Use of high-performance thin layer chromatography by the American Herbal Pharmacopoeia

TLC characterizations are among the key identity tests in most pharmacopoeial monographs. Pharmacopoeial standards are typically used by industry as a basis for meeting QC requirements and current good manufacturing practices (cGMPs). TLC is a relatively low-cost, highly versatile tool for developing specifications for raw materials, as well as for the various preparations for which pharmacopoeial standards are created. In addition to its use in the development of identity tests, TLC is a valuable tool for screening plant samples that pharmacopoeias must review in the development of monographs and botanical reference materials (BRMs). Specifically, HPTLC is the ideal TLC technique for these purposes because of its increased accuracy, reproducibility, and ability to document the results, compared with standard TLC. Because of this, HPTLC technologies are also the most appropriate TLC technique for conformity with GMPs. This article highlights the manner in which HPTLC is used by the American Herbal Pharmacopoeia (AHP) in the development of AHP monograph identity standards, the identification of adulterating species, and the development of AHP-verified BRMs.     

An approach to comparative analysis of chromatographic fingerprints for assuring the quality of botanical drugs

The present study was focused on developing the chemometric methods for analysis of the chromatographic fingerprint to control the quality of botanical drugs, which has gained attention in Asia and other countries. We developed a novel approach to generate a set of fingerprint features, called Fisher components (FCs) that were extracted from the chromatographic fingerprint. The method greatly reduces the dimensionality of the fingerprint vector, and the resulting FCs still retain most discriminatory information of the original fingerprint. Choosing an example of relevance to contemporary botanical drugs, we applied the FCs to a set of Shenmai injection samples. We successfully identified the manufacturers of the samples using two classifiers, linear discriminant analysis (LDA) and k-Nearest Neighbor (k-NN) based on the FCs. We also applied a similarity assessment together with the visual analysis using the FCs to exam the products from different manufacturers. We found that the lot-to-lot consistency of products can be accurately determined using the FCs. Finally, we demonstrated that the application of chemometric methods for chromatographic fingerprinting offers reliability to detect suspected fraud samples. In summary, we demonstrated that the presented approaches could be useful to determine the identity, consistency, and authenticity of Shenmai injection through chromatographic fingerprinting. The methods are equally applicable to other botanical drugs.     

Differentiating the gum resins of two closely related Indian Gardenia species, G. gummifera and G. lucida, and establishing the source of dikamali gum resin using high-performance thin-layer chromatography and ultra-performance liquid chromatography-UV/MS

Dikamali is a gum resin obtained from the leaf buds of Gardenia lucida or G. gummifera. There is controversy regarding the botanical source of this gum resin with some stating it to be from G. lucida while others claim it to be from G. gummifera. Analytical methods including UPLC and HPTLC were developed for the qualitative analysis of Gardenia species and various commercial samples. The separation using a UPLC method was achieved within 12.0 min by using C18 column material, a water/acetonitrile mobile phase, both containing formic acid, a gradient system, and a temperature of 40 degrees C. Extensive studies of dikamali collected from various parts of India in comparison with the gum resins collected from G. lucida and G. gummifera clearly indicated that the botanical source of commercially available dikamali is G. lucida, not G. gummifera. The marker compounds isolated from a market sample of dikamali were present only in the gum resin of G. lucida and the compounds isolated from G. gummifera were not present in any of the dikamali samples, confirming the botanical source of dikamali. This work is of utmost importance, given the ambiguity regarding the botanical source of the gum resin dikamali. LC/MS coupled with electrospray ionization is described for the identification and confirmation of nine compounds from various samples of the gum resin. An HPTLC method was also developed for the fast chemical fingerprint analysis of Gardenia samples.     

