GB 5009.190-2014National Food Safety Standard -- Determination of Indicator Polychlorinated Biphenyls in Foods (English PDF)

3.1 Reagents

3.1.1 N-hexane (C6H14): pesticide residue.

3.1.2 Dichloromethane (CH2Cl2): pesticide residue.

3.1.3 Acetone (C3H6O): pesticide residue.

3.1.4 Methanol (CH3OH): pesticide residue.

3.1.5 Isooctane (C8H18): pesticide residue.

3.1.6 Anhydrous sodium sulfate (Na2SO4): guaranteed reagent. Put the commercially

available anhydrous sodium sulfate into the chromatographic column, and successively elute it twice with n-hexane and dichloromethane; the volume of solvent used for each elution is about twice the volume of anhydrous sodium sulfate. After elution, transfer the anhydrous sodium sulfate into a flask; bake it to dry at 50°C and then bake it for 8~12h at 225°C; cool it and then store it in a dryer.

3.1.7 Sulfuric acid (H2SO4): with content of 95%~98%, guaranteed reagent.

3.1.8 Sodium hydroxide (NaOH): guaranteed reagent.

3.1.9 Silver nitrate (AgNO3): guaranteed reagent.

3.1.10 Chromatographic silica gel (75~250µm). Put the commercially available silica gel

into the glass chromatographic column, and successively elute it twice with n-hexane and dichloromethane; the volume of solvent used for each elution is about twice the volume of silica gel. After elution, transfer the silica gel into flask, and cover the flask mouth with aluminum foil; put it into the oven, and bake at 50°C to dry; then heat up to 180°C and bake for 8~12h; cool it and then put it into a reagent bottle with ground stopper; store it in the dryer.

3.1.11 44% acid silica gel: weigh 100g of activated silica gel, dropwise add 78.6g of sulfuric acid and shake until it is free of cake; put in into the reagent bottle with ground stopper and store it in dryer.

3.1.12 33% alkaline silica gel: weigh 100g of activated silica gel, dropwise add 49.2g of

1mol/L sodium hydroxide solution and shake until it is free of cake; put in into the reagent bottle with ground stopper and store it in dryer.

3.1.13 10% silver nitrate silica gel: dissolve 5.6g of silver nitrate into 21.5mL of deionized water, dropwise add it into 50g of activated silica gel, and shake until it is free of cake; put in into the brown reagent bottle with ground stopper and store it in dryer.

3.1.14 Alkaline aluminum oxide: adopt alkaline aluminum oxide for chromatogram

chromatography, and bake it at 660°C for 6h; put it into the reagent bottle with ground stopper and store it in dryer.

3.2 Standard solutions

3.2.1 Standard solution for the determination of the time window: composed of cognate

can lead to a good cleaning effect. Where brush is adopted for cleaning, special attention shall be paid to that the brush shall not damage the internal surface of glass ware.

5.1 Sample preparation

5.1.1 Pretreatment

5.1.1.1 Pack the site-collected sample with lucifugal material like aluminum foil and brown

bottle, put it in small refrigerator and then transport it to the laboratory; store it in a cryogenic refrigerator below -10°C.

5.1.1.2 Solid sample like fish and meat may be dried by freezing or anhydrous sodium sulfate, and shall be mixed uniformly. Oil and fat may be directly dissolved in n-hexane for purification treatment.

5.1.2 Extraction

5.1.2.1 Before extraction, put an empty cellulose or glass fiber extraction sleeve into the

Soxhlet extractor; use n-hexane + dichloromethane (50+50) as extraction solvent, pre-extract for 8h, and then take it out for air drying.

5.1.2.2 Put 5.0g~10.0g of pretreated sample into the extraction sleeve treated according to

Article 5.1.2.1, add 13C12 labeled quantitative internal standard (Article 3.2.2); cover the sample with glass wool, balance for 30min and then put into Soxhlet extractor; use proper amount of n-hexane + dichloromethane (50+50) as extraction solvent and extract for 18~24h;

control the back flow speed within 3~4 times/h.

5.1.2.3 After extraction, transfer the extraction solution into a round-bottom flask, concentrate it with rotary evaporator to nearly dry. Where the analysis result is calculated according to fat, the fat content of sample shall be determined.

