian Journal of Food Science, 2022; 34 (3) 93 Determination of aspartame and alitame Studies have shown that the effect of aspartame may be related to methanol or its metabolites. As a neuro- toxin, methanol is harmful to human health. One litre of beverage containing aspartame can produce about 56 mg of methanol, and a can of beverage containing aspartame can produce about 22.4 mg of methanol. The Environmental Protection Bureau recommends that the daily intake of methanol should not exceed 7.8 mg (Liu et al., 2012). Moreover, long-term consumption of aspar- tame can cause migraine (Sathyapalan et al., 2015; Zaeem et al., 2016). Some scholars have found that aspartame has become a new type of water pollutant, which can affect the water environment through biochemical reac- tions (Lin et al., 2017; Seo et al., 2016). According to national regulations (GB 28050-2011), additives in food must be clearly marked on the label, but there are still phenomena of incomplete and non-standard content of food labels. It is necessary to strengthen the supervision of the use of synthetic sweeteners. At present, the detection methods of aspartame and alitame mainly include ultra-high performance liquid chromatography (UPLC) (Jin, 2020; Lu et al., 2021; Yu et al., 2018), ion chromatography (Xie et al., 2011; Zhu et al., 2005), high performance liquid chromatography (HPLC) (Chen and Li, 2006), and high performance liquid chromatography tandem mass spectrometry (HPLC-MS) (Tang et al., 2019; Wang et al., 2019; Yang and Chen, 2009), etc. However, most of the above meth- ods require expensive equipment, which is not available in conventional laboratories. In this study, a convenient and reliable HPLC method for the determination of aspartame and alitame was developed and validated. Furthermore, this study measured the concentrations of aspartame and alitame in liquid dairy products and milk-containing beverages collected from Jinan super- markets and milk tea shops. The results could provide a basis for improving market supervision and regulation in China. Materials and Methods Reagents and chemicals Standards: Aspartame (purity asis for improving mar- ket supervision and regulation Solarbio (Beijing, China). Acetonitrile (HPLC grade) was purchased from Damao Chemical Reagent Factory (Tianjin, China), ethanol (superior purity) from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), filter paper (diameter 15 cm) from Hangzhou Special Paper Co., Ltd. (Zhejiang, China), organic filter membrane (0.45 μm) from Beijing high purity Technology Co., Ltd. (Beijing, China), and the experimental water was Milli-Q double distilled water (Bedford, MA, USA). Sample collection and treatment All the samples were selected according to the daily purchasing habits of consumers: 100 samples of dif- ferent types, including 40 liquid dairy products and 60 milk-containing beverages, were collected randomly from supermarkets and milk tea shops in Lixia District of Jinan, China, in August 2019. An aliquot of 5 g (accurate to 0.0001 g) of thoroughly and adequately mixed sample was put into a 50 mL centri- fuge tube (Shimadzu analytical balance AUX120, Kyoto, Japan). Ten milliliters ethanol was added to each sample and then sealed. The centrifuge tube was turned upside down five times without oscillating for milk-containing beverages, or was vortexed for 10 s for liquid dairy prod- ucts. The resulting mixture was left at room tempera- ture for 1 min, and then centrifuged at 4000 rpm for 5 min (Eppendorf centrifuge 5430R, Hamburg, Germany). The supernatant was transferred to a 25 mL volumetric flask. The residue was dissolved with 8.0 mL ethanol/ water (2/1, v/v) and centrifuged at 4000 rpm for 5 min. Subsequently, the supernatant was transferred into the same 25 mL volumetric flask and made up to volume with ethanol/water (2/1, v/v). Finally, the solution was fil- tered through a 0.45 μm organic filter membrane, and 20 µL aliquot solution was injected into the HPLC system. Standard solutions Stock standard solutions of aspartame and alitame were prepared at 205.8 and 200 μg mL–1 by dissolving 2.1 and 2.0 mg of powder in 10 mL of water, respectively. All the stock solutions were stored at 4°C for utilization. A mixed standard solution was produced by transferring 2.5 and 1.7 mL of aspartame and alitame stock solutions into a 10 mL volumetric flask and making up to volume with water. Thereafter, a series of mixed standard solu- tions were obtained by further dilution. The concentra- tions of aspartame in the mixed standard solutions were 0.51, 1.03, 10.29, 34.99, and 51.45 μg mL–1, respectively; the concentrations of alitame were 0.50, 1.00, 10.00, 34.00, and 50.00 μg mL–1, respectively. The mixed stan- dard solutions were stable for 1 month and were stored at 4°C. HPLC conditions A LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) was