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As more herbal drugs associated with Traditional Chinese Medicine enter the European Pharmacopoeia (Ph. Eur.) framework, quality requirements for exporting these materials to Europe are extending beyond botanical identification and the determination of active or analytical markers.<\/p>\n
Safety parameters\u2014including mycotoxins, pesticide residues, elemental impurities and microbiological quality\u2014have become important aspects of herbal drug quality control.<\/p>\n
How does the European Pharmacopoeia address mycotoxin analysis in herbal drugs?<\/p>\n
The answer is:<\/p>\n
Immunoaffinity column clean-up and enrichment, followed by liquid chromatography with fluorescence detection.<\/strong><\/p>\n The relevant Ph. Eur. chapters include:<\/p>\n For laboratories seeking to establish analytical capabilities based on these pharmacopoeial principles, immunoaffinity columns provide an important link between complex herbal extracts and reliable chromatographic analysis.<\/p>\n Herbal drugs are analytically complex matrices.<\/p>\n Roots, rhizomes, stems, leaves, flowers, fruits and seeds may contain large quantities of pigments, polysaccharides, organic acids, volatile constituents, saponins, flavonoids and other secondary metabolites.<\/p>\n If these components are not adequately removed before chromatographic analysis, they may cause:<\/p>\n An immunoaffinity column contains immobilised antibodies that specifically recognise the target mycotoxin.<\/p>\n When a prepared herbal extract passes through the column, the target toxin is selectively captured by the antibodies. A significant proportion of the pigments and other matrix constituents is removed during loading and washing.<\/p>\n Methanol is then used to disrupt the antigen\u2013antibody interaction and elute the purified toxin for HPLC-FLD or a suitably validated LC-MS\/MS method.<\/p>\n The overall workflow can be summarised as:<\/p>\n Extraction \u2192 dilution \u2192 column loading \u2192 selective capture \u2192 washing \u2192 elution \u2192 instrumental analysis<\/strong><\/p>\n For aflatoxin risk control, a VNYA aflatoxin immunoaffinity column can be incorporated into a sample-preparation procedure developed according to the analytical principle of Ph. Eur. chapter 2.8.18.<\/p>\n The chapter focuses on aflatoxin B\u2081. Depending on the applicable requirements, compliance with a limit for the sum of aflatoxins B\u2081, B\u2082, G\u2081 and G\u2082 may also be required.<\/p>\n Mycotoxins may be distributed unevenly within a batch of herbal material. Sampling, homogenisation and grinding can therefore have a direct impact on the final result.<\/p>\n A representative laboratory sample should be thoroughly ground and mixed before the test portion is taken.<\/p>\n The required sample quantity and particle size should be determined according to the currently applicable Ph. Eur. text, the relevant individual monograph and the laboratory\u2019s validated procedure.<\/p>\n The example procedure described in Ph. Eur. chapter 2.8.18 uses a methanol\u2013water mixture to extract aflatoxins from powdered herbal material.<\/p>\n The publicly available reference text describes the following conditions:<\/p>\n Methanol facilitates the extraction of aflatoxins from the herbal matrix. However, a high organic-solvent concentration can reduce antibody activity inside the immunoaffinity column. The extract must therefore be diluted with water before column loading.<\/p>\n In the example workflow, 10.0 mL of the clear filtrate is diluted with 70 mL of water. A 40 mL portion of the diluted solution is then passed through the immunoaffinity column.<\/p>\n When operating a VNYA aflatoxin immunoaffinity column:<\/p>\n The pharmacopoeial example specifies a loading flow rate of approximately 3 mL\/min and states that 5 mL\/min should not be exceeded.<\/p>\n If the sample passes through too quickly, the target analytes may not have sufficient contact time with the immobilised antibodies, potentially reducing recovery.<\/p>\n After sample loading, wash the column with water to remove unbound pigments, salts and other matrix constituents.<\/p>\n The example method uses two 10 mL portions of water, with the washing flow rate not exceeding 5 mL\/min.<\/p>\n Following washing, residual water can be removed using gentle vacuum or by passing air through the column.<\/p>\n Excess water remaining in the column may dilute the methanol eluate and affect elution efficiency or analytical sensitivity.<\/p>\n Apply methanol to the column to disrupt the antibody\u2013analyte interaction and release the retained aflatoxins.<\/p>\n The pharmacopoeial example uses three 0.5 mL portions of methanol, with a defined contact time between the individual elution steps.<\/p>\n The objective is not simply to allow methanol to flow through the column. The solvent must remain in sufficient contact with the immunoaffinity material to release the captured analytes as completely as possible.<\/p>\n Collect the entire eluate, dilute to the specified volume and mix thoroughly. If the solution is not clear, it may be filtered using a validated membrane that does not retain aflatoxins.<\/p>\n The purified eluate is then introduced into the liquid chromatography system.<\/p>\n Ph. Eur. chapter 2.8.18 uses reversed-phase liquid chromatography with fluorescence detection. Post-column derivatisation may be applied to enhance the fluorescence response of aflatoxins B\u2081 and G\u2081.<\/p>\n The published procedure describes several derivatisation options, including:<\/p>\n A calibration curve is used to calculate the concentration of aflatoxin B\u2081 in the herbal drug. Where required, aflatoxins B\u2081, B\u2082, G\u2081 and G\u2082 can also be evaluated as total aflatoxins.<\/p>\n For ochratoxin A risk control, a VNYA ochratoxin A immunoaffinity column can be used to develop a sample-preparation procedure based on the analytical principle of Ph. Eur. chapter 2.8.22.<\/p>\n The publicly available reference text for chapter 2.8.22 describes the following example procedure:<\/p>\n The extraction efficiency of ochratoxin A is influenced by the composition and pH of the extraction solution. These conditions can also affect the subsequent binding of the target analyte to the immunoaffinity antibodies.