5.1.2.4 Fat determination: exactly weigh the mass of round-bottom flask before

concentration, and then exactly weigh the mass of the round-bottom flask again after the solvent is concentrated to dry; the difference of two weighed results is regarded as the fat content of the sample. After the fat content determination, add a small amount of n-hexane to dissolve the residue in flask.

5.1.3 Purification

5.1.3.1 Acidic silica gel column purification

Purification column filling: plug the bottom of glass column with glass wool, and then successively fill in 4g of activated silica gel, 10g of acid silica gel, 2g of activated silica gel and 4g of anhydrous sodium sulfate (see Figure D.1 in Appendix D) from bottom to top. Elute with 100mL of n-hexane in advance.

Purification: completely transfer the concentrated extraction solution to the column; wash the round-bottom flask for 3~4 times with 5mL of n-hexane, and transfer the washing liquid to the column. Where the liquid level falls to the anhydrous sodium sulfate layer, add 180mL of n-hexane to elute, and concentrate the eluent to about 1mL.

Where the complete acid silica gel layer is discolored, it indicates that the fat content in the sample exceeds the load limit of column. After the eluent concentration, prepare a stick of new acidic silica gel purification column, and repeat above operation until the sulfuric acid silica gel is not completely discolored.

5.1.3.2 Composite silica gel column purification

Purification column filling: plug the bottom of glass column with glass wool, and then successively fill in 1.5g of silver nitrate silica gel, 1g of activated silica gel, 2g of alkaline silica gel, 1g of activated silica gel, 4g of acid silica gel, 2g of activated silica gel and 2g of anhydrous sodium sulfate (see Figure D.1 in Appendix D) from bottom to top. Then, elute with 30mL of n-hexane + dichloromethane (97+3) in advance.

Purification: completely transfer the concentrated eluent after being purified according to

Article 5.1.3.1 to the column; wash the round-bottom flask for 3~4 times with 5mL of n-hexane, and transfer the washing liquid to the column. Where the liquid level falls to the anhydrous sodium sulfate layer, add 50mL of n-hexane + dichloromethane (97+3) to elute, and concentrate the eluent to about 1mL.

5.1.3.3 Alkaline aluminum oxide column purification

Purification column filling: plug the bottom of glass column with glass wool, and then successively fill in 2.5g of baked alkaline aluminum oxide and 2g of anhydrous sodium sulfate (see Figure D.1 in Appendix D) from bottom to top. Elute with 15mL of n-hexane in advance.

Purification: completely transfer the eluent concentrated after being purified according to

Article 5.1.3.2 to the column, wash the round-bottom flask for 3~4 times with 5mL of n-hexane, and then transfer the washing liquid to the column. Where the liquid level falls to the anhydrous sodium sulfate layer, add 30mL of n-hexane (2×15mL) to elute the column;

where the liquid level falls to the anhydrous sodium sulfate layer, use 25mL of dichloromethane + n-hexane (5+95) for elution. Concentrate the eluent to nearly dry.

5.1.4 Treatment before machine analysis

Transfer the purified sample solution into small injection tube, and concentrate it under nitrogen flow; wash the round-bottom flask for 3~4 times with a small amount of n-hexane, ratio for the detected ion of each compound replaced by trichlorine to heptachlor shall be greater than 3; otherwise, instrument tuning shall be repeated until meeting the relevant requirements.

5.4 Qualification and quantification of PCBs

5.4.1 Confirmation requirements of PCBs chromatographic peak: the signal to noise ratio of

the detected chromatographic peak shall be greater than 3 (see Figure C.1 or Figure C.3 in

Appendix C).

5.4.2 The abundance ratio of two monitored characteristic ions shall be within the theoretical range, respectively see Table B.1 and Table B.2 in Appendix B.

5.4.3 Inspect the mass spectrogram corresponding to chromatographic peak (see Figure C.2

or Figure C.4 in Appendix C); where the concentration is sufficiently large, the fragment ion (M-70) discarding 2 chlorine atoms shall exist, see Table B.1 in Appendix B.

5.4.4 Inspect the mass spectrogram corresponding to chromatographic peak (see Figure C.2

or Figure C.4 in Appendix C); for chromatographic peak of trichlorinated biphenyl to heptachlor biphenyl, fragment ion (M+70) added with 2 chlorine atoms cannot exist, see

Table B.1 in Appendix B.

