STANDARDIZATION OF HERBAL DRUGS/PRODUCTS: BOON TO INDIAN PHARMA MARKET

About Author:
Swati Manik
Department of Pharmacognosy,
I.S.F. College of Pharmacy, Moga, Punjab
swatimanik@yahoo.in

ABSTRACT:-
There is a great demand of the herbal drugs from both the developing as well as developed nations because of their efficacy, safety and lesser side effects as compared to synthetic molecules. These drugs also offer therapeutics for age-related disorders like memory loss, osteoporosis, immune disorders, etc. for which no modern medicine is available. It shows the promisingcontribution of herbal drugs in both economy and health concern, thus, it is ground enough to invest on their development.As the effectiveness and acceptability of the herbal products greatly depends upon its quality. The lack of standardization is the key factor due to that India despite of its rich traditional knowledge, heritage of herbal medicines and large biodiversity has a dismal share of the export to the world market. Therefore, the quality assurance of herbal drug/products is the need of an hour. Hence, standardization has become an essential component of the herbal drug/products that ensures the benefits of the herbal drugs.
Thus, by standardization of herbal drugs/products the Indian herbal market and even the export of herbal medicines will increase, making “India the Pharma Power House”.

REFERENCE ID: PHARMATUTOR-ART-1643

INTRODUCTION:-
“Health for all” is a dream and a goal which humanity at large shares and strives for, unfortunately, it has been proven without doubt that modern pharmaceuticals are and will remain out of reach for a large proportion of the human population for the near future. This has created an appreciation and a need for the use of other sources of human knowledge to provide common health benefits. Alternative and traditional medicines, largely herbal in nature, are now regarded as important but underutilized tools against disease.

Nature always stands as a golden mark to exemplify the outstanding phenomena of symbiosis. Natural products from plant, animal and minerals have been the basis of the treatment of human disease. The consumption of plant-based medicinesand other botanicals in the West has increased manifold in recent years. About two centuries ago, our medicinal practices were largely dominated by plant-based medicines.However, the medicinal use of herbs went into a rapid decline when more predictablesynthetic drugs were made commonly available. In contrast, many developing nations continued to benefitfrom the rich knowledge of medical herbalism. For example, Ayurvedic medicine in India, Kampo medicine in Japan, traditional Chinese medicine (TCM), and Unani medicine in the Middle East andsouth Asia are still used by majority of people.In the recent era of herbal renaissance, the demand of herbal medicines and other botanicals byWestern communities have been increasing steadily, particularly over the past two decades. Along with herbal medicines, other herbal products such as cosmetics, fragrances, teas, health foods, and nutraceuticals are equally popular and constitute a large proportion of global herbal business.

In olden times, in India vaidyas used to treat patients on individual basis, and prepare drug according to the requirement of the patient. But the scenario has changed now; herbal medicines are being manufactured on the large scale in Pharmaceutical units, where manufactures come across many problems such as the availability of good quality raw material, authentication of raw material, availability of standards, proper standardization methodology of single drugs and formulation, quality control parameters. The use of herbal medicine due to toxicity and side effects of allopathic medicines, has led to sudden increase in the number of herbal drug manufacturers [1].

India has a rich heritage of traditional medicine constituting with its different components like Ayurveda, Siddha, Unani, Homoeopathy and naturopathy. Traditional health care has been flourishing in this country for many centuries [2]. The growing use of botanicals by the public is forcing moves to evaluate the health claims of these agents and to develop standards of quality and manufacture. Most medications before being offered to Consumers undergo rigorous evidence-based Clinical testing; this is not necessarily true for Herbs, due to their long historical clinical use and reliable therapeutic efficacy. Various traditional medicine system, especially Indian system of medicine attracted the global attention, and many big pharmaceutical companies are using traditional medicine as an excellent pool for discovering natural bioactive compounds. With the growing need for safer drugs, attention has been drawn to their quality, efficacy and standards of the traditional Indian medicine [3, 4]. Each traditional system of medicines has their own method of standardization for assuring quality most in human linguistic terms. This method of evaluation has to be taken into consideration in standardization of herbal medicine/drugs [5].

