TLC-densitometric Method Research Articles

Mitragyna speciosa: Hairy Root Culture for Triterpenoid Production and High Yield of Mitragynine by Regenerated Plants

Hairy root cultures of Mitragyna speciosa were established by infection of Agrobacterium rhizogenes ATCC 15834 and maintained in McCown woody plant medium (WPM) supplemented with 0.5 mg/1 naphthaleneacetic acid. The hairy roots were identified for the rooting genes loci of rolA and rolB by polymerase chain reaction. For studying the secondary metabolite production, the n-hexane extract of the hairy roots was prepared and the compounds were isolated by silica gel column chromatography, affording triterpenoids (ursolic acid and oleanolic acid) and phytosterols (beta-sitosterol and stigmasterol). The shoots from the hairy root cultures were regenerated and differentiated to the plantlets. For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron. The mitragynine contents of 5-month-old regenerated plants and in vitro plantlets (germinated from seeds) were determined using the TLC-densitometric method. The regenerated plants contained (14.25 +/- 0.25) mg/g dry wt mitragynine, whereas the in vitro plantlets contained (4.45 +/- 0.09) mg/g dry wt.

Thin-Layer Chromatography-Densitometric Analysis of -Mangostin Content in Garcinia mangostana Fruit Rind Extracts

The fruit rinds of mangosteen (Garcinia mangostana Linn.) have long been used as traditional medicines for treatment of skin infections, wounds, and diarrhea. A simple thin-layer chromatography (TLC)-densitometric method has been developed for the simultaneous quantification of alpha-mangostin in the extracts from unripe and ripe fruit rinds of G. mangostana. It was found in the ranges of 10.48 +/- 0.83 and 16.65 +/- 0.38% (w/w) in the dried unripe and ripe fruit rinds, respectively. The method was validated for linearity, precision, accuracy, limit of detection (LOD), and limit of quantitation (LOQ). The linearity was found over the range of 100-500 ng/spot with regression coefficient 0.999. Intraday and interday precision studies showed the relative standard deviation was <2%. Accuracy of the method was determined by a recovery study conducted at 3 different levels, and the average recovery was 99.49%. The LOD and LOQ were 40 and 100 ng, respectively. The proposed TLC-densitometric method was found to be simple, precise, specific, sensitive, and accurate. This method can be used for routine quality control of raw material of G. mangostana fruit rind, extract, and its products. It also can be applied in quantifying this marker compound in other drugs.

Densitometric analysis of 2-arylpropionate derivatives in pharmaceutical preparations

A TLC-densitometric method has been established for identification and quantification of tiaprofenic acid, ketoprofen, naproxen, dexibuprofen, flurbiprofen, alminoprofen, and ibuprofen in selected pharmaceutical preparations. The separation was performed on TLC silica gel F 254 plates using two mobile phases. UV densitometry was performed at 225 and 270 nm. The method is of high sensitivity; limits of detection and quantitation range from 0.05 to 0.29 μg per band. For individual constituents recovery ranged from 98.71% to 102.65%.

Simultaneous Determination of Paeoniflorin, Albiflorin and Benzoylpaeoniflorin in Radix Paeoniae Alba by TLC

Paeoniflorin, albiflorin and benzoylpaeoniflorin are three representative monoterpene glycosides in Radix Paeoniae Alba, a well-known traditional Chinese medicine with a great important biological activity. In the present paper, the three marker compounds were simultaneously quantified by TLC densitometric methods using high performance thin layer chromatography. The established method was validated in terms of LOD/LOQ, linearity, recovery and repeatability. The method was found to be precise with RSDs for intra-day in the range of 0.78–1.05, 0.67–0.98, 0.93–1.42% and for inter-day in the range of 0.85–1.23, 0.98–1.29, 1.28–1.94% for different concentrations of albiflorin, paeoniflorin and benzoylpaeoniflorin. Instrumental precision was 0.36, 0.41 and 0.45 (% RSD) for albiflorin, paeoniflorin and benzoylpaeoniflorin. Recoveries of abliflorinl, paeoniflorin and benzoylpaeoniflor were 98, 101.95 and 96.25%, respectively. The proposed method is simple, precise, specific, sensitive, and accurate and can be used for routine quality control of the crude drug.

