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1 Translated English of Chinese Standard: GB NATIONAL STANDARD OF GB THE PEOPLE S REPUBLIC OF CHINA National Food Safety Standard-Determination of total mercury and organic-mercury in foods Issued on: September 21, 2015 Implemented on: March 21, 2016 Issued by: National Health and Family Planning Commission Page 1 of 23

2 Table of Contents Foreword Scope... 5 Chapter I Determination of total mercury in foods... 5 The first method Atomic fluorescence spectrometry Principle Reagents and materials Apparatus Analysis procedures Representation of analysis result Precision Other The second Method Cold Atomic Absorption Spectrometric Method Principle Reagents and materials Apparatus Analysis procedures Representation of analysis result Precision Other Chapter II Determination of methylmercury in foods Liquid chromatography - atomic fluorescence spectrometry method Page 2 of 23

3 16 Principle Reagents and materials Apparatus Analysis procedures Representation of analysis result Precision Other Annex A Annex B Page 3 of 23

4 Foreword This Standard replaces GB/T Determination of Total Mercury and Organic-mercury in Foods. Compared with GB/T , the main changes are as follows: - modified the standard name to National Food Safety Standard- Determination of total mercury and organic-mercury in foods ; - deleted double sulphur hydrazone colorimetry method for determination of total mercury, gas chromatography for determination of organic mercury and atomic absorption spectrometry; - added liquid chromatography - atomic fluorescence spectrometry (LC- AFS) for determination of methylmercury. Page 4 of 23

5 National Food Safety Standard - Determination of total mercury and organic-mercury in foods 1 Scope The first chapter of this Standard specified the determination method for total mercury in foods. The first chapter of this Standard is applicable to the determination of total mercury in foods. The second chapter of this Standard specifies the liquid chromatography - atomic fluorescence spectrometry for determination of methylmercury in foods. The second chapter of this Standard is applicable to the determination of methylmercury in foods. Chapter I Determination of total mercury in foods The first method Atomic fluorescence spectrometry 2 Principle After the sample is heated and digested by acid, in acidic medium, mercury in sample is reduced to atomic mercury by potassium borohydride or sodium borohydride. It is brought by carrier gas (argon) into the atomizer. Under irradiation of mercury hollow cathode lamp, ground state mercury atom is stimulated to high energy state. When it is back from high energy state to ground state, it shall emit the characteristic wavelength fluorescence. Its fluorescence intensity is proportional to the mercury content, in quantitative comparison with the standard series solution. 3 Reagents and materials NOTE: Unless otherwise indicated, reagents used in this method are guaranteed reagents; water is the grade one water specified in GB/T Page 5 of 23

6 3.1 Reagents Nitric acid (HNO3) Hydrogen peroxide (H2O2) Sulfuric acid (H2SO4) Potassium hydroxide (KOH) Potassium borohydride (KBH4): analytically pure. 3.2 Reagent preparation Nitric acid solution (1+9): measure 50 ml of nitric acid; slowly add it into 450 ml of water Nitric acid solution (95+5): measure 5 ml of nitric acid; slowly add it into 95 ml of water Potassium hydroxide solution (5g/L): weigh 5.0 g of potassium hydroxide; dissolve it in pure water; set volume to 1000 ml; well mix it Solution of potassium borohydride (5g/L): weigh 5.0 g of potassium borohydride; use 5g/L potassium hydroxide solution to dissolve and set volume to 1000 ml; well mix it; prepare it when required Potassium dichromate nitric acid solution (0.5 g/l): weigh 0.05 g of potassium dichromate to dissolve in 100 ml of nitric acid solution (5+95) Nitric acid-perchloric acid mixed solution (5+1): measure 500 ml of nitrate and 100 ml of perchloric acid; well mix. 3.3 Standard product Mercuric chloride (HgCl2): purity 99%. 3.4 Preparation of standard solution Mercury standard stock solution (1.00 mg/ml): accurately weigh g of dried mercuric chloride; use potassium dichromate nitric acid solution (0.5 g/l) to dissolve and transfer to 100 ml measuring flask; dilute it to scale; well mix; the concentration of this solution is 1.00 mg/ml; store it in 4 C refrigerator in dark conditions; it can be stored for 2 years; or purchase the standard solution substance awarded by national certification standard certificate Mercury standard intermediate solution (10 μg/ml): pipette 1.00 ml of mercury standard solution (1.00 mg/ml) in 100 ml measuring flask; use potassium dichromate nitric acid solution (0.5 g/l) to dilute to scale; well mix; Page 6 of 23

