New rapid validated HPTLC method for the determination of galanthamine in Amaryllidaceae plant extracts

The work reported in this paper aims at developing an accurate, specific, repeatable and robust HPTLC method for the determination of galanthamine in different Amaryllidaceae plant extracts.     

Chemical fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and densitometry

Introduction –  Lawsonia inermis L. is a natural red colouring agent, commonly named “Henna”, which is used to dye skin and hair. The aim of this study was to evaluate the quality of L. inermis that is commercially available as a raw plant material or preparation in order to guarantee good quality products. Objective ?  To develop a simple protocol for the qualification of different samples labelled as L. inermis by using the HPTLC densitometry method and to identify possible adulterations with other plants. Methodology ?  Samples of leaves of L. inermis were extracted with methanol. Two chromatographic methods were developed to determine the chemical fingerprinting of L. inermis. The first was based on HPTLC identification followed by densitometric measurements at 337 nm. The second was based on RP‐HPLC separation with gradient elution and photodiode array detection at 337 nm. Samples of Cassia obovata Collad., and Indigofera tinctoria L., were treated in the same way. Results –  The simplicity of the sample preparation, and the possibility of analysing several samples of herbal products simultaneously in a short time, make HPTLC the method of choice. The HPTLC method was feasible for the comprehensive quality evaluation of herbal products. From the comparison of their “fingerprint”, it was possible to detect substitution of plants that are different from those declared on the label. Conclusion ?  The HPTLC may be used as a rapid method by which to control the quality of raw plant materials and formulations based on the title plant. Copyright © 2008 John Wiley & Sons, Ltd.     

An o-toluidine method for detection of carbohydrates in protein hydrolysates

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.     

Oxyphor R2 and G2: phosphors for measuring oxygen by oxygen-dependent quenching of phosphorescence

Microparticles in the circulation activate the coagulation system and may activate the complement system via C-reactive protein upon conversion of membrane phospholipids by phospholipases. We developed a sensitive and reproducible method to determine the phospholipid composition of microparticles. Samples were applied to horizontal, one-dimensional high-performance thin-layer chromatography (HPTLC). Phospholipids were separated on HPTLC by chloroform:ethyl acetate:acetone:isopropanol:ethanol:methanol:water:acetic acid (30:6:6:6:16:28:6:2); visualized by charring with 7.5% Cu-acetate (w/v), 2.5% CuSO(4) (w/v), and 8% H(3)PO(4) (v/v) in water; and quantified by photodensitometric scanning. Erythrocyte membranes were used to validate the HPTLC system. Microparticles were isolated from plasma of healthy individuals (n = 10). On HPTLC, mixtures of (purified) phospholipids, i.e., lysophosphatidylcholine, phosphatidylcholine (PC), sphingomyelin (SM), lysophosphatidylserine, phosphatidylserine, lysophosphatidylethanolamine, phosphatidylethanolamine (PE), and phosphatidylinositol, could be separated and quantified. All phospholipids were detectable in erythrocyte ghosts, and their quantities fell within ranges reported earlier. Quantitation of phospholipids, including extraction, was highly reproducible (CV < 10%). Microparticles contained PC (59%), SM (20.6%), and PE (9.4%), with relatively minor (<5%) quantities of other phospholipids. HPTLC can be used to study the phospholipid composition of cell-derived microparticles and may also be a useful technique for the analysis of other samples that are available only in minor quantities.     

High-performance thin-layer chromatographic determination of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in urine and comparison with high-performance liquid chromatography

The consumption of N-ethyl-3,4-methylenedioxyamphetamine (MDE, 1), an analogue of ecstasy, can be detected by direct in situ HPTLC-FTIR measurement of the main metabolite N-ethyl-4-hydroxy-3-methoxyamphetamine (HME, 2). HME (2) can, like the other important metabolite 3,4-methylenedioxyamphetamine (MDA, 3) and unchanged MDE (1), be determined quantitatively in urine by HPTLC-UV after two-step automatic development. The results have been compared with those obtained using an HPLC method. The differences were not generally significant. Small deviations were attributable to the different sample preparation methods necessary. The working range for the HPTLC method was between 0.1 and 8.2 μg/ml and for the HPLC method between 0.2 and 60.0 μg/ml. The method standard deviations were 2.66–4.91% (HPTLC) and 0.48–3.67% (HPLC).     

