ISO 23956:2022

INTERNATIONAL ISO
STANDARD 23956
First edition
2022-02
Traditional Chinese medicine —
Determination of benzopyrene in
processed natural products
Médecine traditionnelle chinoise — Dosage du benzopyrène dans les
produits naturels transformés
Reference number
© ISO 2022
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Test methods . 2
4.1 Reagents and apparatus . 2
4.1.1 Reagents . 2
4.1.2 Apparatus . 2
4.2 Sample preparation . 2
4.3 HPLC-FLD method . 3
4.3.1 Chromatographic condition . 3
4.3.2 Identification . 3
4.3.3 Construction of calibration curve . 3
4.3.4 Quantification . 3
4.4 GC-MS method . 4
4.4.1 Chromatographic conditions . 4
4.4.2 Assay . 4
Annex A (informative) Chemical information of benzopyrene . 6
Annex B (informative) National regulations and limitations of benzopyrene .7
Annex C (informative) The example of HPLC pattern about benzopyrene(s) .8
Annex D (informative) Determination of benzopyrene(s) in wood creosote .9
Bibliography .10
iii
Foreword
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iv
Introduction
The International Agency for Research on Cancer (IARC) classified benzopyrene (C H ) as a carcinogen
20 12
[1]
(Group 1) in 2006. Benzopyrene is produced by the incomplete combustion of carbohydrates, proteins
and fats during high-temperature cooking and processing of raw materials. To prevent hazards induced
by benzopyrene, it is important to determine the benzopyrene content in processed natural products
used in traditional Chinese medicine (e.g. processed Rehmannia root, Cyperus rhizome and mume fruit),
which are processed by the application of high temperatures (200 °C to 600 °C).
Some national pharmacopeias have technical guidelines for the determination of benzopyrene
[2,3]
content . This document describes an analytical method, including the device specifications,
chemical reagents, operation procedures and formulae to determine benzopyrene content, considering
each national pharmacopeia, as well as giving references to other standardized testing methods,
maximum limits and risk assessment.
v
INTERNATIONAL STANDARD ISO 23956:2022(E)
Traditional Chinese medicine — Determination of
benzopyrene in processed natural products
1 Scope
This document specifies the method for the determination of benzopyrene content in processed natural
products.
It is applicable to processed natural products such as processed Rehmannia root, processed Cyperus
rhizome, processed ginseng and processed mume fruit. It is not applicable to the analysis of minerals
used in traditional Chinese medicine.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
benzopyrene
C H
20 12
organic compound of pentacyclic hydrocarbons made of pyrene and phenylene group
Note 1 to entry: The chemical information of benzopyrene is described in Annex A. National regulations and
limitations of benzopyrene are given in Annex B.
3.2
polycyclic aromatic hydrocarbon
PAH
compound that contains two or more fused aromatic rings made of only carbon and hydrogen atoms
[SOURCE: ISO 11338-1:2003, 3.3]
3.3
high-performance liquid chromatography
HPLC
technique in analytical chemistry used to separate, identify and quantify each component in a mixture
Note 1 to entry: High-performance liquid chromatography relies on pumps to pass a pressurized liquid solvent
containing the sample mixture through a column filled with a solid adsorbent material. Each component
in the sample interacts differently with the adsorbent material, resulting in different flow rates for different
components, thereby leading to the separation of components as these flow out of the column.
3.4
gas chromatography
GC
analytical technique used to separate and determine the components of complex mixtures based on
partitioning between gas and stationary phases
[SOURCE: ISO 11504:2017, 3.8, modified — definition revised.]
3.5
fluorescence detector
FLD
device used to measure the parameters of fluorescence, its intensity and wavelength distribution of the
emission spectrum after excitation by a specific wavelength of light
Note 1 to entry: These parameters are used to identify the presence and amounts of specific molecules in a
medium. Modern fluorometers are capable of detecting fluorescent molecule concentrations of as low as one
part per trillion. Fluorescence analysis should be orders of magnitude more sensitive than other techniques.
Applications include monitoring in the fields of chemistry, biochemistry, medicine and environmental sciences.
3.6
mass spectrometry
MS
analytical technique that ionizes chemical species and sorts of ions based on their mass-to-charge
ratios
4 Test methods
4.1 Reagents and apparatus
4.1.1 Reagents
4.1.1.1 Benzo[a]pyrene (BaP) standard [high-performance liquid chromatography (HPLC) grade].
4.1.1.2 3-Methylcholanthrene, HPLC grade as an internal standard.
4.1.1.3 n-Hexane, ethanol, dichloromethane and acetonitrile (HPLC grade).
4.1.1.4 Water (or equivalent) referred to as deionized water (DIW).
4.1.2 Apparatus
4.1.2.1 Rotary vacuum evaporator.
4.1.2.2 Visible nitrogen sample concentrator.
4.1.2.3 Solid phase extraction (SPE) tube vacuum manifolds.
4.1.2.4 Rotor-stator homogenizer (mechanical homogenizer).
4.1.2.5 Gas chromatography (GC) or mass spectrometry (MS).
4.2 Sample preparation
a) Add 5 g of sample powder to 100 ml water and then extract for 90 min using an ultrasonic bath
with a cooling system.
b) Add 100 ml of hexane with 1 ml of internal standard (50 μg/kg or 50 μg/l: purity ≥ 96 % for analysis
grade) to a mixture by homogenization for 5 min, and extract the samples mixtures in an ultrasonic
bath for 30 min.
c) Transfer all mixtures into a separating funnel and collect the supernatants.
d) Extract the residue twice with 50 ml of hexane as detailed in step b).
e) Add water (50 ml; ≥ 18,2 MΩ) to the combined hexane fraction for washing and filter the hexane
fraction using anhydrous sodium sulfate. Next, concentrate the hexane fraction at 45 °C using a
rotary vacuum evaporator to approximately 2 ml.
f) Wash a Florisil SPE cartridge with 10 ml of dichloromethane for activation. Clean the cartridge
using 20 ml of hexane to remove dichloromethane.
g) Transfer directly 2 ml of the extracts into the activated Florisil SPE cartridge and elute the extracts
with 20 ml of hexane and dichloromethane mixture (volume ratio of 3:1).
h) Concentrate the eluent using a visible nitrogen sample concentrator at 35 °C. Dissolve the residue
in 1 ml of acetonitrile and filter the eluent through a 0,45-μm polytetrafluoroethylene (PTFE)
membrane filter.
i) Inject 10 μl of the final eluent into the fluorescence detector (FLD) for BaP analysis.
4.3 HPLC-FLD method
4.3.1 Chromatographic condition
— Column: supelcosil LC-PAH (4,6 × 250 mm, 5 μm) or equivalent.
— Column temperature: 35 °C.
— Detector: excitation at 294 nm and emission at 404 nm.
— Mobile phase: acetonitrile and water mixture (volume ratio of 8:2)
— Flow rate: 1 ml/min.
4.3.2 Identification
The retention time of the peak should be consistent with the retention time of the standard
within ± 0,2 % under identical analysis conditions.
4.3.3 Construction of calibration curve
a) Dissolve standard (BaP) and internal standard (3-methylcholanthrene) in acetonitrile (1 μg/ml
each). Store this stock solution at 4 °C. Replace the stock solution every six months.
b) Prepare standard solutions with different concentrations of 3 ng/ml, 5 ng/ml, 1
...