Arriving at this point, you may ask yourself: Do I need to read this chapter? The answer, although not easy, is yes, and this is why: Validation is the formal proof that a method is suitable for its intended use. In other words, at some point someone has to check whether what’s said is true and whether results of a method actually are what they should be. In the simplest case, “validation” is just a quick test of whether “everything works” under the conditions of your laboratory. There is also a much more involved and formal approach required for quality assurance and for work regulated by Good Manufacturing Practice (GMP). Whether you are actually validating a method or trusting someone else to have done it properly makes no difference. In both cases, you should understand the concept. This chapter is about validation of two kinds of HPTLC methods. Qualitative methods are focused on RF values, sequences, and colors of zones. The identity does not have to be known. Visual or densitometric evaluation of chromatograms is preferably done based on images of the HPTLC plate. As reference, a sample on the same or a different plate can be used. An electronic image can serve as reference as well. Quantitative methods determine amounts of known, well-separated substances. Peak areas/heights of analytes are calculated against calibration curves generated from calibration standards on the same plate. Certain methods based on visual estimation of zone intensities or sizes are often called semiquantitative. Those are not considered here. For validation, they have to be treated either like qualitative or like quantitative methods. Quantitative methods for botanicals are typically validated like those for synthetic drugs following an official guideline. We discuss the specifics that come from the off-line principle of TLC and how the requirements can be efficiently fulfilled. The concept of validating qualitative HPTLC methods is new, and we introduce an approach particularly tailored to the needs of the botanical industry. General Information Official Guidelines and Their Applicability The concept of method validation was born as part of quality assurance. It was driven to become a laborious formalism when the pharmaceutical industry was forced to comply with GMP. Today there are many spinoffs, generally referred to as GxPs,where the “x” can mean “laboratory,” “clinical,” “agricultural,” or other. For each process in a GxP environment, there exists a standard operating procedure (SOP) that describes the process in detail and provides the basis for quality control. Keeping (and maintaining) records of all steps is a central element of GxP. Quality of a product can thus be Whenever chemical analyses are performed under GxP, the employed methods must be valid. It is not surprising that also the process establishing this validity for the task at hand—validation—is clearly regulated. Two important documents by the International Conference on Harmonization (ICH; an organization that works toward harmonization of the regulatory authorities of Europe, Japan, and the United States) provide the framework for validation. The “Note for Guidance on Validation of Analytical Methods Q2(R1)” provides definitions, explains the terminology, and gives details on the methodology.1 Table 6–1 summarizes validation parameters for different types of analytical methods. For definitions of the terms, see Appendix A. A similar but less stringent approach is taken by the International Organization for Standardization (ISO) with its document ISO/IEC/EN 17025: “General Require ments for the Competence of Calibration and Testing Laboratories.”2 The most refined program of method validation is that of the Association of Analytical Communities (AOAC) International. There are actually several levels of validation, but ultimately the program results in “official methods of analysis” that have been investigated in collaborative trials including at least 8 to 10 independent laboratories. Details can be found on the AOAC Web site.4 The new cGMP regulation (“c” for current) for the botanical industry, which is derived from “regular” GMP, creates challenges for the quality control of botanicals, because some of the “quality” that is supposed to be ensured/controlled is not even defined yet. We discussed this in Chapters 1 and 4. Many of the required methods are of qualitative nature and guidelines for validation are not available. For the analyst at the bench, all of this is more a burden than a help. Primarily validation represents a good amount of paperwork that can easily grow out of proportion if left to the “regulators.” Compliance with regulation is one thing, but what counts much more at this level are the following questions: Validation can answer all those questions and should therefore be considered a very useful tool. This is the major point we want to make, and we show in the next sections that with reasonable effort, validation of TLC methods is easily accomplished. It is our goal to help you establish a valid method, not to judge the quality of your material. You and the regulatory authorities have to work out acceptance criteria according to which a sample will pass or fail. Two fundamental papers on the subject have been published: one focuses on quantitative TLC methods of the type covered by the ICH.5 The other paper proposes an approach to validation of qualitative methods important to the botanical industry.6 Table 6–2 summarizes the parameters we consider most useful for different types of methods. The terms in the first column have the same meaning as in Table 6–1, only the precision needs further explanation. According to the ICH guidelines, precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. We will consider it at four levels:
6
Validation
| Type of analytical procedure | Qualitative methods* | Quantitative methods′ |
|---|---|---|
| Specificity | + | + |
| Precision (on the plate) | + | + |
| Repeatability | + | + |
| Intermediate precision | + | + |
| Reproducibility‡ | + | + |
| Robustness§ | + | + |
| Accuracy | – | + |
| Detection limit | – | +¶ |
| Quantitation limit | – | + |
| Linearity | – | + |
| Range | – | + |
*Identification, process control, stability tests, batch-to-batch consistency, mix-up.