Standardisation of Gymnema sylvestre r. Br. with reference to gymnemagenin by high-performance thin-layer chromatography

A simple and reproducible HPTLC method for the determination of gymnemagenin (1) in Gymnema sylvestre has been developed. Components were separated on pre-coated silica gel 60 F254 plates with chloroform:methanol (9:1) and scanned using a densitometric scanner in the UV reflectance mode at 290 nm. Linearity of determination of 1 was observed in the range 4-10 microg. The average percentage recovery of 1 from an extract was 99.09 +/- 0.29, and the content of 1 in leaves of the title plant was 1.61% (dry weight).     

Analysis of cocaethylene,benzoylecgonine and cocaine in human urine by high-performance thin-layer chromatography with ultraviolet detection: a comparison with high-performance liquid chromatography

Cocaine and ethanol are frequently used at the same time, resulting in the formation of cocaethylene by transesterification. We studied the capability of high-performance thin-layer chromatography (HPTLC) to simultaneously detect cocaethylene, cocaine and benzoylecgonine in 16 urine specimens of drug addicts, previously tested as positive for benzoylecgonine at immunoenzymatic screening. Accuracy and precision, as well as detection and quantitation limits of the method, were evaluated by comparison with high-performance liquid chromatography (HPLC). HPTLC limit of quantitation was 1.0 μg/ml for the three compounds, whereas HPLC limits were 0.2 μg/ml for benzoylecgonine and cocaine, and 0.1 μg/ml for cocaethylene. The relative standard deviation (RSD) ranged from 1.03 to 12.60% and from 1.56 to 16.6% for intra- and inter-day HPTLC analysis, respectively. In the case of the HPLC method, the RSD for the intra-day precision ranged from 0.79 to 5.05%, whereas it ranged from 1.19 to 10.64% for the inter-day precision. In comparison with HPLC, HPTLC is less expensive and faster, requiring 2–3 h to analyze 10–12 samples on a single plate. In conclusion, HPTLC is suitable for determinations of the three analytes only for samples with high concentrations.     

Sensitive high-performance thin-layer chromatographic method for the estimation of diospyrin, a tumour inhibitory agent from the stem bark of Diospyros montana Roxb.

Diospyrin, a tumour inhibitory agent from the stem bark of Diospyros montana was isolated and characterised. A sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the estimation of diospyrin. The method was validated for precision (intra- and inter-day), repeatability and accuracy. The method was found to be precise, with the RSDs for intra-day in the range of 0.72–1.85% and RSDs for inter-day in the range of 1.06–2.95%, for different concentrations. Instrumental precision and repeatability of the method were found to be 0.086 and 0.937 (% CV), respectively. Accuracy of the method was checked by performing the recovery study at two levels and average percentage recovery was found to be 97.87%. The developed HPTLC method was adopted for the estimation of diospyrin content of the stem bark of D. montana from different regions, which varied from 0.35 to 0.47% (w/w) in the samples.     

Biophysical properties and finger print of Boswellia Sp. Burseraceae

For the first time a finger print analysis via high-performance thin layer chromatography (HPTLC) of Boswellia Sp. Burseraceae was accomplished. A preliminary investigation of the Boswellia Sp. Burseraceae displayed the presence of chemical constituents that could be involved in the production of innovative pharmaceuticals for an array of antiviral, anticancer, and antibacterial uses. Moreover, the finger print analysis would deem useful for establishing HPTLC standardization for natural and herbal photochemical constituents.     

Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram

A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.     

