GB 5009.31-2025National food safety standard - Determination of p-hydroxybenzoic acid esters in foods (English PDF)

Foreword

This document was issued on 16 March 2025 by the State Administration for Market Regulation; Standardization Administration of the PRC and takes effect on 16 September 2025.

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

1 Scope

GB 5009.31-2025 is the Chinese national food safety standard for determining parabens in food. The p-hydroxybenzoic acid esters are permitted preservatives with maximum use levels, and the reason they are watched more closely than most is that the longer-chain members have shown weak oestrogenic activity, so the several esters have to be determined separately rather than as a group. The standard gives the methods in sequence - gas chromatography, liquid chromatography and liquid chromatography-tandem mass spectrometry - each with its principle, reagents and materials, instruments and equipment, analytical steps, expression of results, precision and notes, with annexes giving the chromatograms of the standard solutions and the multiple reaction monitoring conditions. It takes effect on 16 September 2025.

This Standard specifies the determination method for p-hydroxybenzoic acid esters (methyl

paraben, ethyl paraben, propyl paraben, isopropyl paraben, butyl paraben, isobutyl paraben and

heptyl 4-hydroxybenzoate) in foods.

The first method of this Standard, gas chromatography, is applicable to the determination of p-

hydroxybenzoic acid esters in foods (except heat-coagulated egg products, brewed sauce and

seasoning sauce).

The second method of this Standard, high performance liquid chromatography, and the third

method, liquid chromatography-tandem mass spectrometry, are applicable to the determination

of p-hydroxybenzoic acid esters in foods.

Method I - Gas Chromatography

2 Principle

The specimen is extracted with methanol-water, purified and enriched by a mixed strong anion

exchange solid phase extraction column, and concentrated and then, determined by a gas

chromatograph equipped with a hydrogen flame ionization detector, and quantified by the

external standard method.

3 Reagents and Materials

Unless otherwise specified, all reagents used in this Method are analytically pure, and the water

is Grade-1 water specified in GB/T 6682.

3.1 Reagents

3.1.1 Methanol (CH3OH).

3.1.2 Formic acid (HCOOH).

3.1.3 Concentrated ammonia water (NH3  H2O). 25% ~ 28%.

3.2 Preparation of Reagents

3.2.1 Methanol-water solution (2 + 8). measure-take 100 mL of methanol and 400 mL of water,

solutions is 2.0 g/mL, 5.0 g/mL, 10 g/mL, 20 g/mL, 50 g/mL and 100 g/mL.

3.5 Materials

3.5.1 Organic phase microporous filter membrane. with a pore size of 0.22 m.

3.5.2 Mixed strong anion exchange solid phase extraction column. 500 mg/6 mL, or other

columns with equivalent performance.

4 Instruments and Equipment

4.1 Gas chromatograph. equipped with a hydrogen flame ionization detector (FID).

4.2 Electronic balance. respectively with a division value of 0.001 g and 0.0001 g.

4.3 Vortex mixer.

4.4 High-speed centrifuge. maximum speed is not less than 6,000 r/min.

4.5 Ultrasonic oscillator.

4.6 Nitrogen blowing instrument.

4.7 Homogenizer.

4.8 Grinder.

5 Analytical Steps

5.1 Preparation of Specimens

Liquid samples are mixed, semi-solid samples are homogenized with a homogenizer, and solid

samples are thoroughly grinded with a grinder and evenly stirred. The prepared specimens are

placed in a clean specimen container, sealed and labeled. Liquid specimens are stored at 4 C,

and other specimens are stored at 18 C.

5.2 Processing of Specimens

5.2.1 Extraction

5.2.1.1 Fresh fruits, fresh vegetables, baked food fillings and surface batter, vinegar, soy

sauce, liquid compound seasoning, etc.

Weigh-take 5 g (accurate to 0.01 g) of sample into a 50 mL graduated centrifuge tube, add 5

mL of water, conduct vortex for 3 min, add 10 mL of methanol, conduct vortex for 3 min,

conduct ultrasound for 20 min, and at 6,000 r/min, centrifuge it for 3 min. Take all the

supernatant into another 50 mL graduated centrifuge tube, add 10 mL of methanol-water

solution (2 + 8) to the residue, repeat the extraction once, combine the two extracting solutions,

add water to 40 mL, at 6,000 r/min, centrifuge it for 3 min, take all the supernatant for

purification.

5.2.1.2 Fruit and vegetable juice, carbonated beverages, flavored beverages, etc.

Weigh-take 5 g (accurate to 0.01 g) of specimen into a 50 mL graduated centrifuge tube

(carbonated beverages need to be ultrasonically degassed in an ultrasonic cleaner for 10 minutes

before weighing), add 30 mL of methanol-water solution (3 + 7), conduct vortex for 3 minutes,

and conduct ultrasound for 20 minutes. Then, add methanol-water solution (3 + 7) and reach

40 mL, at 6,000 r/min, centrifuge it for 3 minutes, filter the supernatant, take the filtrate for

purification.