Evaluation of a TLC Densitometric Method for Analysis of Azole Antifungal Agents

A new method for identification and quantitative determination of the azole antifungal agents; ketoconazole, bifonazole, fluconazole and itraconazole is described using TLC with densitometric detection. Validation of the method was carried out to confirm its precision (%RSD ranged between 1.31 and 3.45), recovery (99.7–102.4%) and linearity (r = 0.99287–0.99722) within the concentration range under investigation. The experimental conditions obtained enable the method to be used for both qualitative and quantitative pharmaceutical analysis.

エーテル型リン脂質のTLCデンシトメトリーによる定量

A quantification method for analysis of individual ether-type phospholipids is important in studies of the regulation of membrane lipid biosynthesis in Archaea. For ester-type lipid of Bacteria and Eucarya, a densitometric method has been established for simultaneous quantification of individual phospholipids visualized with molybdenum blue reagent on a TLC plate. In this study, we developed a TLC densitometric method for rapid quantitative determination of 6 kinds of main ether-type phospholipids in a methanogenic archaeon and an extremely halophilic archaeon. It has been reported previously that on densitometric quantification the values of molar absorptivities are approximately the same among most ester-type phospholipids. On the other hand, we found significant disparity in the molar absorptivity of archaeal ether-type lipids and serine-containing ester-type lipid. Therefore, analysis should be accomplished by use of each standard mixture. Compared with a previous method (preparative TLC method) that is measurement of inorganic phosphate of silica gel powder scraped off from spots of phospholipids on a TLC plate, the TLC densitometry is accomplished at one tenth the sample size in a short time.

Use of TLC with densitometric detection for determination of impurities in chlorpromazine hydrochloride, trifluoperazine dihydrochloride, promazine hydrochloride, and doxepin hydrochloride

A TLC-densitometric method has been established for determination of impurities in chlorpromazine hydrochloride, trifluoperazine dihydrochloride, promazine hydrochloride, and doxepin hydrochloride. Statistical analysis of the data showed the method is precise and highly sensitive. Results from linear regression analysis of the calibration plots were indicative of good linear relationships over wide ranges of concentration. Application of densitometric UV detection at 254 nm and at the wavelengths of maximum absorbance of the substances resulted in increased detectability of zones in the chromatograms compared with visual inspection under a UV lamp. The conditions described enable objective quantitative evaluation of the limiting concentrations of impurities.

TLC-densitometric investigations of phenylpropanoid glycosides in black horehound (Ballota nigraL.)

Most of the beneficial biological effects (e.g. neurosedative, antioxidant, and antimicrobial) of black horehound ( Ballota nigra L.) are because of its phenylpropanoid glycoside content. A rapid TLC-densitometric method with acceptable accuracy has been established for quantitative analysis of the most characteristic components of black horehound. Experiments were performed to establish the optimum conditions for densitometric measurement and for storage of extracts. Practical application of the elaborated method is reported for five plant extracts.

Development and Validation of TLC-Densitometric Method for Resolution and Determination of Enantiomeric Purity of Ropivacaine, Using Different Cyclodextrins as Chiral Selector

A novel economic procedure for stereoselective separation and determination of R(+) - and S(-)- ropivacaine was described using different thin layer chromatographic plates and different cyclodextrins at different temperatures. The spots were detected either with iodine vapors or UV lamp 254nm, followed by densitometric measurements at 262nm. Comparative study was achieved using different cyclodextrins namely, hydroxypropyl-β-cyclodextrin (HP-β-CD), methyl-β-cyclodextrin (M-β-CD), and Dimethyl-β-Cyclodextrin (DM-β-CD) as chiral selectors. The mobile phase enabling successful resolution of (±) ropivacaine was acetonitrile: water (17:3 v/v) containing 1mM of DM-β-CD at ambient temperature 25±20C. All variables affecting the resolution, such as concentration of different chiral selectors, temperature, and pH were investigated and the conditions were optimized. The procedure provided a linear response over the concentration range of 1.25 - 35 μg/spot for determination of R (+)- and S- (-)enantiomers (r = 0.9998, n = 8), (r = 0.9998, n = 6) with acceptable precision (% RSD < 1.5) and accuracy (% RE= -1.18 to 2.00). Limits of detection and quantification were found to be 0.29μg/spot and 0.96 μg/spot for -(R) and 0.26μg/spot and 0.86μg/spot for -(S) respectively. The developed method was validated and proved to be robust. Ropivacaine sample solution was found to be stable for one weak in methanol. The proposed method was found to be selective and accurate for identification and quantitative determination of enantiomeric purity of ropivacaine in bulk powder and pharmaceutical dosage form. Keywords: Ropivacaine, Enantiomeric purity, Cyclodextrins, TLC-densitometry, Mobile phase additive, Pharmaceutical dosage form