HPTLC determination of vasicine and vasicinone in Adhatoda vasica

A simple high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of the pharmacologically important quinazoline alkaloids vasicine and vasicinone in Adhatoda vasica. The assay combines the separation and quantification of the analytes on silica gel 60 GF254 HPTLC plates with visualisation under UV and scanning at 270 and 281 nm. Using this technique, the alkaloidal content of different parts of the title plant have been determined.     

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low‐cost, high‐throughput analytical methods for quality control. Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Results – Δ⁹‐THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r² = 0.9968. Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of Δ⁹‐THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars. Copyright © 2009 John Wiley & Sons, Ltd.     

Chiral Ligand‐Exchange Resolution of Underivatized Amino Acids on a Dynamically Modified Stationary Phase for RP‐HPTLC

The synthesis of Spi(τ‐dec), derived from the selective alkylation of L‐spinacine (4,5,6,7‐tetrahydro‐1H‐imidazo[4,5‐c]pyridine‐6‐carboxylic acid) at the τ‐nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ‐dec) was successfully employed in the past to prepare home‐made chiral columns for chiral ligand‐exchange high‐performance liquid chromatography. In the present article a new method is described, using Spi(τ‐dec) as a chiral selector in high‐performance thin‐layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ‐dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive. Chirality 26:313–318, 2014. © 2014 Wiley Periodicals, Inc.     

Quantitative high-performance thin-layer chromatography determination of common liposome components and critical parameters influencing the analysis results

The aim of this study was to investigate the performance of a newly devised high-performance thin-layer chromatography (HPTLC) method in quantifying common liposome membrane components, including the five phospholipids (PLs), phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, as well as cholesterol, cholesteryl hemisuccinate, and linoleic acid. Besides strictly keeping to a standardized procedure, three parameters were particularly critical for proper quantification. First, a relative humidity of higher than 60% caused migration distances to increase and reduced the resolution of the PLs on a silica-gel 60 HPTLC plate. Second, PLs underwent oxidative combustion during storage for 2 or 24 hours on an HPTLC plate, with peak losses of up to 25–44%. These losses could be prevented by storage under nitrogen and, to some extent, by the addition of the antioxidant, DL-α-tocopherol. Third, even with automated sample application, the accuracy and consistency of the application volume proved to be an important cause of error and needs routine verification. Considering these parameters, the method was found to accurately and precisely determine the composition of three different liposome preparations. The recovery was 97.2–101.8%, compared to secondary methods, and consistent over different days and with different operators (mean RSD of the recovery: 2.03?±?1.16%, n?=?9). The working range was determined to be 100–300?ng in the case of the PLs (individual limit of determination between 40 and 80?ng) and 20–60?ng in the case of cholesterol (limit of determination: 16?ng).     

High-performance thin-layer chromatography method for inositol phosphate analysis

A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol–25% ammonia solution–water (5:4:1) and substance quantities as low as 100–200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R_F values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.     

2',3'-cyclic nucleotide phosphohydrolase activity determined using an image analyzer detection system

The activity of 2',3'-cyclic nucleotide phosphohydrolase (CNPase) was assayed using high-performance thin-layer chromatography (HPTLC) and an image analyzer detection system. The assay system was used to study a possible inhibitory effect by aminoguanidine on CNPase specific activity. One advantage of using a fixed-time HPTLC system over a real-time spectrophotometric system for an enzyme activity study was that apparent inhibition of the enzyme due to interference of the assay system (chromophore inhibition, etc.) was avoided. In addition, due to the increased accuracy of the image analyzer over conventional methods of TLC plate analysis, a rapid and more accurate measurement of HPTLC plates was possible which required only nanomole amounts of substrate. Also, a digital image of each plate analyzed was stored indefinitely in the computer's memory for future reference. The measurements of CNPase specific activity made using this system compared favorably to those found in recent literature.     