5.2.1.3 Jams, etc.

Weigh-take 5 g (accurate to 0.01 g) of sample into a 50 mL graduated centrifuge tube, add 5

mL of concentrated ammonia water, conduct vortex for 3 min, let it stand for 1 h; add 10 mL of

methanol, conduct vortex for 3 min, conduct ultrasound for 20 min, and at 6,000 r/min,

centrifuge it for 3 min. Take all the supernatant into another 50 mL graduated centrifuge tube.

Add 10 mL of methanol-water solution (2 + 8) to the residue, conduct vortex for 3 min, conduct

ultrasound for 20 min, and at 6,000 r/min, centrifuge it for 3 min. Combine the two extracting

solutions, add water to 40 mL, at 6,000 r/min, centrifuge it for 3 min, and take all the supernatant

for purification.

5.2.2 Purification

Respectively use 5 mL of methanol and 5 mL of water to activate the solid phase extraction

column, transfer the solution to be purified into the activated solid phase extraction column,

successively use 5 mL of water and 5 mL of methanol-water solution (3 + 7) to rinse it and use

6 mL of methanol to elute it. Use 6 mL of 2% formic acid methanol solution to elute the jam

samples, collect the eluate, in a 40 C water bath, use nitrogen to blow it dry. Use 1.0 mL of

methanol to re-dissolve it and filter the membrane for the determination by gas chromatograph.

5.3 Reference Conditions of Instruments

5.3.1 Chromatographic column. weak polar quartz capillary column, column stationary liquid

is (5%) phenyl-(95%) methyl polysiloxane, 30 m  0.25 mm (inner diameter), 0.25 m (film

thickness), or equivalent column.

5.3.2 Inlet temperature. 280 C.

5.3.3 Carrier gas. nitrogen, purity  99.999%; flow rate 1 mL/min, tail blowing 30 mL/min (the

flow rate of carrier gas can be adjusted in accordance with the instrument conditions).

5.3.4 Injection volume. 1 L.

5.3.5 Injection mode. split (split ratio can be adjusted in accordance with chromatographic

conditions) or splitless injection.

5.3.6 Temperature programming. initial temperature 70 C, hold for 2 min; at 20 C/min, raise

to 300 C; hold for 2 min.

5.3.7 Detector. hydrogen flame ionization detector (FID), temperature 300 C.

5.3.8 Gas. hydrogen 35 mL/min; air 400 mL/min (the flow rate of hydrogen and air can be

adjusted in accordance with the instrument conditions).

5.4 Drawing of Standard Curve

Inject the mixed standard working solution of p-hydroxybenzoic acid esters into the gas

chromatograph in order of concentration from low to high. With the mass concentration of the

standard solution as the horizontal axis and the peak area as the vertical axis to draw the

standard curve. See Figure A.1 of Appendix A for the gas chromatogram of p-hydroxybenzoic

acid esters.

5.5 Determination of Specimen Solution

5.5.1 Qualitative determination

In accordance with the conditions described in 5.3, respectively inject the mixed standard

working solution of p-hydroxybenzoic acid esters and the specimen solution into the gas

chromatograph and use the retention time for qualitative determination. Compare the retention

time of the chromatographic peak in the specimen solution with the retention time of p-

hydroxybenzoic acid esters in the mixed standard working solution, and the allowable deviation

shall be less than 0.5%.

5.5.2 Quantitative determination

In accordance with the conditions described in 5.3, inject the specimen solution into the gas

chromatograph to obtain the peak area of the compound under test. In accordance with the

standard curve, obtain the mass concentration of the compound under test in the specimen

solution. The response value of the compound under test in the specimen solution shall be

within the linear range of the standard curve. If the response value exceeds the linear range of

the standard curve, it shall be diluted and re-analyzed.

5.6 Blank Test

Except for not adding specimen, handle it at the same time as the specimen in accordance with

the steps in 5.2.

6 Expression of Analysis Results

The content of p-hydroxybenzoic acid esters in the specimen (in terms of 4-hydroxybenzoic

acid) is calculated in accordance with Formula (1).

Where,

Xi---the content of p-hydroxybenzoic acid esters in the specimen (in terms of 4-hydroxybenzoic

acid), expressed in (mg/kg);

rho---the mass concentration of p-hydroxybenzoic acid esters in the injection solution calculated

from the standard curve, expressed in (g/mL);

rho0---the mass concentration of p-hydroxybenzoic acid esters in the blank test solution calculated

from the standard curve, expressed in (g/mL);

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

m---the mass of the specimen weighed, expressed in (g);

1,000---the unit conversion factor;

fi---the conversion factor of p-hydroxybenzoic acid esters to 4-hydroxybenzoic acid.

