Analysis of synthetic food color additive, sugar, and mycotoxin content in traditional, cereal-based Sobia beverage using high-performance liquid chromatography and mass spectrometry

2.1 Chemicals

Mycotoxin (AF, OTA, T2, and DON) standards were obtained from Trilogy Analytical Laboratory (Missouri, USA), while synthetic food color additive (E-102, E-110, E-122, and E-151) standards were obtained from the SFDA (Riyadh, Saudi Arabia). Other chemicals, including fructose, glucose, sucrose, ammonium acetate, and acetonitrile (high-performance liquid chromatography [HPLC]-grade) were purchased from Sigma Aldrich (St. Louis, MO, USA). Water was treated in a Milli-Q water purification system (Millipore, Molsheim, France) to obtain HPLC-grade water.

2.2 Samples

Sobia samples were obtained from anonymous vendors in western and central provinces of Saudi Arabia. A total of 14 samples were collected, divided by color, and coded with letters and numbers. The letter A was given to samples obtained from west regions, and T for samples obtained from central region. All samples were kept at –20 °C until use.

2.3 Sample preparation

Samples were prepared according to the method described by Ntrallou et al., (2020). In short, 10 mL of Sobia sample were centrifuged at 10,000 rpm for 15 min, then filtered through 0.45 µm syringes before injection into HPLC systems for synthetic food colorant and sugar content analysis.

There is no established procedure or the preparation and extraction of malt- and wheat-based juices for mycotoxin analysis. Thus, the “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) method, with slight modification, was applied to prepare and extract mycotoxins from the Sobia samples (González-Jartín et al., 2019). Specifically, 10 mL of acetonitrile containing 1% formic acid was added to 10 mL of Sobia sample in a 50 mL polypropylene tube and shaken for 30 min using a laboratory shaker. A buffer salt mixture (4 g MgSO4 + 1 g NaCl + 1 g trisodium citrate dehydrate + 0.5 g disodium hydrogen citrate sesquihydrate) was then added, and the tube was shaken vigorously by hand for 1 min. After that, the sample was centrifuged at 10,000 rpm for 5 min. The top acetonitrile layer was extracted and micro-filtered using a 0.2 µm filter and transferred into a new 15 mL centrifugation tube. Following this, 0.1 mL of the extract was added to 0.4 mL of mobile phase A and B [1:1] and transferred to a vial for analysis.

2.4 Synthetic food color additive analysis by HPLC-diode array detector (DAD)

Samples were analyzed for synthetic food color additives using HPLC (Agilent Technologies) with column Zorbax SB-C18 (250 × 4.6 mm; 5 μm) and detector 1260 DAD-VL. A 10 nM ammonium acetate solution was used for mobile phase A, and acetonitrile was used for mobile phase B. The flowrate was 1 mL/min, with an optimized gradient program (A:B) of 95:5 initially and 50:50 after 30 min. Absorbance was monitored at 426 nm for E-102, 482 nm for E-110, 514 nm for E-122, and 613 nm for E-151. Peaks were identified and quantified using the retention times of standard absorption spectra.

2.5 Sugar analysis by HPLC-refractive index detector (RID)

Fructose, glucose, and sucrose were analyzed using a Shimadzu HPLC system (Salamatullah et al., 2021) equipped with a prominence LC-10AB binary pump and a SIL-20A autosampler (Kyoto, Japan). Analysis was conducted using a mobile phase of 85% HPLC-grade aqueous acetonitrile at an isocratic flow rate of 1 mL/min. Compounds of interest were separated using a Shimadzu LC-NH2 column (15 × 4.6 mm; 5 µm) and identified using a RID-10A Shimadzu detector (Kyoto, Japan). Subsequently, 10 µL of each sample was injected into the HPLC system, and the peak retention times of fructose, glucose, and sucrose were compared to standards and analyzed using a Shimadzu LabSolutions LC WorkStation v. 1.22 (Kyoto, Japan).

2.6 Mycotoxin analysis by ultra-HPLC-positively charged electrospray ionization (ESI + )-mass spectrometry (MS)/MS

Aflatoxins, OTA, T-2, and DON were analyzed using a Sciex Triple Quad 6500 LC-MS/MS System equipped with analytical column Kinetex™ Cl8 (100 mm × 2.1 mm; 2.6 µm) at 40 °C. Mobile phase A was 5 mM of ammonium formate in water (0.2% formic acid), and mobile phase B was methanol (0.2% formic acid). The ultra-HPLC-(ESI + )-MS/MS gradient program is detailed in Supplementary Table 1 (Liao et al., 2013).

2.7 Statistical analysis

Statistical calculations were completed using Microsoft Excel, 2019 (Microsoft, Seattle, WA, USA). The experiments were independently analyzed in three times, and data were presented as arithmetical mean ± standard deviation.

