Welcome to the video question and answer from our webinar Comprehensive HPTLC Fingerprinting for Quality Control of Herbal Drugs. In this video, our HPTLC expert Dr. Melanie Broszat answers questions we didn’t get to during the webinar. Below the video, you will find a transcript.
If the SST has passed, selected tracks can be exported to a comparison file in visionCATS. This file can contain HPTLC fingerprints from different plates which means different analysis files. I have done a screen record to show you how it works. Here you can see the user interface of the visionCATS software. All the steps have been performed and here in the track assignment table it has been defined which tracks are used as references, samples, and for system suitability test. In the data view, we can have a look at our obtained results, here you can get the HPTLC fingerprint in the different detection modes and you can see the fingerprints of the SST on the first, middle, and last track. If we then go to the SST tab and if we have defined in our method the acceptance criteria, then the software can check if the reference substances are in the expected range. Then the software will give the status passed or failed – either the SST is within the window or it's not. If the SST has passed, we can go to the Data tab and select our tracks of interest / sample tracks that we want to compare to already existing data. Let's say this fingerprint is of interest and this as well, then we can export them in one detection mode or in several detection modes, both for images and profiles. These are the images after derivatization or if we click here then we can also export all the images that have been captured. Then visionCATS will open those selected tracks and it will add them to the end of the existing comparison file. Here you can see the two selected tracks now and the obtained tracks can be moved and compared to the different fingerprints, in order to compare similarities and differences. This shows the example of Equisetum - there is the good one and next to it the related species that should not be present, which can be seen by the green zone in the bottom part of the chromatogram. This zone present in E. palustre and not in the good one - in the E. arvense.
To establish specifications for a herbal drug, the selected sample population has to be sufficiently large, properly authenticated, and representative for the desired quality. What does this mean? We should first collect as many representatives of a certain plant species as possible from different sources. At least 10 samples should be analyzed and then, if they are all showing a typical pattern, we can also generate a pooled sample that contains all the zones, which can be present or not, to also display the natural variability. We create a combined HPTLC fingerprint, which represents the whole natural variability of a certain plant species. All zones that can be present should be seen within this reference fingerprint. It's not always easy to get many samples of known identity or with a certificate, but the more you have, the more you can get, the more you can ensure that your fingerprint is representative and displays the species the right way. There can also be used herbal reference materials and botanical reference materials that can be purchased from companies. What you can also do, is that you compare with HPTLC fingerprints from the global HPTLC Atlas of the HPTLC Association. Here is an example from the HPTLC Atlas showing the results for different ginger species. There exist different Ginger species from different geographical regions and with this fingerprint method shown by the association you get typical patterns, so typical fingerprints, that you can use to compare your samples with.
The CCBS database provides a rapid search for applications in the field of TLC/HPTLC. The best what you can do if you are looking for mobile phases is doing a literature search. A good source for information is the CAMAG CCBS database. It allows a rapid search for applications in the field of TLC and HPTLC. If you enter your search item here, then you get in this case five entries and the first one gives you the two analytes of interest, separated on a silica gel plate with this mobile phase: toluene - ethyl-acetate - glacial acetic acid. Enter the name and you directly have your answer.
Yes, if the whole plant is used as drug then the fingerprint method will be based on this, but if it's a certain plant part like the ginger roots or another drug that is just a plant part, just the fruits for example, then this is analyzed. In this example the drug is only the leaves and you can also see that the male leaf and female leaf show some differences. The representative fingerprint can be like a merged or pooled fingerprint representing the two. On the right side, you can see the aerial part, the flower, the stem and so on and only the aerial part has the fingerprint in accordance to the drug.
When we are talking about non-target HPTLC then I think about hyphenation with biological tests. A good review was written and published this year by Gerda Morlock. It shows the coupling possibilities with enzymatic tests, with biological tests like bacteria, algae, fungi, adhesive cells, and also non-target chemical tests for example the DPPH to screen for accident antioxidant activities.
Please see the review of Gerda Morlock and have a look at the CCBS literature database. You can also find videos on YouTube. One example demonstrates the planar Yeast Estrogen Screen, a non-target screening for estrogen active compounds in different samples. They are showing it in the video in beer samples, but this can be also applied to herbal samples. For selected assays of interest you can also get further information via email. Please send your request to firstname.lastname@example.org.
This is how chromatography should not take place. The analyte alone and the analyte within the sample should have the same RF values at the end. The matrix should not be disturbing. If the target analyte within the sample matrix is not migrating then a sample preparation step is needed. There are also some possibilities to eliminate matrix on the HPTLC plate by a so-called planar solid phase extraction. What does it mean? If there is disturbing apolar matrix, then we can apply our sample with a lipophilic matrix and get rid of it by a first development up to the front with very apolar solvent. We are usually using heptane and not hexane anymore. Then the apolar matrix is sitting up to here. Our analyte and the other sample components are remaining on the application position. After drying we can then do a second development with our mobile phase. The same is also working for removal of polar matrices. We apply the sample, we develop first with a very polar solvent, dry the plate and then we develop with a less polar mobile phase. We can also apply our sample as a rectangle with the ATS 4 and first do a focusing to get a band instead of an area, and then develop with our mobile phase. This helps to spread your sample on a on a wider surface and then by focusing we can better get narrow bands and then also better separate our analyte out of the matrix.
