Simplified Sulfite Analysis in Food and Beverages Using Ion Chromatography

Introduction In the food and beverage industries, various additives are used during production processes to extend shelf life, increase nutritional content, and for many similar purposes. Some of these additives remain unchanged in the final product, while others can form new compounds. Today, there are still over 300 approved food additives on the market. When these additives are added to commercially packaged food products, they must be labeled with the letter "E" on the content list on the package, and preservatives are also included in the definition of food additives. Sulfites are well-known preservatives in the food and beverage industry. The term "sulfites" stands out as the definition of a group containing sulfur dioxide (SO₂) and chemically related molecules such as sodium sulfite (Na₂SO₃), sodium bisulfite (NaHSO₃) or sodium metabisulfite (Na₂S₂O₅). Sulfites not only prevent microbial growth (food spoilage), but also improve food color, prevent browning and have antioxidant properties. Such properties lead to widespread use in a wide range of foods and beverages including fruits, breakfast cereals, vegetables, seafood, fruit juices, alcoholic and non-alcoholic beverages. While sulfite used in food has beneficial effects on final products, it is known to have some negative effects on human health. Sulfite intake has been associated with various adverse reactions including hypersensitivity, allergic reactions, or vitamin deficiency. Symptomatic reactions may span a wide range from mild skin symptoms to anaphylactic reactions and asthma, depending on various factors. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) Codex Food Labeling Committee (CCFL) have included sulfites in the "FAO / WHO Codex Alimentarius" list among other food components that must be declared as allergens. The acceptable daily intake (ADI) of sulfite determined by the expert committee is [0.7 mg/kg]/body weight. Depending on country conditions, sulfite additives fall into different classifications. While European Union Regulation No. 1169/2011 Annex II lists sulfites as "substances or products causing allergy or intolerance," the United States Food and Drug Administration (FDA) and USDA-FSIS (US Department of Agriculture Food Safety and Inspection Service) classify sulfites not on the allergen list, but on the list of "additives that may cause intolerance." According to EU Regulation No. 1169/2011 and FDA CFR 21- Chapter 101, labeling of sulfite in food and beverages is required when the total concentration value within the product exceeds 10 mg/kg. Various analytical methods have been developed to determine sulfite at this threshold in food products. In this review article, we aim to provide detailed information about the Ion Chromatography (IC) technique presented by Metrohm and different detection types, as well as two new methods developed for sulfite determination in food and beverage matrices, along with the advantages that can be achieved in such analyses.

Sulfite - Chemical Properties

Sulfite can be present in various forms in food and beverages depending on moisture content, pH or temperature conditions. Figure 1 shows many reactions that sulfite can undergo to form other molecules. Sulfites are generally prepared under aqueous conditions by adding an excess amount of sulfur dioxide (SO₂) and a base such as carbonate (CO₃^-2) / bicarbonate (HCO₃-). The resulting salts then release sulfur dioxide in different amounts depending on pH or temperature.   Under acidic conditions (pH 1–2), the most common form of sulfite is sulfur dioxide molecule. Since sulfurous acid (H₂SO₃) is not thermodynamically stable, the typical form of sulfur dioxide is hydrate (SO₂ x H₂ O). In fresh meat products (pH 4.5-6.8), the most stable form is hydrogen sulfite ion (HSO₃^-). In products with low water activity, hydrogen sulfite is in equilibrium with disulfite ion (S₂ O₅ ^2-). At higher pH values (above 6.5), sulfite (SO₃ ^2-) is found in the most stable form. As meat and other processed foods come into contact with air and microbial activity decreases following the onset of drying, sulfite is oxidized to sulfate (SO₄ ^2-) form. This reaction generally occurs as a result of an antioxidation process or enzyme-catalyzed reaction. Due to sulfate formation, it is estimated that approximately 15% of the total sulfite amount can be lost. To prevent such problems, antioxidants or chelating agents are added to food products. Depending on the composition and pH value of foods, sulfites as chemical reactive additives undergo multiple nucleophilic addition and substitution processes, and as a result, their concentrations can decrease to levels of 49%. The smallest percentage of decreasing concentration is related to sulfite oxidation. The main reactions are related to the formation of organic sulfonates. When sulfite additives are added to food, only part of them becomes irreversibly bound to the matrix. The sum of the remaining free and reversibly bound sulfites (total sulfite) becomes the analyte of main interest. Reversibly bound sulfites are present primarily in the form of additive products with carbonyl compounds and hydroxyl sulfonates. From low pH values to pH 8, these adducts are quite stable. Above pH 8.5, these adducts dissociate into free sulfite.