used for the determination of aspartame and alitame and was quantified by diode array detection using an L-2455 diode array detector (Hitachi, Tokyo, Japan) with the wavelength of 200 nm. The sample was separated on a C18 column (4.6 × 150 mm, 5 μm) (Elite, Dalian, China) 94 Italian Journal of Food Science, 2022; 34 (3) Yu J et al. 208 nm. In this experiment, the detection wavelength was carried out at 200 nm based on the ultraviolet full- wavelength scanning spectrum. Mobile phase The composition of the mobile phase plays an import- ant role in elution and chromatographic separation. At the beginning of this project, we planned to perform the HPLC analysis of aspartame and alitame using two types of mobile phases, methanol/water (40/60, v/v) and ace- tonitrile/water (20/80, v/v). It was found that the target peaks overlapped, which may be due to the interference of other impurities in the sample, when the mobile phase methanol/water (40/60, v/v) was used for sample detec- tion. Other mobile phases were tested by changing the ratio of acetonitrile and water, but these mobile phases did not deliver a notable improvement to the chromatog- raphy. Therefore, acetonitrile/water (20/80, v/v) was used as the mobile phase in terms of peak shape, resolution, and overall analysis time. Flow rate The fast flow rate can increase column pressure, while slow flow rate can cause tailing deformation of chromato- graphic peaks. The flow rate of 0.8, 1, and 1.2 mL min–1 was employed for this experiment, and other experimen- tal conditions remained unchanged. When the flow rate was 1.2 mL min–1, the resolution of the chromatographic peak was poor. When the flow rate was 0.8 mL min–1, the retention time (RT) of the chromatographic peak was longer. Considering comprehensively, a flow rate of 1  mL  min–1 was suitable for sample analysis, and the analysis time was 10 min (Figure 1). at 30°C, with a mobile phase of 20% acetonitrile and 80% water at a flowrate of 1 mL min–1, and an injection vol- ume of 20 µL. Method validation The HPLC method was validated using the following parameters: linearity, recovery, precision, LOD, and lim- its of quantification (LOQ). Linearity was evaluated from the five concentration levels of mixed standard solution. Recovery was assessed by spiking the sample with two sweeteners at three fortified concentrations of 0.285, 0.355, 0.425 µg mL–1 in three replicates, and calculated as the ratio of a measured concentration for spiked sam- ple divided by spiked concentration. The precision tests were conducted using a sample added 71 µg of alitame standard to determine for six times in the same day. The precision was based on the relative standard deviation (RSD%). The LOD and LOQ were defined by signal/ noise ratios of 3:1 and 10:1, respectively. Both LOD and LOQ were verified experimentally after injecting blank samples. Results and Discussion Optimization of chromatographic conditions Detection wavelength The maximum absorption wavelength of aspartame was 195.2 nm, and that of alitame was 198.7 nm. According to the literature, analytes were mostly detected at 200 or Au 0.15 0.10 0.05 0.00 0 1 2 3 4 5 6 7 8 9 10 min Aspartame Alitame Figure 1. Chromatogram of aspartame and alitame standard solutions. Italian Journal of Food Science, 2022; 34 (3) 95 Determination of aspartame and alitame especially for aspartame. In our study, under the LOD of this method, no alitame was detected in any sam- ple and it was not identified on the food label. Notably, a total of 21 products were marked with aspartame on the food label, while there were far more than these in actual testing. The samples that detected aspartame accounted for nearly half of the total. Among them, aspartame was detected in 11 samples of 40 liquid dairy products (27.5%), and the concentrations ranged from 2.41 to 103.67 μg g–1; aspartame was detected in 24 sam- ples of 42 milk-containing beverages (57.1%), and the concentrations ranged from 1.81 to 61.49 μg g–1; aspar- tame was detected in eight samples of 18 milk drinks on sale (44.4%), and the concentrations ranged from 2.14 to 21.11 μg g–1. Table 4 summarized determination results of aspartame and alitame in liquid dairy products and milk-containing beverages from the Chinese market. The national standard stipulates a limit of 600 μg g–1 of aspartame in modified milk, protein drinks, and flavored drinks (GB 2760-2014). According to the regression equation, the measured value of aspartame in the sample was calculated, and compared with the national standard, none of them exceeded the standard. Through the detection of liquid dairy products and milk-containing beverages samples, there was no phe- nomenon of excessive use of aspartame and alitame. Although alitame was rarely used in the products in this study, most of the products were