Determination of Benzoic Acid, Sorbic Acid & Saccharin Sodium in Foods | GB 5009.28-2016 Standard Methods (HPLC/GC)

GB 5009.28-2016 National Food Safety Standard: Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods

1. Scope

This standard specifies methods for the determination of benzoic acid, sorbic acid, and saccharin sodium in foods, with the following applicability:

• Method 1 (Liquid Chromatography, LC): Applicable to the determination of benzoic acid, sorbic acid, and saccharin sodium in foods.
• Method 2 (Gas Chromatography, GC): Only applicable to the determination of benzoic acid and sorbic acid in soy sauce, fruit juices, and jams (not applicable to saccharin sodium).

2. Method 1: Liquid Chromatography

Principle

After pretreatment, target analytes are separated by liquid chromatography, detected via ultraviolet (UV) detector, and quantified using the external standard method.

Reagents and Materials

Note: Unless otherwise specified, all reagents are of analytical grade, and water complies with Grade 1 requirements per GB/T 6682.

Key Reagents:

Ammonia solution, sodium hydroxide, sulfuric acid, potassium ferrocyanide, zinc acetate, sodium chloride, tartaric acid, silicone resin, sodium dihydrogen phosphate, potassium dihydrogen phosphate, neutral alumina, ammonium acetate; methanol (HPLC grade).

Reagent Preparation:

• Ammonia solution (1+1): Mix equal volumes of ammonia solution and water; filter through microporous membrane.
• Sodium hydroxide solution (4 g/L): Dissolve 4 g NaOH in water and dilute to 1,000 mL.
• Sulfuric acid solution (0.5 mol/L): Gradually add 30 mL concentrated H₂SO₄ (~70%) to 500 mL water with stirring; cool and dilute to 1,000 mL.
• Potassium ferrocyanide solution (92 g/L): Dissolve 106 g K₄[Fe(CN)₆]·3H₂O in water and dilute to 1,000 mL.
• Zinc acetate solution (183 g/L): Dissolve 220 g Zn(CH₃COO)₂·2H₂O in minimal water, add 30 mL glacial acetic acid, and dilute to 1,000 mL.
• Tartaric acid solution (15%): Dissolve 15 g C₄H₆O₆ in water and dilute to 100 mL.
• Phosphate buffer (pH 7.2): Dissolve 16.72 g NaH₂PO₄ and 2.72 g KH₂PO₄ in water; dilute to 1,000 mL and filter.
• Neutral alumina (100–200 mesh): Dry at 105°C for 2 h, cool, deactivate with 2 mL water per 100 g, mix, seal, and equilibrate for 12 h.
• Ammonium acetate solution (1.54 g/L): Dissolve 1.54 g CH₃COONH₄ in water, dilute to 1,000 mL, and filter.

Reference Standards

• Sodium benzoate (CAS: 532-32-1, purity ≥99%)
• Potassium sorbate (CAS: 590-00-1, purity ≥99%)
• Saccharin sodium (CAS: 128-44-9, purity ≥99%; dry at 120°C for 4 h before use).

Standard Solution Preparation

Stock Solutions:

• Sodium benzoate: Accurately weigh 0.2360 g, dissolve in water, dilute to 200 mL (equivalent to 1.00 mg/mL benzoic acid).
• Potassium sorbate: Accurately weigh 0.2680 g, dissolve in water, dilute to 200 mL (equivalent to 1.00 mg/mL sorbic acid).
• Saccharin sodium: Accurately weigh 0.2340 g (dried), dissolve in water, dilute to 200 mL (1.00 mg/mL).
• Mixed Standard Working Solutions: Dilute stock solutions to prepare calibrators at 0.000, 0.020, 0.040, 0.080, 0.160, and 0.320 mg/mL for each analyte.

3. Method 2: Gas Chromatography

Principle

Acidified samples are extracted with diethyl ether. Benzoic acid and sorbic acid are separated by GC with FID detection and quantified by external standard method.

Reagents and Materials

Note: Unless otherwise specified, all reagents are analytical grade; water complies with GB/T 6682 Grade 1.

• Key Reagents:

Diethyl ether (peroxide-free), petroleum ether (30–60°C boiling range), hydrochloric acid, sodium chloride, anhydrous sodium sulfate.

• Reagent Preparation:

          Hydrochloric acid solution (1+1): Slowly add 100 mL HCl to 50 mL water; cool and dilute to 200 mL.

          Acidified sodium chloride solution (40 g/L): Acidify NaCl solution (40 g/L) with HCl (1+1).

          Petroleum ether-diethyl ether (3+1): Mix 150 mL petroleum ether and 50 mL diethyl ether.

Reference Standards

• Benzoic acid (CAS: 65-85-0, purity ≥99%)
• Sorbic acid (CAS: 110-44-1, purity ≥99%)

Standard Solution Preparation

• Stock Solutions: Accurately weigh 0.2 g each of benzoic acid and sorbic acid (to 0.0001 g), dissolve in methanol, dilute to 100 mL (2.0 mg/mL each).
• Working Solutions: Dilute stock solutions with petroleum ether-diethyl ether (3+1) to 50, 100, 150, 200, and 250 μg/mL for quantification.

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