An HPTLC Method for the Evaluation of Two Medicinal Plants Commercially Available in the Indian Market Under the Common Trade Name Brahmadandi

Brahmadandi is an important medicinal plant used in the Indian system of medicine (Ayurveda) for the treatment of numerous diseases. A literature search revealed that different plants are available on the market under the trade name Brahmadandi viz., roots of Echinops echinatus Roxb, and the aerial parts of Tricholepis glaberrima DC which are sold either in their crude or in powdered form. Currently, no analytical procedures appear to be available for quality control purposes. In the present communication, we report a simple HPTLC method for the quantification of the lupeol content in the aforementioned plant species. The method was validated for precision, repeatability and accuracy. Instrument precision and repeatability of the method were found to be 0.56 and 2.87 %RSD, respectively. Intra-day and inter-day precision of the method was determined to be in the ranges of 0.71–2.02 and 1.03–2.02 %RSD, respectively. Accuracy of the method was evaluated by a recovery study conducted at three different levels. The mean percentage recovery was found to be 100.85%. The developed HPTLC method for estimation of lupeol was found to be simple, precise and accurate and may be useful for routine quality control of the commercial samples of Brahmadandi.     

A Rapid Densitometric Method for the Quantification of Luteolin in Medicinal Plants Using HPTLC

Luteolin, a flavonoid, is reported to occur widely in many medicinal plants. It has been shown to have important biological activities. We report sensitive HPTLC method for the quantification of luteolin from plant material. The method was validated for precision, repeatability and accuracy. The method was found to be precise with RSDs for intraday in the range of 0.77–1.29% and inter–day in the range of 1.02–2.08%. Instrumental precision and repeatability of the method were found to be 0.39 and 0.57 (%CV). Accuracy of the method was checked by recovery study conducted at two different levels and the average percentage recovery was found to be 100.92%. The method was used for quantification of luteolin in three important herbal drugs viz. fruit of Cuminum cyminum, whole plant of Bacopa monnieri, flower of Achillea millefolium. The proposed HPTLC method for the quantification of luteolin was found to be simple, precise, specific, sensitive and accurate and can be used for quality control of raw materials.Revised: 1 October 2003 and 18 February 2004     

A rapid and highly sensitive stability-indicating high-performance thin-layer chromatography technique for the determination of tedizolid phosphate with a classical univariate calibration

No high-performance thin-layer chromatography (HPTLC) techniques have been established for the determination of tedizolid phosphate (TDZP) in pharmaceutical products or physiological fluids. Therefore, a rapid and highly sensitive stability-indicating HPTLC technique has been developed for the determination of TDZP in commercial formulations with a classical univariate calibration. The HPTLC‒densitometry analysis of TDZP was carried out via chloroform‒methanol (90:10, V/V) mobile phase. The determination of TDZP was performed at the wavelength of 300 nm. The proposed HPTLC technique was linear in the range of 10‒2000 ng band^‒1. In addition, the method was found to be highly accurate (% recovery = 98.53‒101.74%), precise (%CV = 0.67‒0.91%), robust (%CV = 0.83‒0.86%), highly sensitive (LOD = 3.41 ng band^‒1, LOQ = 10.23 ng band^‒1) for the determination of TDZP. The proposed technique was also able to detect TDZP in the presence of its degradation products under various stress conditions and it can be considered as a stability-indicating method. The proposed HPTLC technique was applied for the analysis of TDZP in its commercial formulations. The TDZP contents of commercial tablets and injection were determined as 98.41% and 101.23%, respectively. These results suggested that the proposed HPTLC technique can be applied for the routine analysis of TDZP in its commercial products and newly established formulations.

     

Phytochemical Profiling and Quality Control of Terminalia sericea Burch. ex DC. Using HPTLC Metabolomics

