USP has recently introduced HPTLC as one of the techniques to be used in chemical fingerprinting of raw material in herbal products. Industry players share their views on this development and seek government support in implementing this technology as it requires considerable investment. By Usha Sharma
India is one of the main hubs for herbal extracts and exports these products to all over the world. The offerings have a reputation of being good quality products. Today, there are several small, mid-sized and big companies that manufacture ayurvedic medicines and the majority of these companies export products to regulated countries like the US and EU. Hence, any regulatory changes or amendments which become effective in these countries, need to be followed by the Indian herbal manufacturers as well. As Shaheen Majeed, Director, Sami Direct explains, “Indian origin-based dietary supplements are a major portion of the dietary supplements market in the US, hence any regulations affecting the US dietary supplement market, directly affects the Indian herbal industry.”
The dietary supplement market in the US has marked 20 years under Dietary Supplement Health and Education Act of 1994 (DSHEA). The law was set up to regulate the dietary supplement industry as a special class of foods, distinctive from the drug and food regulations. It was intended to ensure consumer access to a variety of safe and effective health supplements. However, the dietary supplement industry recently had to bear the ire of the New York Attorney General who took action against some of the major retailers who sell, among many other products, herbal supplements.
Yet, United States Pharmacopeia (USP), a central body for determination of appropriate tests, methodologies, and standards for drugs as well as food and dietary supplements, has described various test procedures to help the manufacturer identify the raw material, in a bid to avoid any ambiguity in the working of the dietary supplement segment. These tests methods, described in Chapter <563>, deal with the identification of articles of botanical origin. This chapter also discusses the various techniques for identification of botanical raw material, including identification of plant species by ascertaining the macroscopic characteristics as well as histological features, chemotaxonomy, which includes studying the active principles and marker compounds, and DNA finger printing methodology for identifying unprocessed raw material.
However, to provide a more definitive, routine identification methodology, USP has recently introduced High Performance Thin Layer Chromatography (HPTLC) as one of the techniques to be used in chemical fingerprinting raw material. This technique seeks to avoid violation of 21 CFR 111.75 and resulting warning letters from the US FDA. It has been implemented since August 1, 2015; and USP mandates that documentation is necessary to record the results in an auditable manner to comply with current good manufacturing practices (cGMP).
What is HPTLC?
HPTLC is a more advanced technique than traditional Thin Layer Chromatography (TLC). While TLC is able to identify only the presence or absence of certain marker compounds in a raw material or extract, HPTLC can help to establish the identity of the raw material and extract by comparing it from, not just a single marker compound, but in fact the fingerprint of an entire range of natural plant compounds present.
However, HPLTC is an advanced form of technology which requires huge investment. Before introducing HPTLC to test the assay of the herbal drugs, the analysis was done either by Gravimetry, titration or UV method.
Dilip Charegaonkar, Managing Director, Anchrom Enterprises informs, “The new USP chapters describe a set of parameters to be followed in the practice of HPTLC, so that reproducible results can be obtained by anyone performing the test. Chapter <203> provides these parameters to be followed, and Chapter <1064> describes the reasons as to why the parameters were chosen. The chapters together cover every aspect of HPTLC, from sample preparation, sample application, development, humidity control, documentation, system suitability testing etc. HPTLC being a visual technique, there is an emphasis on photo documentation of HPTLC plates.”
Comparing the benefits of HPTLC against TLC, Majeed says, “HPTLC can be used for obtaining the chromatogram for herbal fingerprinting of the raw material, which can help in identifying the species of the raw material. The sampling procedures do not require cumbersome preparation and use a very small amount of the raw material. In contrast to TLC, HPTLC is an automated technique and the recording of the data and its retrieval for future reference, is much simpler than with TLC.”
Dr Pulok K Mukherjee, School of Natural Product Studies, Dept of Pharmaceutical Technology, Jadavpur University also mentions, “HPTLC is extremely versatile and sensitive for the identification of many herbal drugs and is especially useful for the detection of adulterations, often with a very high degree of sensitivity. One of the most obvious orthogonal features of the two techniques is the primary use of reversed phases in HPLC versus unmodified silica gel in HPTLC, resulting in partition chromatography and adsorption chromatography respectively.”
