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Simultaneous determination of sweeteners in beverages by LC-MS/MS a
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Hiroaki Sakai , Azusa Yamashita , Masayoshi Tamura , Atsuo Uyama & Naoki Mochizuki
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Research Laboratories for Food Safety Chemistry, Asahi Group Holdings, Ltd, Moriya, Ibaraki, Japan Published online: 20 Mar 2015.
Click for updates To cite this article: Hiroaki Sakai, Azusa Yamashita, Masayoshi Tamura, Atsuo Uyama & Naoki Mochizuki (2015) Simultaneous determination of sweeteners in beverages by LC-MS/MS, Food Additives & Contaminants: Part A, 32:6, 808-816, DOI: 10.1080/19440049.2015.1018341 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1018341
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Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 6, 808–816, http://dx.doi.org/10.1080/19440049.2015.1018341
Simultaneous determination of sweeteners in beverages by LC-MS/MS Hiroaki Sakai, Azusa Yamashita, Masayoshi Tamura, Atsuo Uyama and Naoki Mochizuki* Research Laboratories for Food Safety Chemistry, Asahi Group Holdings, Ltd, Moriya, Ibaraki, Japan
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(Received 29 August 2014; accepted 5 February 2015) A new method was established for the simultaneous determination of 10 sweeteners and a degradation product in beverages by using LC-MS/MS. An ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm) was used as the LC column and 0.1% each of aqueous formic acid and formic acid in acetonitrile were used as the mobile phase. A simple and rapid determination of sweeteners was possible by diluting with a solvent, and in the case of some samples containing a large amount of foreign matter, after pre-treatment by diluting with solvent and clean-up of the sample using an Oasis HLB cartridge. All the validation results were satisfactory. As the regulations and standards for sweeteners vary from country to country, a field survey of 58 beverages marketed in Japan was performed using the present method. No issues concerning the labelling or food sanitation law were found in the tested samples. Keywords: LC-MS/MS; sweetener; beverage; acesulfame potassium; sucralose; aspartame; diketopiperazines; rebaudioside A
Introduction In recent times, obesity has become a growing problem in many countries including the USA, UK and Australia. Diet is an important contributing factor to obesity, and reducing sugar intake is a preventive and remedial measure for obesity. In Japan, people are increasingly concerned about obesity and have become diet conscious with the aim of improving and maintaining their health and beauty. In this context, low-sugar and low-calorie products that use sweeteners are increasingly replacing sugar-laden foodstuff. Sweeteners are sweeter than sugar and some of them are several hundred times sweeter than sugar. The quantity of sugar used can be greatly reduced by adding a small amount of sweetener, thereby also helping to control both the calorie intake and blood sugar. Some sweeteners are excreted from the body without being metabolised and they contribute few or no calories of their own. In Japan, the use of food additives such as sweeteners is stipulated by the food sanitation law. The safety of food additives is assessed, and food additives are classified as permitted as long as there is no possibility of any harm being caused to human health. Currently in Japan sweeteners such as acesulfame potassium (AK), sucralose (SUC), aspartame (APM) and sodium saccharine (SA) have been approved as additives. As an exception, conventional food additives that have been widely used in Japan are also permitted to be used as food additives. Among them are sweeteners that include stevia extract (with stevioside and rebaudioside A (REB) as the main compounds) and liquorice extract (glycyrrhizic acid (GA) as the main compound). Sweeteners were introduced in Japan around 1890, and sugar, which was an expensive commodity, was
widely substituted with saccharin (Yamamoto 1980). However, in the mid-1900s, saccharin was suspected to be carcinogenic, and in 1971 it was removed from the list of generally recognised as safe (GRAS) substances in the USA. In Japan, its use in food products was banned in 1973 (Food Safety Commission 2011). Subsequently, saccharin was reconfirmed to be safe and its use was permitted again in Japan, and now it is being used in the European Union and countries like the USA and Australia (Food Safety Commission 2011, Nihon Shokuhin Tenkabutsu Kyokai 2012). Dulcin (DU) is another food additive that was used in Japan and the USA. However, it was found to be carcinogenic (Fitzhugh et al. 1951), and was subsequently banned in Japan and other countries. Sodium cyclamate (CYC) is another food additive that was used in Japan and USA in the 1950s. However, as the FDA indicated it to be toxic, it was banned in both the countries in 1969 (Aiso 1972; Kagawa 1994). CYC was later recognised as being safe and is now used in the European Union and Australia. However, it is still banned in Japan and the USA. (Kagawa 1994; Nihon Syokuhin Tenkabutsu Kyokai 2012). AK is being used as a sweetener in at least 90 countries, including Japan (Beppu 2010). Neotame (NE) is used in Japan, the USA, Canada and the European Union (Beppu 2010; Nihon Syokuhin Tenkabutsu Kyokai 2012). Alitame (AL) is used in Australia, China and Malaysia. However, it is not permitted for use in Japan, the USA and the European Union (Nihon Syokuhin Tenkabutsu Kyokai 2012). Therefore, as described above, sweeteners that are permitted for use vary according to the country. Sometimes, even the standards established for permitted sweeteners
*Corresponding author: Email: [email protected] © 2015 Asahi Group Holdings, LTD. Published by Taylor & Francis.
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Food Additives & Contaminants: Part A vary from country to country. Therefore, because of the differences in regulations, imported food products are inspected by various government agencies. With the development of new sweeteners, the number of sweeteners permitted for use in food products is increasing. In many cases, a combination of several sweeteners is added to food products to obtain synergistic effects such as the enhancement of sweetness and improvement of flavour; in other words, the use of sweeteners is being diversified. Furthermore, sweeteners are being increasingly used in a variety of food products. Thus, for the analysis of sweeteners it is essential to perform a pre-treatment step according to the matrix corresponding to the respective food product. A variety of methods involving pre-treatment by solvent extraction, SPE, dialysis and dialysis SPE followed by individual or simultaneous measurement using analytical techniques such as HPLC, LC-MS and LC-MS/MS have been reported for the analysis of sweeteners (Nihon Shokuhin Eisei Kyokai 2003; Matsumoto et al. 2008; Beppu 2010; Tsuruda et al. 2013). Moreover, violations such as the presence of non-permitted sweeteners and exceeding the maximum level have been observed occasionally. Therefore, the development of an efficient and rapid method is essential for the simultaneous analysis of a wide variety of sweeteners. We have therefore used beverages available in Japan in various sweetened food products as samples to measure and simplify the troublesome pre-treatment process. Ten
Figure 1.
Structural formulae of the 11 compounds.
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sweeteners (namely, AK, SA, APM, SUC, NE, REB, GA, CYC, AL and DU) and 5-benzyl-3,6-dioxo-2-piperazineacetic acid (diketopiperazines, DKP) (Tsang et al. 1985), which is the main decomposition product of APM (Figure 1), were analysed in these beverages. A high concentration of DKP in commercial beverages leads to the decomposition of APM; therefore, the estimated APM concentration might be less than the added APM concentration. Various conditions such as instrumental parameters have been established for the rapid and simultaneous determination of sweeteners by LC-MS/ MS. Therefore, we have analysed sweeteners used in various types of beverages sold in Japan, and this study is a compilation of the results.
Materials and methods Samples and reagents In total, 58 commercially available beverage samples (12 carbonated beverages, 22 coffee, five sports drinks, five fruit drinks, seven non-alcoholic cocktails and non-alcoholic shochu-based beverages, and seven cocktails and shochu-based beverages) were analysed. AK, SA, APM, DU and CYC were obtained from Kanto Chemical Co. Inc. (Tokyo, Japan). SUC, REB, GA and DKP were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). NE was obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). AL
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H. Sakai et al.
