Analysis of Antioxidants in Bakery Products following the GB 5009.32-2016 Method
Dean Duan, Senior Scientist
Merck R&D APAC Lab, Shanghai, China
Abstract
The Discovery® HS C18 column was used to separate the 9 antioxidants propyl gallate (PG), 1-(2,4,5-trihydroxyphenyl)-1-bunanone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (Ionox-100), octyl gallate (OG), butylated hydroxytoluene (BHT) and dodecyl/lauryl gallate (DG) in pastries following the Chinese national standard GB 5009.32-2016 method for determination of 9 antioxidants in food. The here-developed method using a Discovery® HS C18 column fulfills the system suitability criteria of the GB 5009.32-2016 method and exceeds the sensitivity criteria.
Section Overview
Introduction
Food antioxidants are additives that prevent or delay the oxidative deterioration of food to improve its stability and extend its storage period. Within the permitted levels for normal consumption, these antioxidants do not pose a health risk. However, individuals who are allergic or have asthma may develop hypersensitive reactions over time.1 The permissible limits for antioxidants vary for different foods in the different countries.2
The Chinese national standard GB 5009.32-2016 method3 describes a C18 HPLC column for the antioxidant measurement. Following this, a Discovery® HS C18 column was used for the here described analysis of 9 antioxidants (Figure 1), propyl gallate (PG), 1-(2,4,5-trihydroxyphenyl)-1-bunanone (THBP), tert-butylhydroquinone (TBHQ), nordihydroguaiaretic acid (NDGA), butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (Ionox-100), octyl gallate (OG), butylated hydroxytoluene (BHT) and dodecyl/lauryl gallate (DG), in a bread roll sample.
Figure 1.Structures of food antioxidant compounds investigated in this study.
Experimental
Standard Preparation
- Antioxidants stock solution (1 mg/mL each): Weigh 10 mg of each of the 9 antioxidants into a 10 mL brown volumetric flask. Add ~8 mL of acetonitrile and sonicate for 5 minutes. Top-up to mark with acetonitrile and mix well.
- Standard solutions for external calibration: Dilute stock solution to 1, 2, 5, 10, 20, 50, 100 and 200 µg/mL with acetonitrile.
Sample Preparation
Saturated solvent solutions: Take 200 mL of acetonitrile and 200 mL hexane into a 500 mL separating funnel. Cap and shake the funnel vigorously for 1 minute and allow it to stand for 5 minutes. Separate the solvents by draining the lower layer (hexane saturated acetonitrile) into a beaker followed by the upper layer (acetonitrile saturated hexane) into another beaker.
- Cut the pastry sample into smaller pieces and grind it into a paste or powder.
- Weigh 1 g (accurate to 0.01 g) of sample into a 50 mL centrifuge tube. For spiked samples, add appropriate quantities of the mixed antioxidants stock solution to the sample and vortex for 1 minute.
- Add 5 mL of a hexane solution saturated with acetonitrile to the sample, vortex for 1 minute, and stand for 10 minutes.
- Add 5 mL of saturated sodium chloride solution to the above.
- Add 5 mL of acetonitrile solution saturated with hexane to the above. Vortex for 2 minutes and centrifuge at 3000 rpm for 5 minutes. Transfer the acetonitrile layer (middle layer) to a 150 mL distillation flask.
- Repeat extraction twice with a 5 mL acetonitrile solution saturated with hexane. Collect all extracts into the same distillation flask.
- Evaporate the extract to dryness using a rotary evaporator under nitrogen in a 40 °C water bath.
- Dissolve the residue in 2 mL of acetonitrile. Filter through a 0.22 μm membrane before HPLC analysis (Table 1).
HPLC-UV Analysis |
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Method Suitability, System Suitability, Calibration
Acceptance Criteria as described in GB 5009.32-2016
- Linear correlation coefficient: R2 > 0.99
- Method recovery: 80 to 120%
- LOQ <20 mg/kg
Results & Discussion
Chromatographic results for a standard injection, a pastry sample and a spike pastry sample are displayed in the Figures 2-4. The specificity/retention times of the 9 antioxidants is shown in Table 2. The calibration curve for propyl gallate (PG) is displayed as example in the Figure 5. Chromatographic data of the pastry sample spiked with 9 antioxidants at 20 mg/kg (Figure 4) is shown in Table 3. The repeatability (n=5) for a standard solution at 20 µg/mL each compound was between 0.19 and 0.51 %RSD (Table 4). The external calibration linearity data from 1 to 200 µg/mL showed R2 values >0.999 (Table 5). The % recovery stayed withing the required 80-120% range (Table 6). Table 7 compares the LOD and LOQ values of this method and the GB 5009.32-2016 method, showing the developed method exceeds the criteria. The determination of antioxidants with an actual bread roll sample sourced from a local store is shown in Table 8. In the investigated unspiked sample a BHA content of 4.82 mg/kg was determined.
Figure 2.Mixed standard solution of 9 antioxidants, 20 μg/mL. For Peak IDs see Table 2.
Figure 3.Chromatogram of an unspiked pastry sample (bread roll).
Figure 4.Spiked pastry sample (bread roll) with 9 antioxidants at 20 mg/kg.
Calibration Data
Calibration in the range of 1-200 µg/mL with 8 standard solutions provided linear calibration curves with R2 values between 0.99958 and 0.99996 (Table 5) meeting the method requirement of R2 > 0.99. As example, the curve for PG is given in Figure 5.
Figure 5.Calibration curve of PG shown as example.
Repeatability
The repeatability for the 9 analytes was between 0.19 to 0.51 %RSD (n=5) for the 20 µg/mL standard solution (Table 5).
Recovery Data
For spiked samples (20 µg/kg) for the 9 analytes, recovery ranged from 77.2 to 115.4% and %RSD for triplicate measurement from 1.40 to 4.12 % (Table 6). Regarding the method criteria for recovery (80 to 120%) only TBHQ showed with 77.2% a recovery slightly below the limit.
Method Sensitivity
The method showed good sensitivities exceeding the requirement of the GB method (Table 7). Determined LODs were <1.1 mg/kg and LOQs of <3.07 mg/kg, exceeding the methods criteria of 20 mg/kg.
Sample measurement
For the investigated unspiked pastry sample only BHA was detected at 4.82 mg/kg (Table 8).
Conclusion
The Discovery® HS C18 column could separate the 9 antioxidants in the pastry matrix efficiently with excellent peak shapes. The shown method meets the acceptance criteria of GB 5009.32-2016 regarding recovery (except for TBHQ) and exceeds the sensitivity requirement with lower LOD and LOQ values.
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References
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