Determination of Free and Total Sulfite in Red Globe Grape by Ion Chromatography

Abstract

Sulfiting agents have been used wide-spread for prolonging shelf life of berries, excessive of sulfur dioxide would cause food allergies to humans. Conventional methods mainly focus on the determination of free or total sulfite in liquid samples, and are not suitable to analyze berries with low level of sulfite. Here formaldehyde method and ion chromatography were coupled to determine the residues of the free and total sulfites in Red Globe grape. Both standard calibration curves showed good correlation between anion peak area and concentration. To free and total sulfite, limits of detection were 0.002 and 0.05 mg/L and recoveries ranged from 88 – 93% and 87 – 98%, respectively. Relative standard deviation values of repeatability and intraday precision for standard anions with the same sample were less than 3%. The method developed was well applicable to be used to determine free and total sulfite in barriers, especially to those with color.

Introduction

Addition of sulfites is a practice widely used in the processing of storage foods like grapes, dried fruits, fruit juices and wine because of their effects in improving the appearance and flavour of food during the process of preparation, storage and distribution by stabilizing the product color and inhibiting discoloration (Mcfeeters and Barish, 2003; Falcone and Maxwell, 1992; Iammarino et al., 2010; Jackowet and Orduña, 2012). However, the over-ingestion of sulfite had shown hazardous to humans, and when the daily sulfite consumption is more than 50 mg/kg body weight it might result in headaches, nausea and diarrhea (Suh et al., 2007). In recent years, the sulfite contents in foods and beverages had been strictly controlled and limited world widely (Zhong et al., 2012). The United States Food and Drug Administration (FDA) require sulfite warning on the label of foods in concentrations of 10 mg/kg sulfite or more (Claudia and Francisco, 2009).

When added into food and beverages, parts of sulphite would react with aldehydes, proteins, ketones, sugars, etc., and parts without changes, accordingly presenting as the bound and free sulphite (Adams, 1997). The former are hydroxymethanesulfonate formed by reaction of carbonyl groups with HSO₃⁻, no more effective as bleaching agent, antioxidant, sterilizing agent or preservative, but contributing as the flavouring agent by inactivating the disagreeable odor of aldehyde, and the latter refers to all the species (SO₂, H₂SO₃, HSO₃⁻ and SO₃²⁻) that may rapidly and quantitatively be converted to SO₂ (Zhao et al., 2006). The sum of them is the total sulfite (Sarudi and Kelemen, 1998; Koch et al., 2010; Brychkovaa et al., 2012). In the quality control of foods, both contents of free and total sulfite are important analytical parameters.

Till to now, many methods have been developed for sulfite determination, and can be classified according to the analytical methods (Filik and Çetintaş, 2012). The first is based on chemical reaction, in which the total sulfite is determined by an acid-base titration of released sulfur dioxide through acidify the sample. A typical example of the titration approach was the Monier-Williams method, which was recommended by the Association of Official Analytical Chemists (AOAC), but this method is an indirect method, and cannot be used for fast or high-throughput analysis (Koch et al., 2010). Furthermore, the method tends to overestimate sulfite levels upon the presence of volatile acidic compounds (Pizzoferrato et al., 1998). The second is based on instruments, in which free and total sulfite are determined directly, including GC (Hamano, 1979; Mitsuhashi et al., 1979), HPLC (Mcfeeters, 2003), electrochemistry (Zhao et al., 2006), and flow injection technique (Claudia, 2009; Navarrro et al., 2010), but all of them are seldom applicable in practice. Spectrophotometry (Segundo and Rangel, 2001; Li and zhao, 2006) is rather widely applied, even if it requires the complicated sample pretreatment and reagent preparation processes. But it cannot meet the more and more critical requirement of food industry for its bad sensitivity limited by the nature of spectrophotometry method. Recent years, ion chromatography (Iammarino et al., 2010; Brychkova et al., 2012; Zhong et al., 2012) was recommended to test free and total sulfite in liquid samples such as wine, fruit juice for its high sensitivity and accuracy.

The sulfite is low-level presence in complex matrices and can be rapidly oxidized to sulfate in aqueous solution, which lead to very hard to accurate quantification of them. To over this problem, various reagents were added as stabilizers to decease the oxidation of the sulfite, including formaldehyde (Massom and Townshend, 1986; Michigami and Ueda, 1994; Masár et al., 2004), acetone, ethanol, mannitol, EDTA, glycerol and tetrachloromercurate (Iammarino et al., 2010). It has been proved the content of the free sulfite would decrease about 25 – 30% after exaction from wine without adding stabilizers.