was obtained from ChromaDex (Irvine, CA, USA). Methanol (for LC-MS), acetonitrile (for LC-MS), 0.1% v/v formic acid–distilled water (for HPLC), and 0.1% v/v aqueous formic acid–acetonitrile (for HPLC) were obtained from Kanto Chemical Co. Formic acid (for LC-MS) was purchased from Wako Pure Chemical Industries. Each of the individual standard solutions was prepared by dissolving 10 mg of the reference standard in solvent and making up to 10 ml to obtain a 1000 µg ml−1 standard solution. However, AK, SA, CYC and AL were made up to 100 ml to obtain 100 µg ml−1 standard solutions. For AK, SA, CYC, NE and SUC, distilled water was used as the solvent, and for AL, DU, REB and GA, a methanol– water (1:1) mixture was used as the solvent. For DKP and APM, methanol and 0.1% aqueous formic acid were used as the solvents, respectively. The mixed standard solution was prepared using the 11 individual standard solutions as follows: 1 ml of the SUC standard solution along with 0.1 ml each of the remaining standard solutions was made up to 10 ml by using 0.1% aqueous formic acid. Sample preparation All the samples were diluted 500 times with 0.1% aqueous formic acid and used as test solutions. However, SUC and GA were diluted 100 times. For carbonic acid-containing samples, about 25 ml of each sample was taken in a 50 ml centrifuge tube and shaken for about 5 min at 150 rpm, followed by de-aeration for about 15 min by ultrasonication and dilution. In coffee samples containing milk, foreign particles such as milk or solid matter were present after dilution. Therefore, the sample was purified by using a SPE cartridge after dilution. The sample (3 ml) was loaded at the rate of one drop per second on an Oasis HLB cartridge, which was conditioned with 3 ml each of methanol and 0.1% aqueous formic acid, respectively, followed by washing with 3 ml of 0.1% aqueous formic acid. Then the sample was eluted with 6 ml of 2% formic acid in methanol. The eluate was collected and concentrated to l ml or less by using a pressured gas blowing concentrator. Then the concentrated sample was made up to 3 ml with 0.1% aqueous formic acid and used as the test solution. When the concentrations of the test samples were outside the calibration curve range, the concentrations were adjusted appropriately. Equipment and instruments A Nexera UHPLC System (Shimadzu Corporation, Kyoto, Japan) and MS/MS model LCMS-8040 (Shimadzu) were used. An ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 µm) HPLC column (Waters Corporation, Milford, MA, USA) was used. An Oasis HLB SPE cartridge
(60 mg/3 cc; Waters) was used. A pressured gas blowing concentrator MG-2200 (Tokyo Rikakikai Co. Ltd, Tokyo, Japan) was also used.
Analytical conditions Elution was performed using 0.1% aqueous formic acid (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The gradient programme was as follows: 10% B (0–2.9 min)–26% B (5 min)–42% B (6.2 min)–70% B (7–8 min)–10% B (8.01–10.5 min). The flow rate of the mobile phase, column temperature and sample injection volume were 0.6 ml min−1, 40ºC, and 20 µl, respectively. ESI (positive and negative) was used as the ion source for MS/MS. The de-solvation line temperature, heat block temperature, nebuliser gas flow rate and flow rate of the drying gas were 300ºC, 500ºC, 3 L min−1 and 15 L min−1.
Results and discussion Results of LC-MS/MS assessment conditions The dissociation of highly polar acidic substances such as AK, SA and CYC was inhibited by keeping the sample and mobile phase under acidic conditions. The separation behaviour of various octadecyl silane columns (ACQUITY UPLC BEH C18, Waters; CAPCELL PAK C18 IF, Shiseido Co. Ltd, Tokyo, Japan; L-column 2, Chemicals Evaluation and Research Institute, Tokyo, Japan; Mastro C18, Shimadzu GLC, Tokyo, Japan; Inertsustain C18, GL Sciences, Tokyo, Japan) was compared. 0.1% each of aqueous formic acid and formic acid in acetonitrile were used as the mobile phase and the gradient conditions were suitably adjusted. The ACQUITY UPLC BEH C18 column was selected for further analysis because it showed better separation of all the components ranging from highly polar AK to weakly polar GA, excellent peak shapes and minimum elution time for all the components (Figure 2). In previous studies the time taken for eluting seven to nine compounds was 20–25 min (Koyama et al. 2005; Asano et al. 2008; Yang & Chen 2009). In the present method, all 11 compounds were eluted within 7 min. Therefore, the proposed method rapidly elutes all the compounds, enabling the simultaneous determination of sweeteners. When the highly versatile electrospray ionisation was used as the ion source, AL and DU ionised well in the positive-ion mode, and the remaining nine compounds were ionised in the negative-ion mode. Conditions of applied voltage in each mode are presented in Table 1. As confirmation ions of SA, CYC and SUC could not be obtained, only fixed ions were set.
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Figure 2. Chromatogram of the standard mixture. The concentrations of the sweeteners were 10 ng ml−1 for AK, SA, CYC and AL; 100 ng ml−1 for APM, DKP, DU, REB, NE and GA; and 1000 ng ml−1 for SUC. Peaks are numbered as follows: 1, AK; 2, SA; 3, CYC; 4, DKP; 5, SUC; 6, APM; 7, DU; 8, AL; 9, REB; 10, NE; and 11, GA.
Table 1.
MRM conditions of the 11 compounds. Quantitative ion
Qualitative ion
Analyte
Polarity
Precursor ion (m/z)
Product ion (m/z)
Collision energy (V)
Product ion (m/z)
Collision energy (V)
AK SA CYC DKP SUC APM DU AL REB NE GA
Negative Negative Negative Negative Negative Negative Positive Positive Negative Negative Negative
162 182 178 261 395 293 181 332 965 377 821
82 106 80 146 359 261 108 129 803 200 351
12 19 25 14 11 9 −24 −21 27 18 45
78 – – 126 – 200 110 159 641 345 193
30 – – 18 – 13 −22 −23 55 10 49
Results of the matrix effects study Previously reported methods (Nihon Syokuhin Eisei Kyokai 2003; Beppu 2010; Tsuruda et al. 2013) were applied to various liquid, semi-solid and solid samples such as sauce, miso (bean paste), peanut butter, jam, takuan (pickled radish) and biscuits, and these also include samples containing very high foreign matter content. In this study, however, we limited the present method to the analysis of liquid samples such as cold beverages and alcoholic drinks. Furthermore, as water was the major constituent in our samples, the foreign matter content was considered to be less than that present in the food products described above (sauce, jam etc.). Therefore, to enable a faster pre-treatment, the matrix effect was reduced by diluting the sample. Furthermore, it was previously confirmed that the samples used in the addition did not contain the 11 compounds under study. Table 2 shows the results of the evaluation of matrix effects (ion suppression or promotion of ionisation). For carbonated drinks, sports drinks, fruit drinks, non-alcoholic cocktails and cocktails, the matrix effect was
obtained from the ratio of sample to which the respective standard was added and the standard mixture (n = 3). The results were favourable with the proportion of matrix effect being determined as 84.7–111.4%, and in all the groups the matrix effect was negligible. For samples subjected to SPE (coffee with milk), the matrix effect was determined from the ratio of sample to which the respective standard was added after pre-treatment and the standard mixture (n = 3). Results were favourable at 95.7–114.5%, and the matrix effect was negligible. However, in Japan, various types of coffee are sold. Thus, a standard addition method that can be applied to a wide range of products was employed.
Results of method validation As discussed in the ‘Results of matrix effects study’ section, in all the groups the matrix effect was negligible. Thus, for validity evaluation with the dilution method, one representative group (cocktail) was used. Linearity was obtained from the correlation coefficient of the calibration
812 Table 2.
H. Sakai et al. Results of the matrix effect study. Compound
Category a
Carbonated drink Sports drinka Fruit drinka Non-alcoholic cocktaila Cocktaila Coffeeb
AK
SA
CYC
SUC
APM
DU
AL
REB
NE
GA
DKP
96.1 84.8 96.9 100.5 84.7 95.7
95.1 88.6 97.1 95.2 101.7 107.2
106.1 103.7 105.3 108.2 98.0 96.9
101.8 96.9 103.3 104.8 100.5 105.2
103.9 103.7 105.2 101.4 97.6 105.8
100.9 101.4 102.3 102.2 97.1 110.8
103.6 106.6 104.5 111.4 100.8 106.1
94.8 100.5 106.2 102.0 94.3 113.5
99.3 105.3 105.6 102.6 99.2 102.0
101.4 100.1 101.9 106.1 89.2 114.5
100.7 99.4 101.5 102.1 99.7 111.3
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Notes: Each number is expressed as a percentage. a Dilution method. b Dilution and SPE method. The spiked levels of the sweeteners were 1 μg ml−1 for AK and AL; 2 μg ml−1 for SA and CYC; 10 μg ml−1 for DU, REB, NE and DKP; 20 μg ml−1 for APM and GA; and 100 μg ml−1 for SUC.
