A Simple LC–MS Multi-Analyte Method to Determine Food Additives and Caffeine in Beverages

Food additives involve a wide group of compounds that differ in their physico-chemical properties. According to their function, they are classified as food colours, preservatives, sweeteners, and antioxidants. The use of food additives is regulated by Regulation (EC) No 1333/2008 of European Parliament and of the Council on food additives, which sets maximum limits for various commodities. To control these regulatory requirements, reliable analytical methods are needed (1,2).

High performance liquid chromatography (HPLC) using reversed-phase columns and neutral or acidic mobile phases are the most common systems for food additive separation. Conventional detectors like ultraviolet-visible(UV–vis) spectrophotometers are often used for the detection of various food additive groups such as colours, preservatives, sweeteners and/or caffeine (3–8). Nevertheless, in some recent studies, mass spectrometry (MS) was preferred for its versatility and better selectivity, and both simple and tandem mass spectrometry were employed (9–13). Although studies introducing multi-detection methods enabling simultaneous analysis of sweeteners, preservatives, and caffeine (6,7), or colours, preservatives, and caffeine (8), were published in the past, LC–MS-based methods suitable for simultaneous determination of all the groups mentionedabove have not yet been published.

This study presents a multi-detection method employing ultra-high performance liquid chromatography (UHPLC) coupled to a simple quadrupole mass spectrometer for the simultaneous determination of several groups of food additives—nine food colours, seven sweeteners, two preservatives, and caffeine in a wide range of beverages.

Materials and Methods

Chemicals: Standards of tartrazine, sunset yellow FCF, azorubine, ponceau 4R, allura red AC, patent blue V calcium salt, brilliant blue FCF, green S, brilliant black BN, sorbic acid, benzoic acid, sodium cyclamate, sodium saccharin, sucralose, neohesperidin dihydrochalcone, and neotame were purchased from Sigma Aldrich (Germany). Anhydrous caffeine and acesulfame K were purchased from Fluka (Germany), aspartame from Supelco (USA). HPLC-grade methanol was obtained from Sigma Aldrich (Germany). Analytical grade ammonium acetate was purchased from Sigma Aldrich (Germany). Water was purified using a Milli-Q Ultrapure water purification system from Millipore (USA).

Samples: In total, 76 samples of beverages were sampled, including 14 soft drinks (including 2 cola type sodas), 19 energy drinks (including five so-called “sugar-free” energy drinks), and 43 alcoholic beverages consisting of 23 liquors, 14 spirits and 6 ciders obtained on the Czech market.

Standard solutions preparation: Stock solutions of 2000 mg/L of each analyte were prepared by dissolution of 10 mg of standard in 5 mL of methanol–water (50:50, v/v) mixture. A standard mixture of 100 mg/L in the same solvent composition was prepared. Calibration solutions in the range from 0.1 to 10 mg/Lwere also prepared. For the analysis of soft and energy drinks, calibration solutions were diluted with deionized water. For alcoholic beverage analysis, calibration solutions in methanol–water (25:75, v/v) were prepared.

Samples preparation: In the case of soft and energy drinks, 40 mL of sample were placed in 100 mL beaker and degassed using the ultrasonic bath for 10 mins. The pH value of the degassed sample was then adjusted to 6 using 1% aqueous solution of ammonium hydroxide. The sample was then transferred to a 50 mL volumetric flask and the volume was adjusted by deionized water. Prior to LC–MS analysis, the sample was filtered through a 0.22-μm syringe filter. If necessary, samples were further diluted with deionized water.

Analyzed alcoholic beverage samples differed in alcohol content, so their composition was adjusted to the level of alcohol–water (25:75, v/v) by dilution using methanol and deionized water. After dilution, the pH was adjusted to the value of 6.

Analytes separation: A Waters Acquity UPLC iClass system (Waters, USA) with a BEH C18 analytical column (2.1 mm × 100 mm, 1.7-μm, Waters, USA) was used for analytes separation. The mobile phase system consisted of 5 mM ammonium acetate aqueous solution (A) and methanol (B). The linear gradient elution was programmed as follows: 0–0.5 min, 2% B; 0.5–1.4 min, 2–15% B; 1.4–2 min, 12–30% B; 2–3 min, 30–60% B; 3–4 min, 60–70% B; 4–5 min, 70–98% B; 5–8 min, 98% B; 8–10 min, 2% B. Mobile phase flow rate was set at 0.4 mL/min, column temperature was 60 °C. The injection volume was 3 μL.