The World Health Organisation (WHO) defined the Herbal drugs as “a finished labelled products that contain active ingredients such as aerial or underground parts of plant or other plant material or combinations thereof, whether in the crude state or as plant preparations”. [6]

Herbal drugs are classified as

Phytomedicines or phytopharmaceuticals sold as over the counter (OTC) products in modern dosage forms such as tablets, capsules & liquids for oral use

Dietary supplements containing herbal products, also called neutraceuticals available in modern dosage forms

Herbal medicines consisting of either crude, semi-processed or processed medicinal plants.

Some time herbs are combined with mineral preparations also. The herbs often exist in crude state and Ayurveda describes method of purification of toxic herbs.

Advantages of Herbal Medicine:-

They have long history of use and better patient tolerance as well as acceptance.

Medicinal plants have a renewable source, which is only hope for sustainable supplies of cheaper medicines for the world growing population.

Availability of medicinal plants is not a problem especially in developing countries like India having rich agro-climatic, cultural and ethnic biodiversity.

Prolong and apparently uneventful use of herbal medicines may offer testimony of their safety and efficacy.

Throughout the world, herbal medicine has provided many of the most potent medicines to the vast arsenal of drugs available to modern medicinal science, both in crude form and as a pure chemical upon which modern medicines are structured.

Limitations of Herbal Medicines:-

Ineffective in acute medical care

Inadequate standardization and lack of quality specifications

Lack of scientific data

Current and future status of Indian herbal medicines:-
India is sitting on a gold mine of well-recorded and well practiced knowledge of traditional herbal medicine. India is one of the 12-mega biodiversity centres having over 45,000 plant species. Its diversity is unmatched due to the presence of 16 different agro-climatic zones, 10 vegetative zones and 15 biotic provinces. The country has 15,000–18,000 flowering plants, 23,000 fungi, 2500 algae, 1600 lichens, 1800 bryophytes and 30 million micro-organisms [7]. India also has equivalent to 3/4 of its land exclusive economic zone in the ocean harbouring a large variety of flora and fauna, many of them with therapeutic properties. About 1500 plants with medicinal uses are mentioned in ancient texts and around 800 plants have been used in traditional medicine.

But, unlike China, India has not been able to capitalize on this herbal wealth by promoting its use in the developed world despite their renewed interest in herbal medicines.

The basic requirements for gaining entry into developed countries market include: (i) Well-documented Ethno-botanical information, (ii) Medicinal plants free from pesticides, heavy metals, etc., (iii) Standardization, based on chemical and activity profile, and (iv) Safety and stability data. However, mode of action studies in animals and efficacy in human will also be supportive. Such scientifically generated data will project herbal medicine in a proper perspective and help in sustained global market.

The major traditional sector pharmas, namely Himalaya, Zandu, Dabur, Hamdard, Maharishi, etc. and modern sector pharmas, namely Ranbaxy, Lupin, Allembic, etc. are standardizing their herbal formulations by chromatography techniques like TLC/HPLC finger printing, etc. There are about 7000 firms in the small-scale sector manufacturing traditional medicines with or without standardization. However, in recent years very few of the pharmaceutical companies is standardizing the herbal medicines using active compounds as markers linked with confirmation of bioactivity of herbal drugs in experimental animal models [8].

The global trade of medicinal and related plant materials was estimated to be of the approximate value 62 billion USD in 2001. China has been successful in acquiring the single largest share in this export market because of its well-designed national policy on the traditional Chinese medicine. Indian share of the world herbal trade is less than 1%. Even here, the export of herbal products is largely in the form of raw herbs with 2/3rd of the export basket comprising raw herbs. China’s share in world herbal market is USD 6 billion while India’s share is only USD1 billion. India followed by China is the largest producer of medicinal plants, having more than 40% of global diversity so ideally it should have the second largest share, but the country lags far behind China owing to its unorganised trade system, lack of standardization of formulations and inadequate policy.

But still India’s export of formulations has reached 16,647.36 Crores till March 2008 and can increase manifold in the coming years if proper standardization techniques are followed [9].

The Indian market is growing at 15-20% per annum (Rs 7,000 million or 150 million) with world demand growing at 1% annually (USD 610 million), the size of export market for medicinal plants appears bigger than the Indian domestic market [10].

For Indian herbal industry, there is a huge export opportunity.