Tradition- and science-based quality control of Chinese medicines--introducing the Phyto-True system

The current QC practice of quantifying presumed active chemicals or arbitrarily selected chemical markers is of doubtful value in assessing multicomponent complex traditional Chinese medicines (CMs) and often leads to an inconsistent or irreproducible research and clinical outcome. Consequently, the first and most important step in the QC of CMs (or other botanical medicines) whose exact active chemical components are unknown is to use analytical techniques that can comprehensively define the totality of the components/attributes making up their identity and quality. One of the most versatile techniques is HPTLC. Using HPTLC, along with other simple techniques such as FTIR spectroscopy and UV-Vis spectroscopy combined with complementary gene expression profiling, we have been able to correctly identify CM materials, detect adulterants, and differentiate closely related materials and botanical species. Our research has resulted in the introduction of the concept and specimens of Phyto-True Reference Material (PTRM), aka Representative Botanical Reference/Research Material (RBRM), now commercially available, and a novel patent-pending technology (Phyto-True system) that can serve as a starting point for the meaningful QC of traditional CMs so far not possible for these complex materials. Examples will be highlighted to demonstrate this new concept.     

Chemical fingerprinting of Equisetum arvense L. using HPTLC densitometry and HPLC

Equisetum arvense L. is a herbaceous medicinal plant, commonly known as horsetail, whose extracts have been reported to possess diuretic and haemostatic properties. The aim of this study was to evaluate the use of fingerprint chromatographic methods on commercially available raw materials or preparations of E. arvense L. in order to ascertain their quality and identify possible adulterants using HPLC and HPTLC densitometry. Two chromatographic methods were used to determine the chemical fingerprints of E. arvense and other allied species. The first was based on HPTLC identification followed by densitometric measurement at 350?nm. The second was based on HPLC separation. The ease of sample preparation and the possibility of simultaneous analysis of several samples in a short time make HPTLC a method of choice for the comprehensive quality evaluation of herbal products.     

Analysis of flavonol aglycones and terpenelactones in Ginkgo biloba extract: A comparison of high-performance thin-layer chromatography and column high-performance liquid chromatography

Advancements in automated high-performance thin-layer chromatography (HPTLC) have made it feasible to assess its use for the quantitative analysis of marker compounds in botanical preparations. We report here the findings of method comparisons for the terpenelactones and flavonol aglycones by column high-performance liquid chromatography (HPLC) with evaporative light scattering and UV detection, and HPTLC with a scanning densitometer. For the HPTLC assay of terpenelactones, total bilobalide, ginkgolide A, and ginkgolide B consistently achieved <70% of the total determined using HPLC, regardless of variations to postchromatographic derivatization time and temperature. Accuracy testing showed the possibility of a matrix interference. In contrast, a good relationship (95%) was determined between HPTLC and HPLC for determination of total flavonol glycosides (calculated from combined quercetin, kaempferol, and isorhamnetin) from an acid-hydrolyzed Ginkgo biloba L. (GBE) sample. The HPTLC flavonol aglycone method also performed well in terms of accuracy (overall average of 96% recovery for the 3 aglycones) and consecutive plate repeatability (overall percent relative standard deviation of 4.4). It is demonstrated that HPTLC can be a time-saving complement to HPLC for routine analysis of the flavonol glycosides in GBE.     

Application of HPTLC Multiwavelength Imaging and Color Scale Fingerprinting Approach Combined with Multivariate Chemometric Methods for Medicinal Plant Clustering According to Their Species

In the current study, multiwavelength detection combined with color scales HPTLC fingerprinting procedure and chemometric approach were applied for direct clustering of a set of medicinal plants with different geographical growing areas. The fingerprints profiles of the hydroalcoholic extracts obtained after single and double development and detection under 254 nm and 365 nm, before and after selective spraying with specific derivatization reagents were evaluated by chemometric approaches. Principal component analysis (PCA) with factor analysis (FA) methods were used to reveal the contribution of red (R), green (G), blue (B) and, respectively, gray (K) color scale fingerprints to HPTLC classification of the analyzed samples. Hierarchical cluster analysis (HCA) was used to classify the medicinal plants based on measure of similarity of color scale fingerprint patterns. The 1-Pearson distance measurement with Ward’s amalgamation procedure proved to be the most convenient approach for the correct clustering of samples. Data from color scale fingerprints obtained for double development procedure and multiple visualization modes combined with appropriate chemometric methods proved to detect the similar medicinal plant extracts even though they are from different geographical regions, have different storage conditions and no specific markers are individually extracted. This approach could be proposed as a promising tool for authentication and identification studies of plant materials based on HPTLC fingerprinting analysis.     