7 sample OR pipette 1 ml ~ 5 ml of liquid sample (accurate to g) into digestion inner tank. Add 5 ml of nitrate to soak overnight. Cover the inner lid. Tighten the stainless steel jacket. Place into thermostatic oven for 4h ~ 5h at 140 C ~ 160 C. Naturally cool it to room temperature in the oven. Then slowly loosen the stainless steel jacket. Take it from the digestion inner tank. Use few amount of water to clean the inner lid. Place on temperature control heating plate or ultrasonic water bath. Drive away brown gas at 80 C or by 2 min ~ 5 min of ultrasonic degassing. Take the digestion inner tank out. Transfer the digestive fluid to 25 ml flask. Use few amount of water to clean the inner tank for 3 times. Combine the detergent in the flask and set volume to scale. Well mix for use. Conduct the blank test at the same time Microwave digestion method Weigh 0.2 g ~ 0.5 g (accurate to g) of solid sample, 0.2 g ~ 0.8 g of fresh sample OR pipette 1 ml ~ 3 ml of liquid sample into digestion inner tank. Add 5 ml ~ 8 ml of nitrate. Cover and place for overnight. Tighten the lid. Digest according to the standard operation procedures of microwave digestion system (see Table A.1 of Annex A for digestion reference conditions). Take it out after cooling. Slowly open the tank to exhaust. Use few amount of water to clean the inner lid. Place on temperature control heating plate or ultrasonic water bath. Drive away brown gas at 80 C or by 2 min ~ 5 min of ultrasonic degassing. Take the digestion inner tank out. Transfer the digestive fluid to 25 ml plastic flask. Use few amount of water to clean the inner tank for 3 times. Combine the detergent in the flask and set volume to scale. Well mix for use. Conduct the blank test at the same time Reflux digestion method Grain Weigh 1.0 g ~ 4.0 g (accurate to g) of sample. Place it in digestion Erlenmeyer flask. Add few glass beads. Add 45 ml of nitric acid, 10 ml of sulfuric acid. Rotate the Erlenmeyer flask to prevent partial carbonization. After mounting condenser tube, low heat it when it starts bubbling. After bubbling stops, heat it for 2h. If during heating, the solution turns brown, it shall add 5 ml of nitrate. Add 5 ml of nitrate, and continue refluxing for 2h till the sample is dissolved by digestion. Generally, it appears light yellow or colorless. After it is cold, carefully add 20 ml of water from the top of condenser tube. Continue heating and refluxing for 10 min. Place it till it becomes cold. Use appropriate amount of water to flush the condenser tube. Combine the flushing fluid into digestive fluid. Filter the digestive fluid into 100 ml flask through glass wool. Use few amount of water to wash the Erlenmeyer flask, filter. Combine the detergent into the flask. Add water to scale. Well mix. Conduct the blank test at the same time. Page 8 of 23

8 Stannous chloride solution (100 g/l): weigh 10 g of stannous chloride and dissolve in 20 ml of hydrochloric acid; heat it in 90 C water bath; slightly oscillate it till stannous chloride is dissolved to transparent state, cool it. Use pure water to dilute it and set volume to 100 ml. Add few metal tin. Store it at cool, dark place. If it is found turbid, re-prepare it Nitric acid solution (1+9): measure 50 ml of nitric acid; slowly add it into 450 ml of water Standard product Mercuric chloride (HgCl2): purity 99% 10.4 Preparation of standard solution Mercury standard stock solution (1.00 mg/ml): accurately weigh g of dried mercuric chloride; use potassium dichromate nitric acid solution (0.5 g/l) to dissolve and transfer to 100 ml measuring flask; set volume; the concentration of this solution is 1.00 mg/ml; store it in 4 C refrigerator in dark conditions; it can be stored for 2 years; or purchase the standard solution substance awarded by national certification standard certificate Mercury standard intermediate solution (10 μg/ml): pipette 1.00 ml of mercury standard solution (1.00 mg/ml) in 100 ml measuring flask; use potassium dichromate nitric acid solution (0.5 g/l) to dilute and set volume; the concentration of this solution is 10 µg/ml; store it in 4 C refrigerator in dark conditions; it can be stored for 2 years Mercury standard solution (50 ng/ml): pipette 0.50 ml of mercury standard intermediate solution (10μg/mL) in 100 ml measuring flask; use 0.5 g/l nitric acid solution of potassium dichromate to dilute to scale; well mix; the concentration of this solution is 50 ng/ml; prepare it when required. 11 Apparatus NOTE: Glassware and polytetrafluoroethylene digestion inner tank all require 24h soaking by nitric acid solution (1+4). Use water to repeatedly wash. Use deionized water to rinse it clean Mercury analyzer (with gas circulation pump, gas drying device, mercury vapor generating apparatus and mercury vapor absorption bottle) or automatic mercury analyzer Balance: inductance is 0.1 mg and 1 mg Microwave digestion system. Page 12 of 23

