GB 5009.215-2016National food safety standard - Determination of Organotin in Food (English PDF)

3.1 Reagents

3.1.1 N-hexane (n-C6H14): re-distilled.

3.1.2 Tetrahydrofuran (CH2CH2OCH2CH2): re-distilled.

3.1.3 Ethyl acetate (CH3COOC2H5): re-distilled.

3.1.4 Cyclohexane (C6H12): re-distilled.

3.1.5 Methanol (CH3OH).

3.1.6 Anhydrous ether (C2H5OC2H5).

3.3.1 Organotin standard substance and its standard solution: please refer to Appendix

A. The purity of dimethyltin and tributyltin is 95%, other than this, the purity of other standard substances shall all be more than 97%.

3.3.2 Internal standard: monomethyltin (MMT) and tripropyltin (TPrT) standard

substance: purity > 98%.

3.4 Preparation of Standard Solutions

3.4.1 Organotin standard stock solution: accurately weigh-take a proper amount of

organotin standard substance, then, place it in a 10 mL beaker. Add methanol - aqueous solution (3.2.4) to dissolve it, then, transfer it to a 10 mL volumetric flask. In addition, dilute it to the scale. Store it in the refrigerator at -20 °C.

3.4.2 Internal standard - standard stock solution: accurately weigh-take a proper

amount of the internal standard, then, place it in a 10 mL beaker. Add methanol - aqueous solution (3.2.4) to dissolve it, then, transfer it to a 10 mL volumetric flask. In addition, dilute it to the scale. Store it in the refrigerator at -20 °C.

3.4.3 Organotin standard and internal standard intermediate solution: measure-take a

proper amount of organotin standard or internal standard stock solution. Use methanol

- aqueous solution (3.2.4) to dilute it by 100 times. Please refer to Appendix for the mass concentration. Store it in the refrigerator at -20 °C.

3.4.4 Organotin standard and internal standard working solution: measure-take a

proper amount of organotin standard or internal standard intermediate solution; place it in a 10 mL volumetric flask. Add methanol - aqueous solution (3.2.4); dilute to the scale. Please refer to Appendix A for the concentration. Store it in the refrigerator at -

20 °C.

4.2 Chromatographic column: DB-1 capillary column or equivalent column; column

length: 30 m; membrane thickness: 0.25 µm; internal diameter: 0.25 mm.

4.3 Tissue homogenizer.

4.4 Oscillator.

4.5 Ultrasonic cleaner.

4.6 Rotary evaporator.

4.7 Nitrogen concentrator.

and drying may be adopted to make freeze-dried powder. Place it at a low temperature of -20 °C for storage.

5.2.1 Sample extraction

5.2.1.1 Fish sample

Accurately weigh-take a proper amount of sample; add 0.15 g of ethylene diamine tetra-acetic acid and 5 mL of 20 g/L sodium chloride solution; shake it up. Add 50 µL of internal standard working solution; add 15 mL of hydrobromic acid - tetrahydrofuran solution (1 + 20) (3.2.5); conduct ultrasound for 5 min. If the sample is wet, add 1 mL of saturated sodium chloride solution; shake it up. Then, add 50 µL of internal standard working solution; add 15 mL of hydrobromic acid - tetrahydrofuran solution (1 + 20) (3.2.5); conduct ultrasound for 5 min.

5.2.1.2 Shellfish sample

Accurately weigh-take a proper amount of sample; add 5 mL of 20% sodium chloride solution; shake it up. Add 50 µL of internal standard working solution; add 15 mL of hydrobromic acid - tetrahydrofuran solution (1 + 20) (3.2.5); conduct ultrasound for 5 min.

5.2.1.3 Liquid sample, for example, wine

Measure-take 10 mL of sample; add 2 g of sodium chloride; shake it up. Add 50 µL of internal standard working solution; add 15 mL of hydrobromic acid - tetrahydrofuran solution (1 + 20) (3.2.5); conduct ultrasound for 5 min.

5.2.2 Purification of sample solution

Add 25 mL of n-hexane, which contains 0.03% tropolone, to the sample solution.

Conduct oscillating extraction for 40 min; conduct centrifugation for 10 min (at 3,000 r/min). Place it still for stratification. Absorb organic phase, then, transfer it to an eggplant-shaped bottle. Add 10 mL of n-hexane to the residue; conduct oscillating extraction for 10 min. Conduct centrifugation for 10 min (at 3,000 r/min); place it still for stratification; extract the organic phase. Combine them in the eggplant-shaped bottle; conduct rotary evaporation and concentration, till it reaches near dryness.

5.2.3 Gel permeation chromatographic purification

5.2.3.1 Filling of gel column: take polystyrene gel; use tetrahydrofuran - ethyl acetate

(1 + 1) solution to soak it overnight. Use glass wool to block the bottom of the glass chromatographic column whose internal diameter is 1.7 cm ~ 1.8 cm. Through wet process, add the previously soaked gel. The gel naturally settles. After it becomes stabilized, the column length is around 15 cm.

5.2.3.2 Purification: add 1 mL of tetrahydrofuran - ethyl acetate (1 + 1) solution to the a) Chromatographic column1: DB-1 column (or equivalent column); column length:

30 m; membrane thickness: 0.25 µm; internal diameter: 0.25 mm.

b) Adopt the mode of no-splitting; sample inlet temperature: 280 °C.

c) Colum temperature program: initial temperature: 50 °C, maintain for 1 min; at

10 °C/min, raise the temperature to 120 °C; at 5 °C/min, raise the temperature to 200 °C; at 10 °C/min, raise the temperature to 280 °C; maintain for 5 min.

d) Carrier gas is high-purity nitrogen (purity > 99.999%).

e) Pulse flame photometric detector reference conditions: pattern: sulfur filter;

temperature: 350 °C; flow rate of fuel gas and oxidant gas: air1 21 mL/min, hydrogen 22 mL/min, air2 11 mL/min; photomultiplier voltage: 550 V; threshold time: 4 ms; gate delay time: 5 ms; excitation voltage: 100 mV.

5.4 Draw a Standard Curve

Accurately weigh-take corresponding food sample, which does not contain organotin, as a proper amount of blank matrix. Add 5 mL of 20% sodium chloride solution;

respectively add 0 µL, 10 µL, 30 µL, 50 µL, 100 µL, 200 µL and 400 µL of organotin mixed standard solution, and 50 µL of internal standard working solution. In accordance with the requirements of the sample extraction and purification process, operate synchronously. Absorb 1 µL of the standard series solution; inject it into the gas chromatograph for analysis. Thus, obtain its chromatogram (please refer to

Appendix B.1 and Appendix B.2); use the retention time for qualitative determination.

5.5 Determination of Sample Solution

Absorb 1 µL of the sample solution, then, inject it into the gas chromatograph. Thus, obtain its chromatogram (please refer to Appendix B.3); use the retention time for qualitative determination. Calculate the peak area ratio or peak height ratio of target chemical compound and internal standard. Use the peak area ratio or peak height ratio of the sample injection volume (ng) of target organotin in the standard series solution and corresponding target organotin and internal standard to draw a linear curve. In accordance with the linear curve, calculate the content of organotin in the sample.