Historical Sulfite Detection Methods

Various methods have been used for sulfite determination in food products. Historically, the first AOAC reference method developed by Monier-Williams dates back to 1927. This method is based on a conventional distillation procedure performed in acidified sample solution. The released SO₂ gas is trapped in a hydrogen peroxide solution and the sulfate released after oxidation is back-titrated using a sodium hydroxide solution. In subsequent years, this method underwent partial modifications through changes in the distillation/extraction procedure as well as the titration step. Other sulfite analysis methods have been developed based on ion exchange or ion exclusion chromatography combined with conductivity, direct current, or pulse amperometric detection. Additionally, a method based on an automated chemical analyzer has been developed, but a significant portion of these methods have been found to be applicable only to specific food matrices (e.g., wine, beer, or dried fruits). Comparison of the Monier-Williams method with IC methods based on acidic distillation or alkaline extraction has provided considerable progress in sulfite analysis method research. Both IC methods examined used electrochemical detection techniques for sulfite analysis, and it was shown that the Monier-Williams method could lead to false positive results compared to both IC methods. Alkaline extraction analysis (pH 9) was found to be easier, faster, and milder compared to the classical acidic distillation method, and it was also found that this method did not release sulfites naturally present in pigments or vegetables such as leeks and radishes. By selecting the aforementioned milder analytical method, multi-laboratory testing was also performed for sulfite analysis in different food products. One of the significant disadvantages of this method was the rapid drop in detector sensitivity that was observed. When a sample was injected for analysis, the applied direct current mode (DC) was found to cause rapid contamination of the electrode, and this contamination showed that the working electrode required frequent manual cleaning. Another disadvantage of this method was evident as insufficient sulfite extraction occurring at pH 9, which prevented the analysis of dark-colored foods. Food and beverage testing laboratories need a robust singular analytical method that provides good sensitivity for sulfite and can be applied to all types of samples. Automating manual steps that require intensive time and labor would provide additional benefits for such users.

Optimized Sulfite Determination - Method 1: Ion Chromatography and Advanced Amperometric Detection:

Metrohm has developed an analytical method applicable to various food matrices to determine total sulfite, eliminating the long and time-consuming steps experienced by the food and beverage industry. The typical instrumentation presented for this method consists of a basic IC system including a high-pressure pump for isocratic analysis, an eluent degassing unit, a separation column and column oven. Using an amperometric detector as the detection technique, automatic sample injections are managed with the aid of an autosampler with low-volume sample injection capacity and additional cooling capability (Figure 2). The system maintains samples at 6°C, enabling accurate sulfite analysis in longer sample series under optimized conditions without significant reduction in sulfite stability and concentration. The newly developed analysis method uses direct current (DC) mode for optimum detection under alkaline sample conditions, while also applying an automatic cleaning procedure to prevent electrode fouling problems that may occur. The device configuration and related reagents used in the optimized sulfite determination method are listed in Table 1 and Table 2. Table 1: System components required for sulfite analysis in various food matrices using IC and amperometric detection technique. Table 2: Reagents used for sulfite analysis in various food matrices using IC and amperometric detection technique.

1.1 Stabilization of Sulfite in Samples

Sulfite is in an unstable form in solution and can easily be oxidized to sulfate. To prevent this reaction, samples and standards are freshly prepared in a stabilization solution before each application. In previously applied methods, a mannitol buffer with pH value of 9 was used as the stabilization solution. In the newly developed method, a formaldehyde solution buffered at pH 10.2 is used. Above pH 8.5, it is known that sulfite adducts dissociate into free SO₃^2-. Alkaline extraction performed at pH 10.2 enables a reproducible and easy analysis process for total sulfite concentration.

1.2 Sample Preparation Procedure

Only 1 gram of homogenized sample is used per analysis. After addition of 29 grams of freshly prepared stabilization solution, the mixture is blended, mixed by hand, and filtered through 0.2 µm filters into 2 ml vials. The 2 ml sample vials are completely filled and capped to prevent any air gaps that could lead to sulfite oxidation.

