Rapid Thin-layer Chromatographic Method Research Articles

Development of a rapid and reproducible TLC method for purity elucidation and related substance identification of metronidazole in metronidazole dosage forms

Introduction: Thin-layer chromatography (TLC) is a quick, inexpensive microscale technique applicable for determination of the number of components in a mixture, monitoring the progress of an organic reaction, verification of substance identity and analysis of the fraction obtained from column chromatography. Aim: The aim of our work is to develop a rapid TLC method for related substance identification, purity elucidation, and control of synthesis of metronidazole in dosage forms and during synthesis of derivatives. Materials and Methods: A rapid and reproducible TLC method for quality determination of metronidazole derivatives is described. The samples and standard were separated on silica gel 60 with fluorescent indicator UV 254 plates with three different mobile phases: A) acetic acid-ammonia-acetone-methylene chloride cyclohexane (1:2:3:3:1), B) toluene-chloroform-methanol (2:7:1), C) toluene-chloroform-ethanol-acetic acid-ammonia (9:8:1:1:1). The visualization of spots was done at 254 nm. Results: As most appropriate was established to be the mobile phase consisting of toluene- chloroform-ethanol-acetic acid-ammonia (9:8:1:1:1). The identification and determination of metronidazole in analyzed drug formulations were proved by the correspondence between the Rf of drug samples and reference standard Rf-0.58. Conclusion: The TLC method for related substance determination of metronidazole in drug formulations is an informative, rapid, and reproducible method. The method may be applied for further analysis and monitoring of synthesis of new metronidazole derivatives.

Chemical Composition and Antioxidant Activity of Essential Oils from Cupressus sempervirens. var. sempervirens, C. sempervirens. cv. Cereiformis and C. sempervirens var. horizentalis

The genus Cupressus belonging to Cupressaceae family has twelve species worldwide. In Iran, Cupressus consists of only one species with two varieties C. sempervirens. var. horizentalis and C. sempervirens. var. sempervirens which has a cultivar namely C. sempervirens. cv. Cereiformis. In this study, the potential antioxidant activities of the oils of different parts of Cupressus sempervirens, C. sempervirens cv. Cereiformis and C. sempervirens var. horizentalis were examined.The qualitative and quantitative compositions of the oils were studied by GC/FID and GC/MS. Then the obtained essential oils, their main components as well as positive controls (Quercetin, DMSO, vitamin E and ascorbic acid) were subjected to different antioxidant tests including Rapid Thin-layer Chromatography method, diphenylpicrylhydrazyl (DPPH) assay, deoxyribose degradation test, and modified deoxyribose degradation test. While all the essential oils and pure compounds showed activity in rapid TLC screening, only the leaf oil of C. sempervirens var. sempervirens (38.4 %) and C. sempervirens cv. Cereiformis (77.9 %) exhibited moderate activity in DPPH assay. In deoxyribose degradation test, the low concentrations (0.05-0.2 μL/mL) of these two plants showed moderate activity while the fruit oil of C. sempervirens var. horizentalis showed the highest activity (17.7 %). These essential oils may be considered as natural preservatives in low concentrations provided that their organoleptic, cytotoxicity and allergic effects would be examined. Thus, complementary studies are recommended to be established. Moreover, it is suggested to examine the antioxidant activity of these oils by using non-polar or less polar solvent systems before any final conclusion.

A Rapid Thin Layer Chromatographic Method for Simultaneous Screening of Albendazole and Ivermectin in Formulations Prescribed for Human or Veterinary Use

An easy and selective thin layer chromatographic method has been developed and experimentally validated for the simultaneous screening of most commonly used anthelmintic drugs i.e. albendazole and ivermectin. Separation of these compounds was attained on silica gel 60 F254 pre-coated thin layer chromatographic plate using an optimized mobile phase of diisopropyl ether:ethyl acetate:glacial acetic acid in the ratio of 7:3:0.1 (v/v), respectively at pH 3.5. The calculated Rf values for albendazole and ivermectin were 0.65 and 0.38, respectively and the LOD was found to be 25 μg/ml and 30 μg/ml for albendazole and ivermectin, respectively. The developed method is selective, sensitive, robust, cost effective, eco-friendly, rapid as well as easy to perform. The developed method was successfully applied for the analysis of albendazole and ivermectin in pharmaceutical preparations marketed as oral suspensions, powder, tablets and injectable of the single or combined dosage forms for human as well as veterinary use. It could also be applied for the simultaneous analysis of both the compounds in other samples.