†Assay of marker substances.
‡Only for multilaboratory validated methods.
§Optional, but highly recommended.
¶Detection of impurities.
Validation and Method Development
Validation in a regulatory sense is a highly formal process. In practice, it is more an iterative approach and cannot be separated from method development. For example, during validation it is formally proved that the target compound is baseline separated from any other constituent of the sample. This must be achieved during method development. In other words, the fact of baseline separation is already established. Whether the corresponding test is repeated during the actual validation or whether the data of the successful experiment from method development are incorporated into the documentation is a matter of agreement. Some so-called prevalidation experiments are essential and have to be completed before validation “begins.” Even if they are dealt with during method development, they become part of the validation documentation. Generally it is not expected that a method developed for a certain purpose will fail during validation, because if it does, it wasn’t properly developed.
Sometimes an “existing” method, such as an official method of a pharmacopoeia, might need to be (re)validated before it can be performed with the equipment of a certain laboratory. Methods are also (re)validated for use with another product or matrix. These are cases in which a formal validation may fail and method modification/optimization must be performed. We want to emphasize that validation includes the entire analytical process from sampling and sample preparation to analysis and calculation or evaluation of the result. This chapter will only deal with the TLC-relevant aspects.
Let us now look in greater detail at some prevalidation experiments and requirements.
Prevalidation Experiments and Requirements
Stability
The stability of the analyte during analysis must be established. Although true for all analytical methods, it is particularly important for TLC due to the off-line nature of the process. In addition, TLC constitutes an open system. Air, light, fumes, dust, temperature, and other factors can change the sample, which is on the surface of a catalytically very active adsorbent. It is generally not a problem, but the possibility of sample degradation must be investigated.
The stability of analyte in solution and on the plate is assessed in parallel in a simple experiment using a single 10× 10–cm HPTLC plate. An exactly weighed portion of the sample is prepared, and the specified amount of the resulting test solution is applied on track 1 as seen in Table 6–1. The remaining test solution is set aside for a certain time (e.g., 3 hours). After that time, another exactly weighed portion (the same mass) is prepared and applied on tracks 2 and 4. Finally, the first sample is applied again, this time on track 3. The experiment can be modified by adding samples that have been in solution overnight, several days, and so forth. Reasonable times should be selected. For example, if 10 samples are prepared in sequence and the extraction takes 30 minutes, the first sample will be in solution for 5 hours before the last sample is ready. If we assume it takes about 1 minute to apply a sample, the first one will be on the plate for 10 minutes, when the last application is complete. But what if we forgot to start chamber saturation? All samples will be on the plate for another 20 minutes prior to chromatography.
After chromatography, the plate is derivatized if necessary and then evaluated. The sample is stable on the plate and in solution over the specified time, if there is no difference between the individual tracks.
In the qualitative analysis of botanicals, the investigation of stability is especially important because of two reasons: the identity of the separated compounds is widely unknown and the number of substances, which “have to be seen” in the sample, is also unknown. When analyzing chemically defined samples, the situation is much simpler. A single compound should give a single zone in the TLC chromatogram. Additional zones are either “genuine” impurities (present before analysis) or degradation products that are generated during analysis. Boxes 6–1 and 6–2 give more details.
Figure 6–1 Stability of stinging nettle in solution and on the plate. MP: ethyl acetate, methanol, water, formic acid (50: 4:4:2.5). D: Natural Products reagent, UV 366 nm. Track 1, sample on the plate 3 hours; track 2, sample on the plate less than 5 minutes; track 3, sample in solution 3 hours; track 4, sample in solution less than 5 minutes. (Courtesy of K. Koll.)