1.3 Amperometric Detection Method

Obtaining sensitive, reproducible and accurate results for total sulfite analysis in various food matrices, as well as providing ease of use to the analyst is important. A Metrohm Amperometric Detector unit is used in this newly developed method. An innovative, rapid and accurate IC method has been developed by operating the amperometric detector in DC mode at 300 mV voltage. Unlike the manual steps used in older methods, an automatic voltage sweep (patent application, EP3786628A1) is applied to the electrodes after each sample analysis. This special procedure takes only two minutes, completely conditions the electrodes, and thus eliminates the need for frequent manual electrode cleaning. Thus, the 8-minute sample analysis followed by the 2-minute voltage sweep procedure is completed in a total of 10 minutes, allowing the next sample analysis to proceed (Figure 4).  

1.4 Tested Samples

In this review, various food samples (chickpea, mustard, cherry, caper, canned garlic, chili pepper and red wine) were examined in terms of their total sulfite contents. During the analyses, samples were prepared according to the "sample preparation procedure" and injected into the IC instrument. At the same time, results were also obtained by spiking known amounts of sulfite into each sample, and percent recovery data were compared. Table 3 shows the increase in sulfite levels in different samples, demonstrating that the applied method is suitable for determining low and high sulfite concentrations.

1.5 Analysis Results

The data obtained after the analyses show that this IC method developed using the new amperometric detection technique demonstrates excellent sensitivity. The Method Detection Limit (MDL) for the sulfite standard solution was determined to be 0.2 mg/kg. The method demonstrates excellent performance not only because of its low detection limit, but also due to good signal stability and reproducibility of results. Unlike methods using ion chromatography in the past, this new sulfite method uses a more alkaline stabilization solution in its composition. This modification enables the determination of total sulfite amounts in almost all sample matrices. Accurate measurement of total sulfite analysis within desired limits is important for manufacturers to prevent microbiological contamination in food and beverages and to control the sulfite stability of products. On the other hand, this determination is vital for food producers and quality control laboratories to substantiate the sulfite content declared in the product (total sulfite amount is a mandatory and legal requirement when exceeding 10 mg/kg). In this study, a high-capacity ion exchange column was used instead of the ion exclusion columns specified in older methods. This column, in addition to short and stable retention times, also provided good peak shapes for sulfite ion. Through the specific eluent mixture applied with the separation column used, very good separation was obtained between sulfite and the complex sample matrix present in different food products. In addition, the newly developed voltage sweep/cleaning procedure for the working electrode (patent application, EP3786628A1) enables analysis of long sample series without operator intervention for manual cleaning. As shown in visualizations A and B in Figure 5, thanks to this voltage sweep/cleaning procedure developed by Metrohm, no fouling of the electrode is observed even after three weeks of use. While this procedure being automatic is advantageous in terms of result reproducibility, it also minimizes analysis delays and analyst workload.     Optimized Sulfite Determination - Method 2: Ion Chromatography and Optimized Conductivity Detection: Another simple and easy-to-use Ion Chromatography method for sulfite determination in food uses the conductivity detection technique. One of the disadvantages of this method compared to the analysis with the advanced amperometric detection mentioned in the previous section is that it has a higher detection limit (MDL 0.5 mg/kg). The device configuration and required reagents used in the sulfite determination method based on optimized conductivity detection are listed in Table 4 and Table 5.

2.1 Method Operating Principle

For samples without high organic content, the sulfite determination method with optimized conductivity detection technique is preferred. A combination of a high-capacity anion column and alkaline eluent allows sulfite to be separated not only from standard anions, but also from other organic acids found in food products. The typical instrumentation presented for this method consists of a basic IC system including a high-pressure pump for isocratic analysis, an eluent degassing unit, a separation column, a column oven and a sequential suppressor system. A conductivity detector is used as the detection technique (Figure 6). The conductivity detector operates based on the detection of conductivity difference produced by sample ions as they pass between two passivized stainless steel blocks located within the detector block after the separation column, within the context of their equivalent conductivity (Figure 7). In conductivity-based separations, sequential suppressor systems (chemical and carbon dioxide suppression) positioned after the separation column and before the detector are used in Metrohm IC systems. Chemical suppression "chemically" modifies the eluent content through ion exchange during flow, obtaining lower background conductivity while increasing the equivalent conductivity of the sample ion to achieve higher peak response. The carbon dioxide suppressor, especially when carbonate eluent compositions are used, removes CO₂ gas that is released after the chemical suppression reaction and dissolved in the eluent. The eluent passing through the sequential suppressor unit is converted to ultra-pure water form before the detector, thereby greatly increasing analysis sensitivity and reproducibility. Metrohm provides unconditional 10-year warranty for anion chemical suppressor units in sequential suppressor systems.