curve by using a standard mixture appropriately diluted with 0.1% aqueous formic acid. Correlation coefficients were 0.9989 or greater in the range of 0.2–10 ng ml−1 for AK and AL, 0.5–20 ng ml−1 for CYC, 1–50 ng ml−1 for DKP, DU and NE, 1–200 ng ml−1 for REB, 5–200 ng ml−1 for APM and GA, and 20–1000 ng ml−1 for SUC, and thus satisfactory results were obtained for all the compounds (Table 3). Repeatability was found from the RSD of the sample to which the respective standard substance was added (n = 6). We tested three different additive concentrations that covered the entire range of the calibration curve. The tests were repeated multiple times over a couple of days. The resulting RSD was 0.8–17.3% (Table 3). The recovery rate of the sweeteners was calculated from the ratio of sample to which the respective standard was added and the standard mixture (n = 3). Additive concentrations and the number of tests were set the same as in repeatability. Therefore, the recovery rate of the sweeteners ranged from 84.7% to 119.0%, which was good (Table 3). For samples that were subjected to SPE (coffee with milk), the calibration curve was created by using the standard addition method. Linearity was obtained from the correlation coefficient of the calibration curve obtained after pre-treatment by using a diluted sample and adding an appropriately diluted standard mixture. The correlation coefficient obtained in the range stated above was at least 0.9985 and good results were obtained for all the compounds (Table 4). Repeatability was found from the RSD of the sample to which the respective standard substance was added (n = 6). Similarly, three levels were set for additive concentrations, and tests were repeated multiple times over a couple of days. The resulting RSD was 1.5–16.8% (Table 4). The recovery rate of the sweeteners was calculated from the ratio of sample to which the respective standard was added prior to pre-treatment and that to which the respective standard was added after pretreatment (n = 3). Additive concentrations and the number
of tests were set the same as in repeatability. Therefore, the recovery rate of the sweeteners ranged from 81.1% to 112.2%, which was good (Table 4). The LOQ was calculated as the concentration at which S/N = 10 and LOD was calculated as the concentration at which S/N = 3 using the chromatogram for the standard mixture (Table 5). Both parameters showed a sensitivity that was more than that of the conventional method (Nihon Syokuhin Eisei Kyokai 2003; Beppu 2010).
Analysis of commercially available products A total of 58 commercially available beverages samples described in the ‘Samples and reagents’ section were analysed by the present method. The results of the measurements performed in the concentration range of each compound are presented in Tables 6 and 7. Of the 58 samples tested, 29 listed the names of the 10 sweeteners in the principal ingredients, while the other 29 did not. The samples in which sweeteners were detected and those for which the names of the sweeteners were mentioned in the principal ingredients are shown in Table 6. Out of the 10 sweeteners, AK, SUC, APM and REB were detected. Among the detected sweeteners, AK and SUC were detected in 24 samples (44.4%) and 21 samples (38.9%), respectively. REB was present in six samples (11.1%), and APM was present in three samples (5.6%). For samples in which the sweeteners were detected, the sweetener mentioned in the principal ingredients matched with the detected sweetener. DKP was detected in three samples, but all of them were those in which APM was detected. For the 29 samples where sweeteners were not mentioned in the principal ingredients, none of the 10 compounds was found. Table 7 shows the sweetener concentration of the various samples. Out of the 58 samples, four sweeteners, namely AK, SUC, APM and REB, were detected only in one sample, namely carbonated drink 1. In the other
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
1 2 1 2
1 2 1 2
1 2 1 2
Day Day Day Day
Day Day Day Day
Day Day Day Day
0.9999 0.2 85.6 88.7 6.7 4.8 1 84.7 86.2 2.7 5.2 10 86.9 89.0 1.5 3.6
AK > 0.9999 0.5 89.0 114.5 15.3 10.7 2 101.7 101.9 6.4 5.0 20 93.3 91.3 1.9 2.5
SA 0.9992 0.5 100.6 93.3 4.4 7.6 2 98.0 98.3 2.8 2.9 20 102.2 99.7 1.0 1.9
CYC > 0.9999 20 107.3 113.0 4.5 2.5 100 100.5 101.7 3.4 3.6 1000 100.5 96.4 1.2 1.2
SUC > 0.9999 5 100.5 103.1 3.9 2.8 20 97.6 99.2 2.5 2.5 200 99.6 97.4 0.9 1.8
APM
1 2 1 2
1 2 1 2
1 2 1 2
Day Day Day Day
Day Day Day Day
Day Day Day Day
> 0.9999 0.2 102.0 91.3 6.2 8.3 1 103.8 104.0 3.6 4.6 10 107.2 88.1 1.8 3.5
AK 0.9998 0.5 100.2 81.1 6.8 16.5 2 95.7 109.4 8.4 4.8 20 106.3 93.7 3.3 4.8
SA 0.9999 0.5 104.5 103.9 7.3 7.6 2 102.1 106.0 3.3 4.3 20 105.2 101.0 2.1 4.2
CYC 0.9997 20 107.3 103.6 4.3 7.2 100 106.1 100.9 1.5 3.4 1000 104.5 106.9 1.9 3.3
SUC 0.9999 5 106.0 101.6 6.2 5.4 20 96.1 104.4 3.5 4.5 200 104.5 99.0 4.0 2.1
APM
Validation results for the established analytical method (dilution and solid phase extraction).
Linearity (r) Spiked level (μg ml−1) Recovery (%)
Compound
Table 4.
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
Linearity (r) Spiked level (μg ml−1) Recovery (%)
Compound
Table 3. Validation results for the established analytical method (dilution).
> 0.9999 1 100.1 90.4 3.8 6.1 10 100.4 103.8 2.5 3.5 50 105.1 98.8 2.8 4.5
DU
> 0.9999 1 96.4 100.2 8.5 5.3 10 97.1 97.0 2.4 5.4 50 98.8 96.3 1.5 1.2
DU
0.9995 0.2 108.9 103.7 6.3 7.1 1 101.0 102.9 1.8 3.7 10 103.2 98.0 2.8 4.7
AL
0.9998 0.2 93.4 101.2 4.3 2.8 1 100.8 102.3 2.2 0.8 10 99.1 100.3 2.0 2.1
AL
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> 0.9999 1 112.2 92.7 5.9 7.3 10 94.3 97.8 5.5 6.3 200 100.4 104.6 6.9 3.5
REB
> 0.9999 1 104.2 111.3 9.4 7.2 10 94.3 91.1 5.9 4.5 200 98.9 99.3 4.3 3.8
REB
> 0.9999 1 108.7 89.4 5.7 5.2 10 101.9 100.7 2.7 6.5 50 101.8 103.7 5.7 5.9
NE
> 0.9999 1 98.8 112.0 8.4 7.8 10 99.2 94.6 6.4 4.4 50 98.9 93.4 3.2 3.7
NE
0.9985 5 99.7 111.2 10.9 2.6 20 106.0 103.2 16.8 16.8 200 87.3 97.7 5.3 11.2
GA
0.9989 5 106.9 119.0 12.5 16.4 20 89.2 91.3 9.1 17.3 200 95.3 101.4 5.8 12.0
GA
> 0.9999 1 101.8 105.7 9.8 4.9 10 100.9 102.5 3.4 3.2 50 105.5 101.9 2.3 4.5
DKP
> 0.9999 1 95.5 105.9 6.9 3.2 10 99.7 95.2 1.5 3.2 50 101.8 94.4 0.8 1.6
DKP
Food Additives & Contaminants: Part A 813
814 Table 5.
H. Sakai et al. Limit of detection (LOD) and limit of quantitation (LOQ).
LOD (μg ml−1) LOQ (μg ml−1)
Table 6.
AK
SA
CYC
SUC
APM
DU
AL
REB
NE
GA
DKP
0.002 0.007
0.02 0.06
0.004 0.01
0.56 1.9
0.004 0.01
0.06 0.19
0.01 0.03
0.005 0.02
0.002 0.008
0.009 0.03
0.09 0.29
Number of samples in which various compounds were detected.
Category
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Carbonated beverage Coffee Sports drink Fruit drink Non-alcoholic cocktaila Cocktailb Total
AK 4 10 2 0 5 3 24
(4) (10) (2) (5) (3) (24)
SUC 4 6 4 1 5 1 21
(4) (6) (4) (1) (5) (1) (21)
APM 3 0 0 0 0 0 3
REB (3)
(3)
3 2 0 1 0 0 6
(3) (2) (1) (6)
DKP
SA
NE
GA
CYC
DU
AL
3 0 0 0 0 0 3
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Notes: Values in parentheses are the number of samples in which the sweeteners used are mentioned in the list of principal ingredients. a Includes non-alcoholic shochu-based beverage. b Includes shochu-based beverage.
