Sohan S.Chitlange

Quality control of herbal medicines is a tedious and difficult job. Herbal medicines differ from that of the conventional drugs and so some innovative methods are coming into being for the sake of quality assessment of herbal drugs.

Fingerprint analysis approach using chromatography has become the most potent tools for quality control of herbal medicines because of its simplicity and reliability. It can serve as a tool for identification, authentication and quality control of herbal drugs.

Introduction

The construction of chromatographic fingerprints plays an important role in the quality control of complex herbal medicines¹. Chemical fingerprints obtained by chromatographic techniques are strongly recommended for the purpose of quality control of herbal medicines, since they might represent appropriately the “chemical integrities” of the herbal medicines and therefore be used for authentication and identification of the herbal products. Based on the concept of phytoequivalence, the chromatographic fingerprints of herbal medicines could be utilized for addressing the problem of quality control of herbal medicines². By definition, a chromatographic fingerprint of a herbal medicine is, in practice, a chromatographic pattern of pharmacologically active and or chemically characteristic constituents present in the extract^3-5. This chromatographic profile should be featured by the fundamental attributions of “integrity” and “fuzziness” or “sameness” and “differences” so as to chemically represent the herbal medicines investigated. This suggest that chromatographic fingerprint can successfully demonstrate both “sameness” and “differences” between various samples and the authentication and identification of herbal medicines can be accurately conducted even if the number and/or concentration of chemically characteristic constituents are not very similar in different samples of herbal medicine. Thus chromatographic fingerprint should be considered to evaluate the quality of herbal medicines globally considering multiple constituents present in the herbal medicines.

Need for development of chromatographic fingerprints

Herbal medicines have a long therapeutic history and are still serving many of the health needs of a large population of the world. But the quality control and quality assurance still remains a challenge because of the high variability of chemical components involved. Herbal drugs, singularly and in combinations, contain a myriad of compounds in complex matrices in which no single active constituent is responsible for the overall efficacy. This creates a challenge in establishing quality control standards for raw materials and standardization of finished herbal drugs⁶. Traditionally only a few markers of pharmacologically active constituents were employed to assess the quality and authenticity of complex herbal medicines. However, the therapeutic effects of herbal medicines are based on the complex interaction of numerous ingredients in combination, which are totally different from those of chemical drugs. Thus many kinds of chemical fingerprint analysis methods to control the quality of herbal drugs have gradually come into being, such as thin layer chromatography, gas chromatography, high performance liquid chromatography etc. chromatographic fingerprint analysis of herbal drugs represents a comprehensive qualitative approach for the purpose of species authentication, evaluation of quality and ensuring the consistency and stability of herbal drugs and their related products. The entire pattern of compounds can then be evaluated to determine not only the presence or absence of desired markers or active constituents but the complete set of ratios of all detectable analytes. The chemical fingerprints obtained by chromatographic and electrophoretic techniques, especially by hyphenated chromatographies, are strongly recommended for the purpose of quality control of herbal medicines, since they might represent appropriately the “chemical integrities” of herbal medicines and therefore be used for authentication and identification of the herbal products.

Chromatographic techniques for fingerprint analysis of Herbal medicines

When Herbal Medicines are concerned, there are always hundreds of components and many of them are in too low amounts. On the other hand, there usually exists variability within the different and even the same herbal materials. Consequently, to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components is not a trivial task. The performance of a chromatographic fingerprint obtained is closely dependent on the chromatographic separation degrees and concentration distribution of all chemical components in the herbal medicine investigated. Fortunately, chromatography offers very powerful separation ability, such that the complex chemical components in herbal medicine extracts can be separated into many relatively simple sub-fractions. Furthermore, the recent approaches of applying hyphenated chromatography and spectroscopy such as High-Performance liquid chromatography-diode array detection (HPLC-DAD), Gas chromatography-mass spectroscopy (GC-MS), capillary electrophoresis-diode array detection (CE-DAD), HPLC-MS and HPLC-NMR, could provide the additional spectral information, which will be very helpful for the qualitative analysis and even for the on-line structural elucidation. With the help of the spectral information the hyphenated instruments show greatly improved performances in terms of the elimination of instrumental interferences, retention time shift correction, selectivity, chromatographic separation abilities, measurement precision. If hyphenated chromatography is further combined with chemometric approaches, clear pictures might be developed for chromatographic fingerprints obtained. A chemical fingerprint obtained by hyphenated chromatography, out of question, will become the primary tool for quality control of herbal medicines^7,8.