In this work, formaldehyde was introduced into the homogenized grape as the stabilizer to decrease the loss of the free sulfite, and ion chromatography (IC) was utilized for direct determination of the residues of sulfite in Red Globe grape. This combination was well applicable to determine free and total sulfite in barriers with high accuracy and sensitivity.

Materials and Methods

Reagents and standards Hydrogen peroxide, sodium hydroxide, sodium sulfate, sodium sulfite, sodium carbonate, sodium bicarbonate, mercuric sodium tetrachloride, ammonium sulfamate and formaldehyde were purchased from Tianjin Chemical Reagent Co. (Tianjin, China). Ultrapure water (specific resistivity 18.2 MΩcm⁻¹) obtained from a CLASSIC UF Water Purification system (PALL, USA) was used for sample preparation, dilution of samples, standard solutions, and IC eluent preparation. Eluent solution was prepared by dissolving appropriate amount of sodium carbonate and sodium bicarbonate in ultrapure water and degassing in an ultrasonic bath for 15 min. The standard solutions and eluent solution were prepared fresh daily, the standard solutions were diluted as appropriated just before use.

Red Globe grape sample treatment Red Globe grape was purchased from Sanping Farm (Xinjiang, China), and quickly transported to the laboratory on the day of the harvest and stored at 0°C overnight before processing. And then, grapes were stored by SO₂ preservative paper obtained from Green Doctor Fresh-Keeping Science & Technology Co. Ltd of Urumqi (Xinjiang, China). The grapes with different storage time were selected for uniform size, maturity and appearance, freed from defects and mechanical damage, and frozen using liquid nitrogen and stored at −80°C for analysis.

Extraction of free sulfite using formaldehyde as stabilizer The extraction of free sulfite was based on the method described previously with few modifications (Masár et al., 2005; Koch et al., 2010). 20 g portion of homogenized sample were dispersed in freshly ultrapure water to make a 50 mL sample solution containing formaldehyde (corresponding to a 10 mmol/l final concentration of formaldehyde), and then the solution was ultrasonic oscillator for 15 min at 0°C. To guarantee a full conversion of free sulfite to hydroxymethanesulfonate (HMS), the solution was further incubated for another 60 min, then the mixture centrifuged at 10,000 r/min for 10 min at ambient temperature, the supernatant was carefully removed and adjusted to 50 mL with ultrapure water. Finally the mixture was diluted 10 times and purified with SPE C18 column (50 mg/1 mL, Dikma, USA) and analyzed by IC as soon as possible. A control sample (without formaldehyde) was prepared in the same way, in parallel.

Determination of total sulfite through hydrogen peroxide oxidation Homogenized samples (20 g) were dispersed into 30 mL sodium hydroxide solution (10 mM). Subsequently the mixture was treated with hydrogen peroxide solution (30% w/w) in ultrasonic oscillator for 15 min, and incubated for another 2 h at ambient temperature to guaranty a full conversion of sulfite to sulfate. Finally the mixture centrifuged at 10,000 r/min for 10 min at ambient temperature, the supernatant was carefully removed and adjusted to 50 mL with ultrapure water. Finally the mixture was diluted 10 times and purified with SPE C18 column and analyzed by IC as soon as possible. A control sample (without H₂O₂) was also prepared in the same way to deduct the amount of sulfate in grape, in parallel.

Ion chromatography The instrument used was an ICS-900 system (Dionex, USA) equipped with an ASRS300 4-mm suppressor, a DS5 conductivity detector. Ion chromatographic condition was used according to our previous study with few modifications (Zhong et al., 2006). The chromatographic separations were carried out on an IonPac AS23 analytical column (4 × 250 mm, particle size 6 µm, stationary phase of supermacroporous resin cross-linked with 55% divinyl-benzene) fitted with an IonPac AG23 guard column (4 × 50 mm, particle size 11 µm, stationary phase of microporous resin cross-linked with 55% divinylbenzene), both from Dionex (Dionex, USA), at room temperature. The mobile phase was 3.84 mM Na₂CO₃ – 6.00 mM NaHCO₃ with isocratic elution at the flow rate of 1.0 mL/min. A self-regenerating suppressor operating at 40 mA was used for suppressed conductivity detection. The in jection volume was 10 µL.

