Rapid Analysis of Soft Drinks Using the ACQUITY UPLC H-Class System with the Waters Beverage Analysis Kit Mark E

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Rapid Analysis of Soft Drinks Using the ACQUITY UPLC H-Class System with the Waters Beverage Analysis Kit Mark E. Benvenuti, Raymond Giska, and Jennifer A. Burgess Waters Corporation, Milford, MA U.S.

APPLICATION BENEFITS INTRODUCTION ■■ Quantitation of six additives The soft drink market is an important worldwide business that generates profits in a seven-minute isocratic run for several major manufacturers. To ensure consistency of products and to satisfy quality control requirements, accurate quantification of additives is essential. ■■ Minimal sample preparation Six additives that are commonly used are sodium benzoate and potassium ■■ Pre-packaged mobile phase and standards sorbate as preservatives; acesulfame K; aspartame and saccharin for sweetness ■■ Environmentally-friendly solvents (diet beverages); and caffeine. Some or all of these compounds may be present depending on the formulation of a particular beverage.

In the manufacturing environment, producers desire simple, rapid analytical techniques with minimal sample preparation. Non-hazardous solvents are also important, both for the safety of laboratory personnel, and to keep disposal costs to a minimum. Isocratic methods are often preferred to keep the method simple, remove the re-equilibration step, and allow for the potential to recirculate mobile phase. Although mobile phase recirculation is not typically used in analytical laboratories, in the manufacturing environment, it is commonly employed to reduce costs and minimize the need for technical involvement.

Here we show the efficacy of the ACQUITY UPLC H-Class System with the Waters® Beverage Analysis Kit, which includes the pre-packaged acetate-buffered ethanol eluent to effect a separation of the above analytes in less than seven minutes. The only sample preparation required is sonication (to remove the carbonation) and filtration.

Waters solutions ACQUITY UPLC ® H-Class System with UV Detection

Beverage Analysis Kit (PN 176002534)

ACQUITY®‚ BEH Phenyl Column (PN 186002885)

key words Soft drink, beverage, mobile phase, soda, caffeine, acesulfame K, aspartame, potassium sulfate, sodium benzoate ACQUITY UPLC H-Class System.

1 E X P E RIMENTAL Standard preparation: A stock standard was prepared by dissolving 0.177 g acesulfame K, LC conditions 0.142 g sodium saccharin, 0.102 g caffeine, 0.272 g sodium benzoate, 0.530 g LC system: A C Q U I T Y U P L C H - C l a s s aspartame, and 0.140 g potassium sorbate in 10% aqueous ethanol, and diluting Runtime: 7.0 min to 100 mL with same. From this, six dilutions were made in 10% aqueous ethanol Column: ACQUITY UPLC BEH to formulate a series of standards with concentrations listed in Table 1. These can Phenyl 1.7 µm, be used for a multi-point calibration. When a single point calibration is preferred, 2.1 X 100 mm Waters’ certified standard mix should be used. (PN 186002885) This standard mix is available as part of the Waters Beverage Analysis Kit. Column temp.: 35 °C A premixed standard containing five of the six analytes is supplied. Aspartame, which degrades over time in solution, is provided as a pre-weighed solid. All that is required for standard preparation is to pour the pre-dissolved liquid mix of Mobile phase: Waters’ Beverage standards into the bottle of solid aspartame, replace the cap, and shake the bottle mobile phase until all the aspartame is dissolved. The final solution of mixed analytes can then (included in Kit be transferred to smaller standard vials for analysis. PN 176002534)

Flow rate: 0.5 mL/min Sample preparation: Injection volume: 1 µL Two diet colas (lime flavored and regular) and two diet fruit-flavored (lemon Detection: UV at 214 nm and orange) carbonated soft drinks were purchased at a local market. They were sonicated to remove carbonation and filtered through a 0.45 µm, 13 mm PVDF disc.

