720005017En.Pdf

[ APPLICATION NOTE ]

A Method for the Rapid and Simultaneous Analysis of Sweeteners in Various Food Products Using the ACQUITY Arc System and ACQUITY QDa Mass Detector

Mark Benvenuti, Gareth Cleland, Jinchuan Yang Waters Corporation, Milford, MA, USA

APPLICATION BENEFITS INTRODUCTION ■■ The ACQUITY® QDa® Mass Detector Sugars are renowned for their sweet taste and are often added to facilitates simultaneous analysis of manufactured foods to enhance human perception of flavor. Due to the sweeteners in a single run. negative health effects of excessive consumption of sugar, alternative non-nutritive sweeteners are commonly used in food and beverage ■■ Quantification of natural and artificial non-nutritive sweeteners in less than products. Examples include soft drinks, table-top sweeteners, chocolates, 10 minutes. dairy products, and many other so-called “diet” foods. In many cases it is a combination of various sweeteners that are used to impart overall ■■ Mass detection provides sweetness to these products. information-rich orthogonal detection for co-eluting compounds. Aspartame, saccharin, acesulfame-K, neotame, and sucralose are approved artificial sweeteners by the U.S. Food and Drug Administration (FDA).1 ■■ The ACQUITY QDa Mass Detector Compounds such as rebaudioside A and stevioside, which originated from provides sensitive and selective the South American Stevia plant, are also becoming more popular in the determination of UV United States. In 2010, the European Food Safety Authority (EFSA), transparent sweeteners. approved the use of stevioside as a sweetener.

The European Union (EU) Directive 94/35/EC, along with four amendments: 96/83/EC, 2003/115/EC, 2006/52/EC and 2009/163/EU, restrict the level of sweeteners in specific types of food. The EU Commission Regulation 1129/2011 lists the maximum level of the sweeteners permissible in various food products. Hence the determination of the amount of these sweeteners in foods is also important in order to ensure consistency in product quality.

The most common method for the detection of sweeteners is HPLC coupled to a UV detector. This configuration enables the detection of some WATERS SOLUTIONS sweeteners such as acesulfame-K, aspartame, saccharin, and neotame. However cyclamate and sucralose cannot be analyzed by UV because they ACQUITY Arc™ System lack chromophores. The ability to analyze all of these sweeteners using a ACQUITY QDa Mass Detector single method with mass detection would be ideal. Waters has developed Empower® 3 Chromatography the ACQUITY QDa Mass Detector to allow food and beverage scientists to Data System incorporate mass detection into their existing chromatographic workflows.

KEYWORDS Non-nutritive sweetener, mass detection, ACQUITY Arc System

1 [ APPLICATION NOTE ]

The ACQUITY QDa not only allows for the detection of all sweeteners in a single run, but also brings improved discrimination to the analysis, thereby eliminating the requirement of baseline separation of all compounds. The combination of Waters® ACQUITY Arc System with the ACQUITY QDa Mass Detector is extremely beneficial for food and beverage manufacturers for the identification and quantification of sweeteners in their products using a single analysis method.

In this application note, a fast, reliable and sensitive method was developed to analyze sweeteners in food and beverage samples.

Acesulfame K Saccharin Cyclamate Sucralose Aspartame

Dulcin Alitame Advantam e Neotame

Stevioside REB A NHDC

Figure 1. Chemical structures of the sweeteners analyzed.

EXPERIMENTAL

UHPLC conditions Time Flow rate %A %B UHPLC system: ACQUITY Arc System (min) (mL/min) Column: Waters CORTECS® T3, 1. Initial 0.55 90 10 3 x 100 mm, 2.7 µm 2. 2.0 0.55 90 10 3. 8.0 0.55 40 60 Column temp.: 40 °C 4. 8.5 0.55 0 100 Injection volume: 2 µL 5. 9.5 0.55 0 100 Flow rate: 0.55 mL/min 6. 9.6 0.55 90 10 Mobile phase A: Water with 0.1% formic acid Table 1. Elution gradient. Mobile phase B: Acetonitrile with 0.1% formic acid Runtime: 8.5 min

A Method for the Rapid and Simultaneous Analysis of Sweeteners in Various Food Products 2 [ APPLICATION NOTE ]

MS conditions Standard preparation MS detector: ACQUITY QDa (Performance) Stock solutions (1000 ppm) of each of the sweeteners were Ionization mode: ESI-, ESI+ prepared in water. From these, a mixed stock of 25 ppm for 10 sweeteners and 50 ppm for REB A and sucralose was Capillary voltage: 0.8 kV prepared in 2:3 (v/v) acetonitrile-water. From this, seven Probe temp: 600 °C additional dilutions were made in 2:3 acetonitrile-water to Cone voltage: 15 V produce eight levels of concentrations from 0.25 to 25 mg/L (ppm) for 10 sweeteners, and 0.5 to 50 mg/L (ppm) for REB A Sampling rate: 2 points/second and sucralose. Acquisition: 100–1000 m/z centroid SIR channels: See Table 2 Sample preparation A total of seven different samples were analyzed for this UV conditions work. The samples included three table top sweeteners, Detector: Waters 2998 PDA Detector a diet candy, a diet chocolate pudding, a diet soft drink and Wavelength: 210–360 nm beverage standard. Sampling rate: 10 points/second The table top sweeteners (~1 g) were dissolved in 100 mL water and further diluted at two additional levels 1:20 and 1:10. Analog channel: 214 nm All three levels were injected to cover the different levels of sweeteners in these samples.