Dr Rahul Singh, Head – CQA (HCD) and R&D (Analytical), Emami explains how HPTLC will be instrumental in enhancing the quality and safety of herbal products and says, “The WHO Guidelines for quality assessment of plant materials are not followed by most of the 8,500 licensed manufacturers of herbal formulations, since most of them are small-scale manufacturers with limited resources and knowledge. However, a few medium and large scale manufacturers undertake some quality control testing of herbal materials. In the case of herbal drugs it is different since no standard pharmacopoeial methods are available for their identity, when they are in multi-herbal formulations and more so when the extracts of herbs have been used in the formulations. However, regular botanical identification and phytochemical testing shall be of immense help if carried out for both raw materials as well as for the formulations. Incorporation of HPTLC method may help to resolve these problems faced by the herbal drug industry.”
Status of HPTLC in various pharmacopoeias as of August 2015
USP: In addition to the two existing chapters <621> “Chromatography” and <201> “Thin-Layer Chromatographic Identification Test” three new chapters have been drafted and published for comment in PF 40(3) and adopted:
<203> High-Performance Thin-Layer Chromatography Procedure for Identification of Articles of Botanical Origin
<1064> Identification of Articles of Botanical Origin by High-Performance Thin-Layer Chromatography Procedure
<202> Identification of Fixed Oils by Thin-layer Chromatography (Note: this chapter describes a TLC method and an HPTLC method).
All chapters will be official in March2015 in the Dietary Supplement Compendium 3 and in October 2015 in the USP 38 NF 33 First Supplement
PhEur: Chapter 2. 2.27 “Thin-Layer Chromatography” describes both TLC and HPTLC conditions. A general chapter 2.8.xx “Qualitative High Performance Thin-Layer Chromatography of Herbal Drugs and Herbal Drug Preparations” was drafted and will be published in the next Pharmeuropa. The content is equivalent to that of USP <203>.
With PhEur 7.0 all monographs for plants include either identification by TLC and HPTLC, or only HPTLC.
ChP: Appendix VI “Thin-Layer Chromatography” describes both TLC and HPTLC conditions. Up to ChP2010 there are no monographs with HPTLC. The 2005 HPTLC Atlas features HPTLC results for 150 plants. A new project “HPTLC Atlas 2015” was launched by the Pharmacopoeia Commission
JP17-II: The general chapter 2.03 Thin-Layer Chromatography features only simple TLC. On December 4, 2014 a proposal to update the chapter to match USP 621 was adopted. That means HPTLC will now be introduced. Further details are not yet know.
IP: Guidance Manual for Monograph Development of Herbs, Processed Herbs/ Herbal Extracts and Herbal Products describes both Thin Layer Chromatography and High Performance Thin Layer Chromatography under chapters 15 & 16 respectively
He elaborates further, “As HPTLC is widely used for botanical identification to monitor the quality of articles of botanical origin on an increasingly globalised level, it seems important to thoroughly standardise the involved analytical procedures. Currently, laboratories in different countries can make the parameter selection widely within the framework of the USP general chapters; however, guidance about how to achieve specific HPTLC results in a reproducible manner is not available. A standardised procedure as proposed in 203 could fill this gap and serve as a basis for harmonisation of results obtained in different laboratories. “
So, is it affordable and justifiable?
Today, India is home to several herbal companies of good repute and global reach. Majority of these big players are largely dependent on small and medium sized players and they do not have strong financial support. So, the question that arises is: How can these small and medium sized players adopt HPTLC if it is not financially feasible?
V Hariharan, CEO and Managing Director, Phytotech Extracts expresses his concern, “I am sure that the new draft guideline of HPTLC is likely to impact exporters of herbal ingredients and herbal drugs. I am afraid that many herbal extracts producers may not have HPTLC as the requisites from major customers are test reports from TLC and HPLC. Who has foreseen that they have to add the HPTLC method of analysis in their testing methods and that too in such a short time? Here, the implementation time is an important factor. MSME sector exporters and companies may not be able to afford investing the amount of Rs 2.5 – Rs 3 million plus immediately for HPTLC as this amount would not have been budgeted.”
