ISO 23956:2022 pdf – 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 20 H 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 18.104.22.168 Benzo[a]pyrene (BaP) standard [high-performance liquid chromatography (HPLC) grade]. 22.214.171.124 3-Methylcholanthrene, HPLC grade as an internal standard. 126.96.36.199 n-Hexane, ethanol, dichloromethane and acetonitrile (HPLC grade). 188.8.131.52 Water (or equivalent) referred to as deionized water (DIW). 4.1.2 Apparatus 184.108.40.206 Rotary vacuum evaporator. 220.127.116.11 Visible nitrogen sample concentrator. 18.104.22.168 Solid phase extraction (SPE) tube vacuum manifolds. 22.214.171.124 Rotor-stator homogenizer (mechanical homogenizer). 126.96.36.199 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.