The candy (5.29 g) and pudding (2.93 g) were dissolved separately in 100 mL water and diluted 1:10. The solutions were filtered through a 0.2 µ PVDF filter and injected. The diet soft drink was degassed through sonnication, filtered as above diluted 1:20 and injected. The beverage standard was diluted 1:20.

RESULTS AND DISCUSSION

The chemical structures for the twelve RT Analyte SIR and Polarity sweeteners analyzed are shown in Figure 1. (min) These sweeteners were separated on a reversed- Acesulfame K 162.0 (-) 1.19 phase column and detected using the ACQUITY Saccharin 182.0 (-) 1.62 QDa Mass Detector. The retention times, along Cyclamate 178.2 (-) 1.94 with the Single Ion Recording (SIR) mass-to- Sucralose 397.1 (-) 4.57 charge ratio (m/z), and detection polarity of these Aspartame 295.1 (+) 4.88 sweeteners are shown in Table 2. Dulcin 181.0 (+) 5.80 Alitame 332.1 (+) 5.93 Advantame 459.2 (+) 7.50 Neotame 379.2 (+) 8.08 Stevioside 641.5 (-) 7.87 REB A 803.5 (-) 7.83 NHDC 611.3 (-) 7.26

Table 2. The SIR channels’ m/z values, the ESI polarity, and the retention times for the 12 sweeteners studied.

A Method for the Rapid and Simultaneous Analysis of Sweeteners in Various Food Products 3 [ APPLICATION NOTE ]

The mass spectrum of each analyte was acquired using a Total Ion Current (TIC) scan (positive and negative). Results are shown in Figure 2. Please note the formate adduct for sucralose was observed. This adduct has been reported elsewhere.1 The mass spectra of the sweeteners REB A and stevioside showed fragment ions as the main species for these compounds (Figure 3).

295.1 162.0 m/z-295.1 Positive-Aspartame m/z-162.0 Negative-AcesulfameK

181.0 182.0 m/z-181.0 Positive-Dulcin m/z-182.0 Negative-Saccharin

332.1 178.2 m/z-178.2 Negative-Cyclamate m/z-332.1 Positive-Alitame

459.2 397.1 395.1 443.1 m/z-397.1 Negative-Sucralose m/z-459.2 Positive-Advantame Formate Adduct

379.2 611.3 m/z-611.3 Negative NHDC m/z-379.2 Positive- Neotame

180.00 360.00 540.00 720.00 900.00 180.00 360.00 540.00 720.00 900.00 m/z m/z

Figure 2. ESI positive and negative mass spectra of 10 sweeteners. The m/z, polarity, and the analyte name are shown in each mass spectrum. The molecular ions are the main ions in these mass spectra, except in sucralose MS, a large formate adduct ion is observed. Chlorine isotope pattern is also observed for sucralose.

641.5 803.5 m/z 641.5 m/z 803.5 Negative: Negative: Stevioside REB A

140000 80000.0 Intensity Intensity

70000 40000.0

0 0.0 180.00 360.00 540.00 720.00 900.00 180.00 360.00 540.00 720.00 900.00 m/z m/z

Figure 3. Mass spectra for REB A and stevioside. The base peaks in these mass spectra are fragment ions.

A Method for the Rapid and Simultaneous Analysis of Sweeteners in Various Food Products 4 [ APPLICATION NOTE ]

The m/z of 641 for stevioside has also been reported by others.2 The 12 SIR chromatograms of a 12 sweetener standard mix in a solvent solution are shown in Figures 4. Calibration curves and regression coefficients are shown in Figure 5. A quadratic fit with 1/x weighting was appropriate for these analytes. The correlation coefficient (R2) is greater than 0.996 for all compounds. Figure 6 is an overlay of the UV trace at 214 nm and the SIR channels at m/z 295.1 and 397.2 of aspartame and sucralose respectively. Sucralose being UV transparent does not appear in the UV trace but has a strong signal at m/z 397.2 showing the strength of mass detection augmenting UV.

4x107 6 m/z-332.1 Positive-Alitam e 1x10 m/z-162.0 Negative- Acesulfame K 2x107 Intensit y Intensit y 0 0 6x107 1.0x106 m/z-182.0 Negative-Saccharin 4x107 m/z-459.2 Positive- Advantam e 5 5.0x10 2x107 Intensit y Intensit y 0.0 0 6 2x10 1.0x108 m/z-379.2 Positive- Neotame m/z-178.2 Negative-Cyclamate 1x106 5.0x107 Intensit y Intensit y 0 0.0 200000 600000 m/z-397.1 Negative- Sucralose 400000 m/z-641.5 Negative-Steviosid e 100000 200000 Intensit y Intensit y 0 0

7 200000 2x10 m/z-295.1 Positive-Aspartame m/z-803.5 Negative- REB A 100000 Intensit y Intensit y 0 0 4x107 m/z-611.3 Negative NHDC m/z-181.0 Positive-Dulcin 500000 2x107 Intensit y Intensit y 0 0 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 Minutes Minutes

Figure 4. SIR chromatograms for sweeteners in a solvent standard mix.