Terminalia sericea is used throughout Africa for the treatment of a variety of conditions and has been identified as a potential commercial plant. The study was aimed at establishing a high-performance thin layer chromatography (HPTLC) chemical fingerprint for T. sericea root bark as a reference for quality control and exploring chemical variation within the species using HPTLC metabo3lomics. Forty-two root bark samples were collected from ten populations in South Africa and extracted with dichloromethane: methanol (1:1). An HPTLC method was optimized to resolve the major compounds from other sample components. Dichloromethane: ethyl acetate: methanol: formic acid (90:10:30:1) was used as the developing solvent and the plates were visualized using 10% sulfuric acid in methanol as derivatizing agent. The concentrations of three major bioactive compounds, sericic acid, sericoside and resveratrol-3-O-β-rutinoside, in the extracts were determined using a validated ultra-performance liquid chromatography-photodiode array (UPLC-PDA) detection method. The rTLC software (written in the R-programming language) was used to select the most informative retardation factor (Rf) ranges from the images of the analysed sample extracts. Further chemometric models, including principal component analysis (PCA) and hierarchical cluster analysis (HCA), were constructed using the web-based high throughput metabolomic software. The rTLC chemometric models were compared with the models previously obtained from ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). A characteristic fingerprint containing clear bands for the three bioactive compounds was established. All three bioactive compounds were present in all the samples, although their corresponding band intensities varied. The intensities correlated with the UPLC-PDA results, in that samples containing a high concentration of a particular compound, displayed a more intense band. Chemometric analysis using HCA revealed two chemotypes, and the subsequent construction of a loadings plot indicated that sericic acid and sericoside were responsible for the chemotypic variation; with sericoside concentrated in Chemotype 1, while sericic acid was more abundant in Chemotype 2. A characteristic chemical fingerprint with clearly distinguishable features was established for T. sericea root bark that can be used for species authentication, and to select samples with high concentrations of a particular marker compound(s). Different chemotypes, potentially differing in their therapeutic potency towards a particular target, could be distinguished. The models revealed the three analytes as biomarkers, corresponding to results reported for UPLC-MS profiling and thereby indicating that HPTLC is a suitable technique for the quality control of T. sericea root bark.     

Efficient and Sensitive Method for Quantitative Determination and Validation of Umbelliferone, Carvone and Myristicin in Anethum graveolens and Carum carvi Seed

An efficient HPTLC method for the analysis of umbelliferone, carvone and myristicin in Anethum graveolens and Carum carvi seed was developed. The method employed HPTLC plates precoated with silica gel 60 F₂₅₄ as the stationary phase. Methanol extracts of seeds from three different sources were used. The calibration plot for umbelliferone, carvone and myristicin were linear with the correlation coefficient of 0.997 ± 0.016, 0.999 ± 0.009 and 0.999 ± 0.013, respectively, which were indicative of good linear dependence of peak area on concentration. The method permits reliable quantification of umbelliferone, carvone and myristicin and showed good resolution and separation. The method was validated as per ICH guidelines. To study the accuracy of the method, recovery studies were performed by the method of standard addition at three different levels and the average percentage recovery was found to be 99.05% for umbelliferone, 100.28% for carvone and 99.8% for myristicin. The proposed HPTLC method for quantitative monitoring of umbelliferone, carvone and myristicin in A. graveolens and C. carvi seed can be used for routine quality testing of these extracts.     

Densitometric Determination of Hecogenin from Agave americana Leaf Using HPTLC

We report a simple TLC densitometric method for the quantification of hecogenin from the leaves of Agave americana using HPTLC. The method was validated for precision, repeatability and accuracy. The method was found to be precise with RSD of 0.78 (intraday) and 0.82 (interday) for different concentrations of hecogenin. Instrumental precision was 0.42 (% RSD) for hecogenin. The content of hecogenin in different samples was estimated by the proposed method and was found to be in the range of 0.05−0.14% w/w in the samples analysed. Accuracy of the method was checked by conducting recovery study at three different levels for hecogenin and the average percentage recovery was 98.98%, 101.92% and 103.33%, respectively. The TLC densitometric method developed for the quantification of hecogenin was found to be simple, precise, specific, sensitive, accurate and can be used in routine quality control. Revised: 7 and 25 April 2006     

Determination and Quantification of Pitavastatin Calcium in Tablet Dosage Formulation by HPTLC Method

Abstract

A simple, sensitive, reliable, and rapid HPTLC method has been developed for the determination of pitavastatin calcium in tablet dosage form. Identification and determination were performed on aluminum backed silica gel 60F₂₅₄ washed with methanol. The mobile phase of ethyl acetate‐methanol‐ammonia‐1 drop formic acid (7:2:0.8) calibration plots were established showing the dependence of response (peak area) on the amount chromatographed. The spot were scanned at 245 nm. The method has a linear range of 50–250 ng/spot. The method was validated for selectivity, repeatability, and accuracy. The method was used for determination of the compound in commercial pharmaceutical dosage forms. It is a more effective option than other chromatographic techniques in routine quality control.     