Chromatographic techniques used for fingerprint analysis of herbal medicines

Thin layer chromatography

Thin layer chromatography (TLC) is frequently used for the analysis of herbal medicines since various pharmacopoeias such as American Herbal Pharmacopoeia (AHP), Chinese Drug Monographs and analysis, Pharmacopoeia of People’s Republic of China etc. still use TLC to provide First characteristic fingerprints of herbs. Rather, TLC is used as an easier method of initial screening with a semiquantitative evaluation together with other chromatographic techniques. High performance TLC has the advantages of many-fold possibilities of detection in analyzing herbal medicines. In addition, HPTLC analysis is rather simple and can be employed for multiple sample analysis. With the help of CAMAG video store system (CAMAG Switzerland) and TLCQA-UV methods, it is possible to get useful qualitative and quantitative information from the developed TLC plate⁹. The advantages of using TLC to construct the fingerprints of herbal medicines are its simplicity, versatility, high velocity, specific sensitivity and simple sample preparation. Thus, TLC is a convenient method of determining the quality and possible adulteration of herbal products. It summarized the progress in forced-flow-planer chromatography (FFPC) and demonstrated the importance of the different techniques like rotation planar chromatography (RPC), overpressured-layer chromatography (OPLC), and electroplanar chromatography (EPC) ¹⁰.

Gas chromatography

Gas chromatography GC is usually used for fingerprint analysis when pharmacologically active components in herbal medicines are volatile chemical compounds. The advantage of GC clearly lies in its high sensitivity of detection for almost all the volatile chemical compounds. This is especially true for the usual FID detection and GC-MS. Furthermore, the high selectivity of capillary columns enables separation of many volatile compounds simultaneously within comparatively short times. However, the most serous disadvantage of GC is that it is not convenient for the analysis of samples of planer and non-volatile compounds^11-13.

High performance liquid chromatography

HPLC is a popular method for the analysis of herbal medicines because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. In general, HPLC can be used to analyze almost all the compounds in the herbal medicines. Reversed- phase (RP) columns may be the most popular columns used in the analytical separation of herbal medicines. It is necessary to notice that the optimal separation condition for the HPLC involves many factors, such as the different compositions of the mobile phases, their pH adjustment, pump pressures etc. Thus, a good experimental design for the optimal separation seems in general necessary^14-16 In order to obtain better separation, some new techniques have been recently developed in research field of liquid chromatography. These are micellar electrokinetic capillary chromatography (MECC)¹⁷, high-speed counter-current chromatography (HSCCC), low- pressure size-exclusion chromatography (SEC)¹⁸, reversed-phase ion-pairing HPLC (RP-IPC-HPLC)^19,20 and strong anion exchange HPLC (SAX-HPLC) ²¹. They will provide new opportunities for good separation for some specific extracts of some herbal medicines.

Hyphenated procedures

In the past two decades, combining a chromatographic separation system on-line with a spectroscopic separation system on-line with a spectroscopic detector in order to obtain structural information on the analytes present in a sample has become the most important approach for the identification and/or confirmation of the identity of target and unknown chemical compounds. For most (trace-level) analytical problems in research field of herbal medicines, the combination of column liquid chromatography or capillary gas chromatography with a UV-vis or a mass spectrophotometer become the preferred approach for the analysis of herbal medicines. Various hyphenated procedures used for the analysis of herbal drugs are HPLC-DAD, CE-DAD, GC-MS, LC-MS, HPLC-MS, HPLC-DAD-MS and LC-DAD-MS. The data obtained from such hyphenated instruments are the so-called two-way data; say one way for chromatogram and the other way for spectrum, which could provide much more information than the classic one-way chromatography. A “total analysis device” has been recently demonstrated in the case of on-line HPLC-UV (DAD)-FTIR-NMR-MS analyses².

Difficulties in development of chromatographic fingerprints for herbal medicines

When herbal drugs are considered for analysis, a large number of chemical components are involved and many of them are in low concentration. Chromatographic instruments and experimental conditions are difficult to reproduce during real analysis. Thus, the baseline and retention time shifts surely will be in existence from one chromatogram to another.  Many other problems associated with chromatographic fingerprints such as the occurrence of abnormal chromatograms from outlying herbal samples or experiments inevitably will be encountered. As a result, in order to obtain reliable chromatographic fingerprints, several data treatments would be needed during fingerprint analysis²².