samples, at most three components were detected. Multiple sweeteners, namely AK, SUC and APM, were detected simultaneously in carbonated drinks 2 and 3. Out of the 58 samples, carbonated drinks 1–3 showed the highest value of the total concentration of sweeteners and among all the samples, carbonated drink 1 showed the highest value of 460.0 µg ml−1. Carbonated drinks 1–3 were the low-sugar and low-calorie products where sugar or sugar syrup was not used. Their sweetness might have been generated only from the sweeteners that were detected. In carbonated drinks 3 and 4, the concentrations of SUC were the highest of all the samples, which were 92.8 and 76.1 µg ml−1, respectively. AK and APM are 200 times sweeter than sucrose, whereas SUC is 600 times sweeter than sucrose. Therefore, the sweetness of carbonated drinks 3 and 4 is considered to be contributed mainly by SUC. Carbonated drinks 4–6 had mentioned the use of sugars (sugar syrup and sugar) in addition to sweeteners and carbonated drinks 7–12 had mentioned the use of only sugars. Furthermore, APM was detected in three carbonated drink samples and DKP was also detected in them (carbonated drinks 1–3). The proportion of DKP was very low (1.2–1.9%) compared with that of APM in all the samples. APM is stable in aqueous solution at pH 3–5 (Homler 1984). The pH of common carbonated drinks is in this range and APM might undergo slight decomposition during the storage of the product. APM undergoes considerable decomposition when the sample has high DKP concentration. However, such samples were not observed in our study. In the case of coffee samples, sweeteners were detected in 10 samples (1–10). All the samples were low-sugar products and seven of them contained sugar in
addition to the detected sweeteners. Three sweeteners, namely AK, SUC and REB, were detected. AK was detected in all the coffee samples (1–10), and SUC and REB were detected in some of the samples. Furthermore, the proportion of AK was markedly high in coffee samples, and its concentration was at least 90% in the samples in which SUC and REB were detected. The aftertaste of AK is compatible with the taste of coffee and therefore AK is often used in coffee. Coffee samples 11–19 were blends or café ole-type products and sweeteners were not detected in any of them. However, the use of sugar was mentioned in their ingredient list. Coffee samples 20–22 were black coffee products. Sweeteners were not detected in them and there was no mention of the presence of sugar. In the case of sports drinks, sweeteners were detected in four out of the five samples. AK and SUC were detected in two of them (sports drinks 1 and 2) and only SUC was detected in the remaining two (3 and 4). The four sports drinks in which sweeteners were detected were low-calorie products and, therefore, they might be containing sweeteners as substitutes for sugar. In the case of fruit drinks, sweeteners (SUC and REB) were detected only in one sample (fruit drink 1). Fruit juice concentration in fruit drinks 2–5 was 10–25% and it was up to 10% in fruit drink 1. Only fruit drink 1 was a low-calorie product. The less the fruit juice concentration, the less is the sugar originating from the fruit juice. Hence, it becomes essential to add sugar to compensate for the sweetness. The greater the quantity of added sugar in the product, the greater is the scope for substituting sugar with a sweetener. In the case of non-alcoholic cocktails, AK and SUC were detected in all the five samples (non-alcoholic
166.6 111.5 171.6 59.8 – – – – – – – – 257.4 236.9 224.8 124.5 148.8 156.6 109.7 109.0 124.2 101.1 – – – – – – –
13.5 53.0 92.8 76.1 – – – – – – – – 10.2 – – 6.5 5.2 – 5.2 7.5 – 9.3 – – – – – – –
SUC 238.9 149.7 88.6 – – – – – – – – – – – – – – – – – – – – – – – – – –
APM 41.0 – – – 22.6 19.3 – – – – – – 19.1 – – – – 4.0 – – – – – – – – – – –
REB 2.9 1.8 1.6 – – – – – – – – – – – – – – – – – – – – – – – – – –
DKP 460.0 314.2 353.0 135.9 22.6 19.3 – – – – – – 286.8 236.9 224.8 130.9 154.0 160.6 114.9 116.5 124.2 110.4 – – – – – – –
Total Coffee 18 Coffee 19 Coffee 20 Coffee 21 Coffee 22 Sports drink 1 Sports drink 2 Sports drink 3 Sports drink 4 Sports drink 5 Fruit drink 1 Fruit drink 2 Fruit drink 3 Fruit drink 4 Fruit drink 5 Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Cocktail 1 Cocktail 2 Cocktail 3 Cocktail 4 Cocktail 5 Cocktail 6 Cocktail 7
Sample number
1 2 3 4 5 6 7
SUC – – – – – 70.3 14.4 18.4 15.9 – 38.1 – – – – 38.8 46.8 20.1 42.6 21.4 – – 12.6 – – – – – –
AK – – – – – 70.9 57.2 – – – – – – – – 215.2 159.4 149.0 135.5 117.0 – – 111.9 144.4 64.2 – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – –
APM – – – – – – – – – – 2.9 – – – – – – – – – – – – – – – – – –
REB
– – – – – – – – – – – – – – – – – – – – – – – – – – – – –
DKP
– – – – – 141.2 71.6 18.4 15.9 – 41.0 – – – – 253.9 206.2 169.0 178.2 138.4 – – 124.5 144.4 64.2 – – – –
Total
Note: Numbers are expressed as µg ml−1; –, not detected; Total, amount of sweeteners (AK, SUC, APM and REB); non-alcoholic cocktail includes non-alcoholic shochu-based beverages; cocktail includes shochu-based beverages.
1 2 3 4 5 6 7 8 9 10 11 12
Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Coffee 1 Coffee 2 Coffee 3 Coffee 4 Coffee 5 Coffee 6 Coffee 7 Coffee 8 Coffee 9 Coffee 10 Coffee 11 Coffee 12 Coffee 13 Coffee 14 Coffee 15 Coffee 16 Coffee 17
drink drink drink drink drink drink drink drink drink drink drink drink
AK
Concentration of various compounds in commercially available beverages.
Sample number
Table 7.
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cocktails 1–5). Moreover, all five samples were low-sugar and low-calorie products. As far as the concentration of the sweetener is concerned, the proportion of AK was high (76–88%). Sweeteners were not detected in the remaining two samples (non-alcoholic cocktails 6 and 7), but here the use of sugar was indicated. Sweeteners were detected in three cocktails (1–3) and only AK or AK and SUC were detected. All the three samples were low-sugar products. There is a tendency to add the four sweeteners (AK, SUC, APM and REB) to low-sugar and low-calorie products, mostly for substituting the sugar or sugar syrup. Moreover, AK and APM were detected in relatively higher concentrations. The concentration of AK in coffee, non-alcoholic cocktails and non-alcoholic shochu-based beverages, and that of APM in carbonated drinks was at least 200 µg ml−1. Among the detected sweeteners, the permitted levels of AK and SUC in cold beverages is set at 0.50 and 0.40 g kg–1, respectively, in Japan, but none of the samples exceeded this limit. SA, NE and GA (glycyrrhiza extract), whose use in beverages is permitted in Japan, and CYC, DU and AL, whose use is not permitted in Japan, were not detected in the samples. In Japan, it is customary to denote the principal ingredients in food in descending order of the percentage by weight. In the present results, all the samples containing multiple sweeteners are mentioned in descending order of content. Moreover, the regulations and standards for sweeteners vary from country to country. Even in Japan there are occasional cases of violations like the detection of nonpermitted sweeteners and the presence of sweeteners at concentrations exceeding the standard value. However, in the present study there were no problems with respect to labelling and food sanitation. Conclusions The simultaneous determination of 10 sweeteners and a decomposition product in beverages was established by using an LC-MS/MS method. A simple and rapid determination of sweeteners was possible by diluting with solvent, and in the case of some samples containing a large amount of foreign matter, after pre-treatment by diluting with solvent and purifying the sample by using an Oasis HLB cartridge. All the validation results were good. As the regulations and standards for sweeteners vary from country to country, a field survey of 58 beverages marketed in Japan was performed by using the present method. Consequently,
no problems regarding labelling or violations of the food sanitation law were observed in the samples. The present method is useful for the quality assurance and field survey of sweeteners in beverages. Disclosure statement No potential conflict of interest was reported by the author(s)
References Aiso K. 1972. Syokuhin Eiseigaku Jiten. Tokyo: Ishiyaku syuppan; p. 249. Asano K, Ando N, Morii K, Yamamoto K. 2008. Analysis of sweeteners in foods by LC/MS/MS. Naraken Hoken Kankyo Kenkyu Cent Nenpo. 42:75–77. Beppu M. 2010. Eisei Shikenho Chukai: Methods of Analysis in Health Science. Tokyo: Kanehara Shuppan; p. 350–367. Fitzhugh OG, Nelson AA, Frawley JP. 1951. A comparison of the chronic toxicities of synthetic sweetening agents. J Am Pharm Assoc. 40:583–586. Food Safety Commission (JPN). 2011. Tenkabutu Hyoukasyo: Calcium Saccharin and Sodium Saccharin. 2nd ed. Tokyo: Food Safety Commission; p. 121–129. Homler BE. 1984. Properties and stability of aspartame. Food Technol. 38:50–55. Kagawa T. 1994. International trend of sweeteners; centering around intensive sweeteners. Food Chemicals. Special Issue 6:60–64. Koyama M, Yoshida K, Uchibori N, Wada I, Akiyama K, Sasaki T. 2005. Analysis of nine kinds of sweeteners in foods by LC/MS. Shokuhin Eiseigaku Zasshi. 46:72–78. Matsumoto H, Hirata K, Sakamaki N, Hagino K, Ushiyama H. 2008. Determination of cyclamate and dulcin by HPLC and LC/MS/MS and systematic analysis of eight types of sweeteners. Tokyoto Kenkou Anzen Kenkyu Cent Kenkyu Nenpo. 59:129–135. Nihon Syokuhin Eisei Kyokai. 2003. Syokuhin Eisei Kensa Shishin: Syokuhin Tenkabutu hen. Tokyo: Nihon Syokuhin Eisei Kyokai; p. 216–220, 233–239. Nihon Syokuhin Tenkabutsu Kyokai. 2012. Shin Sekai no Syokuhin Tenkabutsu Gaisetsu. Tokyo: Nihon Syokuhin Tenkabutsu Kyokai. Tsang WS, Clarke MA, Parrish FW. 1985. Determination of aspartame and its breakdown products in soft drinks by reverse-phase chromatography with UV detection. J Agric Food Chem. 33:734–738. Tsuruda S, Sakamoto T, Akaki K. 2013. Simultaneous determination of twelve sweeteners and nine preservatives in foods by solid-phase extraction and LC-MS/MS. Shokuhin Eiseigaku Zasshi. 54:204–212. Yamamoto S. 1980. Nihon Syokuhin Eiseishi: Meiji hen. Tokyo: Chuou Houki Syuppan; p. 329–334. Yang DJ, Chen B. 2009. Simultaneous determination of nonnutritive sweeteners in foods by HPLC/ESI-MS. J Agric Food Chem. 57:3022–3027.