Fingerprint profiling of acid hydrolyzates of polysaccharides extracted from the fruiting bodies and spores of Lingzhi by high-performance thin-layer chromatography

Modern extraction and planar chromatographic instrumentation were employed for the fingerprint profiling of carbohydrates from an important and popular medicinal mushroom commonly known as Lingzhi. For the first time, the feasibility of employing the high-performance thin-layer chromatography (HPTLC) peak profiles (fingerprints) of carbohydrates for the screening of various Lingzhi species/products was demonstrated. An analytical procedure was developed such that upon acid hydrolysis of the polysaccharides extracted from various Lingzhi samples, fingerprint profiles that reveal the relative amounts of the degradation products, such as mono- and oligosaccharides, can be obtained using HPTLC plates (Si 50000) for separation and 4-aminobenzoic acid as the post-chromatographic derivatization reagent for detection. Also, using automated multiple development (AMD), the acid hydrolyzates from Lingzhi, consisting of simple and more complex sugars, can be separated simultaneously with high degree of automation. An important finding was that unique fingerprint patterns were observed in the monosaccharide profiles between two highly valued Lingzhi species, Ganoderma applanatum and Ganoderma lucidum, under total or partial acid hydrolysis conditions. Additionally, the HPTLC fingerprint profiles of carbohydrates were obtained from the extracts of the spores and fruiting bodies of Lingzhi and compared.     

Determination of vitamin A (retinol) in infant formula and adult nutritionals by liquid chromatography: First Action 2011.15

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," held on June 29, 2011, an Expert Review Panel (ERP) reviewed the method for the "Determination of Vitamins A (Retinol) and E (alpha-Tocopherol) in Foods by Liquid Chromatography: Collaborative Study," published by Jonathan W. DeVries and Karlene R. Silvera in J. AOAC Int. in 2002. After evaluation of the original validation data, an ERP agreed in June 2011 that the method meets standard method performance requirements (SMPRs) for vitamin A, as articulated by the Stakeholder Panel on Infant Formula and Adult Nutritionals. The ERP granted the method First Action status, applicable to determining vitamin A in ready-to-eat infant and adult nutritional formula. In an effort to achieve Final Action status, it was recommended that additional information be generated for different types of infant and adult nutritional formula matrixes at varied concentration levels as indicated in the vitamin A (retinol) SMPR. Existing AOAC LC methods are suited for specific vitamin A analytical applications. The original method differs from existing methods in that it can be used to assay samples in all nine sectors of the food matrix. One sector of the food matrix was powdered infant formula and gave support for the First Action approval for vitamin A in infant and adult nutritional formula. In this method, standards and test samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated by an LC method, using UV detection at 313 or 328 nm for retinol. Vitamin concentration is calculated by comparison of the peak heights or peak areas of retinol in test samples with those of standards.     