Rapid Analysis of Soft Drinks Using the Waters H-Class System with the Waters Beverage Analysis Kit 2 Std Acesulfame K Sodium Caffeine Sodium Aspartame Potassium Saccharin Benzoate Sorbate 1 35.4 28.4 20.4 54.4 106 28 2 70.8 56.8 40.8 108.8 212 56 3 88.5 71 51 136 265 70 4 177 142 102 272 530 140 5 354 284 204 544 1060 280 6 442.5 355 255 680 1325 350

Table 1. Soft drink standards, units are mg/L.

RESULTS AND DISCUSSION A typical separation of the six analytes of interest is shown in Figure 2. All analytes were baseline resolved and well separated. Seven injections of a QC standard at the same level as standard 3, shown in Table 1, were performed and the repeatability data is provided in Table 2. The % RSD for retention time was 0.04% or less for all analytes. When the data were quantified against the six-point calibration curve, the measured amount of each analyte was within 1% of the actual amount for all the analytes. When the data were quantified against standard three as a single point calibration, the measured amount of each analyte was also within 1% of the actual amount. The % RSD for the measured amount was 0.42% or less for all analytes. Calibration curves for the six analytes are shown in Figure 3. R2 values were greater than 0.999 for all compounds. Each standard was injected in triplicate.

1 1- Acesulfame K- 88.5 mg/L 2- Sodium Saccharin- 71.0 mg/L 0.30 AU 3- Sodium Benzoate-136.0 mg/L 4- Potassium Sorbate- 70.0 mg/L 5- Caffeine- 51.0 mg/L 6- Aspartame- 265.0 mg/L 3

6 5

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Minutes

Figure 2. Chromatogram of standard three.

Rapid Analysis of Soft Drinks Using the Waters H-Class System with the Waters Beverage Analysis Kit 3 Analyte RT ( min ) %RSD Theoretical Measured %RSD % Deviation RT Amount Amount Amount AcesulfameK 0.87 0.04 88.5 89.0 0.24 0.56 Sodium Saccharin 1.35 0.03 71.0 71.3 0.21 0.42 Sodium Benzoate 2.92 0.01 136.0 136.3 0.28 0.22 Potassium Sorbate 4.3 0.01 70.0 70.7 0.32 1.00 Caffeine 4.67 0.01 51.0 51.3 0.30 0.59 Aspartame 5.90 0.01 265.0 265.4 0.42 0.15

Table 2. Reproducibility data for seven injections of a QC standard.

2.4x106 1.3x106

2.2x106 1.2x106 2.0x106 AcesulfameK R2= 0.99945 1.1x106 Potassium SorbateR2= 0.999907 1.8x106 1.0x106

5 1.6x106 9.0x10

5 1.4x106 8.0x10

5 1.2x106 7.0x10

5 Area 6.0x10 1.0x106 Area 5.0x105 8.0x105

4.0x105 6.0x105

3.0x105 4.0x105

2.0x105 2.0x105 1.0x105 0.00 40.00 60.00 100.00 140.00 180.00 220.00 260.00 300.00 340.00 380.00 420.00 Amount 0.00 40.00 80.00 120.00 160.00 200.00 240.00 280.00 320.00 360.00 Amount

4.0x106 3.8x106 3.6x106 2.6x106 3.4x106 2 Sodium Saccharin R = 0.999874 6 3.2x106 2.4x10 2 3.0x106 Caffeine R = 0.999912 2.2x106 2.8x106 6 2.6x106 2.0x10

6 2.4x10 1.8x106 2.2x106 1.6x106 2.0x106

6 6 Area 1.8x10 1.4x10 1.6x106 1.2x106 6 1.4x10 Area 1.2x106 1.0x106 1.0x106 8.0x105 8.0x105 6.0x105 6.0x105 4.0x105 4.0x105 2.0x105 2.0x105 0.00 40.00 80.00 120.00 160.00 200.00 240.00 280.00 320.00 360.00 Amount 0.00 40.00 80.00 120.00 160.00 200.00 240.00 Amount