Quantification of Valerenic Acid in Valeriana jatamansi and Valeriana officinalis by HPTLC

A simple, rapid, cost-effective and accurate high performance thin layer chromatographic method has been developed for quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis which is one of the stable compounds of Valeriana officinalis and designated as a key marker compound. Valerenic acid makes substantial contribution to the sedative and spasmolytic activity of the essential oil and extract of Valeriana officinalis. Separation and quantification was achieved by HPTLC using ternary mobile phase of hexane: ethyl acetate: acetic acid (80:20:0.5 v/v) on precoated silica gel 60F₂₅₄ aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent at 700 nm, in absorption-reflectance mode. The calibration curves were linear in the range of (500 ng–2.5 μg). This is the first HPTLC report for the identification and quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis.     

Quantitative Determination of Four Water-Soluble Compounds in Herbal Drugs from Lamiaceae Using Different Chromatographic Techniques

HPTLC-densitometric and HPLC–UV techniques were used for qualitative and quantitative determination of luteolin-7-O-glucuronide, lithospermic acid, rosmarinic acid and caffeic acid in several herbal drugs from the Lamiaceae family: Thymi herba, Serpylli herba, Majoranae herba and Menthae piperitae folium. Unmodified silica gel (HPTLC Si60) and silica gel chemically modified with aminopropyl groups (HPTLC NH₂) were used during the investigation process. Among HPTLC methods the best resolution and selectivity was achieved with mobile phases: diisopropyl ether–acetone–formic acid–water (50:30:10:10, v/v/v/v) and acetone–formic acid (85:15, v/v), respectively. Plates were densitometrically evaluated. Contents of analyzed compounds in the studied aqueous extracts prepared from herbal drugs were established using both techniques. The results from the HPTLC-densitometric analysis have been compared with those from HPLC–UV on a C18 column with acetonitrile–water–formic acid as a mobile phase. The chromatographic methods were validated for linearity, LOD, LOQ, repeatability, intermediate precision and recovery. An analysis of variance showed that the HPTLC-densitometric and HPLC–UV methods are equivalent and sufficiently precise for the estimation of polyphenolic compounds mentioned above, in investigated herbal drugs. All of the suggested methods (HPTLC NH₂, HPTLC Si60 and HPLC RP18) give results with good agreement.     

Detection and characterization of recombinant DNA expressing <Emphasis Type="Italic">vip3A</Emphasis>-type insecticidal gene in GMOs—standard single,multiplex and construct-specific PCR assays

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An application for an Indian patent (1891/DEL2006/17.08.07) comprising a substantive part of this study has been filed. ITRC communication no. 2516.     

Qualitative and quantitative analyses of aloe-emodin,rhein, and emodin in qi yin granules by high-performance thin-layer chromatography

The aim of this study was to perform qualitative and quantitative analyses of aloe-emodin, rhein, and emodin in three prepared samples of compound qi yin granules by high-performance thin-layer chromatography (HPTLC) and to establish an analytical method. TLC was used to qualitatively analyze the three major components of the compound: aloe-emodin, rhein, and emodin. HPTLC was performed to determine the contents of the three components. HPTLC analysis showed that using Anhui Liangchen high-efficiency silica gel G plate was the optimal stationary phase and the upper layer solution of a petroleum ether–ethyl acetate–formic acid (15.5:5:1, V/V) mixed solution was the optimal developing agent. The composition of the samples for testing was basically the same, but the content was different. In summary, this study used HPTLC to qualitatively and quantitatively analyze aloe-emodin, rhein, and emodin in compound qi yin granules. It can lay the foundation for improving the quality control and standards of compound qi yin granules.

     

On testing quality and traceability of virgin olive oil by calorimetry

Extra Virgin olive oils (7 samples) originating from different areas of Tuscany, defective olive oils (5 samples), commercial edible seed oils (4 samples) and two commercial samples of olive oil (one declared ‘extra virgin olive oil’ and one ‘olive oil’) were studied by different calorimetric techniques: high sensitivity isothermal, differential scanning, and modulated scanning calorimetry. The temperature interval (–60) – (+30)°C was explored for monitoring: i) the main features of the liquid↔solid phase transitions, ii) the nucleation and growth rate of the polymorphous crystalline phases of the triacylglicerols, and iii) the melting process. This investigation was planned for verifying the utility and effectiveness of calorimetry for screening quality and origin of olive oil. To this end, the main calorimetric operation modes have been applied, the experimental results reported and their utility for developing an effective and reliable screening protocol discussed.     