PHS Method According to the China National Standard GB/T 5009.34-2003, 20 g portion of homogenized sample were dispersed in freshly ultrapure water, and then 20 mL 0.05 M Na₂ (HgCl₄) solution was added, after incubation for 4 h to completely release bound sulfite from carbonyl adducts, the solution was filtered into a 100 mL volumetric flask. A volume of 5 mL solution was sampled, and then mixed with 5 mL Na₂ (HgCl₄) solution, 1 mL NH₄SO₃NH₂ solution (12 g/L), 1 mL formaldehyde solution (2 g/L) and 1 mL pararosaniline hydrochloride solution in test tube. After incubation 20 min, UV (UV-2450, SHIMADZU, Japan) analysis was performed at 550 nm.

Results and Discussion

Optimized conditions of sample treatment

a) Determination of free sulfite

Sulfites were rather labile and can be quickly degraded during extraction procedure. Masár groups (Masár et al., 2004) introduced formaldehyde as stabilizer to transfer sulfite into HMS by following reaction:   

Here, formaldehyde was also used as stabilizer to analyze grape samples. The amount of formaldehyde and the reaction time were investigated to obtain optimized conditions for the accurate determination of free sulfite in grapes. It was found when the concentration of formaldehyde was in the range 5 – 10 mmol, the peak area of sulfite was increased sharply, and when the adding amount above 10 mmol, the peak area of sulfite kept constantly (Fig. 1 (A)). The reaction time also significantly affected the peak are of free sulfite, and when it reached 60 min, the biggest peak area was gotten (Fig. 1 (B)).

Fig. 1.

(A) Effect of the amount of formaldehyde, and (B) reaction time to the peak area of SO₃²⁻.

Red Globe grape samples just after the harvest or after storage of 0 day and 5 days by SO₂ preservative paper were analyzed with/without formaldehyde treatment in parallel. To the grapes just after harvesting, the results with/without formaldehyde treatment were consistent, the free sulfite were not detected. To samples after storage of 5 days by SO₂ preservative paper, an average amount of free sulfite were 8.62 mg/kg and 7.12 mg/kg (a decrease of 17.4%) with/without formaldehyde treatment, respectively. This result testified formaldehyde treatment can largely avoid the loss of sulfite in the procedure of sample treatment. This result testified the addition of formaldehyde can efficiently avoid the loss of free sulfite.

b) Determination of total sulfite

When the sample was alkalized, the reversibly bound sulfite can be released, and sulfite can be subsequently oxidized to sulfate by hydrogen peroxide (Edmond et al., 2003; Koch et al., 2010). But there are lots of solid in berries, such as peel, pulp etc., which meant the matrix of grapes was much more complex than those of wine and other liquid samples, so the reaction time and amount of oxidant should be optimized to guaranty a full oxidization of sulfite to sulfate by hydrogen peroxide, and the results were summarized in Fig. 2. It showed that the optimized conditions were at pH 11 (Fig. 2 (A)), 120 min of reaction time (Fig. 2 (B)), and 5 mL of H₂O₂ (Fig. 2 (C)). The optimized pH value and the volume of H₂O₂ were similar with previous reports (Masár et al., 2005; Koch et al., 2010), but the reaction time was almost doubled in this work.

Fig. 2.

(A) Effect of pH value, (B) oxidation time, and (C) H₂O₂ volume to the peak area of SO₄2⁻

Chromatographic separation Chromatographic separation was performed under optimized condition (data not showed). The typical chromatogram was shown in Fig. 3. The retention time of sulfate and sulphite were 21.5 min and 22.7 min, respectively. The linearities of the IC method to sulfite and sulfate were evaluated and results were listed in Table 1. The calibration graphs were rectilinear for 0.1 – 40 mg/L (correlation coefficient, r = 0.9998) and 0.01 – 40 mg/L (r = 0.9997) to sulphite and sulphate, respectively. RSD (%) of intraday repeatability (with 3 replicates) were below 2.3%, which testified the accuracy of the method. The LODs were as low as 0.002 and 0.050 mg/L to sulfate and sulfite, respectively (calculated as the concentration corresponding to three times of signal-to-noise ratio). To testify the accuracy of method, the recoveries were analyzed by spiking sulfite and sulfate standards to reach the end concentration of 4, 10, 20 mg/L of into real grape sample, and amounted to 88 – 93% to sulfite and 87 – 98% to sulfate (the data was also shown in Table 1).

