Comprehensive high performance thin layer chromatography (HPTLC) fingerprinting in quality control of herbal drugs, preparations and products

[eng] Quality control of herbals has its roots in the study of morphoanatomic and organoleptic characters. Nevertheless, in the last century, with the evolution of analytical chemistry, the quality control rapidly evolved from elementary tests to the use of sophisticated instruments combined with software for data management. In the current days, many authorities and organizations recommend a suite of tests, featuring many of these instruments, to evaluate quality of herbal products. HPTLC offers a comprehensive set of data that can be used not only for identification but also to evaluate the purity and content of herbal drugs, herbal preparations, and herbal products. The objective of this doctoral thesis was to explore in-depth the capacities of HPTLC and develop applications for quality control of herbals, far beyond simple identification of the herbal drugs, preparations, and products. For that, five studies were developed. In the first study, the quality of herbal drugs, preparations, and products from milk thistle fruit, coneflower root and aerial parts and black cohosh root, regulated under food supplements or medicines were evaluated with existing HPTLC methods. The suitability of these methods, using the entire fingerprint and several detection modes, as a tool for detecting quality problems, mainly adulterations, was confirmed. In the second study, the comprehensive HPTLC fingerprinting concept was developed with the goal of simplifying the quality control process. This concept combines the qualitative and quantitative information of HPTLC images, obtained in a single analysis, to evaluate the identity, purity and content of herbals. The possibilities of applying it to identify an herbal drug, detect mixtures with re¬lated species (purity), and develop a minimum content test of an analytical marker were demonstrated in Angelica gigas root. In the third study, the application of comprehensive HPTLC fingerprinting aimed to go one step beyond in the test for adulterants and to evaluate the use of the HPTLC for purity limit tests. This approach was evaluated with sam¬ples of ginkgo leaf and extracts, commercialized as food supplements in different countries. This study demonstrated that the information contained in the HPTLC finger¬prints was suitable for verifying levels of rutin and quercetin, providing results similar to that of the HPLC limit test. It was also useful for detecting mixtures of ginkgo products not only with rutin and quercetin but also with buckwheat herb and sophora (flower bud or fruit). In the fourth study, it was evaluated the use of comprehensive HPTLC fingerprinting as an alternative method to the current HPLC assay of markers of TCM drugs in the Ph. Eur. The goal of this project was to simplify the determination of content and thus reducing the number of tests to be performed during quality control. For this evaluation, two TCM herbal drugs were chosen by the experts of the TCM working party of the Ph. Eur.: Fritillaria thunbergii bulbs and corydalis rhizome. In both cases, comprehensive HPTLC fingerprinting was proven useful for identification and minimum content testing in one single analysis. The fifth study goes one step beyond in the content determination. While the previous studies focused in the quantification of single markers, this study aimed to apply comprehensive HPTLC fingerprinting to quantify a group of constituents in an herbal drug, as an example of a more holistic assessment of quality. This determination was combined with the tests for purity and identity. To illustrate this concept, Ganoderma lucidum fruiting body was chosen. In this work, HPTLC proved to be a useful technique for routine quality control of herbal drugs, preparations and products. As demonstrated, it can simplify this process by applying the concept of comprehensive HPTLC fingerprinting. A detailed guideline of how to develop, validate and apply comprehensive HPTLC fingerprinting methods for routine quality control of herbals has been elaborated and is also included in the thesis.  [cat] El control de qualitat dels productes a base de plantes té les seves arrels en l'estudi dels caràcters morfoanatòmics i organolèptics. No obstant això, al segle passat, amb l'evolució de la química analítica, el control de qualitat va evolucionar ràpidament des de proves elementals a l'ús d'instruments sofisticats combinats amb programari per a la gestió de dades. Actualment, moltes autoritats i organitzacions recomanen un conjunt de proves, amb molts d'aquests instruments, per avaluar la qualitat dels productes a base de plantes. La HPTLC ofereix un conjunt complet de dades que poden usar-se no només per a la identificació, sinó també per avaluar la puresa i el contingut de drogues i preparats vegetals i productes a base de plantes. L'objectiu d'aquesta tesi doctoral va ser explorar en profunditat les capacitats de la HPTLC i desenvolupar aplicacions per al control de qualitat dels productes de plantes medicinals, molt més enllà de la simple identificació de drogues i preparats vegetals i productes acabats comercialitzats. Per això, es van desenvolupar cinc estudis. En el primer estudi, es va avaluar la qualitat de les drogues vegetals, preparats vegetals i productes a base de plantes del fruit de card marià, l'arrel i la part aèria de equinàcia i l'arrel de cimicífuga, regulats com complements alimentosos o medicaments, amb els mètodes existents de HPTLC. Es va confirmar la idoneïtat d'aquests mètodes, utilitzant l'empremta dactilar completa i diversos formes de detecció, com una eina per a detectar problemes de qualitat, principalment adulteracions. En el segon estudi, es va desenvolupar el concepte d'anàlisi integral de l'empremta dactilar per HPTLC (comprehensive HPTLC fingerprinting) amb l'objectiu de simplificar el procés de control de qualitat. Aquest concepte combina la informació qualitativa i quantitativa de les imatges d’HPTLC, obtingudes en una única anàlisi, per avaluar la identitat, la puresa i el contingut dels productes a base de plantes. La seva aplicabilitat per identificar una droga vegetal, detectar mescles amb espècies relacionades (puresa) i desenvolupar un assaig de contingut mínim d'un marcador analític es van demostrar en l'arrel d'Angelica gigas. En el tercer estudi, l'aplicació de l'anàlisi integral de l'empremta dactilar per HPTLC va tenir com a objectiu anar un pas més enllà en l'assaig de adulterants i avaluar l'ús de l’HPTLC per a l'assaig límit de puresa. Aquest enfocament es va avaluar amb mostres de fulla i extracte de ginkgo, comercialitzats com a complements alimentosos en diferents països. Aquest estudi va demostrar que la informació continguda en les empremtes dactilars per HPTLC era adequada per verificar els nivells de rutina i quercetina, proporcionant resultats similars als de l'assaig límit per HPLC. També va ser útil per detectar mescles de productes de ginkgo no només amb rutina i quercetina, sinó també amb part aèria de blat sarraí i sòfora (botó floral i fruit). En el quart estudi, es va avaluar l'ús de l'anàlisi integral de l'empremta dactilar per HPTLC com un mètode alternatiu a l'actual valoració de marcadors per HPLC en drogues vegetals de la medicina tradicional xinesa (MTC) a la Ph. Eur. L'objectiu d'aquest projecte era simplificar la determinació del contingut i, per tant, reduir el nombre de proves a realitzar durant el control de qualitat. Per a aquesta avaluació, dues drogues vegetals de la MTC van ser elegides pels experts del grup de treball TCM de la Ph. Eur.: bulb de Fritillaria thunbergii i rizoma de coridalis. En tots dos casos, es va demostrar que l'empremta dactilar completa per HPTLC era útil per a la identificació i l'assaig de contingut mínim en una sola anàlisi. El cinquè estudi va un pas més enllà en la determinació del contingut. Si bé els estudis anteriors es van centrar en la quantificació de marcadors individuals, aquest estudi va tenir com a objectiu aplicar l'anàlisi integral de l'empremta dactilar per HPTLC a la quantificació d'un grup de components en una droga vegetal, com un exemple d'una avaluació més holística de la qualitat. Aquesta determinació es va combinar amb els assajos d'identitat i puresa. Per il·lustrar aquest concepte, es va triar el carpòfor de Ganoderma lucidum. En aquest treball, s'ha demostrat que la HPTLC és una tècnica útil per al control de qualitat rutinari de drogues i preparats vegetals i productes a base de plantes, i que es pot simplificar aquest procés aplicant el concepte d'anàlisi integral de l'empremta dactilar per HPTLC. S'ha elaborat una guia detallada (inclosa a la tesi) sobre com desenvolupar, validar i aplicar mètodes d'anàlisi integral de l'empremta dactilar per HPTLC per al control de qualitat rutinari de productes a base de plantes.