Development and Validation of a TLC-Densitometric Method for the Simultaneous Quantitation of Strychnine and Brucine from Strychnos spp. and its Formulations

A simple, sensitive, and specific thin-layer chromatography densitometric method has been developed for the simultaneous quantitation of strychnine and brucine. These two marker compounds are quantitated in the seeds of Strychnos nux-vomica, Strychnos ignatii, and its formulations. The method involves densitometric evaluation of strychnine and brucine after resolving it by high-performance TLC on silica gel plate with toluene-ethyl acetate-diethyl amine-methanol (7:2:1:0.3 v/v) as the mobile phase. The method is validated for precision (interday and intraday), repeatability, and accuracy. The relationship between the concentration of standard solutions and the peak response is linear within the concentration range of 160 to 480 ng/spot for strychnine and 80 to 480 ng/spot for brucine. Instrumental precision is found to be 0.54 and 0.78 (% CV), and repeatability of the method is 1.01 and 1.06 (% CV) for strychnine and brucine, respectively. Accuracy of the method is checked by recovery study conducted at three different levels and the average percentage recovery is found to be 99.13% for strychnine and 100.16% for brucine. The proposed HPTLC method for the simultaneous quantitation of strychnine and brucine is found to be simple, precise, specific, sensitive, and accurate, and it can be used for routine quality control of raw material of Strychnos spp. It also can be applied in quantitating any of these marker compounds in other formulations.

PENETAPAN KADAR TRIPROLIDINA HIDROKLORIDA DAN PSEUDOEFEDRINA HIDROKLORIDA DALAM SEDIAAN SIRUP OBAT INFLUENZA SECARA KROMATOGRAFI LAPIS TIPIS DENSITOMETRI

The determination of pseudoephedrine hydrochloride and triprolidine hydro-chloride in influenza syrup medicine has been performed using TLC densitometric method. Pseudoephedrine hydrochloride and triprolidine hydrochloride were extracted using chloroform at pH 12 from the syrup, and separated using HPTLC silica Kie-selguhr glass plates 60 F 254, 20x10 cm2 as stationary phase, and a mixture of methanol, ammonia and chloroform (40:2:30) as mobile phase. The plates were ana-lyzed using Camag TLC Scanner 3 with UV-detector at 257 nm for pseudoephe-drine hydrochloride and at 290 nm for triprolidine hydrochloride. The results showed that the linearity, limit of detection, and limit of quantitation of the method for pseudoephedrine hydrochloride were 0.9999, 0.0064 µg, and 0.2124 µg respectively; while for triprolidine hydrochloride were 0.9999, 0.0076 µg, and 0.0254 µg respec-tively. The coefficient of variance (CV) of repeatability for the two substances were less than 2.0%; and the recovery values for pseudoepherine hydrochloride and triprolidine hydrochloride were 99.98 + 1.05% and 99.73 + 1,54% respectively. The result showed that the samples analysed contained pseudoephedrine hydrochloride 94.36% of the labeled ammount, and triprolidine hydrochloride 94.44% of the la-beled ammount. Key words : pseudoephedrine hydrochloride, triprolidine hydrochloride, TLC densitometric method.

Densitometric Determination of Fenbendazole in Veterinarian Suspension: Validation of the Method

A simple and rapid densitometric method has been developed for determination of fenbendazole in veterinarian suspension. After extracting the samples with 0.1 N HCL in methanol, the solutions were spotted on precoated silica gel TLC plates, which were eluted with a mixture of dichloromethane‐ethyl acetate–formic acid‐methanol (12:1.0∶0.6∶0.6, v/v). Quantitative evaluation was performed by measuring the absorbance reflectance of the fenbendazole spots at λ=293 nm. The TLC densitometric method is cheap, selective, precise, and accurate, and can be used for routine analysis of suspension in veterinary, pharmaceutical industry quality control laboratories.