3.8x106 3.6x106

3.6x106 3.4x106 3.4x106 2 3.2x106 3.2x106 Sodium Benzoate R = 0.999895 3.0x106 2 3.0x106 Aspartame R = 0.999915 2.8x106 2.8x106 2.6x106 2.6x106 2.4x106 2.4x106 2.2x106 2.2x106 2.0x106 2.0x106 1.8x106 1.8x106

Area 6 6 1.6x10 1.6x10 Area

6 1.4x106 1.4x10

1.2x106 1.2x106

1.0x106 1.0x106

8.0x105 8.0x105

5 6.0x10 6.0x105

5 4.0x10 4.0x105 2.0x105 2.0x105

0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 0.00 200.00 400.00 600.00 800.00 1000.00 1200.00 Amount Amount

Figure 3. Calibration curves for the six analytes.

To demonstrate the performance of the method for the types of products that would routinely be analyzed with this method, four products were purchased. The chromatograms of these products are shown in Figures 4 to 7. Figure 4 shows that the lime-flavored diet cola contained four of the six analytes: acesulfame K, potassium sorbate, caffeine, and aspartame. The plain diet cola shown in Figure 5, also contained four analytes but instead of potassium sorbate, sodium benzoate was used as the preservative. Sodium benzoate was also used in the diet-lemon and orange-flavored beverages, as shown in Figures 6 and 7, respectively. Of the four products, the orange-flavored beverage was the only product that did not contain caffeine.

Rapid Analysis of Soft Drinks Using the Waters H-Class System with the Waters Beverage Analysis Kit 4 1 1- AcesulfameK- 99.3 mg/L 4- Potassium Sorbate- 99.7 mg/L 5- Caffeine- 106.9 mg/L 6- Aspartame- 329.0 mg/L 0.30 AU 0.30

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Figure 4. Chromatogram of Minutes a lime-flavored diet cola.

1- AcesulfameK- 134.3 mg/L 3- Sodium Benzoate 187.7 mg/L 0.35 AU 0.35 5- Caffeine- 97.0 mg/L 3 6- Aspartame- 223.2 mg/L

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Figure 5. Chromatogram Minutes of a plain diet cola.

1- Acesulfame K- 82.4 mg/L 3- Sodium Benzoate- 438.2 mg/L 0.60 AU 5- Caffeine-152.8 mg/L 6- Aspartame- 234.4 mg/L

1 5

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Figure 6. Chromatogram of a Minutes lemon-flavored diet beverage.

Rapid Analysis of Soft Drinks Using the Waters H-Class System with the Waters Beverage Analysis Kit 5 CONCLUSIONS 1 This application note has shown a rapid, simple method for the 1- Acesulfame K- 161.8 mg/L 3- Sodium Benzoate-179.6 mg/L

0.45 AU analysis of soft drink additives. Implementation of this procedure in 6- Aspartame- 230.3 mg/L a manufacturing environment has the capacity to improve overall 3 workplace efficiency.

■■ The pre-prepared Waters Beverage Analysis Kit removes the requirement for the preparation of any mobile phase, wash 6 solvents, or standards.

■■ Minimal sample preparation is required, only sonication 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 and filtration. Minutes ■■ The isocratic method ensures ease-of-use and eliminates Figure 7. Chromatogram of an orange-flavored non-caffeinated diet beverage. column re-equilbration.

■■ The use of non-hazardous solvents ensures safety of In terms of the product labeling, the observations listed above analysts and consumers and provides a more environmentally matched the ingredient list from each of the products. Caffeine is the friendly solution. only compound out of the six that has the concentration listed on the ■■ product label. Caffeine quantification was within 1.1% of the stated Accurate and reproducible results can be obtained in label claim for all three of the caffeinated beverages. seven minutes.

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