Precoated TLC Plates Research Articles

Pharmacognostical Study and Development of Quality Control Parameters for Sarcostemma brevistigma Wight and Arn

Objective: To develop pharmacognostical and quality control parameters for drug Sarcostemma brevistigma Wight and Arn. (Family: Asclepiadaceae) commonly known as Somlata, is rasayana drug. Materials and Methods: Stems of S. brevistigma were collected from Jamnagar, Dwarka and Bhuj in the month of November 2008 and studied for macroscopical and microscopical characters. Quantitative microscopy and physico-chemical parameters were also performed. A pregnane derivative mono- O -acetyl bregenin was isolated from stem extract and characterized by U.V., IR, NMR, Mass spectroscopy. HPTLC method was developed to quantify mono- O -acetyl bregenin and generate fingerprint profile for the drug. HPTLC was performed using precoated TLC plate (silica gel 60 F 254 ) as a stationary phase and Chloroform : Methanol (9.4 : 0.6) as a mobile phase. The plate was scanned at 366nm. Results: Macroscopically and microscopically all the samples of stems were found to be similar but differed in quantitative parameters. Stems were green, cylindrical in shape having longitudinal ridges and nodes. T.S. of stems showed papillose epidermis, rosettes of calcium oxalate, non lignified fibres and latex cavities in cortex, striated cells of inner cortex and perimedullary phloem. Stone cells and abundant rosettes of calcium oxalate were found in section passing through the node. Powder showed stone cells, rosettes, fragments of pitted xylem vessels and non lignified fibres. Mono- O -acetyl bregenin was isolated by column chromatography. HPTLC chromatogram was developed and blue fluorescent spot was visualised at R f 0.5 under UV at 366nm. Mono- O -acetyl bregenin content was found to be 0.0339% w/w, 0.0399% w/w, 0.0227% w/w in samples collected from Jamnagar, Dwarka and Bhuj respectively.

Method Development & Comparative Statistical Evaluation of HPLC & HPTLC Method for Simultaneous Estimation of Cefodrixil Monohydrate & Ambroxol Hydrochloride

Rapid, precise, accurate, specific, and sensitive high performance liquid chromatographic method and high performance thin layer chromatographic methods have been developed for simultaneous determination of cefadroxil monohydrate and ambroxol hydrochloride in their tablet formulation. The HPLC method was standardised using Purospher BDS C18 column (25cm X 4.6mm, 5μm) with mobile phase constituted of 0.5M ammonium acetate buffer- acetonitirile (50:50v/v), pH adjusted to 7 using orthophosphoric acid delivered at the flow rate of 1.0 mlmin-1 and detection was performed at 247nm. For HPTLC analysis separation was carried out on precoated TLC plates, coated with silica gel 60F-254 and using mobile phase methanol- potassium dihydrogen phosphate (0.067M) (35:65v/v) scanned at 254nm with CAMAG TLC scanner controlled by Cats Software. Different analytical performance parameters such as linearity, accuracy, precision, repeatability, robustness LOD and LOQ were determined according to International conference of Harmonization ICH Q2B guidelines. Results of developed methods were compared with reported method using ANOVA. © 2011 IGJPS. All rights reserved.

Quantification of Glycyrrhizin in Glycyrrhiza Glabra Extract by Validated HPTLC Densitometry

Glycyrrhiza glabra Linn (Family-Fabaceae) is active as an anti-allergic, anti-inflammatory, spasmolytic, mild laxative, antistress, antidepressive, antiulcer, liver protective, estrogenic, emmenagogue, and antidiabetic substance, and is widely used in the Indian system of medicine. The major bioactive constituent is glycyrrhizin. A simple HPTLC method has been developed to control the quality of raw as well as finished glycyrrhiza using glycyrrhizin as the bioactive marker. The solvent system was optimized to chloroform-methanol-water (65 + 36 + 7.5, v/v/v). Extract and standard were dissolved in 70% methanol and applied on a precoated TLC plate. After development, the plate was scanned at 254 nm to create a chromatogram, then the quantity of glycyrrhizin was determined in the extract. The method was validated in terms of specificity, linearity, precision, LOD, and LOQ. Linearity range was found to be 0.96-4.80 microg per spot. The linearity relationship was described by the equation: Y = 612.706 + 1.091X (with r = 0.99904 and SD = 2.52%), where Y is the area under curve and X is the amount of glycyrrhizin (ng). The amount of glycyrrhizin found in the extract was 9.1%. Thus, the method provides a rapid and cost-effective quality measure for Glycyrrhiza glabra hydroalcoholic extract.

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.

Isolation and Estimation of Clerodendrin a in Clerodendrum Phlomidis and Premna Integrifolia Root

Clerodendrin A, a chemical marker was isolated from the root of Clerodendrum phlomidis (Verbanaceae). We here report first time, the presence of clerodendrinAin another Verbanaceae drug, Premna integrifolia. Further, HPTLC method was developed to quantify isolated clerodendrinAin C. phlomidis and P. integrifolia roots using n-hexane: ethyl formate (7:3) as mobile phase, pre-coated TLC plates (silica gel 60 F 254 ) as stationary phase and H 2 SO 4 as derivatizing agent. The method was validated in terms of linearity, precision, repeatability and accuracy. The limit of detection and limit of quantification was determined on the basis of signal to noise ratio. Content of clerodendrin A was found to be 0.073 and 0.04% w/w in C. phlomidis and P. integrifolia respectively. The developed HPTLC method was easy, simple, and precise.