5.4.5 The retention time of confirmed PCBs shall be within the time window predetermined through analyzing the standard solution for the determination of the time window. Standard solution for the determination of the time window is composed of cognate compounds of the first and last peaks of chloride-replaced PCBs on DB-5ms chromatographic column. Analyze the standard solution of window determination (1µL) by adopting determined chromatographic condition and full-scan mass spectrum collection mode, and determine the time window according to the retention time section of each PCBs family. The retention time sections of three PCBs families are overlapped on DB-5ms chromatographic column, thus, the characteristic ion of each PCBs family shall be detected within the same time window. To guarantee the selectivity and sensitivity of analysis, the detected characteristic ions in the same window shall be as little as possible during the time window determination.

5.5 Expression of analysis result

5.5.1 In this standard, isotope dilution technology is adopted for the quantification of

PCB28, PCB52, PCB118, PCB153, PCB180, PCB206 and PCB209; internal standard method is adopted for the quantification of other target compounds and the recovery calculation of quantitative internal standard. 20 target compounds determined in this standard covers a majority of PCBs industrial products, including 3 compounds from each family from trichlorinated biphenyl to Octa-PCB, and respectively one from nonachlor biphenyl and decachlorobiphenyl; see Table A.4 in Appendix A. A 13C12 labeled compound is adopted as the quantitative internal standard of each family, see Table A.2 in Appendix A. There are two internal recovery standards to calculate the recovery of quantitative internal standard, see ms - the quantity of added quantitative internal standard, ng;

H - the peak height of quantitative internal standard;

RRFn - the relative response factor of target compound to quantitative internal standard;

m - the sample quantity, g.

Since sample matrix, sampling quantity, injection volume, recovery of quantitative internal standard, chromatographic separation condition, electrical noise level and instrument sensitivity may affect the detection limit of sample, the noise level shall be obtained from actual sample spectrogram. Where the result of certain target compound is reported as not detected, the detection limit of sample shall be simultaneously reported.

6.1 Initial precision and accuracy test

Before analyzing the actual sample, the laboratory shall reach acceptable precision and accuracy level. Verify the reliability of analysis method through analyzing the labeled sample.

Take at least 3 portions of blank sample with matrix similar to that of the actual sample, and respectively add standard solution of precision and accuracy test, see Table A.5 in Appendix A;

respectively add quantitative internal standard solution. Analyze the prepared labeled sample through the method same as that of the actual sample, calculate the recovery of target compound and quantitative internal standard. The determined value of target PCBs of each portion shall be within 75%~120% of the adding quantity, RSD< 30%. The average recovery of quantitative internal standard shall be within 50%~120%, and the recovery of quantitative internal standard shall be within 30%~130%.

The above standard shall be reached before analyzing the actual sample. Where the extraction or purification method of the sample is modified and the analysis operator is changed, the above test shall be repeated until reaching the above standard. The above test shall be carried out for the laboratory every 6 months until reaching the above standard requirements.

Where the standard reference substance with matrix similar to that of the sample is available, the quantitative internal standard solution may be replaced by standard reference substance in precision and accuracy test.

6.2 Recovery of quantitative internal standard

Before sample extraction, add quantitative internal standard to calibrate the loss of target compound in sample extraction and purification process. The recovery of quantitative internal standard shall be within 30%~130%. Where the recovery of quantitative internal standard of sample analysis result fails to meet the above requirements, the extraction, purification and machine analysis of sample shall be repeated.

6.3 Method blank

For each batch, a method blank test shall be carried out for each 15 samples at most.

6.4 Quality-controlled sample

For each batch, a quality-controlled sample shall be provided for each 15 samples at most.

The quality-controlled sample may be standard reference substance or labeled sample with given concentration. The determined value of the target compound shall be within 75%~125% of the standard value.

6.5 Retention time window

Analyze the standard solution for the determination of the time window weekly to determine the accuracy of the retention time window. Where the chromatographic column is changed or cut, or the chromatographic parameter is changed, the retention time window shall be calibrated with standard solution for the determination of the time window.