2.2 Preparation of Calibration Standards

Stock solutions of 1000 ppm were used for standard anions. Sulfite was prepared in desired amounts from the relevant salts of tartrate, malate and oxalate. Due to sulfate impurity in sodium sulfite salt, solutions were prepared separately for sulfite ion.

2.3 Sample Preparation Procedure

In alcoholic beverage samples such as beer or wine, samples are gravimetrically diluted with ultra-pure water in 1:10 and 1:50 ratios before injection. Since the 858 IC Sample Processor system used as an autosampler in the analyses does not have a cooling feature, only a few capped sample bottles at room temperature were placed on the autosampler to minimize sulfite oxidation. Samples are automatically passed through an integrated inline ultra-filtration device and injected into the IC system. The automatic inline filtration process saves time for analysts working in laboratories and laboratory operating costs. With a 0.2 µm diameter membrane inside the inline ultra-filtration cell, multiple samples can be automatically filtered.

2.4 Analysis Results

For wine producers, product consistency and quality are two extremely important parameters. Yeast fermentation performance and efficiency are closely monitored during the fermentation process. Additionally, through wine analysis, it is possible to evaluate nutrients and other additives that may have potentially harmful effects on efficiency and production during the fermentation process. In this study, the standard anions and organic acids present in the red wine (Table 6) and white wine (Table 7) samples used as examples, as well as the sulfite results, were examined.

General Conclusion

Sulfite is a commonly added preservative in food and beverage products to improve shelf life, extend antioxidative properties, and enhance or preserve product color. Due to the variety of allergic reactions reported following sulfite exposure, regulations regarding these additives have been implemented and their monitoring has been required. Regulatory laws in the EU and the US state that sulfite must be labeled when its concentration in food products exceeds 10 mg/kg. The concentration declaration is related to the total sulfite concentration within the sample matrix. Previously used methods for sulfite determination in foods can produce false positive results (Monier-Williams) or fall short of providing a solid analytical procedure. Metrohm offers food and beverage industry customers two superior Ion Chromatography-based method options for sulfite analysis in food and beverage matrices. The first method operating with amperometric detection allows precise detections below the legal limit through accurate quantification at levels down to 0.2 mg/kg. In addition to its low detection limit, this method stands out with high signal stability and reproducibility, and also provides unique analysis stability and uninterrupted operation periods with the newly developed integrated fully automatic working electrode cleaning procedure. This method is the most preferred method for foods spanning a wide range with various sulfite contents. The second method operating with conductivity detection has been specifically developed to detect higher sulfite amounts above the existing legal limit, particularly in samples with low organic load. Metrohm Ion Chromatography Systems provide a wide range of options from entry-level routine instruments; to highly automated modular platforms for trace analysis, research and method development. Combining ease of use and excellent reliability with maximum flexibility; our IC systems offer superior solutions capable of meeting the needs of all quality control and research laboratories in many industries, including food and beverages.

References

1.Metrohm White Paper WP 065EN_1615280884 "Simplified sulfite determination in foods and beverages using ion chromatography". 2.Metrohm Application Work AW IC ES6-0009-112019. "Determination of total sulfite in food and beverages by IC with DC mode electrochemical detection". 3.Metrohm Application Work AW US6-0249-062017 "Analysis of Chloride, Phosphate, Malate, Sulfite, Tartrate, Sulfate, and Oxalate in Red and White-Wine". 4.Kim, H.; Conca, R.; Richardson, M. Determination of sulfur dioxide in grapes: Comparison of the MonierWil-liams method and two ion exclusion chromatographic methods. Journal of Association of Official Analytical Chem-ists 1990, 73, 983–989. DOI:10.1093/ jaoac/73.6.983.