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg^?1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg^?1. Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg^?1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n?=?6), mean intra-day precision 3.2 % (n?=?3 within a plate; n?=?2 repeated within a day), mean inter-day precision 3.7 % (n?=?3), mean reproducibility over the whole procedure including sample preparation 4.1 % (n?=?2), and mean recovery 106.9 % (n?=?5 different concentrations; n?=?4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

Simultaneous Determination of Caffeine and Paracetamol in Commercial Formulations Using Greener Normal-Phase and Reversed-Phase HPTLC Methods: A Contrast of Validation Parameters

There has been no assessment of the greenness of the described analytical techniques for the simultaneous determination (SMD) of caffeine and paracetamol. As a result, in comparison to the greener normal-phase high-performance thin-layer chromatography (HPTLC) technique, this research was conducted to develop a rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in commercial formulations. The greenness of both techniques was calculated using the AGREE method. For the SMD of caffeine and paracetamol, the greener normal-phase and reversed-phase HPTLC methods were linear in the 50–500 ng/band and 25–800 ng/band ranges, respectively. For the SMD of caffeine and paracetamol, the greener reversed-phase HPTLC approach was more sensitive, accurate, precise, and robust than the greener normal-phase HPTLC technique. For the SMD of caffeine paracetamol in commercial PANEXT and SAFEXT tablets, the greener reversed-phase HPTLC technique was superior to the greener normal-phase HPTLC approach. The AGREE scores for the greener normal-phase and reversed-phase HPTLC approaches were estimated as 0.81 and 0.83, respectively, indicated excellent greenness profiles for both analytical approaches. The greener reversed-phase HPTLC approach is judged superior to the greener normal-phase HPTLC approach based on numerous validation parameters and pharmaceutical assays.     

Study on triterpenoic acids distribution in Ganoderma mushrooms by automatic multiple development high performance thin layer chromatographic fingerprint analysis

Ganoderma--"Lingzhi" in Chinese--is one of the superior Chinese tonic materia medicas in China, Japan, and Korea. Two species, Ganoderma lucidum (Red Lingzhi) and G. sinense (Purple Lingzhi), have been included in the Chinese Pharmacopoeia since its 2000 Edition. However, some other species of Ganoderma are also available in the market. For example, there are five species divided by color called "Penta-colors Lingzhi" that have been advocated as being the most invigorating among the Lingzhi species; but there is no scientific evidence for such a claim. Morphological identification can serve as an effective practice for differentiating the various species, but the inherent quality has to be delineated by chemical analysis. Among the diverse constituents in Lingzhi, triterpenoids are commonly recognized as the major active ingredients. An automatic triple development HPTLC fingerprint analysis was carried out for detecting the distribution consistency of the triterpenoic acids in various Lingzhi samples. The chromatographic conditions were optimized as follows: stationary phase, precoated HPTLC silica gel 60 plate; mobile phase, toluene-ethyl acetate-methanol-formic acid (15 + 15 + 1 + 0.1); and triple-development using automatic multiple development equipment. The chromatograms showed good resolution, and the color images provided more specific HPTLC fingerprints than have been previously published. It was observed that the abundance of triterpenoic acids and consistent fingerprint pattern in Red Lingzhi (fruiting body of G. lucidum) outweighs the other species of Lingzhi.     

Identification of the plant origin of the botanical biomarkers of Mediterranean type propolis

Propolis is a honeybee product which bees produce by collecting resins from various botanical sources. The chemical composition of propolis is directly dependant on the availability of resinous plant materials in different geographic regions. This study was undertaken to evaluate the resinous plant sources used by bees to produce Mediterranean type propolis. Although this propolis type has already been the subject of numerous studies, its major botanical source had not yet been identified. In this study, using GC-MS analysis, we identify the resin of the common cypress, Cupressus_sempervirens, as the major plant source of the characteristic diterpene fingerprint profile of Mediterranean propolis.     

Lipid analysis by thin-layer chromatography--a review of the current state

High-performance thin-layer chromatography (HPTLC) is a widely used, fast and relatively inexpensive method of separating complex mixtures. It is particularly useful for smaller, apolar compounds and offers some advantages over HPLC. This review gives an overview about the special features as well as the problems that have to be considered upon the HPTLC analysis of lipids. The term "lipids" is used here in a broad sense and comprises fatty acids and their derivatives as well as substances related biosynthetically or functionally to these compounds. After a short introduction regarding the stationary phases and the methods how lipids can be visualized on an HPTLC plate, the individual lipid classes will be discussed and the most suitable solvent systems for their separation indicated. The focus will be on lipids that are most abundant in biological systems, i.e. cholesterol and its derivates, glycerides, sphingo- and glycolipids as well as phospholipids. Finally, a nowadays very important topic, the combination between HPTLC and mass spectrometric (MS) detection methods will be discussed. It will be shown that this is a very powerful method to investigate the identities of the HPTLC spots in more detail than by the use of common staining methods. Future aspects of HPTLC in the lipid field will be also discussed.     