9 15 Other When sample s sampling mass is 0.5 g, the constant volume is 25 ml, the detection limit of the method shall be mg/kg, the quantification limit of the method shall be mg/kg. Chapter II Determination of methylmercury in foods Liquid chromatography - atomic fluorescence spectrometry method 16 Principle After the methylmercury in foods is pipetted by 5 mol/l hydrochloric acid solution with the help of ultrasonic wave, use C18 reverse phase column separation. Chromatography effluent shall go into online UV digestion system to react with strong oxidizer potassium persulfate under UV irradiation. Inorganic mercury shall become methylmercury. In acidic environment, the inorganic mercury shall react with potassium borohydride to generate mercury vapor online. It shall be determined by atomic fluorescence spectrometer, determined by retention time and quantified by peak area of external standard method. 17 Reagents and materials NOTE: Unless otherwise indicated, reagents used in this method are guaranteed reagents; water is the grade one water specified in GB/T Reagents Methanol (CH3OH): chromatographically pure Sodium hydroxide (NaOH) Potassium hydroxide (KOH) Potassium borohydride (KBH4): analytically pure Potassium persulfate (K2S2O8): analytically pure. Page 15 of 23

10 Ammonium acetate (CH3COONH4): analytically pure Hydrochloric acid (HCl) Ammonia (NH3 H2O) L- Cysteine (L-HSCH2CH (NH2) COOH): analytically pure Reagent preparation Mobile phase (5% methanol mol/l ammonium acetate + 0.1% L-cysteine): weigh 0.5 g of L-cysteine, 2.2 g of ammonium acetate; place them in 500 ml flask; use water to dissolve; add 25 ml of methanol; use water to set volume to 500 ml; after being filtered by 0.45µm organic membrane, carry out ultrasonic degassing in ultrasonic water bath for 30 min; prepare it when required Hydrochloric acid solution (5 mol/l): measure 208 ml of hydrochloric acid; dissolve it and dilute to 500 ml Hydrochloric acid solution 10% (volume ratio): measure 100 ml of hydrochloric acid; dissolve it and dilute to 1000 ml Potassium hydroxide solution (5 g/l): weigh 5.0 g of potassium hydroxide; dissolve it and dilute to 1000 ml Sodium hydroxide solution (6 mol/l): weigh 24 g of sodium hydroxide; dissolve it and dilute to 100 ml Potassium borohydride solution (2 g/l): weigh 2.0 g of potassium borohydride; use potassium hydroxide solution (5 g/l) to dissolve and dilute to 1000 ml; prepare it when required Potassium persulfate solution (2 g/l): weigh 1.0 g of potassium persulfate; use potassium hydroxide solution (5 g/l) to dissolve and dilute to 500 ml; prepare it when required L- Cysteine solution (10 g/l): weigh 0.1 g of L- Cysteine and dissolve in 10 ml of water; prepare it when required Methanol solution (1+1): weigh 100 ml of methanol; add it into 100 ml of water; well mix Standard product Mercuric chloride (HgCl2): purity 99% Methylmercury chloride (HgCH3Cl): purity 99%. Page 16 of 23

11 See Sample extraction Weigh 0.50 g ~ 2.0 g of sample (accurate to g). Place it in a 15 ml plastic centrifuge tube. Add 10 ml of hydrochloric acid solution (5 mol/l). Place it overnight. Carry out 60 min ultrasonic bath extraction at room temperature. Shake it for several times during extraction. Carry out 15 min centrifugation at 8000 r/min below 4 C. Accurately pipette 2.0 ml of supernatant liquid into a 5 ml measuring tube. Add sodium hydroxide solution (6 mol/l) drop by drop so as to make sample solution s ph 2~7. Add 0.1 ml of L- Cysteine solution (10 g/l). Use water to set volume to scale. Use 0.45 µm organic membrane to filter for determination. Carry out the blank test at the same time. NOTE: When adding sodium hydroxide solution (6 mol/l), it shall add slowly to prevent the late heat diffusion generated by acid-base neutralization and make temperature rise quickly, which shall cause mercury compounds volatilization and determination value too low Instrument reference conditions Reference conditions for liquid chromatography Reference conditions for liquid chromatography are as follows: - Column: C18 column (column length is 150 mm, inner diameter is 4.6 mm, particle size is 5 μm), C18 pre-column (column length is 10 mm, inner diameter is 4.6 mm, particle size is 5 μm). - Flow rate: 1.0 ml/min. - Injection volume: 100 μl Reference conditions for atomic fluorescence detection Reference conditions for atomic fluorescence detection are as follows: - Negative high voltage: 300 V; - Mercury current: 30 ma; - Atomization method: cold atoms; - Carrier liquid: 10% hydrochloric acid solution; - Carrier liquid flow rate: 4.0 ml/min; - Reducing agent: 2 g/l potassium borohydride solution; Page 18 of 23

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