Comparative Pharmacognostic Study of Clerodendrum phlomidis and Premna integrifolia

Objective: A comparative study was designed to develop physico-chemical parameters for roots of Clerodendrum phlomidis and Premna integrifolia (Family: Verbenaceae), commonly known as Arni. Materials and Methods: Roots of C. phlomidis and P. integrifolia were studied for macro and microscopical characters. Clerodendrin-A, a chemical marker was isolated from the root of C. phlomidis . HPTLC method was developed to generate fingerprint profiles for the two roots and to quantify isolated clerodendrin-A (in C. phlomidis and P. integrifolia roots) using n-hexane: ethyl formate (7:3) as a mobile phase, precoated TLC plates (silica gel 60 F 254 ) as a stationary phase and H 2 SO 4 as derivatizing agent. Results and Conclusion: Morphologically both roots resemble each other except for their color and size. Microscopically they can be differentiated by presence of rhytidoma in roots of P. integrifolia . Further, starch grains are found distributed in C. phlomidis , only in xylem parenchyma and xylem rays, where as in P. integrifolia all tissues except cork show starch. Clerodendrin-A isolated from C. phlomidis was also found to be present in P. integrifolia . Considering clerodendrin-A as a chemical marker, the present study was designed to develop HPTLC method for generation of distinct chemoprofile and quantification of clerodendrin-A in roots of C. phlomidis and P. integrifolia . HPTLC study of n-hexane fraction (diterpenoid rich) of the two roots performed using silica gel 60 F 254 as a stationary phase, n-hexane: ethyl formate (7:3) as a mobile phase and H 2 SO 4 as derivatizing agent revealed presence of a clerodendrin-A, a major diterpenoid at R f 0.26 (violet color). Clerodendrin- A concentration was found to be 0.073% w/w in C. phlomidis and 0.04% w/w in P. integrifolia .

Development of Physico-chemical Parameters for Indigenous Drug, Bharangi: Clerodendrum serratum and Premna herbacea

Objective: To develop physico-chemical parameters for indigenous drugs Clerodendrum serratum and Premna herbacea commonly known as Bharangi. Materials and Methods: Roots of C. serratum and P. herbacea were studied for macro and microscopical characters. HPTLC method was developed to generate fingerprint profiles for the two roots and to quantify I²-sitosterol (in P. herbacea root) using n-hexane: ethyl formate (7:3) as a mobile phase, precoated TLC plates (silica gel 60 F 254 ) as a stationary phase and H 2 SO 2 as derivatizing agent. Further, presence of sugar, D-mannitol in Clerodendrum serratum was confirmed with the use of paper chromatography. Results: Roots of C. serratum are hard, woody, tortuous, earthy brown and bear rootlets. Stratified cork, cortex, phloem and wood, all filled with starch are characteristic features of transverse section of C. serratum root. Roots of P. herbacea are brown or earthy brown, woody, shows swollen nodes and exfoliated surface exposing the inner orange colored tissue. T.S. of the root shows cork with yellowish orange content, cortex constituted of collenchymatous parenchyma, phloem and wood portion. Presence of starch grains and stone cells in powdered root of C. serratum differentiate it from that of P. herbacea . I²-sitosterol (0.012% w/w) was found to be present in the root P. herbacea only. Sugar D-mannitol was present in Clerodendrum serratum only. Conclusion: The present study provides the first report regarding comparative study and identification parameters for C. serratum and P. herbacea . Further, we report presence of I²-sitosterol in P. herbacea root and HPTLC method to develop distinct chemo-profile for both the roots.

Enantiomeric resolution of amino acids by thin-layer chromatography

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase TLC plate, without using impregnated plates or a chiral mobile phase, is described. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide were spotted on a reversed phase pre-coated TLC plate. Enantiomers of glutamate and aspartate were separated most effectively with solvent consisting of 25% acetonitrile in triethylamine-phosphate buffer (50 mM, pH 5.5) (v/v). Separation of l- and d-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline and alanine were separated with 35% of acetonitrile solvent, and those of methionine, valine, phenylalanine and leucine with 40% of acetonitrile solvent. The possibility of using TLC for quantitative determination of amino acid enantiomers was shown by the quantitative recovery of d- and l-alanine from the TLC plate in the range of 0.56–4.48 nmol.

Solute effects on reversed-phase thin-layer chromatography a linear free energy relationship analysis

RM w values determined by Dross et al. using RP-18 precoated TLC plates and aqueous methanol mobile phases have been analysed using our general LFER to give the equation, RM w = 0.259 + 0.239 R 2 − 0.662 π 2 H − 0.666 α 2 H − 3.006 Σβ 2 O + 3.603V x n = 76, ϱ = 0.9891, S.D. = 0.206, F = 635 The solute descriptors are, R 2 an excess molar refraction, π 2 H the dipolarity/polarizability, Σα 2 H and Σβ 2 O the overall or effective hydrogen-bond acidity and basicity, and V x the McGowan characteristic volume. The coefficients in (i) are very similar to those for the correlation of water-octanol partition coefficients, confirming the suggestion that RP-TLC extrapolated RM values can be used as a measure of lipophilicity. However, for a number of solutes, particularly aromatic and heterocyclic nitrogen bases, RM w values do not correlate either with i, or with log P oct, and hence care should be taken in the application of the RP-TLC method to these compounds. More limited data by Podgorny and Kowalska on RP-18 plates with aqueous tetrahydrofuran (THF) mobile phases suggests that it is not always necessary to use extrapolated RM w values, but that RM values with 80% water-20% THF can be used to estimate log P oct.