6x106 6 3x10 2 0 Acesulfame K R - 0.9994 8x106 4x106 2 0 Cyclamate R - 0.9995 5.0x106 6 2.5x10 2 0.0 Saccharin R - 0.9998 2x106 1x106 2 0 Sucralose R - 0.9994 3.0x107 1.5x107 2 0.0 Aspartame R - 0.9985 1.0x108 5.0x107 2 0.0 Dulcin R - 0.9993 1.0x108 5.0x107 2

Peak Area 0.0 Alitame R - 0.9995 1.4x106 5 7.0x10 2 0.0 NHDC R - 0.9982 1.0x108 7 5.0x10 2 0.0 Advantame R - 0.9990 800000 400000 2 0 REB A R - 0.9965 1.2x106 5 6.0x10 2 0.0 Stevioside R - 0.9972 Figure 5. Calibration plots for 12 1.8x108 sweeteners. A quadratic fit with 1/x 9.0x107 Neotame R2- 0.9993 weighting was used for all compounds. 0.0 The analyte and the correlation coefficient 0.00 12.00 24.00 36.00 48.00 (R2) are shown in each individual plot. R2 Concentration (mg/L) is greater than 0.996 for all compounds.

A Method for the Rapid and Simultaneous Analysis of Sweeteners in Various Food Products 5 [ APPLICATION NOTE ]

ANALYSIS OF SAMPLES The seven samples were prepared as previously 0.04 A: UV at 214 nm described and analyzed in duplicate. The quantitation results are listed in Table 3. The 0.02 first table-top sweetener contained sucralose (14.27 mg/g). The second table-top sweetener 0.00 was found to contain aspartame (16.05 mg/g) B: Aspartame (25 ppm) 6x106 and saccharin (4.17 mg/g). The third table-top m/z: 295.1 sweetener listed Reb A as the main sweetener. 3x106 However, besides the Reb A (27.72 mg/g), we note Intensity a small amount of stevioside (0.03 mg/g) was 0 present although it is not listed as an ingredient. 300000 C: Sucralose (50 ppm) With the discrimination and sensitivity of mass m/z: 397.1 detection, it was easily apparent that both of 150000 these co-eluting sweeteners were present in the sample, even though the stevoiside was 0 present at a much lower level and was not labeled 4.20 4.50 4.80 5.10 5.40 Minutes to be present. This is another example of the power of mass detection to detect low levels of Figure 6. Overlay of UV chromatogram at 214 nm and the SIR chromatograms of aspartame analytes. The beverage standard was certified as (m/z 295) and sucralose (m/z 397.1). 150 mg/L acesulfame K and 100 mg/L saccharin. Our values of 137.95 mg/L for acesulfame K and 97.66 mg/L for saccharin agree well here.

Sample Acesulfame K Aspartame REB A Saccharin Stevioside Sucralose Units Table-top Sweetener 1 – – – – – 14.27 mg/g Table-top Sweetener 2 – 16.05 – 4.17 – – mg/g Table-top Sweetener 3 – – 27.72 – 0.03 – mg/g Diet candy – – – – – 0.21 mg/g Diet Pudding 0.17 – – – – 0.10 mg/g Diet Soft Drink 80.14 294.51 – – – 87.33 mg/L Beverage Standard 137.95 – – 97.66 – – mg/L

Table 3. Quantification results of the seven samples tested in this study.

A Method for the Rapid and Simultaneous Analysis of Sweeteners in Various Food Products 6 [ APPLICATION NOTE ]

CONCLUSIONS References This application note has described a method for the separation, detection 1. M. Wu et al. “Direct large volume injection ultra-high performance liquid chromatography-tandem mass and quantification of twelve natural and artificial non nutritive sweeteners spectrometry determination ofartificial sweeteners in less than 14 minutes (including equilibration time) using the sucralose and acesulfame in well water,“ Journal of ACQUITY Arc System coupled with the ACQUITY QDa Mass Detector. Chromatography A, 1359 (2014) 156–161. 2. D. Yang and B. Chen. “Simultaneous Determination The ACQUITY QDa Mass Detector enables a single method to analyze of Nonnutritive Sweeteners in Foods by both UV transparent and non-transparent sweeteners, and this method HPLC/ESI-MS” J. Agric. Food Chem. 2009, 57, pp. 3025. can be incorporated into existing workflows – with or without UV detection. Mass detection provides a much higher level of analyte discrimination and it does not require baseline separation of the analytes with different masses. This helps minimize method development times and removes the need for separate injections of individual standards to verify compound identifications.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. Waters, The Science of What’s Possible, ACQUITY, UPLC, QDa, and Empower are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners. T: 1 508 478 2000 F: 1 508 872 1990 ©2017 Waters Corporation. Produced in the U.S.A. June 2017 720005017EN AG-PDF www.waters.com