added with aspar- tame. As a new type of sweetener in recent years, there is no guarantee that long-term consumption of synthetic sweeteners is safe. Therefore, the type of sweetener added should be identified on the food label, especially aspar- tame (containing phenylalanine). It is recommended that the market management department implement label Method validation After verification, the standard curve was plotted using the peak area as the ordinate and the concentrations as the abscissa. The linearity was good using the standard solutions at five concentration levels (Table 1). According to the experimental results, the average recovery values of aspartame and alitame were 93.0–107.3% and 97.2– 98.2%, respectively. The repeatability expressed as RSD (%) was 1.1% for aspartame and 2.5% for alitame. The data corresponding to the recoveries and precisions were detailed in Tables 2 and 3, and met the needs of analysis with great reliability and repeatability. The LODs were 0.52, 0.48 μg g–1 for aspartame and alitame, respectively, and the LOQs for same analytes were 1.72 and 1.58 μg g–1, respectively. Lu Y et al. established an analytical method for the detection of nine kinds of sweeteners and pre- servatives in baked products by UPLC, and the LODs of aspartame and alitame in this method were 3.0 mg kg–1, which were higher than our method (Lu et al., 2021). In the study of UPLC detection of sweeteners in beverages, the LODs of aspartame and alitame were 0.75 mg  kg–1 (Jin, 2020). For the liquid chromatography coupled with mass spectrometry techniques, the LODs of the two sweeteners were sufficient for trace analysis, which were 1.0 and 0.2 ug L–1, respectively (Wang et al., 2019). Given the requirements of the equipment and the performance of the method, our method has the advantage of being simple, rapid, and low cost. Analysis of aspartame and alitame in liquid dairy products and milk-containing beverages Among the 100 samples in this survey, except for milk drinks on sale, the remaining 82 beverages all have label instructions. Milk tea is immensely popular among young people for its smooth and silky taste, but so far, many businesses lack the label of the ingredient list, Table 1. Linear equation and linear range of aspartame and alitame. Analyte Linear equation R Linearity (μg mL–1) Aspartame y = 11485x + 2613 0.9993 0.51–51.45 Alitame y = 9200x − 4606 0.9996 0.50–50.00 Table 2. Recoveries and RSD values of aspartame and alitame in samples. Aspartame Alitame Spiked (µg mL–1) Average recovery % RSD % n=3 Spiked (µg mL–1) Average recovery % RSD % n=3 0.285 93.0 2.7 0.285 98.2 1.6 0.355 100.1 2.0 0.355 97.8 0.4 0.425 107.3 1.9 0.425 97.2 1.2 Table 3. Precision test results. Analyte Measured value (µg g–1) Average (µg g–1) RSD % 1 2 3 4 5 6 Aspartame 595.53 591.05 587.69 581.18 586.44 578.53 586.73 1.1 Alitame 262.58 274.41 267.68 256.39 259.00 262.60 263.78 2.5 96 Italian Journal of Food Science, 2022; 34 (3) Yu J et al. Table 4. Concentrations of aspartame and alitame in liquid dairy products and milk-containing beverages. Group N(a) Food labels Sweeteners Labels containing sweeteners Detected sample Concentrations of sweeteners in detected samples (μg g–1) Mean P50(c) P90(c) P95(c) Max Liquid dairy products 40 40 Alitame 0 8 –(b) 11 – – – – – Aspartame 31.66 8.23 72.10 87.88 103.67 Milk-containing beverages (pre-package) 42 42 Alitame 0 13 – 24 – – – – – Aspartame 24.32 16.27 60.64 61.23 61.49 Milk-containing beverages (made-on-site) 18 0 alitame 0 0 – 8 – – – – – Aspartame 5.77 3.00 11.33 16.22 21.11 (a) Number of samples (b) – indicates did not detect the Acesulfame in these samples (c) 50th, 90th, and 95th percentiles of the distribution management on milk drinks, and indicate the types of sweeteners used, which is conducive to protecting the legitimate rights and interests of consumers. Conclusions In this paper, a rapid and reliable HPLC method for the simultaneous determination of aspartame and alitame in liquid dairy products and milk-containing beverages from the Chinese market was developed and validated, which was applied for the analysis of 100 products. Since the use of artificial sweeteners has increased, con- tinued monitoring and strengthening the regulation of the sweeteners used in food is essential. In addition, the analysis data showed that 14 samples with food labels were not labelled with aspartame. The mislabelling leads to uncertainty over both the contents and their concentrations, and increases the likelihood of exces- sive intake of artificial sweeteners. Thus, the results of this survey involve public health issues, and require the attention of the Chinese government to strengthen market supervision and regulation. However, there are still some uncertainties that should be considered. 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