<\/p>\n According to the example method, 5.0 mL of the clear supernatant is thoroughly mixed with 30 mL of pH 7.4 buffer. The complete solution is then passed through the immunoaffinity column.<\/p>\n Dilution and pH adjustment create suitable conditions for the immunochemical binding reaction.<\/p>\n If the organic-solvent content, ionic strength or pH of the loading solution exceeds the tolerance of the antibodies, column-binding performance may decrease.<\/p>\n A successful extraction procedure must therefore achieve two objectives:<\/p>\n Pass the adjusted sample solution through the column at a stable and controlled flow rate.<\/p>\n The pharmacopoeial example uses a flow rate of approximately 3 mL\/min and specifies that 5 mL\/min should not be exceeded.<\/p>\n During this step, ochratoxin A is selectively captured by the antibodies inside the column, while a large proportion of the unbound matrix components passes through.<\/p>\n After sample loading, wash the column first with pH 7.4 buffer and then with two portions of water.<\/p>\n This process helps remove non-specifically retained substances and water-soluble matrix components.<\/p>\n After washing, remove excess water from the column before methanol elution.<\/p>\n The pharmacopoeial example uses three 0.5 mL portions of methanol to elute the captured ochratoxin A.<\/p>\n The combined eluate is then evaporated to dryness under nitrogen at approximately 40\u00b0C and reconstituted in the specified solution before chromatographic analysis.<\/p>\n Compared with the aflatoxin procedure, the concentration and reconstitution steps increase the analyte concentration and prepare the sample for sensitive liquid chromatographic detection.<\/p>\n Careful control of temperature and evaporation endpoint is important. Excessive heating, prolonged evaporation or sample splashing may result in analyte loss.<\/p>\n Ph. Eur. chapter 2.8.22 uses reversed-phase gradient liquid chromatography with fluorescence detection.<\/p>\n The publicly available reference procedure recommends:<\/p>\n A calibration curve prepared with ochratoxin A standards is used for quantification.<\/p>\n The concentration in the original herbal drug is calculated using the sample mass, extraction volume, aliquot subjected to immunoaffinity clean-up and final reconstitution volume.<\/p>\n A pharmacopoeial procedure should not automatically be assumed to work identically for every herbal drug.<\/p>\n Different herbal materials vary considerably in their pigment content, water absorption, lipid content, acidity, alkalinity and secondary-metabolite composition.<\/p>\n Even when the same immunoaffinity column is used, extraction efficiency, clean-up performance and analyte recovery may differ between matrices.<\/p>\n When applying a VNYA immunoaffinity column to a new herbal matrix, laboratories should evaluate at least:<\/p>\n Chemically different materials\u2014such as liquorice root, ginger, senna pods, Fritillaria<\/em> bulb and spine date seed\u2014should be evaluated separately. Validation data from one herbal matrix should not automatically be considered representative of all herbal drugs.<\/p>\n Instrumentation determines which analytes a laboratory can measure. Sample preparation often determines whether the resulting data will be clean, stable and reliable.<\/p>\n VNYA aflatoxin and ochratoxin A immunoaffinity columns can support the development, optimisation and validation of mycotoxin methods for herbal drugs before HPLC-FLD or LC-MS\/MS analysis.<\/p>\n Their potential value within the workflow includes:<\/p>\n As more TCM-related and other herbal drugs enter the European Pharmacopoeia framework, companies must look beyond the question of which herbal species have been included.<\/p>\n They must also understand the quality-control principles and analytical workflows applied to those materials.<\/p>\n For aflatoxin B\u2081 and ochratoxin A, the technical direction is clear:<\/p>\n The target mycotoxin is selectively purified and enriched from the complex herbal extract using an immunoaffinity column, followed by quantitative liquid chromatographic analysis.<\/strong><\/p>\n VNYA looks forward to working with herbal drug manufacturers, botanical extract producers, pharmaceutical quality-control laboratories and independent testing organisations to develop reliable sample-preparation and mycotoxin-testing solutions for different herbal matrices.<\/p>","protected":false},"excerpt":{"rendered":" As more herbal drugs associated with Traditional Chinese Medicine enter the European Pharmacopoeia (Ph. Eur.) …<\/p>\n\n
Why does the Ph. Eur. method use immunoaffinity columns?<\/h6>\n
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Using a VNYA aflatoxin immunoaffinity column for herbal drug analysis<\/h6>\n
Step 1: Prepare a representative sample<\/h6>\n
Step 2: Extract the aflatoxins<\/h6>\n
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Step 3: Dilute the extract and load the column<\/h6>\n
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Step 4: Wash the immunoaffinity column<\/h6>\n
Step 5: Elute the captured aflatoxins<\/h6>\n
Step 6: Analyse by HPLC-FLD<\/h6>\n
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Using a VNYA ochratoxin A immunoaffinity column for herbal drug analysis<\/h6>\n
Step 1: Extract the herbal sample<\/h6>\n
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Step 2: Adjust the column-loading conditions<\/h6>\n
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Step 3: Load the VNYA ochratoxin A immunoaffinity column<\/h6>\n
Step 4: Wash with buffer and water<\/h6>\n
Step 5: Elute, concentrate and reconstitute<\/h6>\n
Step 6: Analyse by HPLC-FLD<\/h6>\n
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What should be evaluated when applying VNYA IACs to different herbal matrices?<\/h6>\n
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The role of VNYA immunoaffinity columns in the analytical workflow<\/h6>\n
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