Further Validation of a Rapid Screening Semiquantitative Thin-Layer Chromatographic Method for Marketed Antimalarial Medicines for Adoption in Malawi.

A recently developed semiquantitative thin-layer chromatographic (SQ-TLC) assay has been employed in postmarketing surveillance of antimalarial medicines used in Malawi prior to HPLC assay. Both methods gave analogous results in a significant majority of the samples, with a good correlation (r = 0.9012) for the active pharmaceutical ingredients of the dosage forms assayed. Artemether-containing medicines had the highest percentage (92.67%) of samples with comparable results for both assays. The lowest percentage (66.67%) was observed in artesunate-containing medicines. The SQ-TLC method was validated for specificity, accuracy, precision, linearity, and stability according to the International Conference on Harmonisation guidelines, with the results falling within acceptable limits. For specificity, retention factor values of the test samples and reference standards were comparable, while accuracy and precision of 91.1 ± 5.7% were obtained for all samples. The method was linear in the range 1.0–2.0 µg/spot with a correlation coefficient of r = 0.9783. Stability tests also fell within acceptable limits. In this study, we present the validation of the SQ-TLC method and propose its adoption as a rapid screening tool for field estimation of the quality of antimalarial and other essential medicines in Malawi and other parts of the developing world prior to a more accurate HPLC assay.

Validated thin‐layer chromatographic method for the identification and monitoring of the effect of the extraction method on the yield and phytochemical constituents of Egyptian Withania somnifera leaves

A sensitive, reliable, simple and rapid thin-layer chromatographic method has been developed for routine analysis of withanolide S content for the purpose of quality control assessment of chemotype III of Withania somnifera. The new method was used first to compare the accumulation of withanolide S in different parts of the plant, which was found to be the highest in the leaves extract (0.21% w/w). Second, to investigate different extraction parameters that improve the extraction efficiency of withanolides from the leaves using conventional and ultrasound-assisted extraction methods. The extraction efficiency was expressed via total withanolide content and withanolide S content.

Development of Microwave-Assisted Extraction of Trigonelline Biomarker fromTrigonella foenum-graecumSeeds Followed by High-Performance Thin-Layer Chromatographic and High-Performance Liquid Chromatographic Analyses

An efficient and fast microwave-assisted extraction (MAE) technique was developed for extracting trigonelline as an indicative biomarker for the quality control of Trigonella foenum-graecum seeds. The MAE procedure was optimized and compared with other conventional extraction techniques. The optimal conditions of MAE were 50% methanol as solvent, solid—liquid ratio 1:20 g mL−1, irradiation power 40%, and two extraction cycles, 3 min each. The proposed extraction technique produced a maximum yield of 7.89 mg g−1 trigonelline in 6 min which was 1.28 and 2.20 times more efficient than 3 h of heat reflux and 15 h of maceration extraction, respectively. Furthermore, rapid high-performance thin-layer chromatographic and high-performance liquid chromatographic methods for the quantification of trigonelline were established and validated. The methods were found to be simple, sensitive, precise, accurate, and specific for the estimation of trigonelline in T. foenum-graecum seeds extract and overcame disadvantages ...