Validated TLC densitometric method for the quantification of paroxetine hydrochloride in solid dosage form

A simple, specific, accurate and precise high performance thin layer chromatography method has been developed for the estimation of paroxetine hydrochloride in tablet dosage forms. The quantification was carried out at 296 nm. Developed method was validated in terms of linearity, accuracy, precision, repeatability and specificity. Limit of detection and limit of quantification of paroxetine hydrochloride were found to be 60 ng/spot and 160 ng/spot, respectively. The linearity range for paroxetine hydrochloride was found to be 160-960 ng/spot with correlation coefficient of 0.995. Content of paroxetine hydrochloride in two tablet dosage forms was found to be 100.10 and 100.20%, respectively.

Novel TLC Densitometric Method for Quantification Of Solasodine in Various Solanum Species, Market Samples and Formulations

This paper describes a novel TLC densitometric method for the determination of solasodine in various Solanum species (Family Solanaceae). Solasodine does not give absorption in UV range. Hence, ion pair complex of solasodine and acid dye was spotted on TLC plates. A developing solvent with an organic acid ensured in situ color development of the complex which could be successfully quantified at 461 nm. Linearity was found to be in the range of 79.2–495 ng spot−1 with correlation coefficient of 0.995. This method is reproducible, specific, eliminates post derivatization steps and the problem of background interference. The method was completely validated and applied to determine solasodine content in various herb samples, herb extract and their formulations. Accuracy of the method was found to be 98.54 ± 2.8%. No matrix interference was observed.

Quantitative Analysis of Barakol Content in Senna siamea Leaves and Flowers by TLC-Densitometry

Objective: To develop a TLC-densitometric method for the determination of barakol content in Senna siamea leaf and flower extracts, and to compare the barakol content in mature leaves, young leaves and young flowers of the plant which are consumed as a vegetable in curry. Materials and Methods: The extraction of pure barakol was performed by boiling the fresh young leaves of S. siamea with 0.5% sulfuric acid followed by chloroform extraction. The extract was further purified and recrystallized from absolute ethanol. Authentic sample of barakol was used for the validation of the TLC-densitometric method. Chromatography was performed on a TLC aluminium plate precoated with silica gel 60 F₂₅₄as a stationary phase and chloroform-methanol (85:15 v/v) as a solvent system. Fifteen percent ethanolic extracts of mature leaves, young leaves and flowers of S. siamea were analyzed and compared for barakol content using the validated TLC-densitometric method. Both the validation and analysis of barakol by TLC-densitometry were carried out at the absorbance mode of 366 nm. Results: Barakol was extracted as pure lemon-yellow crystals from young S. siamea leaves with 0.1% yield. Linearity was found over the range of 200–900 ng/spot (r² = 0.997). The developed method gave high precision (%RSD < 0.50) and accuracy (average 101.12%). The limit of detection and limit of quantitation were 8 and 50 ng, respectively. Barakol content in young leaves, mature leaves and young flowers were 1.67, 0.78 and 1.43% dry weight, respectively. R_f value of the barakol in young leaves, young flowers and authentic sample was the same: 0.45 ± 0.03. Conclusion: The TLC-densitometric method was simple, precise and convenient; hence it is an effective procedure for the simultaneous determination of barakol in plant extracts.

Simultaneous determination of neomycin sulfate and polymyxin B sulfate by capillary electrophoresis with indirect UV detection

A simple and rapid capillary electrophoresis method, with indirect UV detection, for the simultaneous determination of neomycin sulfate and polymyxin B sulfate in pharmaceutical formulations was developed. Critical parameters such as pH, buffer composition and concentration, voltage and injection time have been studied to evaluate, how they affect responses, such as resolution and migration times. Separation was performed on a fused silica capillary with 50 μm i.d. and 27 cm total length at an applied voltage of 6 kV with a 15 mM phosphate run buffer (pH 5.0) containing 40 mM N-(4-hydroxy-phenyl)acetamide and 50 mM tetradecylammonium bromide (TTAB). The detection wavelength was set at 280 nm. Quantitative analysis was validated by testing the reproducibility of the method, giving a relative standard deviation less than 0.4 and 2.4% for the repeatability of migration time and corrected peak area, respectively. Accuracy was tested by spiking eye–ear formulations with standards and the recoveries of neomycin sulfate and polymyxin B sulfate were found to be between 97.44–103.18% and 96.85–101.68%, respectively. Linearity of neomycin sulfate and polymyxin B sulfate were obtained in the ranges of 17–682 and 24–608 μg/mL, respectively, with r 2 values above 0.999. The established TLC–densitometric method was applied to evaluate the proposed CE method, and comparable results were obtained by using CE with much shorter analysis time and a small quantity of solvents consumed. The developed method is also the first report on the simultaneous determination of neomycin sulfate and polymyxin B sulfate in pharmaceutical preparations by CE.