Dynamic NMR investigation of two new interconvertible diasteriomeric epimers of natural 2-benzyl-2-hydroxybenzofuranone derivative from Pterocarpus marsupium

The first example of a pair of interconvertible diastereomeric epimers 2α/2β-hydroxy-2-p-hydroxybenzyl-3(2H)-benzofuranone-7-C-β-d-glucopyranoside isolated from the heartwood of Pterocarpus marsupium is reported. The predominance of 1a over 1b was supported by dynamic exchange rates and activation parameters obtained from NMR studies. The mechanism of this unique phenomenon is thought to be operative by the formation of diketone as suggested by deuterium exchange.     

Evaluation of traditional Chinese herbal medicine: Chaihu (Bupleuri Radix) by both high-performance liquid chromatographic and high-performance thin-layer chromatographic fingerprint and chemometric analysis

Chaihu (Bupleuri Radix), roots of Bupleurum chinense and B. scorzonerifolium, is an authentic Chinese Materia Medica in the Chinese Pharmacopoeia. Some other species such as the roots of B. falcatum, B.bicaule and B. marginatum var. stenophyllum similar to Chaihu can also be occasionally found in local raw herb markets. The quality of 33 lots of authenticated Chaihu samples vs. 31 lots of commercial samples was evaluated by both high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) and high-performance thin-layer chromatography (HPTLC) analyses of its principal bioactive components (saikosaponins). The pre-treated data acquired from both HPLC fingerprints and HPTLC fluorescent images were processed by chemometrics for similarity and pattern recognition, including Artificial Neural Networks (ANNs), k-nearest neighbor (k-NN) and an expert’s panel. It was apparent that k-NN classifier exhibited good performance with sufficient flexibility for processing HPTLC fingerprint images which were otherwise not easily dealt with by other algorithms due to the shift of R_f values and varying hue/saturation of the band colours between different TLC plates. These two chromatographic fingerprint methods can be considered complementary measure of quality control. The roots of Chaihu from different species of the genus Bupleurum could readily be distinguished from each other so that commercial samples can easily be classified. Chaihu collected from several major herbal distribution centers was found to belong to B. chinense with great variation in the content of its major saikosaponins.     

New insights into sugar characterization in complex plant matrices by high-performance thin-layer chromatography

High-performance thin-layer chromatography (HPTLC) is an innovative, green, reliable, and rapid method for the characterization of complex sugar mixtures. For research into sustainable nontimber forest products, Adansonia digitata dry fruit pulp in this proof-of-concept study was used to demonstrate this method due to its reported high nutritional value and prebiotic activity. Chromatographic separations were performed on (20?×?10?cm²) HPTLC glass plates with diol stationary phase, using a 15-step gradient by mixing solutions acetone/acetonitrile 1:1, and ultrapure water for enzymatic degradation products. Instead, monosaccharide moieties were separated on HPTLC silica gel 60?F₂₅₄ over isocratic mode with 80?mm of eluent front composed of acetonitrile/acetic acid/water 63/33/5?v/v/v. Identification and quantification were performed by densitometry acquisitions at 400?nm after opportune derivatization. Our research highlighted that this technique can be used as a standard method to gain new insights into inulin determinations, with several advantages over existing conventional liquid chromatography. The development of innovative methods for the characterization of biopolymers is crucial, for food and nutraceutical industry and for quality control of phytochemicals. Characterization of such materials, up until now, has been done by mass difference and so accurate analytical methods were lacking.     

Quality control and original discrimination of Ganoderma lucidum based on high-performance liquid chromatographic fingerprints and combined chemometrics methods

In this paper, the feasibility and advantages of employing high-performance liquid chromatographic (HPLC) fingerprints combined with chemometrics methods for quality control of the cultured fruiting bodies of Ganoderma lucidum were investigated and demonstrated for the first time. In order to compare the HPLC fingerprints chromatograms between G. lucidum from different origins, the similarities of all the 60 samples and relative peak areas of 19 characteristic compounds were firstly calculated respectively. Then different pattern recognition procedures, including hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least squares-discrimination analysis (PLS-DA) and soft independent modeling of class analogy (SIMCA) were applied to classify the G. lucidum samples according to their cultivated origins. Consistent results were obtained to show that G. lucidum samples could be successfully grouped in accordance with the province of origin. Furthermore, four marker constituents were screened out to be the most discriminant variables, which could be applied to accurate discrimination and quality control of G. lucidum by quantitative analysis. Finally, the chemical properties of those samples were also investigated to find out the differences of quality between them. Ranked in decreasing order, the quality of the G. lucidum can be arranged as Jinzhai/Huangshan, Shandong followed by Zhejiang samples. Our results revealed that the developed method has potential perspective for the original discrimination and quality control of G. lucidum.     