SMEs do not have enough financial backing to installed such advanced technologies. Vikas Jain, Director, MMV Nutrient Products informs, “The herbal raw material is prone to a lot of variations due to many factors. Seasonal variation is the most important one. Our herbal drug manufacturers are mainly SMEs, who don’t have sophisticated technology like HPTLC to ensure that their products meet this specification.”
Beside considering the financial assistance in adopting HPTLC technology companies also need to address other issues which are linked with the technology upgradation. Jain informs, “One needs to plan for purchasing of such expensive instruments. They need to plan a lot of allied things such as the right standards of the ingredients, qualified and well trained staff who would be proficient to operate the HPTLC and study the results and so on.”
When companies make such a huge investment they would like to see return on investment as well. Rationalising the benefits behind making investments for advanced technologies like HPTLC, Hariharan highlights, “Once HPTLC is in place working would be quite easy and cost effective. Some of the independent laboratories and a few companies may have invested on HPTLC and one could seek the help from them to get the product tested initially as investing a large amount for an HPTLC may not be feasible immediately. The testing time and the expenses are not high with HPTLC.”
However, the industry remains sceptical about the suggested changes, as Hariharan points out, “US FDA may come out with new test regulations from time to time for herbal drugs. They may ask the companies to implement the same within a very short time and the companies may envisage such methods of analysis and be prepared for it. Let it not be a shock as they have financial implications for purchase of instruments and so on.”
Moving towards a new era
Quality assurance of herbal medicine is an important factor and considered as a basic requirement for herbal as well as other drug development industries. There are several problems which influence the quality of herbal drugs. Rationlising the importance of HPTLC, Hariharan emphasises, “The proposed guideline of USP, focusing on HPTLC for herbal drugs, is a welcome one. The accuracy of identification of impurities and the active ingredients are well established. Quite a few overseas customers have identified many impurities in the extracts due to faulty production techniques or the use of adulterated solvents or may be like certain Chinese producers, some intentionally add adulterants to reduce cost of production.”
Jain stresses on why herbal manufacturers need to have HPTLC and how it will help them in expanding their business footprints, “The problem faced by the buyers of these herbal drugs are having a presence of minute quantity of active principle in the product. HPLTC would be useful for its detection. Another issue is that at times lack of knowledge regarding dosage calculation due to varied concentration of active constituent from same amount of raw material may also result in different values. So, to avoid this, it is very necessary to make the HPTLC fingerprints of herbal drugs compulsory according to the guidelines.
Clarifying the necessity for switching from traditional methods to HPTLC, Hariharan also says, “As assay levels are not so accurate and also non-identification of individual components by these techniques, the customers have started analysing assay by HPTLC which was more accurate, then after they started demanding for that. However, the initial identification of the relevant ingredient is being done by TLC method. Subsequently, the demand has come to identify the residual solvents, heavy metals and thereafter pesticides. Many companies get these done by Gas chromatography (GC) from other laboratories who have already invested in GC. These have now become mandatory for any herbal extract or drugs.”
Voice of experience
Dr PD Sethi, Pharmaceutical Analyst, shares his experience and recalls the techniques used earlier, “The guideline describes standardised HPTLC procedure for end use. Considering my personal experience of almost four decades with the use of TLC/ HPTLC for identification and quantitative analysis of pharma formulations, I can confidently say that these are the best drafted, scientifically sound, practical guidelines. The detailed guidelines shall enable even a novice analyst to follow the procedure with adequate control of various variables affecting the separation within permissible limits.”
Agreeing to Sethi, Majeed comments, “While USP has issued the draft guidance of using HPTLC as one of the techniques for identification of botanical raw materials and extracts, this technique, because of its robustness, ease of sampling and low running costs, can make HPTLC an essential tool for the industry. With the eyes of the industry, and the media on herbal supplements, utilising the best ‘fit for purpose’ testing tools must become common practice.”
Rising to the challenge
Sethi concludes, “I feel we should accept this challenge and prepare ourselves to meet these specifications if we intend to export our medicines. In fact, it shall be logical if the USP guidelines are included in Ayurvedic Pharmacopoeia of India as well as in Indian Pharmacopoeia which contains numerous monographs on herbal drugs. Any relaxation in the specification of herbal medicines for use in India as compared to specifications for export shall imply a compromise with the health of our people.