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Simultaneous determination of sweeteners in beverages by LC-MS/MS a
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Hiroaki Sakai , Azusa Yamashita , Masayoshi Tamura , Atsuo Uyama & Naoki Mochizuki
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Research Laboratories for Food Safety Chemistry, Asahi Group Holdings, Ltd, Moriya, Ibaraki, Japan Published online: 20 Mar 2015.
Click for updates To cite this article: Hiroaki Sakai, Azusa Yamashita, Masayoshi Tamura, Atsuo Uyama & Naoki Mochizuki (2015) Simultaneous determination of sweeteners in beverages by LC-MS/MS, Food Additives & Contaminants: Part A, 32:6, 808-816, DOI: 10.1080/19440049.2015.1018341 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1018341
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Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 6, 808–816, http://dx.doi.org/10.1080/19440049.2015.1018341
Simultaneous determination of sweeteners in beverages by LC-MS/MS Hiroaki Sakai, Azusa Yamashita, Masayoshi Tamura, Atsuo Uyama and Naoki Mochizuki* Research Laboratories for Food Safety Chemistry, Asahi Group Holdings, Ltd, Moriya, Ibaraki, Japan
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(Received 29 August 2014; accepted 5 February 2015) A new method was established for the simultaneous determination of 10 sweeteners and a degradation product in beverages by using LC-MS/MS. An ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm) was used as the LC column and 0.1% each of aqueous formic acid and formic acid in acetonitrile were used as the mobile phase. A simple and rapid determination of sweeteners was possible by diluting with a solvent, and in the case of some samples containing a large amount of foreign matter, after pre-treatment by diluting with solvent and clean-up of the sample using an Oasis HLB cartridge. All the validation results were satisfactory. As the regulations and standards for sweeteners vary from country to country, a field survey of 58 beverages marketed in Japan was performed using the present method. No issues concerning the labelling or food sanitation law were found in the tested samples. Keywords: LC-MS/MS; sweetener; beverage; acesulfame potassium; sucralose; aspartame; diketopiperazines; rebaudioside A
Introduction In recent times, obesity has become a growing problem in many countries including the USA, UK and Australia. Diet is an important contributing factor to obesity, and reducing sugar intake is a preventive and remedial measure for obesity. In Japan, people are increasingly concerned about obesity and have become diet conscious with the aim of improving and maintaining their health and beauty. In this context, low-sugar and low-calorie products that use sweeteners are increasingly replacing sugar-laden foodstuff. Sweeteners are sweeter than sugar and some of them are several hundred times sweeter than sugar. The quantity of sugar used can be greatly reduced by adding a small amount of sweetener, thereby also helping to control both the calorie intake and blood sugar. Some sweeteners are excreted from the body without being metabolised and they contribute few or no calories of their own. In Japan, the use of food additives such as sweeteners is stipulated by the food sanitation law. The safety of food additives is assessed, and food additives are classified as permitted as long as there is no possibility of any harm being caused to human health. Currently in Japan sweeteners such as acesulfame potassium (AK), sucralose (SUC), aspartame (APM) and sodium saccharine (SA) have been approved as additives. As an exception, conventional food additives that have been widely used in Japan are also permitted to be used as food additives. Among them are sweeteners that include stevia extract (with stevioside and rebaudioside A (REB) as the main compounds) and liquorice extract (glycyrrhizic acid (GA) as the main compound). Sweeteners were introduced in Japan around 1890, and sugar, which was an expensive commodity, was
widely substituted with saccharin (Yamamoto 1980). However, in the mid-1900s, saccharin was suspected to be carcinogenic, and in 1971 it was removed from the list of generally recognised as safe (GRAS) substances in the USA. In Japan, its use in food products was banned in 1973 (Food Safety Commission 2011). Subsequently, saccharin was reconfirmed to be safe and its use was permitted again in Japan, and now it is being used in the European Union and countries like the USA and Australia (Food Safety Commission 2011, Nihon Shokuhin Tenkabutsu Kyokai 2012). Dulcin (DU) is another food additive that was used in Japan and the USA. However, it was found to be carcinogenic (Fitzhugh et al. 1951), and was subsequently banned in Japan and other countries. Sodium cyclamate (CYC) is another food additive that was used in Japan and USA in the 1950s. However, as the FDA indicated it to be toxic, it was banned in both the countries in 1969 (Aiso 1972; Kagawa 1994). CYC was later recognised as being safe and is now used in the European Union and Australia. However, it is still banned in Japan and the USA. (Kagawa 1994; Nihon Syokuhin Tenkabutsu Kyokai 2012). AK is being used as a sweetener in at least 90 countries, including Japan (Beppu 2010). Neotame (NE) is used in Japan, the USA, Canada and the European Union (Beppu 2010; Nihon Syokuhin Tenkabutsu Kyokai 2012). Alitame (AL) is used in Australia, China and Malaysia. However, it is not permitted for use in Japan, the USA and the European Union (Nihon Syokuhin Tenkabutsu Kyokai 2012). Therefore, as described above, sweeteners that are permitted for use vary according to the country. Sometimes, even the standards established for permitted sweeteners
*Corresponding author: Email: [email protected] © 2015 Asahi Group Holdings, LTD. Published by Taylor & Francis.
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Food Additives & Contaminants: Part A vary from country to country. Therefore, because of the differences in regulations, imported food products are inspected by various government agencies. With the development of new sweeteners, the number of sweeteners permitted for use in food products is increasing. In many cases, a combination of several sweeteners is added to food products to obtain synergistic effects such as the enhancement of sweetness and improvement of flavour; in other words, the use of sweeteners is being diversified. Furthermore, sweeteners are being increasingly used in a variety of food products. Thus, for the analysis of sweeteners it is essential to perform a pre-treatment step according to the matrix corresponding to the respective food product. A variety of methods involving pre-treatment by solvent extraction, SPE, dialysis and dialysis SPE followed by individual or simultaneous measurement using analytical techniques such as HPLC, LC-MS and LC-MS/MS have been reported for the analysis of sweeteners (Nihon Shokuhin Eisei Kyokai 2003; Matsumoto et al. 2008; Beppu 2010; Tsuruda et al. 2013). Moreover, violations such as the presence of non-permitted sweeteners and exceeding the maximum level have been observed occasionally. Therefore, the development of an efficient and rapid method is essential for the simultaneous analysis of a wide variety of sweeteners. We have therefore used beverages available in Japan in various sweetened food products as samples to measure and simplify the troublesome pre-treatment process. Ten
Figure 1.
Structural formulae of the 11 compounds.
809
sweeteners (namely, AK, SA, APM, SUC, NE, REB, GA, CYC, AL and DU) and 5-benzyl-3,6-dioxo-2-piperazineacetic acid (diketopiperazines, DKP) (Tsang et al. 1985), which is the main decomposition product of APM (Figure 1), were analysed in these beverages. A high concentration of DKP in commercial beverages leads to the decomposition of APM; therefore, the estimated APM concentration might be less than the added APM concentration. Various conditions such as instrumental parameters have been established for the rapid and simultaneous determination of sweeteners by LC-MS/ MS. Therefore, we have analysed sweeteners used in various types of beverages sold in Japan, and this study is a compilation of the results.