Separation and quantitation of alkylphosphocholines and analogues of different liposome formulations by HPTLC

High-performance thin-layer chromatographic (HPTLC) analysis of non UV-active phospholipids in biological matrixes is a common method for separation, detection, and quantitation. Liposomes containing new alkylphosphocholines and analogues with enhanced cytostatic activity had been prepared. The liposomal formulations were designed to enable the intravenous application of the alkylphosphocholines and analogues and to reduce dose-limiting toxicities observed after oral administration. For quality control the liposomes were analyzed by HPTLC for content of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), cholesterol, alkylphosphocholines, and analogues and their related compounds (main degradation products). Due to the differences in lipophily of the compounds, different mobile phases were necessary to achieve separation. Automated Multiple Development was used to reduce the number of plates and to improve the selectivity and the capacity of the chromatographic system to separate the described alkylphosphocholines and analogues from DPPG and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in one chromatographic system.     

Quantitative high-performance thin-layer chromatography of lipids in plasma and liver homogenates after direct application of 0.5-microliter samples to the silica-gel layer.

Lipid profiles were determined by high-performance thin-layer chromatography (HPTLC) after direct application of 0.5 microliter plasma from capillary blood to the silica-gel layer. Coefficients of variation for the fluorescence measurements were 2.1% for phosphatidylcholine. The recovery of known amounts of lipid was 96--100%. A linear relationship between peak area and amount of lipid was found in the nmole range, corresponding to the amount of lipid in 0.125--0.75 microliter Lipid-Trol, which served as the standard reference sample. The plasma lipids of healthy subjects and of patients suffering from various illnesses were analyzed using reference methods and HPTLC. Identical values were obtained for cholesterol esters, triacylglycerols and phosphatidylcholine. Free cholesterol values determined by HPTLC were slightly lower (7%). The correlation between data obtained by reference methods and HPTLC was as follows: cholesterol, r = 0.938; cholesterol esters, r = 0.964; triacylglycerols, r = 0.985; phosphatidylcholine, r = 0.938. The separaiton and quantitation of liver lipids using HPTLC after direct application of the tissue homogenate to the silical-gel layer was carried out. Comparison with reference methods revealed that HPTLC gave higher cholesterol values (24%). The triacylglycerol concentrations, however, were identical under both methods and correalted satisfactorily (r = 0.959).     

Pterostilbene: A Highly Reliable Quality-Control Marker for the Ayurvedic Antidiabetic Plant `Bijasar'

Summary In the Indian Ayurvedic system of medicine the heart wood of Bijasar (Lat: Pterocarpus marsupium; Fam: Leguminaceae) has been extensively used to treat diabetes mellitus. Detailed chemical and HPTLC analysis has been performed to identify a reliable marker for quality control of Pterocarpus marsupium and to check commercial samples for authenticity and purity. The two anti-diabetic principles viz. pterostilbene (1) and marsupsin (2) were isolated by extensive chromatographic screening of its ethyl acetate extract. HPTLC studies of these two compounds revealed that levels of pterostilbene are several times more than that of marsupsin both in plant samples and in commercial formulations. The HPTLC method gave reproducible results and response was linearly dependent on amount analysed in the range 200–500 ng. Pterostilbene can therefore be regarded as a reliable marker for evaluation of the quality of Pterocarpus marsupium.     

