GB 5009.26-2023National food safety standard - Determination of N-nitrosamines in foods (English PDF)

Foreword

This document was issued on 6 September 2023 by the State Administration for Market Regulation; Standardization Administration of the PRC and takes effect on 6 March 2024.

It is a GB standard without the /T suffix: compliance is mandatory in China.

1 Scope

GB 5009.26-2023 covers the laboratory determination of N-nitrosamines in food, and lays out four independent analytical routes for the same analyte, N-dimethylnitrosamine, so a laboratory can use the instrument it owns. Each route is built the same way: principle, reagents and materials, instruments and apparatuses, analysis steps, expression of results, precision, and a closing clause on detection and quantitation limits. The first and third routes carry the sample through steam distillation, absorb the nitrosamine into dichloromethane, separate it by liquid-liquid extraction and measure it by gas chromatography with tandem mass spectrometry; the second drops distillation for acetonitrile extraction with a dispersive filler cleaning the matrix; the fourth ends at a thermal energy analyser, the detector still in service where no mass spectrometer is available. Three of the four add a deuterated internal standard before work-up, which keeps recovery honest when a fatty meat matrix holds back analyte during distillation. Nitrosamines form in cured and smoked products from nitrite and amines and matter at concentrations no taste panel would notice, so a shipment is accepted or rejected on a number two laboratories have to agree on — hence the repeatability clause and the appendix drawing the steam distillation device. Written for food testing laboratories, meat product processors, and the regulators who arbitrate a failed result.

This standard specifies the method for the determination of N-dimethylnitrosamine

(NDMA) in foods.

This standard applies to the determination of N-dimethylnitrosamine in meat and meat

products, aquatic animals and their products.

Method 1.Steam distillation-gas chromatography-mass spectrometry/mass

spectrometry

2 Principle

This method uses N-dimethylnitrosamine-D6 as the internal standard. The internal

standard is added to the sample; after steam distillation, the N-dimethylnitrosamine in

the sample is absorbed by dichloromethane and separated by liquid-liquid extraction,

which is determined by gas chromatography-mass spectrometry/mass spectrometry

(GC-MS/MS) and quantified by the internal standard method.

3 Reagents and materials

Unless otherwise specified, all the reagents in this method are analytical reagents, and

the water is grade-1 water specified by GB/T 6682.

3.1 Reagents

3.1.1 Dichloromethane (CH2Cl2). chromatographic pure.

3.1.2 Concentrated sulfuric acid (H2SO4). 18.4 mol/L.

3.1.3 Isooctane (C8H18). chromatographic pure.

3.1.4 Anhydrous sodium sulfate (Na2SO4).

3.1.5 Sodium chloride (NaCl). guaranteed reagent.

3.2 Preparation of reagents

Sulfuric acid solution (1+3). Measure 30 mL of concentrated sulfuric acid; slowly pour

it into 90 mL of cold water; stir to fully dissipate heat; mix carefully after cooling.

3.3 Standards

3.3.1 N-dimethylnitrosamine standard solution (C2H6N2O, CAS number. 62-75-9). N-

dimethylnitrosamine methanol solution with a mass concentration of 1 000 ug/mL, or

standard products certified by the nation and awarded a standard material certificate.

3.3.2 N-dimethylnitrosamine-D6 internal standard solution (NDMA-D6, C2D6N2O,

CAS number. 17829-05-9). the mass concentration is 1 000 ug/mL, and the solvent is

methanol.

3.4 Preparation of standard solutions

3.4.1 N-dimethylnitrosamine standard stock solution (100 ug/mL). Accurately draw 1.0

mL of N-dimethylnitrosamine standard solution (1 000 ug/mL); put it in a 10 mL

volumetric flask; use dichloromethane to adjust the volume to the mark; mix well.

Transfer the solution to a brown glass container; store at -18 °C in the dark for 6 months.

3.4.2 N-dimethylnitrosamine standard intermediate solution (1 ug/mL). Accurately

draw 1.0 mL of N-dimethylnitrosamine standard stock solution (100 ug/mL); place it

in a 100 mL volumetric flask; use dichloromethane to adjust the volume to the mark;

mix well. Transfer the solution to a brown glass container; store at -18 °C in the dark

for 3 months.

3.4.3 N-dimethylnitrosamine-D6 internal standard stock solution (100 ug/mL).

Accurately draw 1.0 mL of N-dimethylnitrosamine-D6 standard solution; place it in a

10 mL volumetric flask; use dichloromethane to adjust the volume to the mark; mix

well. Transfer the solution to a brown glass container; store at -18 °C in the dark for 6

months.

3.4.4 N-dimethylnitrosamine-D6 internal standard intermediate solution (1 ug/mL).

Accurately draw 1.0 mL of N-dimethylnitrosamine-D6 internal standard stock solution;

place it in a 100 mL volumetric flask; use dichloromethane to adjust the volume to the

mark; mix well. Transfer the solution to a brown glass container; store at -18 °C in the

dark for 3 months.

3.4.5 N-dimethylnitrosamine standard and internal standard mixed series working

solutions. Respectively and accurately draw 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL and 2.0

mL of N-dimethylnitrosamine standard intermediate solution (1 ug/mL); place them in

the 10 mL volumetric flasks; add 0.4 mL of internal standard intermediate solution (1

ug/mL) to each; use dichloromethane to adjust the volume to the mark; mix well. The

mass concentrations of N-dimethylnitrosamine standard series working solutions are 10

ug/L, 20 ug/L, 50 ug/L, 100 ug/L and 200 ug/L, among which the mass concentrations

of the internal standards are all 40 ug/L. Prepare when necessary.

4 Instruments and apparatuses

4.1 Gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS).

4.2 Rotary evaporator.

4.3 All-glass steam distillation device or fully automatic steam distillation device (see

Figure A.1 in Appendix A).

4.4 Nitrogen-blowing instrument.

4.5 Electric balance. sensitivity of 0.001 g.

4.6 Ice maker.

4.7 Cooling water preparation machine.

4.8 Water bath.

4.9 10 mL graduated test tube.

4.10 100 uL ~ 1 000 uL pipette or graduated pipette.

5 Analysis steps

5.1 Sample pretreatment

5.1.1 Sample preparation

Take the edible part of the representative sample and mash it; prepare it into a uniform

sample; put it into a clean container; seal it and mark it well. Freeze and store the sample

at -18 °C for future use.

5.1.2 Extraction

Accurately weigh 20 g (accurate to 0.01 g) of the sample; add 40 uL of N-

dimethylnitrosamine internal standard intermediate solution (1 ug/mL); add 100 mL of

water and 50 g of sodium chloride into the distillation bottle (tube); mix thoroughly. Air

tightness inspection is required before and after loading the sample. Add 50 mL of

dichloromethane and 0.5 mL of isooctane into a 250 mL Erlenmeyer flask; extend the

outlet of the condenser tube (cooling water temperature controlled at 10 °C ~ 15 °C)

below the dichloromethane liquid surface; place the Erlenmeyer flask in an ice bath;

turn on the distillation device for heating and distillation (reference conditions. steam

power of the automatic distillation device set to 50%); collect 200 mL ~ 250 mL of

condensate (including 50 mL of dichloromethane extract); then, turn off the heating

device and stop distillation.

corresponding N-dimethylnitrosamine mass concentration calculated from the

standard curve, in micrograms per liter (ug/L);

V - the final constant volume of the test solution, in milliliters (mL);

1 000 - conversion coefficient;

m - sample mass, in grams (g).

Retain 2 digits after the decimal point for the calculation results.