Materials and methods Samples and reagents In total, 58 commercially available beverage samples (12 carbonated beverages, 22 coffee, five sports drinks, five fruit drinks, seven non-alcoholic cocktails and non-alcoholic shochu-based beverages, and seven cocktails and shochu-based beverages) were analysed. AK, SA, APM, DU and CYC were obtained from Kanto Chemical Co. Inc. (Tokyo, Japan). SUC, REB, GA and DKP were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). NE was obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). AL
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was obtained from ChromaDex (Irvine, CA, USA). Methanol (for LC-MS), acetonitrile (for LC-MS), 0.1% v/v formic acid–distilled water (for HPLC), and 0.1% v/v aqueous formic acid–acetonitrile (for HPLC) were obtained from Kanto Chemical Co. Formic acid (for LC-MS) was purchased from Wako Pure Chemical Industries. Each of the individual standard solutions was prepared by dissolving 10 mg of the reference standard in solvent and making up to 10 ml to obtain a 1000 µg ml−1 standard solution. However, AK, SA, CYC and AL were made up to 100 ml to obtain 100 µg ml−1 standard solutions. For AK, SA, CYC, NE and SUC, distilled water was used as the solvent, and for AL, DU, REB and GA, a methanol– water (1:1) mixture was used as the solvent. For DKP and APM, methanol and 0.1% aqueous formic acid were used as the solvents, respectively. The mixed standard solution was prepared using the 11 individual standard solutions as follows: 1 ml of the SUC standard solution along with 0.1 ml each of the remaining standard solutions was made up to 10 ml by using 0.1% aqueous formic acid. Sample preparation All the samples were diluted 500 times with 0.1% aqueous formic acid and used as test solutions. However, SUC and GA were diluted 100 times. For carbonic acid-containing samples, about 25 ml of each sample was taken in a 50 ml centrifuge tube and shaken for about 5 min at 150 rpm, followed by de-aeration for about 15 min by ultrasonication and dilution. In coffee samples containing milk, foreign particles such as milk or solid matter were present after dilution. Therefore, the sample was purified by using a SPE cartridge after dilution. The sample (3 ml) was loaded at the rate of one drop per second on an Oasis HLB cartridge, which was conditioned with 3 ml each of methanol and 0.1% aqueous formic acid, respectively, followed by washing with 3 ml of 0.1% aqueous formic acid. Then the sample was eluted with 6 ml of 2% formic acid in methanol. The eluate was collected and concentrated to l ml or less by using a pressured gas blowing concentrator. Then the concentrated sample was made up to 3 ml with 0.1% aqueous formic acid and used as the test solution. When the concentrations of the test samples were outside the calibration curve range, the concentrations were adjusted appropriately. Equipment and instruments A Nexera UHPLC System (Shimadzu Corporation, Kyoto, Japan) and MS/MS model LCMS-8040 (Shimadzu) were used. An ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 µm) HPLC column (Waters Corporation, Milford, MA, USA) was used. An Oasis HLB SPE cartridge
(60 mg/3 cc; Waters) was used. A pressured gas blowing concentrator MG-2200 (Tokyo Rikakikai Co. Ltd, Tokyo, Japan) was also used.
Analytical conditions Elution was performed using 0.1% aqueous formic acid (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The gradient programme was as follows: 10% B (0–2.9 min)–26% B (5 min)–42% B (6.2 min)–70% B (7–8 min)–10% B (8.01–10.5 min). The flow rate of the mobile phase, column temperature and sample injection volume were 0.6 ml min−1, 40ºC, and 20 µl, respectively. ESI (positive and negative) was used as the ion source for MS/MS. The de-solvation line temperature, heat block temperature, nebuliser gas flow rate and flow rate of the drying gas were 300ºC, 500ºC, 3 L min−1 and 15 L min−1.
Results and discussion Results of LC-MS/MS assessment conditions The dissociation of highly polar acidic substances such as AK, SA and CYC was inhibited by keeping the sample and mobile phase under acidic conditions. The separation behaviour of various octadecyl silane columns (ACQUITY UPLC BEH C18, Waters; CAPCELL PAK C18 IF, Shiseido Co. Ltd, Tokyo, Japan; L-column 2, Chemicals Evaluation and Research Institute, Tokyo, Japan; Mastro C18, Shimadzu GLC, Tokyo, Japan; Inertsustain C18, GL Sciences, Tokyo, Japan) was compared. 0.1% each of aqueous formic acid and formic acid in acetonitrile were used as the mobile phase and the gradient conditions were suitably adjusted. The ACQUITY UPLC BEH C18 column was selected for further analysis because it showed better separation of all the components ranging from highly polar AK to weakly polar GA, excellent peak shapes and minimum elution time for all the components (Figure 2). In previous studies the time taken for eluting seven to nine compounds was 20–25 min (Koyama et al. 2005; Asano et al. 2008; Yang & Chen 2009). In the present method, all 11 compounds were eluted within 7 min. Therefore, the proposed method rapidly elutes all the compounds, enabling the simultaneous determination of sweeteners. When the highly versatile electrospray ionisation was used as the ion source, AL and DU ionised well in the positive-ion mode, and the remaining nine compounds were ionised in the negative-ion mode. Conditions of applied voltage in each mode are presented in Table 1. As confirmation ions of SA, CYC and SUC could not be obtained, only fixed ions were set.
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Figure 2. Chromatogram of the standard mixture. The concentrations of the sweeteners were 10 ng ml−1 for AK, SA, CYC and AL; 100 ng ml−1 for APM, DKP, DU, REB, NE and GA; and 1000 ng ml−1 for SUC. Peaks are numbered as follows: 1, AK; 2, SA; 3, CYC; 4, DKP; 5, SUC; 6, APM; 7, DU; 8, AL; 9, REB; 10, NE; and 11, GA.
Table 1.
MRM conditions of the 11 compounds. Quantitative ion
Qualitative ion
Analyte
Polarity
Precursor ion (m/z)
Product ion (m/z)
Collision energy (V)
Product ion (m/z)
Collision energy (V)
AK SA CYC DKP SUC APM DU AL REB NE GA
Negative Negative Negative Negative Negative Negative Positive Positive Negative Negative Negative
162 182 178 261 395 293 181 332 965 377 821
82 106 80 146 359 261 108 129 803 200 351
12 19 25 14 11 9 −24 −21 27 18 45
78 – – 126 – 200 110 159 641 345 193
30 – – 18 – 13 −22 −23 55 10 49
Results of the matrix effects study Previously reported methods (Nihon Syokuhin Eisei Kyokai 2003; Beppu 2010; Tsuruda et al. 2013) were applied to various liquid, semi-solid and solid samples such as sauce, miso (bean paste), peanut butter, jam, takuan (pickled radish) and biscuits, and these also include samples containing very high foreign matter content. In this study, however, we limited the present method to the analysis of liquid samples such as cold beverages and alcoholic drinks. Furthermore, as water was the major constituent in our samples, the foreign matter content was considered to be less than that present in the food products described above (sauce, jam etc.). Therefore, to enable a faster pre-treatment, the matrix effect was reduced by diluting the sample. Furthermore, it was previously confirmed that the samples used in the addition did not contain the 11 compounds under study. Table 2 shows the results of the evaluation of matrix effects (ion suppression or promotion of ionisation). For carbonated drinks, sports drinks, fruit drinks, non-alcoholic cocktails and cocktails, the matrix effect was
obtained from the ratio of sample to which the respective standard was added and the standard mixture (n = 3). The results were favourable with the proportion of matrix effect being determined as 84.7–111.4%, and in all the groups the matrix effect was negligible. For samples subjected to SPE (coffee with milk), the matrix effect was determined from the ratio of sample to which the respective standard was added after pre-treatment and the standard mixture (n = 3). Results were favourable at 95.7–114.5%, and the matrix effect was negligible. However, in Japan, various types of coffee are sold. Thus, a standard addition method that can be applied to a wide range of products was employed.