Traditional medicines and chromatographic fingerprints-

Due to low toxicity and known pharmacological activity, traditional medicine, containing mainly of herbal drugs, has been popularly and extensively used for many centuries. However, it is not easy to conduct quality control and assurance of herbal medicines because of high variability of chemical components involved. Thus, as pointed out by World Health Organization (WHO), since the quality and quantity of safety and efficacy data on traditional medicine are far from sufficient to meet the criteria needed to support its use worldwide and there is still a lack of adequate or accepted research methodology for evaluating traditional medicine upto now, traditional medicine has not been officially recognized in most countries. Under this situation, the U.S.ÎõDA does not definitely emphasize the developments of very clear pictures of all chemical components from herbal medicines, and thus, the fingerprinting approach has been recently recognized and accepted for quality assessment of traditional medicines.  The concept of phytoequivalence was developed in Germany in order to ensure consistency of herbal products. According to this concept, a chemical profile, such as chromatographic fingerprint, for a herbal product should be constructed and compared with the profile of a clinically proven reference product. Chinese State Food and Drug Administration have said to regulate the compositions of liquid injections with herbal ingredients using stringent quality procedures such as chemical assay and standardization. Fingerprints of herbal medicinal liquid injections are compulsorily carried out for this purpose. In addition, among the various experimental techniques, chromatographic methods are highly recommended for finding out fingerprints of herbal products because of the high separation ability of chromatography.

Chemometric approaches and data processing for chromatographic fingerprint of herbal medicines

Due to complexity of the chromatographic fingerprint and the irreproducibility of chromatographic instruments and experimental conditions, several chemometric approaches such as variance analysis, peak alignment, correlation analysis, and pattern recognition were employed to deal with the chromatographic fingerprint. Many mathematical algorithms are used for data processing in chemometric approaches. The basic principles for this approach are variation determination of common peaks/ regions and similarity comparison with similarity index and linear correlation coefficient. Similarity index and linear correlation coefficient can be used to compare common pattern of the chromatographic fingerprints obtained. In general, the mean or median of the chromatographic fingerprints under study is taken as the target and both are considered to be reliable. To facilitate the data processing, a software named Computer Aided Similarity Evaluation (CASE) has been developed. All programs of chemometric algorithms for CASE are coded in METLAB5.3 based on windows. Data loading, removing, cutting, smoothing, compressing, background and retention time shift correction, normalization, peak identification and matching, variation determination of common peaks/regions, similarity comparison, sample classification, and other data processes associated with the chromatographic fingerprint can be investigated with this software²².

Conclusion:

The problem of quality assurance of herbal medicines has been solved to a great extent with the help of chromatographic fingerprint analysis. The variation determination of common peaks/regions in a set of chromatographic fingerprints could provide useful qualitative and quantitative information on the characteristic components of herbal medicines investigated. On the other hand, whether the real samples were identified as the herbs with the same quality grade could be determined successfully by way of comparing the chromatographic fingerprints with the similarity index and linear correlation analysis. Furthermore, pattern recognition can be used to discriminate different kinds of samples of herbal medicines investigated²⁰. Thus chromatographic fingerprint analysis serves as a promising quality control tool for herbal medicines.

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About Authors:

Mr.Sohan S.Chitlange (M.Pharm. Pharmaceutical Chemistry)
Asst. Professor, Dept.of Pharm.Chemistry, Padm. Dr.D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune.
sohanchitlange@rediffmail.com

Mr.Santosh S. Bhujbal (M.Pharm. Pharmacognosy)
Asst.Professor, Dept.of Pharmacognosy, Padm.Dr.D. Y. Patil  Institute of Pharmaceutical.Sciences & Resreach, Pimpri, Pune

Mr.Amol A. Kulkarni (M.Pharm. Pharmaceutical Chemistry)
Lecturer, Dept.of Pharm.Chemistry, Padm. Dr.D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune.
amolkulkarni89@rediffmail.com

Mohammed Imran (M.Pharma. Sem.IV Quality Assurance)
Dept.of Pharm.Chemistry ; imransa99@rediffmail.com

Prajakta S. Kulkarni (M.Pharm. Pharmaceutical Chemistry)
Dept.of Pharm.Chemistry, prajakta3084@gmail.com