GB 23200.92-2016Food safety national standard -- Determination of pentachlorophenol residues in animal-derived foods by liquid chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Methanol (CH3OH). Chromatographic Purification.

4.1.2 acetonitrile (CH3CN). pure chromatography.

4.1.3 Triethylamine (C6H15N).

4.1.4 Formic acid (HCOOH). Chromatographic pure.

4.1.5 Ammonium acetate (CH3COONH4).

4.1.6 Ammonia (NH3 · H2O).

4.2 Solution preparation

4.2.1 acetonitrile - water solution (7 3). accurately measure 70mL acetonitrile and 30mL water, mix and shake.

4.2.2 5% triethylamine acetonitrile - water solution. accurately measure 50 mL of triethylamine, transferred into 1000 mL volumetric flask, with acetonitrile - water solution (7 3) Constant volume to the scale, mixed evenly.

4.2.3 5% ammonia solution. the amount of.200mL concentrated ammonia (25%), transferred into the 1000 mL volumetric flask, water volume to the scale, mixed Evenly.

4.2.4 4% formic acid methanol solution. the amount of 10 mL of formic acid, transferred into the 250 mL volumetric flask, with methanol volume to the scale, mixed evenly.

4.2.5 2% formic acid methanol aqueous solution. take a certain amount of 4% formic acid methanol solution and water volume mixed evenly.

4.2.6 5 mmol/L ammonium acetate aqueous solution. accurately weighed 0.3854 g ammonium acetate, dissolved in water, transferred to 1000 mL volumetric flask, set Capacity to scale, mixed evenly.

4.3 Standards

4.3.1 Pentachlorophenol sodium salt (CAS No.. 131-52-2). purity >=94.5%, or equivalent.

4.4 Standard solution preparation

4.4.1 Standard stock solution (200mg/L). Weigh the equivalent of 10 mg (accurate to 0.1 mg) pentachlorophenol standard, with a the alcohol was dissolved and set in a 50 mL brown volumetric flask. This stock solution can be stored for up to 12 months under -18 ° C.

4.4.2 standard working solution. standard working solution according to the need before use with blank sample matrix solution preparation. Store in 4 °C refrigerator.

4.5 Materials

4.5.1 Solid phase extraction (SPE) column. Oasis MAX, 60 mg, 3 mL, or equivalent. Use 5 mL of methanol and 5 mL of water before use activation.

4.5.2 Nitrogen. Purity >=99.99%.

6.1 Preparation of the sample

During the sample preparation operation, it is necessary to prevent the sample from being contaminated or the change in the content of the residue.

6.1.1 Animal muscle, liver, kidney, fish, shrimp and crab

A typical sample of about 500 g was taken from all the samples taken and mashed evenly in a tissue masher and divided into two portions.

Into a clean container, sealed and marked with a mark.

6.1.2 Milk samples

A typical sample of about 500 g was taken from all the samples taken and mixed thoroughly and divided into two portions, packed in a clean container, Seal, and mark the mark.

Note. The above sample sampling site according to GB 2763 Appendix A implementation.

6.2 Sample storage

Samples were stored at -18 ° C or lower.

7.1 Extraction

7.1.1 Muscle, fish, liver, kidney, shrimp and crab

Weigh the homogeneous sample 2 g (accurate to 0.01 g), placed in 10 mL screw cap polypropylene centrifuge tube, add 6 mL of 5% triethylamine Acetonitrile - aqueous solution. Muscle and fish samples were homogenized for 2 min; liver, kidney, shrimp and crab samples were mixed for 1 min and extracted by ultrasonic for 5 min.

3 000 r/min centrifugation 5 min, the supernatant was collected in a graduated centrifuge tube. The residue after centrifugation was treated with about 6 mL of 5% triethylamine in acetonitrile-Aqueous solution The above extraction step was repeated once, and the supernatant was combined and mixed.

7.1.2 Milk

Weigh the homogeneous sample 2 g (accurate to 0.01 g), placed in a 10 mL graduated screw cap polypropylene centrifuge tube, add 10 mL 5% tri Amine in acetonitrile - aqueous solution, vortex mixed for 1 min, ultrasonic extraction 5 min, 5 000 r/min centrifugation 5 min, collecting supernatant in a scale Centrifuge tube.

7.2 Solid phase extraction purification

The resulting solution from 7.1 was transferred to a pre-treated MAX solid phase extraction column to pass the solid solution at a rate of about 1 drop/sec.

Phase extraction column, discard the effluent. Followed by elution with 5 mL of 5% aqueous ammonia solution, 5 mL of methanol, 5 mL of 2% formic acid in methanol-water After the lotion is completely passed through the column, the effluent is discarded and the solid phase extraction column is evacuated with a vacuum pump for more than 5 min. Washed with 4 mL of 4% formic acid in methanol the eluate was collected with a clean 15 mL scale tube and concentrated at 0 ° C in a water bath at 40 ° C to 1 mL. The mixture was fixed to 2 mL with water and mixed. Dissolve the solution was filtered through a 0.22 µm organic filter for analysis.

