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HP-TLC fingerprint: A picture is worth a thousand words!

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Dr Saikat Mallick, Asst Lab Manager, Anchrom Enterprises India, gives an insight on HP-TLC Fingerprint, which has been newly introduced regulation for chromatogram evaluation, of complex and variable natural products

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Dr Saikat Mallick

Chromatography methods are extremely popular for separation and quantification of components in mixtures, as well as for their identification when coupled with a MS detector. Particularly so, in synthetic chemistry labs where a lot of information is available on the inputs in terms of identity, purity, impurities etc. and a reasonable  prediction can be made about the output. Identifying a pure component in synthetic chemistry samples is not difficult, today.

With ‘articles of botanical origin’ like herbal extracts and medicines, foods, neutraceuticals, essential oils, spices, vegetable oils etc etc. the analysis requirements are very different. Here, one has to certify authenticity and non-adulteration (purity) of a ‘sample’ containing numerous and unknown components, without knowing the components! Just stating a sample name e.g. Ashwgandha is not enough because there are so many variables inherent in the ‘Ashwagandha sample’ such as – which part of the plant, age, storage, geographical origin, altitude, species, sub-species, chemotype, phenotype etc.  Then there is the method of extraction or trituration or treatment (eg. Shodhan or purification as in Ayurveda) etc. which too can alter the phytochemical composition of the ‘botanical sample.’

With so many variables, that too in a complex matrix, the simplest and best approach for identification of a single botanical material for quality control is to have a ‘Botanical Reference Material’ (BRM) and then compare its HP-TLC fingerprint, with the sample. If there are e.g. six varieties of saffron, then there would be 6 saffron BRMs and 6 HP-TLC fingerprints viz. Saffron (Iran), Saffron (Spain), Saffron (Kashmir) etc. These fingerprints could confirm e.g. if the premium label ‘Kashmir’ saffron is genuine or cheap Iranian saffron. About 50 samples of saffron could be checked for identity and adulteration in a day, at a very nominal cost, using HP-TLC. No other chromatography method gives any visual information about the sample. Leave alone a ‘fingerprint.’

‘HP-TLC fingerprint’ is a visual representation of the phytochemical composition obtained by the technique of High Performance Thin Layer Chromatography (HP-TLC). One does not need to know the names of the phytochemicals present! So called ‘markers’ i.e. components whose presence has been previously identified, have no role in fingerprint identification which is the primary test for identification of an article of botanical origin. Inorganic samples like ‘bhasma’ cannot be HP-TLC fingerprinted. Here is an example of HP-TLC fingerprint.

The number, distance, colour and intensity of all bands in a sample is its fingerprint.

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Figure 4-15: HPTLC fingerprints of hawthorn species (Crataegus leaf and flowers). MP: ethyl acetate, methanol, formic acid, water (50:3:3:6) D: Natural Products reagent, UV 366nm. Tracks 1 and 7, C. Laevigata; tracks 2 to 5, C. monogyna (4, strongly fermented sample); track 6, standards (with increasing Rf: rutin, vitexin – 2”- O-rhamnoside, chlorogenic, hyperoside, vitexin); track 8, C. laevigata, “Punicea”; track 9, C. x macrocarpa; track 10, C.azarolus; track 11, C.pentagyna. Ref: HPTLC Analysis of Medicinal Plants by Dr Reich

HP-TLC fingerprint is very easy to obtain by the simple HP-TLC technique, at a low cost with high throughput. A modern s/ w controlled HP-TLC can fingerprint dozens of different samples per day.  Software control is a must because HP-TLC is performed using five independent steps, using five stand alone instruments. Only with the common controlling link i.e. modern HP-TLC software, regulatory and cGLP requirements can be complied with. Pure substances may be chromatographed on same plate during fingerprinting for System Suitability Test.

The industrial quality images can be used for quantification or far better quantification can be achieved by scanning densitometry. Evaluation of reference fingerprint vs sample’s fingerprint must be done on same computer display only and in no other way. The fingerprint is ideally used in botanical quality control of individual materials. In case of formulations, the fingerprint cannot be, often, related to the raw materials used. But checking batch to batch consistency, stability testing is possible. Fingerprints are also useful to monitor changes of in-process materials, particularly in those Ayurvedic preparations, with complex preparations.

To sum up, HP-TLC Fingerprint is a newly introduced regulation for chromatogram evaluation, of complex and variable natural products, by comparing the phytochemical composition represented in photographic image form, for the primary purpose of identification and secondary purpose of detecting adulteration and dilution of materials of botanical origin. This is a very recent development and will become a very commonly used procedure in the next few years, in the botanical (herbal and food) industries.

Further reading

  • HPTLC for Analysis of Medicinal Plants by Dr Reich (ISBN 3-13-141601-7/ ISBN 978-3-13-141601-8)
  • USP Chapters 202, 203, 1064, 2251
  • PhEur Chapter 2.8.25
  • WHO Expert Committee on specifications for Pharma Preparations (Report 51)

Contact Details:
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