As the global market is big and expanding, the Indian herbal industry is focusing on exports. From a global perspective, critical challenges for herbal industry are:

· Regulatory concerns
o    Quality
o    Efficacy
o    Safety
o    Standardization

· Consumer perceptions

· Competition

Need of standardization:-
Traditional medicine implies knowledge and practice of herbal healing for the prevention, diagnosis, and elimination of physical, mental, or social imbalance [11]. The costs for health care are rising at an alarming rate throughout the world. At the same time, the world market for phyto-pharmaceuticals is growing steadily. Herbs, used as medicine, are also regulated under different categories throughout the world.

It is a common observation that people diagnosed with incurable chronic disease states such as diabetes, arthritis, and AIDS turned to herbal therapies for a sense of control and mental comfort from taking action [12]. Herbal product studies cannot be considered scientifically valid if the product tested has not been authenticated and characterized in order to ensure reproducibility in the manufacturing of the product. Several studies have indicated quantitative variations in marker constituents in herbal preparations. Moreover, many dangerous and lethal side effects have recently been reported, including direct toxic effects, allergic reactions, effects from contaminants, and interactions with drugs and other herbs. [13]

Recent surveys reported in the American news media indicated that a large percentage of the public would like to see products supported by clinical research. This means consumers are increasingly demanding products of known quality [14]. There is a strong demand and need to accelerate the research in phytomedicine. Standardized herbal products of consistent quality and containing well-defined constituents are required for reliable clinical trials and also to provide consistent beneficial therapeutic effects. Pharmacological properties of an herbal formulation depend on phytochemical constituents present therein. 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. Without consistent quality of a phytochemical mixture, a consistent pharmacological effect is not expected. Standardization is the first step for the establishment of a consistent biological activity, a consistent chemical profile, or simply a quality assurance program for production and manufacturing [15].

Therefore, the European Union has defined three categories of herbal products:

Those containing constituents (single compounds or families of compounds) with known and experienced therapeutic activity that are deemed solely responsible for clinical efficacy.

Those containing chemically defined constituents possessing relevant pharmacological properties those are likely to contribute to the clinical efficacy.

Those in which no constituents have been identified as being responsible for the therapeutic activity [16].

A system to ensure that every packet of medicine that is being sold has the correct substances in the correct amount and will induce its therapeutic effect this is known as standardization. It is very important that a system of standardization is established for every plant medicine in the market because the scope for variation in different batches of medicine is enormous. Plant material may vary in its phytochemical content and therefore in its therapeutic effect according to different places of collection, with different times in a year for collection, with collection at the same time and places but in different years and with different environmental factors surrounding the cultivation of a particular medicinal plant. Adding to this variability is the fact that in herbal medicine several plants are used together in the same preparation. This means that there should be a quality control test for the entire preparation to ensure quality of the product.

World Health Organization (WHO) encourages, recommends and promotes traditional herbal remedies in national health care programmes because these drugs are easily available at low cost, safe and people have faith in them.

Alternative medicine is the need of the day, but improper authentication of herbs, adulterations by microorganism, pesticide residue has made standardization of herbal drug of primary importance. At present no official standard are available for the herbal preparation. Manufactures those who are doing some testing of their formulation, have fixed their own parameter, most of them are only preliminary in nature. It is very difficult to identify the presence of all the ingredients as claimed in a formulation. Hence, the first important task is to evolve such parameter by which the presence of the entire ingredient can be got identified various Chromatographic and Spectrophotometric methods and evaluation of physicochemical properties can be tried to evolve pattern for identifying the presence of different ingredient.

Herbal formulations are difficult to standardize as the plant is a biosynthetic laboratory for chemical compounds like glycosides, alkaloids etc. These exert physiological and therapeutic effect. So it will be very difficult if we want to estimate each and every ingredient in term of their chemical constituent. But if few major constituents having particular therapeutic action indicated in the labelled can be pinpointed then these constituents should be estimated quantitatively along with the other parameters through which presence of all ingredients can be confirmed. Combined, well coordinated efforts from scientific workers of different disciplines are required for this purpose.