[spa] El control de calidad de los productos a base de plantas tiene sus raíces en el estudio de los caracteres morfoanatómicos y organolépticos. Sin embargo, en el siglo pasado, con la evolución de la química analítica, el control de calidad evolucionó rápidamente de las pruebas elementales al uso de instrumentos sofisticados combinados con software para la gestión de datos. Actualmente, muchas autoridades y organizaciones recomiendan un conjunto de pruebas, con muchos de estos instrumentos, para evaluar la calidad de los productos a base de plantas. La HPTLC ofrece un conjunto completo de datos que pueden usarse no sólo para la identificación, sino también para evaluar la pureza y el contenido de drogas y preparados vegetales y productos a base de plantas. El objetivo de esta tesis doctoral fue explorar en profundidad las capacidades de HPTLC y desarrollar aplicaciones para el control de calidad de los productos de plantas medicinales, mucho más allá de la simple identificación de drogas vegetales, preparados vegetales y productos finales comercializados. Para eso, se desarrollaron cinco estudios. En el primer estudio, se evaluó la calidad de las drogas vegetales, preparados vegetales y productos a base de plantas del fruto del cardo mariano, la raíz y la parte aérea de equinácea y la raíz de cimicífuga, regulados como complementos alimenticios o medicamentos, con los métodos existentes de HPTLC. Se confirmó la idoneidad de estos métodos, utilizando la huella digital completa y varios modos de detección, como una herramienta para detectar problemas de calidad, principalmente adulteraciones. En el segundo estudio, se desarrolló el concepto de análisis integral de la huella dactilar por HPTLC (comprehensive HPTLC fingerprinting) con el objetivo de simplificar el proceso de control de calidad. Este concepto combina la información cualitativa y cuantitativa de las imágenes de HPTLC, obtenidas en un único análisis, para evaluar la identidad, la pureza y el contenido de los productos a base de plantas. Su aplicabilidad para identificar una droga vegetal, detectar mezclas con especies relacionadas (pureza) y desarrollar un ensayo de contenido mínimo de un marcador analítico se demostraron en la raíz de Angelica gigas. En el tercer estudio, la aplicación del análisis integral de la huella dactilar por HPTLC tuvo como objetivo ir un paso más allá en el ensayo de adulterantes y evaluar el uso de la HPTLC para el ensayo límite de pureza. Este enfoque se evaluó con muestras de hoja y extracto de ginkgo, comercializados como complementos alimenticios en diferentes países. Este estudio demostró que la información contenida en las huellas dactilares por HPTLC era adecuada para verificar los niveles de rutina y quercetina, proporcionando resultados similares a los del ensayo límite por HPLC. También fue útil para detectar mezclas de productos de ginkgo no sólo con rutina y quercetina, sino también con parte aérea de trigo sarraceno y sófora (botón floral y fruto). En el cuarto estudio, se evaluó el uso del análisis integral de la huella dactilar por HPTLC como un método alternativo a la actual valoración de marcadores por HPLC en drogas vegetales de la medicina tradicional china (MTC) en la Ph. Eur. El objetivo de este proyecto era simplificar la determinación del contenido y, por lo tanto, reducir el número de pruebas a realizar durante el control de calidad. Para esta evaluación, dos drogas vegetales de la MTC fueron elegidas por los expertos del grupo de trabajo TCM de la Ph. Eur.: bulbo de Fritillaria thunbergii y rizoma coridalis. En ambos casos, se demostró que la huella digital completa de HPTLC era útil para la identificación y el ensayo de contenido mínimo en un solo análisis. El quinto estudio va un paso más allá en la determinación del contenido. Si bien los estudios anteriores se centraron en la cuantificación de marcadores individuales, este estudio tuvo como objetivo aplicar el análisis integral de la huella dactilar por HPTLC a la cuantificación de un grupo de componentes en una droga vegetal, como un ejemplo de una evaluación más holística de la calidad. Esta determinación se combinó con los ensayos de identidad y pureza. Para ilustrar este concepto, se eligió el carpóforo de Ganoderma lucidum. En este trabajo, se ha demostrado que la HPTLC es una técnica útil para el control de calidad rutinario de drogas y preparados vegetales y productos a base de plantas, y que se puede simplificar este proceso aplicando el concepto de análisis integral de la huella dactilar por HPTLC. Se ha elaborado una guía detallada (incluida en la tesis) sobre cómo desarrollar, validar y aplicar métodos de análisis integral de la huella dactilar por HPTLC para el control de calidad rutinario de productos a base de plantas

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