Analytes detection: A simple quadrupole MS system (QDa, Waters, USA) was used for the detection of target analytes. Electrospray ionization (ESI) was used, while this MS system was operated in selected ion recording (SIR) mode with ion polarity switching, allowing the detection in both positive and negative ionization mode within a single run. Nitrogen gas was used as both desolvation and cone gas, and the probe temperature was set at 600 °C. Capillary voltage was set to 0.8 kV for both positive and negative ionization mode. Cone voltage was set at 10 V. Detector gain was set to 1. Acquisition and data processing were carried out using Masslynx v.4.1 software (Waters, USA). Monitored m/z values of target analytes together with retention times under experimental conditions are summarized in Table 1.

Validation: The Method was validated by the analysis (six replicates) of soft drink 10 (lemon soda, 9.5 % sugar content) to which targeted analytes were added at levels of 1 or 5 mg/Ldepending on the limit of quantification (LOQs) which, together with recoveries and repeatabilities are summarized in Table 2.

Results and Discussion

As the set of target analytes includes compounds largely differing in their polarity and presence of ionisable groups, universal, trifunctionally bonded (ethylene bridged) BEH C18 (100 × 2.1 mm; 1.7 µm) ultra-high performance liquid chromatography column was employed for separation. Poor peak shapes and low responses were obtained for preservatives when using ammonium formate as a mobile phase modifier. Also, low retention times for tartrazine and acesulfame K were obtained when solvent of lower polarity than methanol was used as a mobile phase B. A wide range of mobile phases were tested, and the best results regarding peak shapes (low width, minimal tailing) and short separation time (4.5 min), were obtained when using a 5-mM aqueous solution of ammonium acetate and methanol gradient.

In the next step of the method development, detectability of target analytes was tested. Interestingly, lower peak intensities were observed for some food colours such as brilliant blue FCF, and azorubine, when the standard was injected in acidic solution of pH 3 (most of the tested drinks had similar pH). The peak intensity increased with growing pH until the range of 6–8, see Figures 1(a) and 1(b), illustrating the influence of this parameter for brilliant blue FCF and azorubine, respectively. It is assumed that more ionic forms of these analytes were present in the samples, as the pH value of 3 is close to their pKa. To optimize analysis conditions, a pH value of 6 was chosen as suitable for the preparation of both calibration solutions and samples.

In the case of alcoholic beverages, the possible impact of alcoholic strength of injected sample on analyte responses was investigated. Compared to an aqueous solution, equal or higher responses were obtained when using a mixture of ethanol and water as a solvent for standard preparation. The most distinct difference was observed in the case of green S, the signal of which, when injected in 25% (v/v) ethanol was higher by 48.4%; after this point, the signal intensity slowly dropped. To standardize sample conditions, dilution of ethanol content 25% (v/v) with distilled water and ethanol, and pH adjustment to the value of 6 was always performed prior to injection.

The matrix effects when analysing real life samples were relatively low, recoveries of targeted analytes ranged from 83.8 to 103.0%. Repeatability of injections, expressed as relative standard deviation, ranged from 0.4 to 4.5%. Limits of quantification (LOQs) ranged from 0.1 to 3.7 mg/L, these values are two to three orders of magnitude lower than limits set by Regulation (EC) No 1333/2008. All validation parameters are summarized in Table 2. The example of chromatographic separation is demonstrated in Figure 2, on standard mixture in distilled water (pH 6).

Beverage samples analysis: The method developed was applied to the analysis of 76 samples of beverages collected at the Czech market. The sample set included 14 soft drinks, 19 energy drinks and 43 alcoholic beverages.

In the case of soft drinks, up to four different sweeteners were detected. Samples “Fruit Soda” 1, 2, and 3 (see Table 3) produced by the same company, differed in flavouring component but contained almost the same concentrations of sweeteners; the ratio probably best simulates the taste profile of natural sugar. Sweeteners were also detected in Fruit Soda 5 and Cola Type Soda 1, which also contained saccharides. The presence of preservatives was confirmed in Fruit Soda samples 4 and 5. Caffeine was detected in Cola Type Soda samples, as expected. An example chromatogram is presented in Figure 3 for Fruit Soda 3. Remaining targeted analytes were neither detected in remaining soda samples nor label-declared.

In energy drinks, caffeine was present in large quantities, in line with declarations on the drink labelling. In total, three different sweeteners (always two in combination) were detected in seven samples, of which, five were labelled as “sugar-free”; in the remaining two, sugar was present. Preservatives were detected in 10 out of 19 energy drink samples. The results of food additive determination in soft and energy drink samples are summarized in Table 3 – only positive findings are presented here.

