The Science Behind an HPTLC Test
In the world of dietary supplements and botanical materials, accurate identity testing is essential for product safety, regulatory compliance, and consumer confidence. One of the most effective tools in this area is Thin Layer Chromatography (TLC), particularly in its high-performance form (HPTLC). This white paper provides an overview of HPTLC, compendial HP methods, selecting a fit-for-purpose method, the acceptance criteria, and the advantages and limitations of compendial methods.
What is HPTLC?
High Performance Thin Layer Chromatography (HPTLC) is a planar chromatographic technique used to compare botanical samples against reference standards. Through comparing the banding patterns of a sample against reference standards, HPTLC enables researchers to confirm the identity of a Plant, Fungi or Algae material.
What is a compendial TLC method?
A compendial HPTLC method is a standardized method that is validated for a specific source of material within a defined scope. The methods are published in authoritative sources such as the United States Pharmacopeia (USP), Food Chemicals Codex (FCC), or European Pharmacopoeia (EP or PhEur).
These methods are validated for specific botanicals or extracts and include detailed acceptance criteria to guide evaluation. Using compendial methods helps ensure regulatory compliance, reproducibility, and transparency in testing.
For example, the method for Melissa leaf (lemon balm) dry extract specifies solvent types and concentrations that must be adhered to for proper testing. Specifically, hot water or hydroalcoholic solvent is used in the production of this type of extract. To meet the scope for this pharmacopeial method, the sample would need to fall under these parameters to ensure that the testing is fit for purpose.
Selecting a fit for purpose method
To choose the right testing method, certain details must be provided:
- Genus species (Latin binomial)
- Plant, Fungi, or Algae part
- Extraction process, solvents, and standardization steps
- Specification or Certificate of Analysis (CoA)
These details ensure the scope of the method aligns with the sample’s characteristics.
Acceptance criteria and evaluation
Every compendial method includes clearly defined acceptance criteria. A System Suitability Test (SST) confirms the chromatographic system's accuracy and performance for specific analysis. HPTLC data is analyzed based on Retention Factor (Rf) values, color, and intensity of bands. Results are documented as “conforms” or “does not conform” on the CoA, along with monograph references, acceptance criteria, observations and plate images.
Examples of chromatography
The following chromatographic examples illustrate the diverse range of botanical materials and product types analyzed using HPTLC. As previously discussed, critical factors such as the plant part, extraction processes, and solvents play a vital role in determining accurate final results
Melissa leaf extract
The client required identification testing for lemon balm extract, produced using a 1:1 ethanol-to-water ratio. The selected method followed the European Pharmacopoeia Melissa Leaf Extract – Identification guidelines. The sample chromatogram bands aligned with the acceptance criteria for the test solution, confirming the result as “conforms.”
Monk fruit extract
In this scenario, the client was looking to confirm the identity of their monk fruit extract. The method Eurofins used was FCC Monk Fruit Extract - Identification: Thin-Layer Chromatography.
- Lane 1: USP Mogroside V RS (Standard Solution A)
- Lane 2: USP Monk Fruit Extract (Standard Solution B)
- Lane 3: Sample
In the above image, the sample (lane 3) corresponds with Standard Solution A (lane 1), which means it meets that portion of the acceptance criteria. The sample solution also corresponds with the placement and color of Standard Solution B (lane 2). Therefore, the sample matched reference standards in the FCC method and received a “conforms” result.
Aloe ferox leaf juice
The client inquired about testing for Aloe ferox leaf juice. To address this, Eurofins selected the European Pharmacopoeia 11.0 Aloes, Cape – Identification method. The expected fluorescent bands for the test solution were present in the sample, confirming the result as “conforms.”
Reishi mushroom powder
Here, the client inquired regarding a compendial method for Reishi mushroom powder. The method selected was USP Ganoderma Lucidum Fruiting Body Powder - Identification A TLC.
- Lane 2: Ganoderic acid A (Standard Solution A)
- Lane 3: Ergosterol (Standard Solution B)
- Lane 4: USP Ganoderma Lucidum Fruiting Body Powdered Extract RS (Standard Solution C)
- Lanes 5-6: Sample
While the sample showed the presence of some bands matching the USP Ganoderma Lucidum Fruiting Body Powdered Extract RS, it lacked the required ergosterol band. The result was “does not conform.” It was later found that the material was an extract rather than crude fruiting body powder, making it out of scope for this compendial method.