It is the cardinal responsibility of the regulatory authorities to ensure that the consumers get the medication, which guarantee purity, safety, potency and efficacy and this duty can be discharged by rigidity following various standards of quality prescribed for raw materials and finished products in pharmacopoeias controlling manufacturing formulate through the use of formularies and manufacturing operation through statutory imposed “Good manufacturing practices”. All these procedure logically would be apply to all type of medication whether included in modern system of medicine or one of the traditional system such as Ayurvedic system of medicine.

Unfortunately the Ayurvedic pharmacopoeias and the formulary have been exempted from the standard attained by present day Modern medicine, which is continuously undergoing metabolic changes and improvements in the standard of purity, safety and efficacy. Thus maintaining the quality of Ayurvedic medication becomes the sole responsibility of the manufacture. The quality control of crude drugs and herbal formulations is of paramount importance in justifying their acceptability in modern system of medicine.

Furthermore, the constituents responsible for the claimed therapeutic effects are frequently unknown or only partly explained. These complex positions of quality aspects of herbal drugs are further complicated by the use of combination of herbal ingredients. It is not uncommon to have as many as five different herbal ingredients in one product. Thus batch to batch variation starts from the collection of raw material itself in the absence of any reference standard for identification. These variations multiply during storage and further processing.

The task of lying down standards for quality control of herbal crude and their formulation involves biological evaluation for a particular disease area, chemical profiling of the material and lying down specification for the finished product. Therefore, in case of herbal drugs and product, the word “Standardization” should encompass entire field of study from cultivation of medicinal plant to its clinical application.

Plant material and herbal remedies derived from them represent substantial portion of global market and in this respect internationally recognized guidelines for their quality assessment and quality control are necessary. In order to overcome certain inevitable shortcoming of the Pharmacopoeial monograph other quality control measures must be explored.

Key Parameters for Herbal drugs/products:-

Standardization

Quality evaluation

Standardization: -

Adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity respectively by adding excipients or by mixing herbal drug extracts.

Also used to describe all measures which are taken during the manufacturing process and Quality Control leading to a reproducible quality [17]

Quality evaluation: - A systematic examination of the extent to which an entity (part or product) is capable of meeting specified requirements. The result of quality evaluation may be used for qualification, approval and registration or accreditation purposes. It is absolutely required for authenticity, purity, safety. It comprises of Quality Control and Quality Assurance [17]

Efficient Standardisation and Quality control of Herbal products can be achieved by adopting a 6 point protocol consisting of:

* Standardisation and authentification of ingredients
(Triple ‘P’ based screening)

* SOP development for manufacturing process of Herbal Products
(Extraction, Distillation, Calcination, Fermentation etc)

* Standardization of Formulations
(Triple ‘P’ based screening, especially Chemical and Chromatographic Profiling, Marker compounds, Quantitative studies)

* Stability (shelf life) studies
(Shelf life evaluation based on physic-chemical, microbial and HPTLC/HPLC Fingerprints)

* Efficacy
(Pharmacological screening data and documented traditional use)

* Safety
(Toxicological data and documented experience) [17].

The European Medicines Agency(EMEA) defines marker compounds as “chemically defined constituents of a herbal drug which are of interest for control purposes, independent of whether they have any therapeutic activity or not”. Examples of markers are the valerenic acids in Valeriana officinalis and piperine in Piper longum etc. [16, 18, and 19].

Marker compounds are pure, single isolated compounds, secondary metabolites mostly with terpene, steroid, alkaloid, flavonoid, aromatic & heteroaromatic frameworks having alcoholic, carbonyl, olefinic, acid, ester & amide functionalities. Marker compounds of ingredients not necessarily the markers for formulations. Some undergo transformations and decompose, vanish or generate new markers. Marker compounds specific to Multiherbal Formulations need to be isolated. Formation of new markers depends on the various Manufacturing processes.

Marker compounds may be used to help identify herbal materials, set specifications for raw materials, standardize herbal formulations during all aspects of manufacturing processes and obtain stability profiles [20].

Generally three types of Markers:-
* Chemical markers
* Bio markers
* Bio-active markers (Therapeutic markers)

Markers compounds new classification:-

* Chemically modified markers generated by
- Dealkylation
- Deacetylation
- Dehydration
- Isomerisation

* Hybrid markers generated by
- Condensation
- Esterification
- Glycosilation

* Pseudo markers generated by
- Degradation
- Decomposition
- Polymerisation

* Impurity markers - These are negative markers formed by
- Pyrolysis

Impurity markers-These are particularly useful for lavana, ksara, bhasma which are prepared by burning the plant ingredients.