7.3 Instrument reference conditions

7.3.1 Liquid Chromatographic Reference Conditions

A) Column. ZORBAX Eclipse XDB-C18, 1.8 µm, 50 x 2.1 mm (id), or equivalent;

B) mobile phase. methanol 5mmol/L ammonium acetate solution, gradient elution (gradient time schedule in Table 1);

C) Flow rate. 250 µL/min;

D) Column temperature. 30 ° C;

E) Injection volume. 30 µL.

Table 1 Gradient elution conditions for liquid chromatography

Time (min) Flow rate (L/min) 5mmol/L Ammonium acetate solution (%) Methanol (%) 0.00 250 60 40 1.00 250 0 100 7.00 250 0 100 7.50 250 60 40 12.00 250 60 40

7.3.2 Mass spectrometry reference conditions

A) ionization mode. electrospray ionization anion mode (ESI-);

B) Mass spectrometry scanning mode. Multiple reaction monitoring (MRM);

C) Resolution. Unit resolution;

D) See Appendix A for other reference mass spectrometric conditions.

E) Quantitative ion pair. 262.7 > 262.7, qualitative ion pair. 264.7 > 264.7,266.7 > 266.7,268.7 > 268.7.

7.4 Determination

7.4.1 Qualitative determination

The MRM was monitored by 4 pairs of parent ions. Under the same experimental conditions, the retention time of the test substance in the sample was compared with that of the matrix standard solution of the retention time deviation within ± 2.5%; and the sample of each component qualitative ion relative abundance and concentration close to the matrix mixed standard working dissolved the relative abundance of the corresponding qualitative ions in the liquid, the deviation does not exceed the range specified in Table 2, it can be determined that there is a corresponding to be measured.

Table 2 The maximum allowable deviation of relative ion abundance when qualitative confirmation

Relative ion abundance > 50% > 20% to 50% > 10% to 20% <=10% Maximum permissible deviation ± 20% ± 25% ± 30% ± 50%

7.4.2 Quantitative determination

In the best working conditions of the instrument, the standard solution of the mixed working solution of the matrix, with the peak area as the ordinate, the matrix mixed standard working solution Liquid concentration for the abscissa to draw the standard working curve, with the standard working curve of the sample quantitative, the sample solution to be measured the response value should be in the linear range of the instrument determination. The multi-reaction monitoring (MRM) chromatogram of pentachlorophenol reference material is shown in Figure B.1 in Appendix B.

7.5 Blank experiment

In addition to the sample, according to the above determination steps.

10.1 Quantitation limits

The quantification limit for pentachlorophenol was 1.0 µg/kg.

10.2 Recovery rate

When adding levels of 1.00, 2.00, 4.00 µg/kg, the back of pentachlorophenol in pork, liver, kidney, milk, fish, shrimp and crab See Appendix C for yield data.

Appendix A

(Informative)

Reference to mass spectrometry conditions A) Air curtain gas pressure (CUR). 20.00 psi (nitrogen);

B) electrospray voltage (IS). - 4 500 V;

C) ion source temperature (TEM). 550 ° C;

D) atomization gas pressure 35 psi (nitrogen);

E) Auxiliary gas pressure 60 psi (nitrogen);

F) Other mass spectral parameters are shown in Table A1.

Schedule A1 is the primary reference to mass spectrometry parameters Compounds Mother ion (M/z) Ion (M/z) Dwell time (Msec) DP EP CE (EV) CXP Pentachlorophenol 262.7 * 262.7 * 100-75 -10 -23 -5 264.7 264.7 100 266.7 266.7 100 268.7 268.7 100 Note. The ions in Table A1 with * ions for the quantitative ions; for different mass spectrometry equipment, instrument parameters may be different, should be determined before the mass spectrometry parameters to the most good.

Non-commercial statement. The reference mass spectrometry conditions listed in Appendix A are performed on the API4000QTrap LC/MS, where the test instrument model is only listed for reference, does not involve commercial purposes, encourage standard users to try different manufacturers or models of equipment.

Appendix B

(Informative)

Standard multi-reaction monitoring (MRM) chromatograms

Figure B1 Multiple reaction monitoring (MRM) chromatograms of 5.0 µg/L pentachlorophenol

Appendix C

(Informative)

The recovery of pentachlorophenol in different substrates

Table C.1 Recovery of pentachlorophenol in different substrates

Material species

Add concentration

(Mg/kg)

Recovery rate range (%) Pork Pig Pig Pork Kidney Milk Fish Meat Shrimp and Crab Pentachlorophenol 1.0 94.9-109 75.8-104 88.0-108 79.0-98.7 80.0-94.1 79.2-104 66.7-94.1 2.0 82.1-108 81.0-97.5 80.6-103 87.1-98.5 78.4-90.5 77.4-89.6 77.0-99.5 4.0 91.6-96.7 83.4-90.2 74.7-89.3 92.8-99.0 79.8-92.6 76.8-87.9 83.0-97.2 Appendix D (Normative appendix) Laboratory repeatability requirements

Table D.1 Laboratory repeatability requirements

Measured component content Mg/kg Precision <=0.001 36 > 0.01 > 1 14 Appendix E (Normative appendix) Inter-laboratory reproducibility requirements

Table E.1 Inter-laboratory reproducibility requirements

Measured component content Mg/kg Precision <=0.001 54 > 0.01 > 1 19