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.

Spectrophotometric and spectrodensitometric determination of paracetamol and drotaverine HCl in combination

Thin-layer chromatography, first derivative, ratio spectra derivative spectrophotometry and Vierordt's method have been developed for the simultaneous determination of paracetamol and drotaverine HCl. TLC densitometric method depends on the difference in Rf values using ethyl acetate:methanol:ammonia (100:1:5 v/v/v) as a mobile phase. The spots of the two drugs were scanned at 249 and 308 nm over concentration ranges of 60–1200 μg/ml and 20–400 μg/ml with mean percentage recovery 100.11% ± 1.91 and 100.15% ± 1.87, respectively. The first derivative spectrophotometric method deals with the measurements at zero-crossing points 259 and 325 nm with mean percentage recovery 99.25% ± 1.08 and 99.45% ± 1.14, respectively. The ratio spectra first derivative technique was used at 246 and 305 nm with mean percentage recovery 99.75% ± 1.93 and 99.08% ± 1.22, respectively. Beer's law for first derivative and ratio spectra derivative methods was obeyed in the concentration range 0.8–12.8 and 0.4–6.4 μg/ml of paracetamol and drotaverine HCl, respectively. Vierordt's method was applied to over come the overlapping of paracetamol and drotaverine HCl in zero-order spectra in concentration range 2–26 and 2–40 μg/ml respectively. The suggested methods were successfully applied for the analysis of the two drugs in laboratory prepared mixtures and their pharmaceutical formulation. The validity of the methods was assessed by applying the standard addition technique. The obtained results were statistically agreed with those obtained by the reported method.

Iridoids ofStachysspecies growing in Hungary

Stachys species contain iridoids, for example harpagide, acetylharpagide, aucubin, harpagoside, and ajugoside. The iridoid composition and content of leaves, stems, and inflorescences of ten Stachys species have been compared by use of a TLC-densitometric method. Harpagoside, harpagide, and acetylharpagide proved to be the main iridoids in Stachys officinalis L., St. sylvatica L., St. grandiflora Host., St. macrantha (C. Koch.) Jalas I., St. alpina L., St. palustris L., St. recta L., St. byzantina Koch., and St. germanica L. No iridoids were detected in St. annua L.

Extraction and TLC Desitometric Determination of Triterpenoid Acids (Arjungenin, Arjunolic Acid) from Terminalia arjuna Stem Bark Without Interference of Tannins

The stem bark of Terminalia arjuna Linn. (fam: Combretaceae), commonly known as Arjuna in Indian systems of medicine, is a reputed drug used for various cardiac disorders. T. arjuna stem bark is reported to contain different groups of chemical constituents including phenolics, tannins, saponins and triterpenoid acids. From our earlier experience with tannin containing herbal drugs, we are aware that tannins interfere in the extraction of certain compounds and hence in their quantification. In the present experiment, we report a sample preparation method to overcome the interference of the tannins by adsorbing them with carboxy methyl cellulose, which facilitates the efficient extraction of the triterpenoid acids. Further we established TLC densitometric methods for the quantification of two of the triterpenoid acids of T. arjuna stem bark viz., arjungenin and arjunolic acid using HPTLC. The methods were validated in terms of accuracy, precision and repeatability. The calibration curve showed linearity in the range of 160–480 ng spot−1 and 160–560 ng spot−1 for arjungenin and arjunolic acid respectively. The percentage of arjungenin and arjunolic acid were found to be 0.324% w/w and 0.524% w/w in the stem bark by this modified method of extraction, which was many times higher than when compared to that using the extraction method without CMC (0.018% and 0.049% respectively). The study reiterates the importance of sample preparation in the quantification of non polar phytochemicals from herbal raw materials, such that the compounds of interest are extracted efficiently, overcoming the interference of other compounds like tannins in the matrix of plant material.