Fig. 3.

Typical ion chromatogram.

a Red Globe grape sample; b standard solution of sulfate and sulfite. Identified peak anions: 1 sulfate; 2 sulfite

Table 1. Regression equation and correlation coefficient, linear range, LOD, RSD and recoveries of IC and PHS

Method	Anions	Regression equation	Correlation coefficient	Linear range (mg/L)	LOD (mg/L)	RSD (%)	Recoveries (%)
IC	SO32−	Y = 3.532 × 10−2 X + 0 . 5320 × 10−2	0.9998	0.1 – 40	0.05	2.34	88a	93b	89c
	SO42−	Y = 4 . 981 × 10−2 X + 5 . 398 × 10−2	0.9997	0.01 – 40	0.002	1.67	87a	98b	96c
PHS	SO32−	Y = 6.009 × 10−2 X − 9 . 112 × 10−4	0.9996	0.2 – 4.0	0.1	2.20	81d	96e	94f

The amount of added standards: a, 4 mg/L; b, 10 mg/L; c, 20 mg/L; d, 0.6 mg/L; e, 2.0 mg/L; f, 4.0 mg/L.

Comparison of PHS and IC method As a comparison, a series of grape samples with different storage time were analyzed using the method proposed here and China National Standard GB/T 5009.34 – 2003, PHS method, used to determine the total sulfite in Red Globe grape; the results were listed in Table 1. By IC method, limits of detection to free sulfite and total sulfite (sulfate) were 0.05 mg/L and 0.002 mg/L, respectively. As comparison, PHS method only can be used to detect total sulfite with the limit of detection of 0.1 mg/L. It should be pointed that the complicated sample pretreatment and reagent preparation processes of PHS are much more time consuming, which almost double than that of IC method.

Changes of the free and total sulfite accumulation in grapes during storage The Red Globe grape samples with different storage time were analyzed using the method proposed in this study, and changes of the free and total sulfite accumulation were shown in Fig. 4. PHS method was also used to determine the amount of total sulfite. Table 2 illustrated the quantitative results of total sulfite derived from two methods. It was found that reliabilities of IC method were acceptable. Except o day, the relative standard deviation (RSD) of IC method was less than 3.3%, meanwhile RSD of PHS method was less < 2.6%. This result testified the high reliability of IC method.

Fig. 4.

amount of free sulfite, total sulfite and bound sulfite in Red Globe grape during different storage time at 0°C

Table 2. IC method and pararosaniline hydrochloride 51 spectrophotometry method determining the total sulfit

Storage Time (d)	IC		PHS
	(mg/kg, n=3)	RSD (%)	(mg/kg, n=3)	RSD (%)
0	1.67 ± 0.13	7.78	1.83 ± 0.22	12.0
5	14.28 ± 0.33	2.31	13.42 ± 0.25	1.86
7	17.22 ± 0.32	1.86	16.18 ± 0.18	1.11
15	18.99 ± 0.41	2.16	17.62 ± 0.36	2.04
30	23.14 ± 0.46	1.99	22.24 ± 0.24	1.08
60	23.94 ± 0.54	2.26	23.14 ± 0.76	3.28
110	27.34 ± 0.72	2.63	27.23 ± 0.28	1.03

(Mean ± Standard Deviation, mg/kg)

From Fig.4. it can be seen that the accumulation of both free and total sulfites were increased sharply at the first week, and then increased slowly down, the accumulation of free and total sulfites reached 19.97 mg/kg and 27.18 mg/kg till 110 days, respectively. It shows there exited big distance between the results of the accumulation between free and total sulfites, which meant there were parts of bound forms of sulfite existing. Since there were many components, including sugar, aldehydes and ketons, would react with SO₂, and transferred into binding compounds, which may have a significant effect on bound sulfites level (Jackowetz, 2012). Conclusion In this work, an accurate and reliable analytical method had been developed and validated for the suitable and quantitative determination of free and total sulfites in Red Globe grape. Relative standard deviation values of repeatability were less than 3%. Most importantly, LOD of IC increased 1 order of magnitude than that of the spectrophotometry to total sulfite, the time consumption was only half of the time PHS needed. The proposed method in this article can be applied to the determination of low-level sulfite in the barriers with color, and can meet the critical requirement of the increasingly stringent food safety standards around the world.

Acknowledgements The authors gratefully acknowledge national natural science foundation of China, grants no. 31160341 and 31060227.

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