High-performance thin-layer chromatography method for the quantification of quetiapine fumarate and its related genotoxic impurities using green solvents

A novel high-performance thin-layer chromatographic (HPTLC) analytical method has been developed and optimized for the quantification of quetiapine fumarate (QF) and its two genotoxic impurities in drug substance and drug product. The desired separation was achieved on 60F₂₅₄ pre-coated HPTLC plates using combination of green solvents, ethyl acetate‒ethanol‒n-heptane (5:1:4, V/V) as developing solvents. The detection wavelength used for quantification was 229 nm. QF and its two related genotoxic impurities, namely, 2-chloroaniline and 2-aminodiphenylsulfide, were well resolved from one another with retention factor values of 0.13 ± 0.02, 0.57 ± 0.02 and 0.76 ± 0.02, respectively. The optimized method was validated according to the guidelines laid down by the International Council for Harmonisation. The linearity was determined in the range of 100–600 ng/spot for QF and 10‒60 ng/spot for its two related genotoxic impurities; R² ≥ 0.993. The method exhibited precision along with good accuracy, where 0.51, 0.86 and 1.86. The percentage recoveries obtained for 2-chloroaniline and 2-aminodiphenylsulfide were 99.04‒101.04%. The developed method can be successfully used for the analysis of drug samples.

     

Development of a HPTLC method to profile the phytochemicals in Allanblackia parviflora (tallow tree) kernel and seed cakes

HPTLC is a useful and practical analytical tool to characterize plant compositions. This study was focused on exploring the results of high-performance thin-layer chromatography (HPTLC) analysis, particularly as a useful tool for the authentication of Allanblackia parviflora seed and kernel cakes. Bulked samples from sixteen different Ghanaian communities were analysed by HPTLC and their fingerprints were compared. The optimum experimental conditions were established: sample weight of 2.0 g, methanol:water (80:20 v/v) as extraction solvent, 30 min extraction time and twice extraction, ethyl acetate:methanol:water (100:16.5:13.5 v/v) as mobile phase, vanillic acid as derivatisation agent and 7 min of plate heating time after derivatisation. The HPTLC profile generated from extracts across 16 communities and 157 trees was very reproducible and demonstrates the robustness of the technique in characterising the profile.

     

Differential content of secondary metabolites in diploid and tetraploid cytotypes of Siegesbeckia orientalis L.

Siegesbeckia orientalis L. is an annual herb widely distributed throughout the world and has many medicinal properties. In Chinese traditional system, it is popularly known as Xi-Xian and used for its anti-inflammatory properties. In the present study, two cytotypes (diploid and tetraploid) have been investigated for their secondary metabolites. The different plant parts have been explored in terms of total phenolics, total flavonoids, DPPH radical scavenging acitivity and total antioxidant capacity. Out of different plant parts, leaves have the maximum amount of secondary metabolites and antioxidant potential. HPTLC technique has been applied to quantify six marker compounds in the two cytotypes. Tetraploid cytotype has been compared with diploid cytotype, which shows that tetraploid has the maximum amount of studied secondary metabolites with high antioxidant potential.     

XAD-2 HPTLC Method of Identification and Determination of Some Synthetic Food Colourings

Abstract

An HPTLC method for identification and determination of 12 synthetic dyes (Amaranth, Allura red, Brilliant black BN, Brilliant blue E, Carmoisine, Erythrosine, Indigotine, Patent blue V, Ponceau 4R, Quinoline yellow, Sunset yellow, Tartazine) has been developed. Amberlite XAD-2 was used as a stationary phase for column chromatography. Elution was performed with various acidified alcohols (i-propanol, n-propanol, n-butanol) and recoveries between 81.5 - 100.2% were obtained. The low limit of detection is in the range of 4 – 10 ng. The method was tested on commercial products as: carbonated soda, candies, chewing gum, identical natural flavours, etc.