* Markers generation by Isolation & spectroscopic techniques are used such as Gravity columns, Preparative layers; Flash / MPLC / HPLC; Liquid – Liquid Chromatography like DCCC / HSCSS; IR/UV/1D and 2D NMR, Mass and their quantification by chromatographic techniques like TLC; HPTLC / AMD; HPLC; GC & GC-MS; TLC – FID/FPD; Capillary Electrophoresis [17]

Fingerprint Analysis for Standardization-
It has potential to determine authenticity and reliability of chemical constituents of herbal drug and formulations [21]. It addresses the systematic and comprehensive nature of herbal medicines, so it has been internationally accepted as one of the efficient methods to control the quality of herbal medicines [22]. Chromatographic and Spectroscopic technologies are still two main methods for establishing the fingerprint, including TLC, HPLC, GC, CE, IR, NIR, NMR, as well as DNA fingerprinting. Nowadays, more and more hyphenated technologies are used such as GC-MS, HPLC-MS, HPLC-DAD-MS, and LC-NMR. Especially, the hyphenation of MS with HPLC or GC has been a very useful means to the chemical constituents’ analysis, quality control and metabolite studies, etc. [23-27]. Sometimes, a single fingerprint is inadequate for the effective analysis of complex herbal medicine, so multiple chromatographic fingerprints with different test conditions in the same or different separation principles have been proposed.

CONCLUSION:-
The pharmacopoeial standards in Ayurvedic Pharmacopoeia of India are not adequate enough to ensure the quality of plant materials. Herbal drugs/products standardization is massively wide and deep. The drugs are primarily dispensed as water decoction or ethanolic extract or in the form of fresh plant parts, juice or crude powder. Thus medicinal plant parts should be authentic and free from harmful materials like pesticides, heavy metals, microbial or radioactive contamination, etc. The extracts should then be checked for indicated biological activity in an experimental animal model(s) as polyherbal formulations contain more than 2 herbs so their combined effect should be studied. It should be then standardized on the basis of active principle or major compound(s) along with fingerprints using chromatographic and spectroscopic methods and stabilization with a minimum shelf-life of over a year, undergo regulatory or limited safety studies in animals. Determination of the probable mode of action will explain the therapeutic profile. The herbal medicines developed in this mode should be dispensed as prescription drugs or even OTC products depending upon disease consideration and under no circumstances as health foods or nutraceuticals.

Herbal medicine is making dramatic comeback and increasing number of patients are visiting alternative medicine clinics as the side effects of synthetic medicine are alarming. Under the prevailing circumstances futher investigations into the concept of polyherbal formulations should be undertaken. It can be concluded that polyherbal formulations should not be dismissed only on the basis that they do not withstand modern research. Ayurveda and herbal medicine has roots in medicinal herbs and they have been practiced for centuries.There is need for development of techniques which includes both traditional methods of evaluation and modern methods of evaluation. This will improve the quality of the drug and also motivates the practitioners to get more involved in the standardization process.

Since marketed polyherbal formulation(s) contains numbers of ingredients so by optimizing the formulation the no. of ingredients can be minimized and the no. of chemical constituents contained within it making Standardization process easier and there will be no need for unnecessary having so many drugs for an ailment and their adverse effects can be reduced. So by Standardization of Herbal drugs/products, their acceptance by the consumers will increase thereby increasing the Indian herbal market and the export of Indian Herbal Products to the world market making India the Pharma Power House till 2020.