As I work at CAMAG, I am not neutral. Let's give you some facts to answer this question. There is an increasing demand for 21 CFR Part 11. CAMAG’s visionCATS software has this option. Furthermore CAMAG is the only provider for handling all steps with one software. The visionCATS software controls the instruments and handles the whole TLC and HPTLC procedure from A to Z, ensuring full traceability for cGMP regulated environments.
This is something you cannot answer in general. Depending on the regulations, you have different requirements and I would recommend to look what is specified for the LC methods and also what is specified for the different analytical tests. There are different criteria for limit tests and for quantification of a content. But I can tell you about usual CVs that we obtain, when we are not doing a derivatization step. Derivatization is an additional step, which will increase the final error. Whenever possible a quantification should be done prior to derivatization and in the best case by scanning densitometry in UV- and/or VIS-range. What also works quite well is the measurement of the native fluorescence with the TLC Visualizer prior to derivatization. With the scanning densitometry, and in some cases also with the TLC Visualizer, you can get CVs smaller than 2% for validated methods.
There I can only give you our internal recommendation as there is no official statement from any regulation. We recommend for the same day and within the same lab a ∆ RF of 0.02 and for different days and between different labs ∆ RF of 0.05.
In my opinion, the reason is, that HPTLC is not known enough and therefore it is not recognized by the regulations yet. I think, that HPTLC has to be taught at any university worldwide because if analysts know about the benefits, and advantages, they can also consider this as a useful technique and the more people are knowing about it, the more also it will be recognized by the regulations. Analysts can make the difference here, and also the professors and the teachers. Show your students that HPTLC exists and then the next generation will bring it to the industry, and then it will be considered as a useful technique. HPTLC is not the gold standard for any analytical task, but the good analyst selects himself/herself. He/she has the method competence and knows about the different techniques - which one is best for this task, which one is best for this target and then we can pick the best method for each task. If HPTLC is not in the minds, if the people do not know about the existence of HPTLC and just have some ideas about old-fashioned TLC, then how can they choose it? Again, analysts can make the difference here: spread the advantages and teach about the disadvantages. Then people can make a decision on when HPTLC is useful, when it is a good choice and when other techniques are the better choice.
First, I want to show you the two options that we can provide for TLC-MS coupling. We have the TLC-MS Interface for doing it more or less manually. This is a stand-alone and not software-controlled device, which can be used for TLC/HPTLC at any lab with or without LC-MS-system. At least you need a pump that is connected here and then you can move the plate to your zones of interest. They are eluted, the eluent can be collected into a vial, and then you can go to the mass spectrometry site for analysis or you can directly couple TLC/HPTLC with the LC- MS in your existing LC-MS environment and then get directly your signal from the eluted zone. Next year, we will launch the HPTLC PRO Module MS-Interface, which will be software-controlled by visionCATS. The pump is already integrated and the user can select the zones of interest via the software. The elution head is moving to those positions and elutes the zones either into a vial for collecting the fractions or directly to a mass spectrometer. The principle for both cases is to elute the zones of interest and then transfer the dissolved zone to your mass spectrometer or into a vial. For direct connection there is a filter at the Rheodyne valve that can be exchanged and which prevents the transfer of silica gel to your mass spectrometer. It's eluting the zones of interest and if it's connected to the mass spectrometer you immediately get the signal of the selected zone. To answer the question When a MS equipment is combined, do we see the MS spectrum of each spot or all spots together? This depends on how good the separation is. If your analyte is well separated, then you just get the signal of this separated analyte. If it is eluted with other co-eluting substances, then in a full scan you get all the signals from all the different substances. If you have a tandem mass spectrometer, an MS-MS-system, you get by MRM mode just your analyte of interest. Everything is possible – you can couple to a single MS, to tandem MS and to a High-Resolution-MS. The limitation is - it has to be an MS that can be connected also to HPLC, because the solvent is present and the solvent has to be evaporated in the source. Therefore, the typical sources used are APCI and ESI sources. There exist also other TLC/HPTLC-MS coupling techniques like DART-MS or MALDI-MS, but CAMAG provides only the elution-based coupling, so with the TLC-MS Interface or with the HPTLC PRO Module MS-Interface. Here is a short video that shows the handling of the stand-alone device. The user positions the plate below the red laser cross to have the zone of interest in the middle, the elution head is lowered, then you switch the valve and then you get your signal on the screen and within, let's say less than a minute for each zone, you are done.
There are some ready-to-use reagents from some chemical companies. CAMAG is not selling reagents, we are an instrument supplier, but we offer the recipes for many different reagents. The chemicals can be purchased via the large suppliers like Merck and others. Here you see an extract from the information sheet of the Derivatizer, which is our automated spraying device to replace the manual spraying. The HPTLC PRO Module DERIVATIZATION, expected to be launched in early 2022, will then also be software-controlled and can be connected to the HPTLC PRO System to have the HPTLC process fully automated. The recipes for further reagents can also be found via the CCBS and the CBS journal, and we have application notes at camag.com and you can find recipes in literature. I highly recommend this book from Jork and co-authors, which is called Thin-Layer Chromatography – Reagents and Detection Methods. This is really a good source for derivatization reagents and also gives you the information about the reaction mechanism, if it was known during the time they have published it.