White willow bark powder
For this example, the client wanted to confirm that their White Willow Bark powder meets USP standards for identity. The method used was USP Salix Species Bark Powder - Identification A, HPTLC for Articles of Botanical Origin.
- Lane 2: Salicin (Standard Solution A)
- Lane 3: USP Salix Species Bark Dry Extract (Standard Solution B)
- Lane 4: USP Salix Species Bark Dry Extract (*Hydrolyzed)
- Lane 5: Sample Solution A
- Lane 6: Sample Solution B (*Hydrolyzed)
The two sample solutions for this test were prepared differently with Sample Solution A (lane 5) dissolved in solution while Sample Solution B (lane 6) was prepared with hydrolysis. The Salicin band in lane 5 should be of lesser intensity than the Salicin Standard Solution A (lane 2). Therefore, it doesn’t meet the acceptance criteria, leading to a “does not conform” result. The test didn’t show any effects of the hydrolysis preparation versus dissolving in solution.
After a discussion with the client, it was revealed that the material processing didn’t fit the scope of the USP method used.
Astaxanthin from Haematococcus pluvialis
The client wanted to confirm their Astaxanthin product was made from Haematococcus pluvialis algae. In this situation, Eurofins used the USP monograph for Haematococcus Pluvialis Powder.
- Lane 4: USP Astaxanthin Esters from Haematococcus pluvialis RS (Standard Solution)
- Lane 5: Sample
The acceptance criteria and system suitability for the method indicate that there should be five bands - two in pairs, with the lowest RF band being the most intense. The sample failed to display the necessary bands, and identity could not be confirmed. The result was “does not conform.”
After an investigation of the results, with consideration to what the client's goal was, the ultimate outcome of this was out of specification for Haematococcus pluvialis algae. The identity could not be confirmed based on this USP method.
Next steps for non-conforming results
When a test result is “does not conform”, the technical experts at Eurofins Botanicals assists in reviewing sample information, verifying method applicability and identifying potential causes. Often, follow-up includes supplier inquiries or further testing, possibly leading to method development tailored to the sample’s unique attributes. Transparency is key, and clients are advised to be as informed as possible about their materials to ensure accurate, compliant results.
Advantages of compendial HPTLC testing
Compendial HPTLC methods provide a reliable, standardized framework for identity testing, ensuring compliance, accuracy, and transparency in analytical results:
- The data that's generated using a standardized pharmacopeial method is readily accepted by quality and regulatory auditors, which helps with compliance.
- The monograph methods have been peer reviewed by expert committees.
- The clear acceptance criteria that are a component of every monograph method provide transparency in how the samples are evaluated. Information from every test performed is provided on the CoA.
- The scope of the method is a way to ensure there's fit for purpose testing performed on every sample.
- The standardization that comes with a pharmacopeial method ensures that, from day to day, from different analysts, the results will be reproducible and accurate according to the criteria and validation of that method.
Limitations of compendial testing
While the scope is an advantage of compendial ID testing, it can also be a limiting factor. It may not account for variability across every region, harvest, season, and different types of extract. The scope is only as broad as the validation data used to develop the method, and the materials, reference standards and inputs that went into developing and validating the method.
Conclusion
Compendial HPTLC methods serve as a reliable foundation for identity testing, providing standardized, regulator-approved protocols that ensure consistency and compliance. However, their predefined scope may require manufacturers to pursue additional method development and expert collaboration to address complex analytical challenges. By leveraging advanced data insights and strategic expertise, companies can refine their approaches, enhance accuracy, and overcome limitations. Ultimately, integrating rigorous testing with tailored solutions empowers manufacturers to uphold the highest standards of product quality and safety—driving innovation while maintaining regulatory confidence.
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Additional Resources:
Botanical Reference Materials: A Critical Guide for Supplement Quality and Regulatory Compliance
Adulteration in Botanicals: A Growing Concern for Purity and Safety