REFERENCES:-
1.   A. Agarwal. Critical issues in Quality Control of Herbal Products. Pharma Times. 37(6). 2005: pp. 09-11
2.   P.K. Mukherjee. Exploring Botanicals in Indian System of Medicine-Regulatory Perspectives. Clinical Research   regulatory affairs 20 (3). 2003 : 249-264
3.   C. Marwick. Growing use of medicinal botanicals forces assessment by drug regulators   medical news and perspectives, JAMA 273(8). 1995: 607–610
4.   S. Agarwal and R.H. Singh. Proceedings of International Congress, Ayurveda, 28–30th January 2002, Abstract No. 209: 221: 2002
5.   K.J. Kumar. Bottle Necks in Standardization of Traditional System of Medicines, Research J. Of Med. Plant DOI: 10.3923/rjmp 2011.
6.   Florey, H. W., Chain, E. and Florey, M. E., 1949. Vol. 1. The antibiotic, Oxford University Press. New York. P p. 576-628.
7.   Drugs and Pharmaceuticals – Industry Highlights Incorporating Patent Information, CDRI, Lucknow, 1998, vol. 21, pp. 33–34.
8.   V.P. Kamboj. Herbal medicine, Curr Sci 78(1): 35-39. 2000.
9.   Report of the Task Force, Ministry of Commerce & Industry, December 12, 2008.
10.   Department of Indian System of Medicine and Homeopathy Draft National Policy 2001: www.indianmedicine.nic.in
11.   O. Akerele. Summary of WHO Guidelines for the Assessment of Herbal Medicines. In HerbalGram 28: 13-16. 1992
12.   JS Brown, SA Marcy. The use of botanicals for health purposes by members of a prepaid health plan. Res Nurs Health. 1991;14: 339
13.   S. Bent, R. Ko. Commonly used herbal medicines in the United States: a review. Am. J. Med. 116, 478. 2004.
14.   V. S. Srinivasan. Challenges and scientific issues in the standardization of botanicals and their preparations. United States Pharmacopeia's dietary supplement verification program—A public health program. Life Sci. 78, 2039. 2006.
15.   G. Ulrich-Merzenich, H. Zeitler, D. Jobst, D. Panek, H. Vetter, H. Wagner. Phytomedicine 14, 70 (2007).
16.   EMEA. Position paper on the risks associated with the use of herbal products containing Aristolochia species. EMEA/HMPWP/23/00 (2000).
17.   Dr. U. V. Mallavadhani. Standardisation and Quality Control of Multiherbal Formulations. Herbal drugs & Bio-Remedies Cell, Regional research laboratory, Bhubaneswar – 751013, India
18.   E. Ernst. Herb-drug interactions: potentially important but woefully under-researched. Eur. J. Clin. Pharmacol. 56,   523. 2000.
19.   E. Ernst. Adulteration of Chinese herbal medicines with synthetic drugs: a systematic review. J. Int. Med. 252,   107. 2002.
20.   N.J. Lazarowych, and P. Pekos. Use of fingerprinting and marker compounds for identification and standardization of botanical drugs: Strategies for applying pharmaceutical HPLC analysis to herbal products, Drug Inf. J 32: 497-512. 1998.
21.   T. Rajkumar, and B.N. Sinha. Chromatographic finger print analysis of budmunchiamines in Albizia amara by HPTLC technique, Int. J. Res. Pharm. Sci 1(3): 313-316. 2010.
22.   C. C. Teo, S. N. Tan, J.W.H. Yong, C. S. Hew, E. S. Ong. 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: 34- 40. 2008.
23.   P. Drasar, and J. Moravcova. Recent advances in analysis of Chinese medical plants and traditional medicines, J. Chromatogr. B 812: 3-21 2004.
24.   W.Yang, M. Ye, M. Liu, D. Kong, R. Shi, X. Shi, K. Zhang, Q. Wang, and Z. Lantong. A practical strategy for the characterization of coumarins in Radix Glehniae by liquid chromatography coupled with triple quadrupolelinear ion trap mass spectrometry, J. Chromatogr. A 1217: 4587-4600. 2010.
25.   X. M. Liang, Y. Jin, Y.P. Wang, G.W. Jin, Q. Fu, and Y.S. Xiao. Qualitative and quantitative analysis in quality control of traditional Chinese medicines, J. Chromatogr. A 1216: 2033-2044. 2009.
26.   J. L. Zhou,L.W. Qi1, and P.Li. Herbal medicine analysis by liquid chromatography/time-offlight mass spectrometry, J. Chromatogr. A 1216: 7582-7594. 2009.
27.   Z.D. Zeng, Y.Z. Liang, F.T. Chau, S. Chen, D.K.W. Mok, C.O. Chan. Mass spectral profiling: An effective tool for quality control of herbal medicines, Anal. Chim. Acta 604: 89- 98. 2007.