HPTLC Methodology Validation

CAMAG Laboratory: Method Development in Practice - Validation of HPTLC methods for the identification of botanicals

There is a great need for appropriate testing methods since the current Good Manufacturing Practices (cGMPs) for Dietary Supplements have become effective in the United States. The analysis of botanicals is challenging because plant based materials are complex mixtures of compounds which exhibit natural variability. Identification methods must be specific to distinguish the presence of wrong species.

HPTLC Methodology Validation

HPTLC is a valuable tool for reliable identification because it can provide chromatographic fingerprints that can be visualized and stored as electronic images. To fully take advantage of this unique feature inherent to HPTLC, reproducible results and images must be ensured.

Dr Reich
Dr. E. Reich, Head of the CAMAG laboratory in Muttenz

Reproducibility is improved if suitable instrumentation is used, a standardized HPTLC methodology is implemented, and methods have been developed and validated according to the following concept [1].

The validation process and its elements

Based on a clearly defined analytical goal the methodology validation process starts with the selection/optimization or with the development of a method [2]. Following a series of stability evaluating experiments a validation protocol is elaborated. Data obtained in the validation experiments are evaluated and compared with the acceptance criteria of the validation protocol. If all criteria are met the method can be regarded as valid.

Validation Process
Validation process

Method selection

Criteria for selecting a suitable method are determined by the analytical goal but also include safety factors, time requirements, and simplicity.

Optimization of method

During optimization all TLC methods are converted into HPTLC methods and a standardized methodology is applied. At this point botanical reference materials (BRM) of known adulterants are included to ensure sufficient specificity of the method.


The stability of the analyte on the plate, in solution, and during chromatography as well as the stability of the visualized chromatogram are to be investigated. Stability of the sample during chromatography is investigated by two-dimensional (2D) development. Therefore one portion of the BRM is prepared and applied as spot at the lower right corner of a plate. The plate is developed, dried, turned 90° to the right, and developed a second time. If the sample is stable during chromatography, all components can be detected on the line connecting the application position and the intersection of the two solvent fronts. Spots located off this line indicate the formation of artifacts. Methods that produce artifacts have to be improved. If visualization of the fingerprint requires a derivatization step, the stability of result must be evaluated over time (e.g. up to one hour).

Eleuthero StableAngelica, Not Stable
Investigation of stability during chromatography for Eleuthero (stable, left) and Angelica (not stable, right)

Validation protocol

The validation protocol is a key instrument for structuring, managing and documenting the validation process. The following elements must be included:

  1. Goal of the method to be validated
  2. General acceptance criteria
  3. Personnel
  4. Detailed description of the method
  5. Validation
    1. Material
    2. Stability
    3. Specificity
    4. Precision
      1. Repeatability
      2. Intermediate precision
      3. Reproducibility (optional)
    5. Robustness
  6. Results, releases, signatures

HPTLC Fingerprint


Authenticated samples of the target species, if possible from different origin, and samples of known adulterants are chromatographed on the same plate. Based on electronic images the sequence (number, color, intensity,and position) of the zones in each fingerprint is evaluated by visual comparison. A method is specific, if an authentic sample gives a fingerprint similar to that of the BRM and any adulterated sample fails the similarity test.


The precision of a qualitative analysis (generation of HPTLC fingerprint) can be expressed as precision of the positions of separated zones (hRF-values). We propose to look at three levels: repeatability, intermediate precision, and reproducibility. Precision is acceptable if the variability of the hRF-values of three markers does not exceed DhRF1 across each plate, DhRF2 for repeatability, and DhRF5 for intermediate precision and reproducibility [1].

Note: It is recommended to use the Automatic Developing Chamber 2 that controls the plate activity.


Effects of many experimental parameters can be evaluated already during method development. As the HPTLC result is generally affected by the relative humidity in the laboratory it is important to evaluate the range over which the method performs as expected.

Fingerprint Comparison
Comparison of the fingerprints of Hoodia gordonii obtained at different relative humidity (1: 3% RH, 2: 33% RH, 3: 47% RH, 4: 54% RH, 5: 75% RH)

Further information is available on request from the author.

[1] E. Reich, A. Schibli, A. DeBatt, J AOAC INT 91 (2008) 13
[2] E. Reich, A. Schibli, High-performance thin-layer chromatography for the analysis of medicinal plants, Thieme Medical Publishers Inc., New York, 2007.

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