About Author:
Alimuddin Saifi
N.K.B.R. College of Pharmacy & Research Centre,
Meerut
asaifi2005@gmail.com
Abstract
Herbal drugs are accepted as important therapeutic agents for the treatment of many diseases. The development of authentic analytical methods which can reliably profile the phytochemical composition, including quantitative analyses of marker/bioactive compounds and other major constituents, is a major challenge to scientists. Pharmacognostical analysis of medicinal herbs remain challenging issues for analytical chemists, as herbs are a complicated system of mixtures. Analytical separation techniques for example high performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS), High Performance Thin Layer Chromatography (HPTLC) etc. among the most popular methods of choice used for quality control of raw material and finished herbal product.
Reference Id: PHARMATUTOR-ART-1522
Introduction
Chromatography represents the most versatile separation technique and readily available.Plant materials are separated and purified by using various chromatographic techniques. Herbal medicine is a complicated system of mixtures. Thus, the methods of choice for identification of ‘botanical drug’ are mainly intended to obtain a characteristic fingerprint of a specific plant that represent the presence of a particular quality defining chemical constituents. For such purposes, chromatographic techniques such as high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography – mass spectrometry (GC-MS) and thin layer chromatography (TLC) were used widely as reported in numerous publications1.
Thin Layer Chromatography (TLC)
Thin layer chromatography is simply known as TLC. It is one of the most popular and simple chromatographic technique used of separation of compounds. In the phytochemical evaluation of herbal drugs, TLC is being employed extensively for the following reasons:
1. It enables rapid analysis of herbal extracts with minimum sample clean-up requirement,
2. It provides qualitative and semi quantitative information of the resolved compounds.
3. It enables the quantification of chemical constituents. Fingerprinting using HPLC and GLC is also carried out in specific cases.
In TLC fingerprinting, the data that can be recorded using a high-performance TLC (HPTLC) scanner includes the chromatogram, retardation factor (Rf) values, the color of the separated bands, their absorption spectra, λ max and shoulder inflection/s of all the resolved bands.
All of these, together with the profiles on derivatization with different reagents, represent the TLC fingerprint profile of the sample. The information so generated has a potential application in the identification of an authentic drug, in excluding the adulterants and in maintaining the quality and consistency of the drug.2
TLC was the common method of choice for herbal analysis before instrumental chromatography methods like GC and HPLC were established.
Even nowadays, TLC is still frequently used for the analysis of herbal medicines since various pharmacopoeias such as American Herbal Pharmacopoeia (AHP)3, Chinese drug monographs and analysis, Pharmacopoeia of the People’s Republic of China etc. still use TLC to provide first characteristic fingerprints of herbs4.
Rather, TLC is used as an easier method of initial screening with a semi quantitative evaluation together with other chromatographic techniques. As there is relatively less change in the simple TLC separation of herbal medicines than with instrumental chromatography, only a brief summary is given here, and for further details about TLC the readers could consult references5-6.
High Performance Thin Layer Chromatography (HPTLC)
HPTLC technique is widely employed in pharmaceutical industry in process development, identification and detection of adulterants in herbal product and helps in identification of pesticide content, mycotoxins and in quality control of herbs and health Food. It has been well reported that several samples can be run simultaneously by use of a smaller quantity of mobile phase than in HPLC. It has also been reported that mobile phases of pH 8 and above can be used for HPTLC. Another advantage of HPTLC is the repeated detection (scanning) of the chromatogram with the same or different conditions. Consequently, HPTLC has been investigated for simultaneous assay of several components in a multi-component formulation. With this technique, authentication of various species of plant possible, as well as the evaluation of stability and consistency of their preparations from different manufactures. Various workers have developed HPTLC method for phytoconstituents in crude drugs or herbal formulations such as bergenin, catechine and gallic acid in Bergenia cilliata and Bergenia lingulata7.
High Performance Liquid Chromatography (HPLC)
Over the past decades, HPLC has received the most extensive application in the analysis of herbal medicines. Reversed-phase (RP) columns may be the most popular columns used in the analytical separation of herbal medicines.
Preparative and analytical HPLC are widely used in pharmaceutical industry for isolating and purification of herbal compounds. There are basically two types of preparative HPLC: low pressure HPLC (typically under 5 bar) and high pressure HPLC (pressure >20 bar).
The important parameters to be considered are resolution, sensitivity and fast analysis time in analytical HPLC whereas both the degree of solute purity as well as the amount of compound that can be produced per unit time i.e. throughput or recovery in preparative HPLC.
In preparative HPLC (pressure >20 bar), larger stainless steel columns and packing materials (particle size 10-30 μm are needed. The examples of normal phase silica columns are Kromasil 10 μm, Kromasil 16 μm, Chiralcel AS 20 μm whereas for reverse phase are Chromasil C18, Chromasil C8,YMC C18. The aim is to isolate or purify compounds, whereas in analytical work the goal is to get information about the sample. This is very important in pharmaceutical industry of today because new products (Natural, Synthetic) have to be introduced to the market as quickly as possible. Having available such a powerful purification technique makes it possible to spend less time on the synthesis conditions2.
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Liquid Chromatography- Nuclear Magnetic Resonance (LC-NMR)
LC-NMR improves speed and sensitivity of detection and found useful in the areas of pharmacokinetics, toxicity studies, drug metabolism and drug discovery process.