Results of method validation As discussed in the ‘Results of matrix effects study’ section, in all the groups the matrix effect was negligible. Thus, for validity evaluation with the dilution method, one representative group (cocktail) was used. Linearity was obtained from the correlation coefficient of the calibration
812 Table 2.
H. Sakai et al. Results of the matrix effect study. Compound
Category a
Carbonated drink Sports drinka Fruit drinka Non-alcoholic cocktaila Cocktaila Coffeeb
AK
SA
CYC
SUC
APM
DU
AL
REB
NE
GA
DKP
96.1 84.8 96.9 100.5 84.7 95.7
95.1 88.6 97.1 95.2 101.7 107.2
106.1 103.7 105.3 108.2 98.0 96.9
101.8 96.9 103.3 104.8 100.5 105.2
103.9 103.7 105.2 101.4 97.6 105.8
100.9 101.4 102.3 102.2 97.1 110.8
103.6 106.6 104.5 111.4 100.8 106.1
94.8 100.5 106.2 102.0 94.3 113.5
99.3 105.3 105.6 102.6 99.2 102.0
101.4 100.1 101.9 106.1 89.2 114.5
100.7 99.4 101.5 102.1 99.7 111.3
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Notes: Each number is expressed as a percentage. a Dilution method. b Dilution and SPE method. The spiked levels of the sweeteners were 1 μg ml−1 for AK and AL; 2 μg ml−1 for SA and CYC; 10 μg ml−1 for DU, REB, NE and DKP; 20 μg ml−1 for APM and GA; and 100 μg ml−1 for SUC.
curve by using a standard mixture appropriately diluted with 0.1% aqueous formic acid. Correlation coefficients were 0.9989 or greater in the range of 0.2–10 ng ml−1 for AK and AL, 0.5–20 ng ml−1 for CYC, 1–50 ng ml−1 for DKP, DU and NE, 1–200 ng ml−1 for REB, 5–200 ng ml−1 for APM and GA, and 20–1000 ng ml−1 for SUC, and thus satisfactory results were obtained for all the compounds (Table 3). Repeatability was found from the RSD of the sample to which the respective standard substance was added (n = 6). We tested three different additive concentrations that covered the entire range of the calibration curve. The tests were repeated multiple times over a couple of days. The resulting RSD was 0.8–17.3% (Table 3). The recovery rate of the sweeteners was calculated from the ratio of sample to which the respective standard was added and the standard mixture (n = 3). Additive concentrations and the number of tests were set the same as in repeatability. Therefore, the recovery rate of the sweeteners ranged from 84.7% to 119.0%, which was good (Table 3). For samples that were subjected to SPE (coffee with milk), the calibration curve was created by using the standard addition method. Linearity was obtained from the correlation coefficient of the calibration curve obtained after pre-treatment by using a diluted sample and adding an appropriately diluted standard mixture. The correlation coefficient obtained in the range stated above was at least 0.9985 and good results were obtained for all the compounds (Table 4). Repeatability was found from the RSD of the sample to which the respective standard substance was added (n = 6). Similarly, three levels were set for additive concentrations, and tests were repeated multiple times over a couple of days. The resulting RSD was 1.5–16.8% (Table 4). The recovery rate of the sweeteners was calculated from the ratio of sample to which the respective standard was added prior to pre-treatment and that to which the respective standard was added after pretreatment (n = 3). Additive concentrations and the number
of tests were set the same as in repeatability. Therefore, the recovery rate of the sweeteners ranged from 81.1% to 112.2%, which was good (Table 4). The LOQ was calculated as the concentration at which S/N = 10 and LOD was calculated as the concentration at which S/N = 3 using the chromatogram for the standard mixture (Table 5). Both parameters showed a sensitivity that was more than that of the conventional method (Nihon Syokuhin Eisei Kyokai 2003; Beppu 2010).
Analysis of commercially available products A total of 58 commercially available beverages samples described in the ‘Samples and reagents’ section were analysed by the present method. The results of the measurements performed in the concentration range of each compound are presented in Tables 6 and 7. Of the 58 samples tested, 29 listed the names of the 10 sweeteners in the principal ingredients, while the other 29 did not. The samples in which sweeteners were detected and those for which the names of the sweeteners were mentioned in the principal ingredients are shown in Table 6. Out of the 10 sweeteners, AK, SUC, APM and REB were detected. Among the detected sweeteners, AK and SUC were detected in 24 samples (44.4%) and 21 samples (38.9%), respectively. REB was present in six samples (11.1%), and APM was present in three samples (5.6%). For samples in which the sweeteners were detected, the sweetener mentioned in the principal ingredients matched with the detected sweetener. DKP was detected in three samples, but all of them were those in which APM was detected. For the 29 samples where sweeteners were not mentioned in the principal ingredients, none of the 10 compounds was found. Table 7 shows the sweetener concentration of the various samples. Out of the 58 samples, four sweeteners, namely AK, SUC, APM and REB, were detected only in one sample, namely carbonated drink 1. In the other
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
1 2 1 2
1 2 1 2
1 2 1 2
Day Day Day Day
Day Day Day Day
Day Day Day Day
0.9999 0.2 85.6 88.7 6.7 4.8 1 84.7 86.2 2.7 5.2 10 86.9 89.0 1.5 3.6
AK > 0.9999 0.5 89.0 114.5 15.3 10.7 2 101.7 101.9 6.4 5.0 20 93.3 91.3 1.9 2.5
SA 0.9992 0.5 100.6 93.3 4.4 7.6 2 98.0 98.3 2.8 2.9 20 102.2 99.7 1.0 1.9
CYC > 0.9999 20 107.3 113.0 4.5 2.5 100 100.5 101.7 3.4 3.6 1000 100.5 96.4 1.2 1.2
SUC > 0.9999 5 100.5 103.1 3.9 2.8 20 97.6 99.2 2.5 2.5 200 99.6 97.4 0.9 1.8
APM
1 2 1 2
1 2 1 2
1 2 1 2
Day Day Day Day
Day Day Day Day
Day Day Day Day
> 0.9999 0.2 102.0 91.3 6.2 8.3 1 103.8 104.0 3.6 4.6 10 107.2 88.1 1.8 3.5
AK 0.9998 0.5 100.2 81.1 6.8 16.5 2 95.7 109.4 8.4 4.8 20 106.3 93.7 3.3 4.8
SA 0.9999 0.5 104.5 103.9 7.3 7.6 2 102.1 106.0 3.3 4.3 20 105.2 101.0 2.1 4.2
CYC 0.9997 20 107.3 103.6 4.3 7.2 100 106.1 100.9 1.5 3.4 1000 104.5 106.9 1.9 3.3
SUC 0.9999 5 106.0 101.6 6.2 5.4 20 96.1 104.4 3.5 4.5 200 104.5 99.0 4.0 2.1
APM
Validation results for the established analytical method (dilution and solid phase extraction).
Linearity (r) Spiked level (μg ml−1) Recovery (%)
Compound
Table 4.
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
Spiked level (μg ml−1) Recovery (%)
RSD (%)
Linearity (r) Spiked level (μg ml−1) Recovery (%)
Compound
Table 3. Validation results for the established analytical method (dilution).
> 0.9999 1 100.1 90.4 3.8 6.1 10 100.4 103.8 2.5 3.5 50 105.1 98.8 2.8 4.5
DU
> 0.9999 1 96.4 100.2 8.5 5.3 10 97.1 97.0 2.4 5.4 50 98.8 96.3 1.5 1.2
DU
0.9995 0.2 108.9 103.7 6.3 7.1 1 101.0 102.9 1.8 3.7 10 103.2 98.0 2.8 4.7
AL
0.9998 0.2 93.4 101.2 4.3 2.8 1 100.8 102.3 2.2 0.8 10 99.1 100.3 2.0 2.1
AL
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> 0.9999 1 112.2 92.7 5.9 7.3 10 94.3 97.8 5.5 6.3 200 100.4 104.6 6.9 3.5
REB
> 0.9999 1 104.2 111.3 9.4 7.2 10 94.3 91.1 5.9 4.5 200 98.9 99.3 4.3 3.8
REB
> 0.9999 1 108.7 89.4 5.7 5.2 10 101.9 100.7 2.7 6.5 50 101.8 103.7 5.7 5.9
NE
> 0.9999 1 98.8 112.0 8.4 7.8 10 99.2 94.6 6.4 4.4 50 98.9 93.4 3.2 3.7
NE
0.9985 5 99.7 111.2 10.9 2.6 20 106.0 103.2 16.8 16.8 200 87.3 97.7 5.3 11.2
GA
0.9989 5 106.9 119.0 12.5 16.4 20 89.2 91.3 9.1 17.3 200 95.3 101.4 5.8 12.0
GA
> 0.9999 1 101.8 105.7 9.8 4.9 10 100.9 102.5 3.4 3.2 50 105.5 101.9 2.3 4.5
DKP
> 0.9999 1 95.5 105.9 6.9 3.2 10 99.7 95.2 1.5 3.2 50 101.8 94.4 0.8 1.6
DKP
Food Additives & Contaminants: Part A 813
814 Table 5.
H. Sakai et al. Limit of detection (LOD) and limit of quantitation (LOQ).
LOD (μg ml−1) LOQ (μg ml−1)
Table 6.
AK
SA
CYC
SUC
APM
DU
AL
REB
NE
GA
DKP
0.002 0.007
0.02 0.06
0.004 0.01
0.56 1.9
0.004 0.01
0.06 0.19
0.01 0.03
0.005 0.02
0.002 0.008
0.009 0.03
0.09 0.29
Number of samples in which various compounds were detected.