High-performance thin layer chromatography for quality control of multicomponent herbal drugs: example of cangzhu xianglian san

Due to their complexity, multicomponent herbal drugs pose enormous analytical challenges for quality control (QC). Although they may have traditionally been used for hundreds of years, the information about their chemical composition is often still limited. Selecting suitable markers to monitor the identity and potency of the mixture is, therefore, difficult. There is also the possibility of natural variability for each plant. This paper illustrates a pragmatic and practical approach to QC of a multicomponent herbal drug by HPTLC. Cangzhu xianglian xan (CXS), composed of the herbal drugs Coptis rhizome, Aucklandia root, and Atractylodes rhizome (30 + 20 + 60, w/w/w), is used as an example. A characteristic fingerprint can be generated for CXS with toluene-ethyl acetate-methanol-isopropanol-water (60 + 30 + 20 + 15 + 3, v/v/v/v/v) mobile phase on HPTLC silica gel 60 conditioned with ammonia. While the corresponding monograph of the Chinese Veterinary Pharmacopoeia focuses only on the detection of berberine, one of the principal components of Coptis rhizome, the proposed method of identification determines the presence of all three components in the drug after derivatization with anisaldehyde reagent. The same method can also be used to quantitatively determine the content of berberine by scanning densitometry. This paper provides details about the validation of the qualitative and quantitative determinations.     

Holographic electron density shape theorem and its role in drug design and toxicological risk assessment

Each complete, boundaryless molecular electron density is fully determined by any nonzero volume piece of the electron density cloud. This inherent feature of molecules, called the "holographic" property of molecular electron densities, provides a strong foundation for the local, quantum chemical shape analysis of various functional groups, pharmacophores, and other local molecular moieties. A proof is presented for the relevant molecular shape theorem, the "holographic electron density shape theorem", and the role of this theorem in quantum chemical, quantitative shape-activity relations (QShAR) is discussed. The quantum chemical methods of molecular shape analysis can be extended to ab initio quality electron densities of macromolecules, such as proteins, as well as to local molecular moieties, such as functional groups or pharmacophores, based on the transferability and additivity of local, fuzzy density fragments and the associated local density matrixes within the framework of the ADMA (Adjustable Density Matrix Assembler) approach. In addition to new results on chemical bonding and the development of macromolecular force methods, the new methodologies are also applicable to QShAR studies in computer-aided drug discovery and in toxicological risk assessment.     

High-performance thin-layer chromatographic fingerprints of isoflavonoids for distinguishing between Radix Puerariae Lobate and Radix Puerariae Thomsonii

The roots of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth have been officially recorded in all editions of Chinese Pharmacopoeia under the same monograph 'Gegen' (Radix Puerariae, RP). However, in its 2005 edition, the two species were separated into both individual monographs, namely 'Gegen' (Radix Puerariae Lobatae, RPL) and 'Fenge' (Radix Puerariae Thomsonii, RPT), respectively, due to their obvious content discrepancy of puerarin, the major active constituent. In present paper, the fingerprint of high-performance thin-layer chromatography (HPTLC) combining digital scanning profiling was developed to identify and distinguish the both species in detail. The unique properties of the HPTLC fingerprints were validated by analyzing ten batches of Pueraria lobata and P. thomsonii samples, respectively. The common pattern of the HPTLC images of the roots of Pueraria spp. and the respective different ratios of the chemical distribution can directly discern the two species. The corresponding digital scanning profiles provided an easy way for quantifiable comparison among the samples. Obvious difference in ingredient content and HPTLC patterns of the two species questioned their bio-equivalence and explained that recording both species separately in the current edition of Chinese Pharmacopoeia (2005 edition) is reasonable due to not only the content of major constituent, puerarin, but also the peak-to-peak distribution in the fingerprint and integration value of the total components. Furthermore, the HPTLC fingerprint is also suitable for rapid and simple authentication and comparison of the subtle difference among samples with identical plant resource but different geographic locations.