High-performance thin-layer chromatographic determination of lamotrigine in serum

A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of lamotrigine (LTG) in serum is reported. The method involves extraction of the drug by ethyl acetate followed by separation on TLC silica plates using a mixture of toluene-acetone-ammonia (7:3:0.5), as eluting solvent. Densitometric analysis was carried out at 312 nm with lamotrigine being detected at Rf of 0.54. The analytical method has excellent linearity (r=0.998) in the range of 20-300 ng/spot. This assay range is adequate for analyzing human serum, as it corresponds to lamotrigine concentrations measured in human serum from epileptic patients. The method was validated for sensitivity, selectivity, extraction efficiency, accuracy and intra and inter-day reproducibility. The limit of detection and limit of quantification were found to be 6.4 and 10.2 ng, respectively. Good accuracy is reported in the range of 92.06-97.12% and high precision with %CV in range of 0.53-2.59. The method was applied for determination of serum lamotrigine levels in epileptic patients and in pharmacokinetic study of lamotrigine administered orally to rabbits.     

High performance thin layer chromatography (HPTLC), an improved technique for screening lichen substances

Abstract: High performance thin layer chromatography (HPTLC) is a method that can be used for screening lichen substances. It is as simple to use as standard TLC, but has many advantages: It is more sensitive, it is possible to run more samples in a shorter period of time, and the amount of solvent used is much smaller. The material needed and the methods used are described in detail. Horizontal chromatogram development was used. Since two of the solvents used in system B have been substituted, and since the properties of the HPTLC plates are slightly different, our results are not entirely in accordance with the standardized TLC method. A revised table for the identification of 69 lichen substances (obtained from 62 taxa) is accordingly presented.     

High performance thin layer chromatography as a method to authenticate Hoodia gordonii raw material and products

Hoodia gordonii which contains the perceived active molecule, P57, is a plant used in many weight loss products that are highly susceptible to adulteration due to increased public demand and limited availability. Rapid and simple methods for authentication and confirmation of the presence of P57 are desirable for the quality control of H. gordonii raw material and products. High performance thin layer chromatography (HPTLC) analysis of several H. gordonii raw material samples collected from different locations as well as weight loss products was carried out on silica gel plates and developed in a mobile phase of toluene:chloroform:ethanol (40:40:12.5 v/v/v). Liebermann–Burchard (LB) reagent was used as derivatising agent since it is specific for glycosides and triterpenes (such as P57) and the plates were viewed under UV light at 365 nm. This method produced good separation of the compounds in complex mixtures with well-defined bands including that of the P57 band (R_f 0.42), which was confirmed by liquid chromatography coupled to mass spectrometry (LC–MS) after preparative thin layer chromatography (TLC). All the HPTLC results obtained for the H. gordonii raw materials and products were confirmed with quantitative LC–MS analyses, which confirmed the qualitative reliability of the HPTLC method. The HPTLC method was used successfully to develop a chemical fingerprint for authentication and reliable confirmation of the presence of P57 in H. gordonii raw material and products.     

Liposomal formulations from phospholipids of Greek almond oil. Properties and biological activity

The seeds of the almond tree [(Prunus dulcis (Mill.) D. A. Webb. (syn. Prunus amygdalus)] were collected in two different periods of maturity and were studied for their lipid content. The total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and were analyzed by HPTLC coupled with a flame ionization detector (HPTLC/FID) and GC-MS. The oils were found to be rich in neutral lipids (89.9% and 96.3% of total lipids) and low in polar lipids (10.1% and 3.7% of total lipids) for the immature and mature seed oils, respectively. The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of phospholipids. GC-MS data showed that the main fatty acid for both oils was 9-octadecenoic acid (oleic acid). The unsaturated fatty acids were found as high as 89.4% and 89.7%, while the percentage of the saturated fatty acids was found 10.6% and 10.3% for the immature and mature seed oils, respectively. Liposomes were prepared from the isolated phospholipids using the thin lipid film methodology, and their physical properties were characterized. Cytotoxicity was found absent when assayed against normal and cancerous cell lines. These new formulations may have future applications for encapsulation and delivery of drugs and cosmetically active ingredients.