Illustration.

0.9078---the conversion factor of methyl paraben to 4-hydroxybenzoic acid;

0.8312---the conversion factor of ethyl paraben to 4-hydroxybenzoic acid;

0.7665---the conversion factor of propyl paraben and isopropyl paraben to 4-hydroxybenzoic

acid;

0.7111---the conversion factor of butyl paraben and isobutyl paraben to 4-hydroxybenzoic acid;

0.5845---the conversion factor of heptyl 4-hydroxybenzoate to 4-hydroxybenzoic acid.

The calculation result shall retain 3 significant figures.

7 Precision

The absolute difference between two independent determination results obtained under

repeatability conditions must not exceed 10% of the arithmetic mean.

8 Others

When the sample weight is 5 g and the constant volume is 1.0 mL, the detection limits of the

seven p-hydroxybenzoic acid esters are all 0.6 mg/kg and the quantification limits are all 2

mg/kg.

Method II - High Performance Liquid Chromatography

9 Principle

The specimen is extracted with methanol-water, purified and enriched by a mixed strong anion

exchange solid phase extraction column, and determined by a high performance liquid

chromatograph equipped with a photodiode array detector or a UV detector, and quantified by

the external standard method.

10 Reagents and Materials

Unless otherwise specified, all reagents used in this Method are analytically pure, and the water

is Grade-1 water specified in GB/T 6682.

10.1 Reagents

10.1.1 Methanol (CH3OH). chromatographically pure.

10.1.2 Phosphoric acid (H3PO4).

10.1.3 Formic acid (HCOOH).

10.1.4 Concentrated ammonia water (NH3  H2O). 25% ~ 28%.

10.1.5 Acetonitrile (CH3CN). chromatographically pure.

10.2 Preparation of Reagents

10.2.1 Methanol-water solution (3 + 7). same as 3.2.2.

10.2.2 Methanol-water solution (2 + 8). same as 3.2.1.

10.2.3 0.1% phosphoric acid aqueous solution. measure-take 1.0 mL of phosphoric acid, add

water to reach a constant volume of 1,000 mL, mix it well and reserve for later use.

10.2.4 2% formic acid methanol solution. same as 3.2.3.

10.3 Standard Substances

Same as 3.3.

10.4 Preparation of Standard Solutions

10.4.1 Standard stock solution of p-hydroxybenzoic acid esters (1.00 mg/mL). same as 3.4.1.

10.4.2 Mixed standard intermediate solution of p-hydroxybenzoic acid esters (100 g/mL).

same as 3.4.2.

10.4.3 Mixed standard working solution of p-hydroxybenzoic acid esters. respectively pipette

0.02 mL, 0.05 mL, 0.10 mL, 0.20 mL, 0.50 mL, 1.0 mL, 2.0 mL and 5.0 mL of the mixed

standard intermediate solution of p-hydroxybenzoic acid esters into a 10 mL volumetric flask,

and use methanol-water solution (3 + 7) reach a constant volume to obtain mixed standard

working solution respectively with a mass concentration of 0.2 g/mL, 0.5 g/mL, 1.0 g/mL,

2.0 g/mL, 5.0 g/mL, 10 g/mL, 20 g/mL and 50 g/mL. Prepare it immediately before use.

10.5 Materials

Same as 3.5.

11 Instruments and Equipment

11.1 High performance liquid chromatography analyzer. equipped with a photodiode array

detector or a UV detector.

11.2 Electronic balance. respectively with a division value of 0.001 g and 0.0001 g.

11.3 Vortex mixer.

11.4 High-speed centrifuge. maximum speed is not less than 6,000 r/min.

11.5 Ultrasonic oscillator.

11.6 Homogenizer.

11.7 Grinder.

12 Analytical Steps

12.1 Preparation of Specimens

Process in accordance with 5.1.

12.2 Processing of Specimens

12.2.1 Extraction

12.2.1.1 Fresh fruits, fresh vegetables, baked food fillings and surface batter, vinegar, soy

sauce, brewed sauce, seasoning sauce and liquid compound seasoning, etc.

Process in accordance with 5.2.1.1.

12.2.1.2 Fruit and vegetable juice, carbonated beverages and flavored beverages, etc.

......

Appendix A Gas Chromatogram of Standard Solution of P-hydroxybenzoic Acid Esters

... 22

Appendix B Liquid Chromatogram of Standard Solution of P-hydroxybenzoic Acid

Esters... 23

Appendix C Liquid Chromatography-mass Spectrometry Multiple Reaction

Monitoring (MRM) Diagram of Standard Solution of P-hydroxybenzoic Acid Esters

... 24

National Food Safety Standard - Determination of P-

hydroxybenzoic Acid Esters in Foods