The Application Notebook
Volume 0, Issue 0
Sweeteners are low or zero-calorie sugar substitutes that are added in drinks to provide the sweet taste of sucrose or table sugar.
Njies Pedjie, PerkinElmer
Sweeteners are low or zero-calorie sugar substitutes that are added in drinks to provide the sweet taste of sucrose or table sugar. The consumption of artificial sweeteners provide practically no calories. On the other hand, the intake of sucrose and the calories that derive from its metabolism is one of the leading causes of obesity and its related health problems including heart disease and diabetes. People with diabetes are unable to properly metabolize sucrose causing an abnormally high concentration of it in the blood stream with damaging effects on blood vessels and other vital body organs.
This application note presents a fast and robust liquid chromatography method to test widely used artificial sweeteners, such as acesulfame potassium, saccharine, and aspartame as well as additives such as caffeine and potassium benzoate. The method was developed to achieve very high throughput at a low flow rate to reduce the testing time and the solvent usage. The throughput was compared to that of a conventional HPLC analysis with a 5 µm particle, 250 × 4.6 mm C-18 column (PerkinElmer Brownlee Analytical Cat # N9303514). The method performance data including precision and linearity are presented, as well as the results of the method applied to two popular soft drinks and two popular coffee sweeteners.
A PerkinElmer Flexar® FX-15 UHPLC system fitted with a Flexar FX PDA photodiode array detector provide the UHPLC platform for this application. The separation was achieved using a Restek® Pinnacle DB C18, 3 µm, 100 × 2.1 mm column (Cat # 9414312). The run time was 3.5 min with a back pressure of 6050 psi (417 bar). A working standard solution containing 200 µg/mL of acesulfame potassium, potassium benzoate, aspartame, and 100 µg/mL of saccharine and caffeine was used for repeatability. Linearity was determined across the range of 2–200 µg/mL concentration. Using water as solvent, 0.5 g/mL of the cola drinks and 2 mg/mL of the two sugar substitutes were prepared.
Figure 1: Chromatogram from the analysis of the sweeteners and additives standard.
When the conventional HPLC column was used at 1 mL/min all the peaks eluted within 12 min. By using a UHPLC column, the run time was dramatically reduced from 12 min to 3.5 min.
Excellent method performance was achieved. For each additive, the linearity of the analysis achieved an R-squared value of 0.999 and precisions values ranging from 0.9–1.5% RSD.
This UHPLC method resulted in more than threefold reduction in run time and 80% reduction in solvent usage. Both of the soft drinks tested were sweetened with aspartame: 181 mg/12 oz for Cola Drink 1 and 157 mg/12 oz for Cola Drink 2. The level of caffeine in drinks was similar to the label claim of 45 mg/12 oz for Cola Drink 1 and 35 mg/12 oz for Cola Drink 2. Similar amounts of potassium benzoate were found in both drinks: 71 mg/12 oz for Cola Drink 1 and 74 mg/12 oz for Cola Drink 2. PerkinElmer's Chromera® software offers many data acquisition and processing features: spectral library creation, peak purity, and spectra 3D and contour maps, which are powerful tools for interrogating the information content of a 3D photodiode array chromatogram. The spectra library function allowed the storage of standard peaks spectra that were later used for peak identification confirmation in the sample.
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