Category
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Carbonated beverage Coffee Sports drink Fruit drink Non-alcoholic cocktaila Cocktailb Total
AK 4 10 2 0 5 3 24
(4) (10) (2) (5) (3) (24)
SUC 4 6 4 1 5 1 21
(4) (6) (4) (1) (5) (1) (21)
APM 3 0 0 0 0 0 3
REB (3)
(3)
3 2 0 1 0 0 6
(3) (2) (1) (6)
DKP
SA
NE
GA
CYC
DU
AL
3 0 0 0 0 0 3
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Notes: Values in parentheses are the number of samples in which the sweeteners used are mentioned in the list of principal ingredients. a Includes non-alcoholic shochu-based beverage. b Includes shochu-based beverage.
samples, at most three components were detected. Multiple sweeteners, namely AK, SUC and APM, were detected simultaneously in carbonated drinks 2 and 3. Out of the 58 samples, carbonated drinks 1–3 showed the highest value of the total concentration of sweeteners and among all the samples, carbonated drink 1 showed the highest value of 460.0 µg ml−1. Carbonated drinks 1–3 were the low-sugar and low-calorie products where sugar or sugar syrup was not used. Their sweetness might have been generated only from the sweeteners that were detected. In carbonated drinks 3 and 4, the concentrations of SUC were the highest of all the samples, which were 92.8 and 76.1 µg ml−1, respectively. AK and APM are 200 times sweeter than sucrose, whereas SUC is 600 times sweeter than sucrose. Therefore, the sweetness of carbonated drinks 3 and 4 is considered to be contributed mainly by SUC. Carbonated drinks 4–6 had mentioned the use of sugars (sugar syrup and sugar) in addition to sweeteners and carbonated drinks 7–12 had mentioned the use of only sugars. Furthermore, APM was detected in three carbonated drink samples and DKP was also detected in them (carbonated drinks 1–3). The proportion of DKP was very low (1.2–1.9%) compared with that of APM in all the samples. APM is stable in aqueous solution at pH 3–5 (Homler 1984). The pH of common carbonated drinks is in this range and APM might undergo slight decomposition during the storage of the product. APM undergoes considerable decomposition when the sample has high DKP concentration. However, such samples were not observed in our study. In the case of coffee samples, sweeteners were detected in 10 samples (1–10). All the samples were low-sugar products and seven of them contained sugar in
addition to the detected sweeteners. Three sweeteners, namely AK, SUC and REB, were detected. AK was detected in all the coffee samples (1–10), and SUC and REB were detected in some of the samples. Furthermore, the proportion of AK was markedly high in coffee samples, and its concentration was at least 90% in the samples in which SUC and REB were detected. The aftertaste of AK is compatible with the taste of coffee and therefore AK is often used in coffee. Coffee samples 11–19 were blends or café ole-type products and sweeteners were not detected in any of them. However, the use of sugar was mentioned in their ingredient list. Coffee samples 20–22 were black coffee products. Sweeteners were not detected in them and there was no mention of the presence of sugar. In the case of sports drinks, sweeteners were detected in four out of the five samples. AK and SUC were detected in two of them (sports drinks 1 and 2) and only SUC was detected in the remaining two (3 and 4). The four sports drinks in which sweeteners were detected were low-calorie products and, therefore, they might be containing sweeteners as substitutes for sugar. In the case of fruit drinks, sweeteners (SUC and REB) were detected only in one sample (fruit drink 1). Fruit juice concentration in fruit drinks 2–5 was 10–25% and it was up to 10% in fruit drink 1. Only fruit drink 1 was a low-calorie product. The less the fruit juice concentration, the less is the sugar originating from the fruit juice. Hence, it becomes essential to add sugar to compensate for the sweetness. The greater the quantity of added sugar in the product, the greater is the scope for substituting sugar with a sweetener. In the case of non-alcoholic cocktails, AK and SUC were detected in all the five samples (non-alcoholic
166.6 111.5 171.6 59.8 – – – – – – – – 257.4 236.9 224.8 124.5 148.8 156.6 109.7 109.0 124.2 101.1 – – – – – – –
13.5 53.0 92.8 76.1 – – – – – – – – 10.2 – – 6.5 5.2 – 5.2 7.5 – 9.3 – – – – – – –
SUC 238.9 149.7 88.6 – – – – – – – – – – – – – – – – – – – – – – – – – –
APM 41.0 – – – 22.6 19.3 – – – – – – 19.1 – – – – 4.0 – – – – – – – – – – –
REB 2.9 1.8 1.6 – – – – – – – – – – – – – – – – – – – – – – – – – –
DKP 460.0 314.2 353.0 135.9 22.6 19.3 – – – – – – 286.8 236.9 224.8 130.9 154.0 160.6 114.9 116.5 124.2 110.4 – – – – – – –
Total Coffee 18 Coffee 19 Coffee 20 Coffee 21 Coffee 22 Sports drink 1 Sports drink 2 Sports drink 3 Sports drink 4 Sports drink 5 Fruit drink 1 Fruit drink 2 Fruit drink 3 Fruit drink 4 Fruit drink 5 Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Non-alcoholic cocktail Cocktail 1 Cocktail 2 Cocktail 3 Cocktail 4 Cocktail 5 Cocktail 6 Cocktail 7
Sample number
1 2 3 4 5 6 7
SUC – – – – – 70.3 14.4 18.4 15.9 – 38.1 – – – – 38.8 46.8 20.1 42.6 21.4 – – 12.6 – – – – – –
AK – – – – – 70.9 57.2 – – – – – – – – 215.2 159.4 149.0 135.5 117.0 – – 111.9 144.4 64.2 – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – –
APM – – – – – – – – – – 2.9 – – – – – – – – – – – – – – – – – –
REB
– – – – – – – – – – – – – – – – – – – – – – – – – – – – –
DKP
– – – – – 141.2 71.6 18.4 15.9 – 41.0 – – – – 253.9 206.2 169.0 178.2 138.4 – – 124.5 144.4 64.2 – – – –
Total
Note: Numbers are expressed as µg ml−1; –, not detected; Total, amount of sweeteners (AK, SUC, APM and REB); non-alcoholic cocktail includes non-alcoholic shochu-based beverages; cocktail includes shochu-based beverages.
1 2 3 4 5 6 7 8 9 10 11 12
Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Carbonated Coffee 1 Coffee 2 Coffee 3 Coffee 4 Coffee 5 Coffee 6 Coffee 7 Coffee 8 Coffee 9 Coffee 10 Coffee 11 Coffee 12 Coffee 13 Coffee 14 Coffee 15 Coffee 16 Coffee 17
drink drink drink drink drink drink drink drink drink drink drink drink
AK
Concentration of various compounds in commercially available beverages.
Sample number
Table 7.
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Food Additives & Contaminants: Part A 815
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816
H. Sakai et al.
cocktails 1–5). Moreover, all five samples were low-sugar and low-calorie products. As far as the concentration of the sweetener is concerned, the proportion of AK was high (76–88%). Sweeteners were not detected in the remaining two samples (non-alcoholic cocktails 6 and 7), but here the use of sugar was indicated. Sweeteners were detected in three cocktails (1–3) and only AK or AK and SUC were detected. All the three samples were low-sugar products. There is a tendency to add the four sweeteners (AK, SUC, APM and REB) to low-sugar and low-calorie products, mostly for substituting the sugar or sugar syrup. Moreover, AK and APM were detected in relatively higher concentrations. The concentration of AK in coffee, non-alcoholic cocktails and non-alcoholic shochu-based beverages, and that of APM in carbonated drinks was at least 200 µg ml−1. Among the detected sweeteners, the permitted levels of AK and SUC in cold beverages is set at 0.50 and 0.40 g kg–1, respectively, in Japan, but none of the samples exceeded this limit. SA, NE and GA (glycyrrhiza extract), whose use in beverages is permitted in Japan, and CYC, DU and AL, whose use is not permitted in Japan, were not detected in the samples. In Japan, it is customary to denote the principal ingredients in food in descending order of the percentage by weight. In the present results, all the samples containing multiple sweeteners are mentioned in descending order of content. Moreover, the regulations and standards for sweeteners vary from country to country. Even in Japan there are occasional cases of violations like the detection of nonpermitted sweeteners and the presence of sweeteners at concentrations exceeding the standard value. However, in the present study there were no problems with respect to labelling and food sanitation. Conclusions The simultaneous determination of 10 sweeteners and a decomposition product in beverages was established by using an LC-MS/MS method. A simple and rapid determination of sweeteners was possible by diluting with solvent, and in the case of some samples containing a large amount of foreign matter, after pre-treatment by diluting with solvent and purifying the sample by using an Oasis HLB cartridge. All the validation results were good. As the regulations and standards for sweeteners vary from country to country, a field survey of 58 beverages marketed in Japan was performed by using the present method. Consequently,
no problems regarding labelling or violations of the food sanitation law were observed in the samples. The present method is useful for the quality assurance and field survey of sweeteners in beverages. Disclosure statement No potential conflict of interest was reported by the author(s)
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