6.6 Standard calibration solution

5 concentration levels of standard calibration solution are used for initial calibration. Where the RSD of RRF is less than 20%, it indicates that the calibration is successful. During the analysis process, a confirmatory test shall be carried out every 12h. Where machine analysis is carried out with CS3 in standard calibration solution, the analysis result shall be within

±20% of its fixed value, and the recovery of quantitative internal standard shall be within

75%~125%.

11.1 Sample extraction

11.1.1 Solid sample: weigh 5~10g of sample (accurate to 0.1g), and put it into a conical flask with plug; add quantitative internal standard PCB198, and then extract for 2h on water bath oscillator with proper amount of n-hexane + dichloromethane (50+50) as extraction solution; the water bath temperature is 40°C, and the oscillating speed is 200r/min.

11.1.2 Liquid sample (except oil and fat sample): weigh 10g of sample (accurate to 0.1g), and put it into a centrifuge tube with plug; add quantitative internal standard PCB198 and

0.5g of sodium oxalate; add 10mL of methanol and shake well; add 20mL of ethyl ether + n-hexane (25+75) and extract for 20min through oscillation; centrifuge for 5min at 3000r/min;

take supernatant and pass it through the glass column filled with 5g of anhydrous sodium sulfate; add 20mL of ethyl ether + n-hexane (25+75) to the residue; repeat the above procedure, and then combine the extraction solution.

11.1.3 Transfer the extraction solution into a round-bottom flask, concentrate it with rotary

evaporator to nearly dry. Where the analysis result is calculated according to fat, the fat content of sample shall be determined.

11.1.4 Sample fat determination: exactly weigh the weight of round-bottom flask before

concentration, and then exactly weigh the weight of the round-bottom flask and residue after the solvent is concentrated to dry; the difference of two weighed results is regarded as the fat content of the sample.

11.2 Purification

11.2.1 Sulfuric acid purification

Transfer the concentrated extraction solution into a 10mL test tube; wash the round-bottom flask for 3~4 times with about 5mL of n-hexane, and then combine the washing liquid into a concentrated solution; scale the volume to the scale with n-hexane, add 0.5mL of concentrated sulfuric acid, and shake for 1min; centrifuge for 5min at a rotation speed of

3000r/min to separate the sulfuric acid layer and organic layer. If the upper-layer solution still has color, it indicates that the fat is not completely removed; then add certain amount of concentrated sulfuric acid and repeat the above operation until the upper-layer solution is colorless.

11.2.2 Alkaline aluminum oxide column purification

Purification column filling: add a small amount of glass wool into the bottom of glass column, and then successively add 2.5g of baked alkaline aluminum oxide and 2g of anhydrous sodium sulfate from the bottom; elute with 15mL of n-hexane in advance.

Purification: transfer the concentrated solution (Article 11.2.1) into the chromatographic column; wash the round-bottom flask for 3~4 times with 5mL of n-hexane, and transfer the washing liquid into the chromatographic column. When the liquid level falls to the anhydrous sodium sulfate layer, add 30mL of n-hexane (2×15mL) for elution; where the liquid level falls to the anhydrous sodium sulfate layer, use 25mL of dichloromethane + n-hexane (5+95) for elution. Concentrate the eluent with rotary evaporator to nearly dry.

11.3 Sample solution concentration

Transfer the sample solution (11.2.2) into a sample injection bottle, wash the round-bottom flask for 3~4 times with a small amount of n-hexane, and combine the washing liquid into the sample injection bottle; concentrate it to 1mL under nitrogen flow and maintain it for GC analysis.

12.1 Chromatographic conditions

12.1.1 Chromatographic column: DB-5ms column, 30m×0.25mm×0.25µm or equivalent

chromatographic column.

12.1.2 Injection port temperature: 290°C.

12.1.3 Temperature programming: adopt a start temperature of 90°C and maintain for

0.5min; heat up to 200°C at a speed of 15°C/min, and maintain for 5 min; heat up to 250 °C at a speed of 2.5°C/min, and maintain for 2min; and then heat up to 265°C at a speed of

20°C/min, and maintain for 5 min.

12.1.4 Carrier gas: high-purity nitrogen (with purity >99.999%); the column pressure is

67kPa (equivalent to 10psi).

12.1.5 Injection volume: splitless injection of 1µL.

12.1.6 Chromatographic analysis: qualify according to retention time while quantify based

on the comparison between the sample and the standard peak height or peak area.

12.2 Qualitative analysis of PCBs

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