In addition to alcohol-free products, a set of 43 different alcoholic beverages were analysed, consisting of 23 liquors, 14 spirits, and six ciders (see Table 4). Overall, six different food colours were determined in 10 liquors and one spirit sample. Colours are used in combinations in order to achieve a specific desired shade of the final product (for example, the green colour in Liquor sample 1 was the result of a mixture of brilliant blue FCF and tartrazine). Table 4 summarizes the results for samples containing some of the targeted additives. Additionally, sorbic acid was used for preservation of Liquors 2 and 3. The chromatographic analysis of Liquor 10 is demonstrated in Figure 4.

In analysed samples, the targeted additives did not exceed the maximum level set for some of them by Regulation (EC) No 1333/2008 (see Tables 3 and 4). The limits of quantification (LOQs) achieved in this study were, as mentioned above, lower than these regulatory limits, thus the developed method is suitable for a rapid and reliable control of beverages. It’s worth noting that, in all cases, the information on the use of food additives was provided on the label.

Conclusion

A multi-analyte UHPLC–MS method for simultaneous determination of nine food colours, seven sweeteners, two preservatives and caffeine has been implemented and validated. Contrary to studies presented so far, this method allows a single-run determination in only 10 min, and with regards to a simple sample preparation, it represents an effective tool for beverage quality control. An analysis of a large set of 69 beverages was achieved. Although a single quadrupole is less selective than more expensive tandem mass analyzers, it still provides a better selectivity and versatility compared to conventional detectors. The achievable limits of quantification are low enough for a reliable control of regulatory limits of targeted food additives.

Acknowledgements

This work was supported by Technology Agency of the Czech Republic (Project No TJ2000238), METROFOOD-CZ research infrastructure project (MEYS Grant No: LM2018100) and the project NPU I LO1601.

References

1. P. Amchova, H. Kotolova, and J. Ruda-Kucerova, Reg. Toxicol. and Pharm.73, 914–922 (2015).
2. M. Carocho, P. Morales, and I.C.F.R. Ferreira, Food and Chem. Toxicol.107, 302–317 (2017).
3. S. Bonan, G. Fedrizzi, S. Menotta, and C. Elisabetta, Dyes and Pigments99, 36–40 (2013).
4. H. Wu, J. Guo, J. Du, H. Tian, C. Hao, Z. Wang, and J. Wang, Food Chem.141, 182–186 (2013).
5. Y. Shen, X. Zhang, W. Prinyawiwatkul, and Z. Xu, Food Chem.157, 553–558 (2014).
6. C.M. Lino and A. Pena, Food Chem.121, 503–508 (2010).
7. O. Kritsunankul and J. Jakmunee, Talanta84, 1342–1349 (2011).
8. B. Asci, S.D. Zor, and O.A. Donmez, Int. J. Anal. Chem.5, 1–8 (2016).
9. I. Ferrer and E.M. Thurman, J. Chromatogr. A.1217, 4127–4134 (2010).
10. C. Chang and T. Yeh, J. Food and Drug Anal.22, 318–328 (2014).
11. A.C. Goren, G. Bilsel, A. Simsek, M. Bilsel, F. Akcadag, K. Topal, and H. Ozgen, Food Chem.175, 273–279 (2015).
12. J. Gui, C. Zhang, Y. Zhang, L. Zhang, and F. Liu, Chin. J. Anal. Chem.44(3), 361–366 (2016).
13. C. Tsai, C. Kuo, and D.Y. Shih, J. Food and Drug Anal.23, 453–462 (2015)

Ales Krmela is a PhD student at the Department of Food Analysis and Nutrition, University of Chemistry and Technology, Prague (UCT Prague). His main focus is on utilization of various LC–MS-based instrumental techniques for the analysis of biologically active compounds and food additives in foodstuffs.

Aliaksandra Kharoshka is a PhD student at the Department of Food Analysis and Nutrition, UCT Prague. Her main focus is on determination of food additives in foodstuffs.

Vera Schulzova is an associate professor at UCT Prague. She is the metrologist of ISO 17025/2018 accredited laboratory. Her work focuses on food analysis and the monitoring of biologically active compounds in foodstuffs and food supplements.

Jana Pulkrabova is a professor of Food Chemistry and Analysis and a Head of the Department of Food Analysis and Nutrition at UCT Prague. Her research group studies groups of organic pollutants in food, internal and external environment, and human biological monitoring.

Jana Hajslova is a professor at UCT Prague. She is the head of ISO 17025/2018 accredited laboratory and also heads a research group concerned with separation science in the field of food/environmental analysis. She is the chair of a series of prestigious international symposia, Recent Advances in Food Analysis (RAFA, www.rafa2021.eu).