The combination of chromatographic separation technique with NMR spectroscopy is one of the most powerful and time saving method for the separation and structural elucidation of unknown compound and mixtures, especially for the structure elucidation of light and oxygen sensitive substances. The online LC-NMR technique allows the continuous registration of time changes as they appear in the chromatographic run automated data acquisition and processing in LC- NMR improves speed and sensitivity of detection. The recent introduction of pulsed field gradient technique in high resolution NMR as well as three-dimensional technique improves application in structure elucidation and molecular weight information. These new hyphenated techniques are useful in the areas of pharmacokinetics, toxicity studies, drug metabolism and drug discovery process8.
Liquid Chromatography- Mass Spectroscopy (LC-MS)
LC-MS has become method of choice in many stages of drug development. Recent advances includes electrospray, thermospray, and ionspray ionization techniques which offer unique advantages of high detection sensitivity and specificity, liquid secondary ion mass spectroscopy, later laser mass spectroscopy with 600 MHz offers accurate determination of molecular weight proteins, peptides. Isotopes pattern can be detected by this technique9.
Gas Chromatography (GC-MS)
It is well-known that many pharmacologically active components in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by gas chromatography is very important in the analysis of herbal medicines. The GC analysis of the volatile oils has a number of advantages. Firstly, the GC of the volatile oil gives a reasonable “fingerprint” which can be used to identify the plant. The composition and relative concentration of the organic compounds in the volatile oil are characteristic of the particular plant and the presence of impurities in the volatile oil can be readily detected. Secondly, the extraction of the volatile oil is relatively straightforward and can be standardized and the components can be readily identified using GC–MS analysis9.
GC equipment can be directly interfaced with rapid scan mass spectrometer of various types. GC and GC-MS are unanimously accepted methods for the analysis of volatile constituents of herbal medicines, due to their sensitivity, stability and high efficiency. Especially, the hyphenation with MS provides reliable information for the qualitative analysis of the complex constituents. The flow rate from capillary column is generally low enough that the column output can be fed directly into ionization chamber of MS. The simplest mass detector in GC is the Ion Trap Detector (ITD). In this instrument, ions are created from the eluted sample by electron impact or chemical ionization and stored in a radio frequency field; the trapped ions are then ejected from the storage area to an electron multiplier detector. The ejection is controlled so that scanning on the basis of mass-to-charge ratio is possible. The ions trap detector is remarkably compact and less expensive than quadrapole instruments.GC-MS instruments have been used for identification of hundreds of components that are present in natural and biological system11.
Supercritical Fluid Chromatography (SFC)
Supercritical fluid chromatography is a hybrid of gas and liquid chromatography that combines some of the best features of each. SFC permits the separation and determination of a group of compounds that are not conveniently handled by either gas or liquid chromatography. SFC has been applied to a wide variety of materials including natural products, drugs, food and pesticide. These compounds are either nonvolatile or thermally labile so that GC procedures are inapplicable or contain no functional group that makes possible detection by the spectroscopic or electrochemical technique employed in LC12.
References:
1. Sim CO, Hamdan MR,Ismail Z and Ahmad MN, Assessment of Herbal Medicines by Chemometrics – Assisted Interpretation of FTIR Spectra, Journal Of Analytica Chimica Acta, 2004, 1-14
2. Bhutani KK, Finger-Printing of Ayurvedic Drugs, The Eastern Pharmacist, 2000; 507: 21-26.
3. R. Upton, International Symposium on Quality of Traditional Chinese Medicine with Chromatographic Fingerprint, Guangzhou, 2001, i 2-1.
4. Yi-Zeng Lianga, Peishan Xieb, Kelvin Chanc, Review: Quality control of herbal medicines; Journal of Chromatography B, 812 (2004) 53–70.
5. H. Wagner, S. Bladt, V. Rickl, Plant Drug Analysis: A Thin Layer Chromatography Atlas, second ed., Springer-Verlag, 1996.
6. A. Baerheim Svendsen, J. Planar Chromatogr. Modern TLC 2 (1989) 8.
7. Nikam P. H., Kareparamban J., Jadhav A. and Kadam V., Future Trends in Standardization of Herbal Drugs, Journal of Applied Pharmaceutical Science 02 (06); 2012: 38-44.
8. Patil PS, Rajani S. An Advancement of Analytical Techniques in Herbal Research J.Adv.Sci.Res. 2010, 1(1); 08-14.
9. Liang YZ, Xie P, Chan K, J., Quality control of herbal medicines, Chromatogr B, 2004; 812: 53–70.
10. Guo F.Q., Huang L.F., Zhou S.Y., Zhang T.M., Liang Y.Z., Comparison of the volatile compounds of Atractylodes medicinal plants by headspace solid-phase microextraction-gas chromatography–mass spectrometry.Anal. Chim. Acta 570: (2006) 73-78
11. Teo C.C., Tan S.N., Yong J.W. H., Hewb C. S., and Ong E. S.Evaluation of the extraction efficiency of thermally labile bioactive compounds in Gastrodia elata Blume by pressurized hot water extraction and microwaveassisted extraction. J. Chromatogr. A 1182: 2008 34– 40.
12. Matthew C, Henry R. Supercritical fluid chromatography, Pressurized liquid extraction, and supercritical fluid extraction. Anal Chem 2006; 78: 3909.
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