3.1 Synthetic food color additive analysis by HPLC-DAD

The results of the analysis of 14 Sobia beverage samples by HPLC-DAD for the presence of four synthetic food colorants are presented in Fig. 2 and Table 1. The data show that all red Sobia samples contained varying concentrations of E-122, ranging from 6.58 to 42.73 µg/mL. These results indicate that the intended use of E-122 in Sobia beverages is to impart a red color (see Figs. 3 and 4). Among all samples, Red A2 contained E-151 colorant at a high concentration (152.97 µg/mL) (Table 1). The only dark red Sobia sample analyzed in this study contained E-110 colorant at a concentration of 0.45 µg/mL. Most white Sobia samples showed low concentrations of E-102, ranging from 0.22 to 1.37 µg/mL, while yellow Sobia drinks showed no added synthetic food colorants (Fig. 3 and Table 1).

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Fig. 2

HPLC Chromatogram of synthetic food color standards.

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Fig. 3

Association between Sobia and food colorant additives (A) red Sobia and brilliant black (left) and carmoisine (right) (B) yellow Sobia (C) white Sobia and tartrazine (D) dark red Sobia and sunset yellow.

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Fig. 4

HPLC Chromatogram of synthetic food colorant, carmoisine, detected in red Sobia T sample.

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Tartrazine is one of the most common additives used in the production of juices and drinks in the kingdom of Saudi Arabia (Ahmed et al., 2021). Ahmed et al. report the high intake of juices and drinks containing E-102 and E-110 by male (303–442 mL/day) and female (283–314 mL/day) Saudi children and the use of E-110 in juices and drinks at concentrations of 0–225 µg/mL (Ahmed et al., 2023). These findings confirm that the consumption of Sobia drinks with added colorants could increase exposure rates to synthetic food colorants, potentially compromising the health of Saudi Arabian consumers.

3.2 Sugar analysis by HPLC-RID

An analysis of sugar (fructose, glucose, and sucrose) content was undertaken to evaluate the nutrient energy content of Sobia. The results of HPLC-RID analysis revealed that fructose and glucose (monosaccharides) were found in all samples at low to moderate concentrations ranging from 0.26 to 37.69 mg/mL (Fig. 5 and Table 2). Sucrose was detected in all Sobia samples at varying concentrations, from 0.3 to 149.67 mg/mL. The sugar content of Hardaliye, a fermented beverage produced from grapes, was found to be 120–320 mg/mL and not less than 200 mg/mL in warm areas (Aydoğdu et al., 2014), indicating that the ripening of grapes in the production of fermented beverages could increase the total sugar content. However, Sobia production does not involve any fruit ripening. The use of sucrose, then, is for the purposes of increasing the fermentation rate and enhancing flavor, regardless of the total resultant energy intake in the form of free sugars.

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Fig. 5

HPLC Chromatogram of fructose, glucose, and sucrose standards.

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3.3 Mycotoxin analysis by ultra-HPLC (ESI + )-MS/MS

The results of mycotoxin analysis by ultra-HPLC-MS/MS (Fig. 6 and Table 3) show that AFs (B1, B2, G1, and G2) and OTA were not detected in all Sobia samples. Conversely, DON was found in most samples, with concentrations ranging from 1.15 to 38.5 ng/mL, and T-2 toxin was detected in Red A2, White A, and White A4, with concentrations of 1.4, 1.14, and 0.6 ng/mL, respectively. These findings are in agreement with analyses of alcoholic and non-alcoholic beverages on the European market (Carballo et al., 2021). The data show that DON is the mycotoxin most frequently detected in Sobia beverages, while T-2 is the least. Further, Al-Taher et al. (2013) found that T-2 was detected in only 11% of samples, with a mean level of 0.3 ng/mL. United States Food and Drug Administration regulations set the limit for DON concentration in cereals and cereal-based products at 1,000 ng/mg. European regulations are stricter, at 50–200 ng/mg (Alshannaq & Yu, 2017). For ready-to-use foods, such as juices and drinks, there are currently no regulations concerning DON or T-2 content. The Joint Food and Agriculture Organization (FAO)/WHO Expert Committee on Food Additives (JEFCA) and the Scientific Committee on Food recommend a total daily intake of T-2 (including hydrolized T-2) and DON of 100 ng/kg and 1,000 ng/kg, respectively (FAO, 2001). Thus, the presence of DON and T-2 in juices and drinks must be actively regulated.

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Fig. 6

(A) Chemical structures of analyzed mycotoxins (B) U-HPLC-(ESI + )-MS/MS chromatogram of the analyzed mycotoxins standards.

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