A 2D chromatogram can be used to investigate the stability of the analyte during chromatography (including the drying step). The sample solution is applied as spot at the lower right corner of a 10×10–cm plate (10 mm from each edge). The plate is developed and dried according to the method. Then the plate is turned 90 degrees to the right and developed a second time with a fresh portion of the developing solvent. If necessary, the plate is derivatized, then evaluated. The sample is stable during chromatography if all zones are located on the diagonal connecting the application position with the intersection of the two solvent fronts (Figure 6–4). Any deviation of zones from that diagonal indicates a decomposition product.a
Box 6–1 Stability of Samples
Let us look at two examples to understand the stability issue better. Figure 6–2 shows chromatograms of three eleutherosides. These compounds can be obtained in high purity and they are stable in methanolic solution at least over 1 week. Only when stored improperly do the eleutherosides degrade significantly. We “know” that each eleutheroside should give only one zone. If two or more zones are seen, something is wrong. How does that knowledge help us with the analysis of a botanical? We do not know how many zones or which to expect in the sample, but the stability tests (on the plate and in solution) will show similarity or differences of the same sample over time. Any additional or missing zone or changes in intensity or color will indicate a problem. As seen in Figure 6–3, Eleuthero is stable at least for 1 week in solution. If the solution is stored improperly over a long time (1 month), changes are apparent. What is the significance of this finding? Whereas a test solution is usually discarded after analysis, it is common practice to use the same reference solution (BRM) over a long time. Whether this solution is still the same as inthe initial test remains to be proved.
Figure 6–2 Stability of three eleutherosides in solution. (A) Fresh. (B) After 1 week in solution. (C) Improperly stored over 1 month. MP: chloroform, methanol, water (70:30:4). D: sulfuric acid reagent, white light. For discussion, see Box 6.1.
Figure 6–3 Stability of Eleuthero in solution. (A)Fresh. (B) After 1 week in solution. (C) Improperly stored over 1 month. MP: chloroform, unknown compounds, it might be acceptable to have some methanol, water (70:30:4). D: sulfuric acid degradation products, as long as it can be established that reagent, white light. For discussion, see Box 6.1.
A decision about the significance of such finding has to be made in each case. For assays, instability cannot be toltolerated. For fingerprints, which generally consist of many unknown compounds, it might be acceptable to have some degradation products, as long as it can be established that their formation is predictable. Box 6.2 gives more details.
aThere are some cases where the “diagonal” is slightly curved instead of a straight line. This can happen if the drying step is incomplete because of highly polar components in the developing solvent.
Figure 6–4 Investigation of stability during chromatography (schematic) with 2D chromatography. The green substance is not stable and shows two degradation products off the diagonal.
There is usually some waiting time between the completion of chromatography and the detection!documentation step. If derivatization is part of the method, there is another waiting period. During these waiting times, the “chromatogram” on the plate can change. To investigate the stability of the chromatographic result, the sample is chromatographed according to the method and derivatized if necessary. The chromatogram is either documented or densitometrically evaluated repeatedly, for example, after 2, 5, 10, 15, 20, and 30 minutes. Figure 6–6 shows three different results. The result of the first method initially changes quickly, but after 20 minutes it becomes stable. It can be concluded either to wait 30 minutes before detection or to take the image immediately after derivatization. The results of the second method do not reach a stable level. The changes affect different compounds to a different degree. The third example represents stable results. No change is seen over 30 minutes.
This test can be used to determine not only proper waiting times but also robustness of a method.
Robustness
Robustness is the ability of a method to tolerate variations of parameters without significant changes in the result. To test robustness, small deliberate changes are made to the method and the resulting effects are evaluated. It is important to plan a robustness test anticipating what can happen in the laboratory when the method is in routine use.
Box 6–2 2D Chromatograms
The stability test by 2D chromatography is based on the following assumptions:
- A stable substance will have the same RF value in both of the two developments. This results in the formation of a straight line connecting the application position with the intersection of the two mobile phase fronts. Stable components of a mixture—including impurities—will all be on this line.
- Any artifact generated during the first development is most likely to behave differently in the second chromatographic step. It will be located aside of the line.
In Fig. 6–5A and B,we see that different mobile phases yield different results in the analysis of a hypericin reference sample that is supposed to be a pure compound. The first mobile phase gives two red zones for hypericin, the second only one. The question is, does the material contain impurities separated only by the first mobile phase? If this were so, the 2D test of the validation should show all zones (main component and impurities) lined up on the diagonal. In the experiment, all zones are located off the diagonal (Fig. 6–5C). They are artifacts and indicate the incompatibility of hypericin with this chromatographic system. The 2D chromatogram obtained with the second mobile phase shows only one red zone (Fig. 6–5D).
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