Stability-indicating High Performance Thin Layer Chromatography /densitometry estimation of Formoterol fumarate dihydrate in bulk and capsules

Novel, simple, rapid and reliable High-Performance Thin-Layer Chromatographic (HPTLC) methods were developed and validated for the analysis of Formoterol Fumarate Dihydrate (FFD) in bulk and in in-house tablet formulation. HPTLC quantitation of FFD was done at UV detection of 281 nm and analysis was performed on (10 cm 10 cm) aluminium sheets precoated with silica gel 60-F 254 (E. Merck) as stationary phase and ethyl acetate: methanol: triethylamine (3.2:1.5:0.3 v/v ) as mobile phase. Quantitation by HPTLC method was performed over the concentration range of 400 - 900 ng/band. The HPTLC method resulted into compact and well resolved band for FFD at retention factor ( R f ) of 0.51 0.3. Linear regression analysis data for calibration of HPTLC method represented good linear relationship with regression coefficient; r 2 = 0.999. The developed methods were validated for precision, robustness, ruggedness, accuracy, sensitivity as per guidelines laid by International Conference on Harmonisation (ICH). FFD was subjected to acid and alkali hydrolysis, oxidation, neutral, photo and thermal degradation. The drug undergoes degradation under acidic, basic, oxidation, and thermal conditions. This indicates that the drug is susceptible to acid, base, oxidation and thermal conditions. The degraded product was well resolved from the pure drug with significantly different R f value. Statistical analysis proved that the developed methods were precise, robust, sensitive and accurate and can be used effectively for the analysis of FFD in bulk and pharmaceutical formulations.

Thin-layer Chromatographic Identification of Chinese Propolis Using Chemometric Fingerprinting

Poplar tree gum has a similar chemical composition and appearance to Chinese propolis (bee glue) and has been widely used as a counterfeit propolis because Chinese propolis is typically the poplar-type propolis, the chemical composition of which is determined mainly by the resin of poplar trees. The discrimination of Chinese propolis from poplar tree gum is a challenging task. To develop a rapid thin-layer chromatographic (TLC) identification method using chemometric fingerprinting to discriminate Chinese propolis from poplar tree gum. A new TLC method using a combination of ammonia and hydrogen peroxide vapours as the visualisation reagent was developed to characterise the chemical profile of Chinese propolis. Three separate people performed TLC on eight Chinese propolis samples and three poplar tree gum samples of varying origins. Five chemometric methods, including similarity analysis, hierarchical clustering, k-means clustering, neural network and support vector machine, were compared for use in classifying the samples based on their densitograms obtained from the TLC chromatograms via image analysis. Hierarchical clustering, neural network and support vector machine analyses achieved a correct classification rate of 100% in classifying the samples. A strategy for TLC identification of Chinese propolis using chemometric fingerprinting was proposed and it provided accurate sample classification. The study has shown that the TLC identification method using chemometric fingerprinting is a rapid, low-cost method for the discrimination of Chinese propolis from poplar tree gum and may be used for the quality control of Chinese propolis.

Validated high-performance thin-layer chromatographic methods for the quantitative estimation of bioactive constituents in Methiorep premix, a feed additive polyherbal formulation

Herbal formulations have reached extensive acceptability as therapeutic agents for several diseases. The development of an authentic analytical method which can reliably profile the phytochemical composition, including quantitative analysis of marker/bioactive compounds and other major constituents, is a major challenge to scientists. With the realization of the growing popularity and demand for phytopharmaceuticals, standardization is becoming a mandatory part for the regulation of herbal drug industry. Simple, precise, and rapid high-performance thin-layer chromatographic methods were developed for the estimation of l-3,4-dihydroxyphenylalanine (l-DOPA) (I) and rutin (II), the two bioactive phytoconstituents in Methiorep premix, a phytoadditive formulation containing herbal ingredients that mimics methionine-like activity. Separation of I and II was carried out on silica gel F254 thin-layer chromatography (TLC) with butan-1-ol-acetic acid-water (8:2:2, v/v) and ethyl acetate-butan-1-ol-formic acid-water...

HPTLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ANALYSIS OF EMODIN AND CHRYSOPHANOL IN Cassia tora LINN METHANOLIC EXTRACT

Anthraquinone glycosides are the major constituents of Cassia tora Linn (Caesulpinaceae) among which emodin and chrysophanol were found to be the active glycosides present as major constituents. The objective of the present article is to develop a simple, precise, and rapid thin-layer chromatographic method for the simultaneous estimation of emodin and chrysophanol in Cassia tora extract and to validate the method as per ICH guidelines. TLC analysis was performed on precoated silica gel 60 F254 plates with Toluene: ethyl acetate (9:1) as mobile phase. On scanning at 435 nm emodin and chrysophanol had shown good resolution with Rf values “0.31 ± 0.02 and 0.85 ± 0.03,” respectively. The method was validated as per ICH guidelines. The percentage content of emodin and chrysophanol was found to be 0.037 and 0.067%, respectively. The linearity range was found to be 10–300 µg/band and average recovery values were found to be 94 and 95% for emodin and chrysophanol, respectively. The developed method did not involve any derivatisation procedure and thus was found to be simple, precise, and reproducible for the routine analysis of emodin and chrysophanol.

Separation and identification of some non-steroidal anti-inflammatory drugs using TLC and HPLC-MS

A simple, rapid and reliable thin-layer chromatographic method has been developed for the identification of drugs. A mixture of 12 non-steroidal anti-inflammatory drugs was separated on silica gel 60 F254 plates using ethyl acetate-toluene-methanol-acetic acid 40:40:1:1 (v/v) as mobile phase. These drugs were also separated on C8 chemically modified diatomaceous earth (diatomite) using acetonitrile-water-H3PO4 85%, 50:50:1 (v/v). A spot visualization was performed in UV light at 254 nm. The piroxicam was one of the 12 drugs studied, and it was separated and identified by thin-layer chromatography (TLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) in urine. The limits of detection are from 0.005 to 0.5 μg per spot, depending on the measured substance.

TLC-densitometric method for quantification of oxaprozin and its degradation products in pharmaceutical preparations

A new, simple and rapid thin-layer chromatographic method with densitometric detection has been developed and validated for analysis of oxaprozin and its degradation products. Chromatography was performed on silica gel plates with n -hexane-chloroform-glacial acetic acid 4:1:1 ( v/v ) as mobile phase. Densitometric analysis of oxaprozin was performed in absorbance mode at 286 nm. The method was validated for linearity, accuracy, and precision. Oxaprozin was subjected to acidic and alkaline hydrolysis. The degradation products were well resolved from the pure drug with significantly different R F values. LC-MS-MS analysis revealed that oxaprozin decomposes to produce 1,2-diphenylethanol, 4-iminobutanoic acid, and propan-1-imine.

A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

3'-[(18)F]fluoro-3'-deoxythymidine ([(18)F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [(18)F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [(18)F]FLT both in tumor cell lysates and tumor cells. The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [(18)F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured. The apparent Michaelis-Menten kinetic parameters for [(18)F]FLT are K(m) = 4.8 ± 0.3 μM and V(max) = 7.4 pmol min(-1) per 1 × 10(6) cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates. The apparent K (m) of [(18)F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [(18)F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells.

Rapid TLC Method for Estimation of Mevalonic Acid in the Leaves of Medicinal Plants

A simple, rapid, specific and sensitive thin layer chromatographic (TLC) method has been developed for the quantitative estimation of mevalonic acid (MVA) in leaves of medicinal plants; Artemisia annua, Psorelia corylifolia, Vinca rosea, Withania somnifera and Barleria proinites. The assay procedure involved conversion of MVA to its lactone, mevalonolactone (MVAL). Mevalonic acid was extracted from the leaf tissues of plants. Separation of MVAL was carried out on silica gel 60 F254 TLC plates using benzene:acetone (3:2) as the mobile phase. The densitometric determination of MVAL was performed at 600 nm after derivatization with anisaldehyde reagent in absorption–reflectance mode. The method was validated over the linearity range of 100–500 ng spot−1 and correlation coefficient for the calibration curve was >0.99. The average recovery of MVAL, used as internal standard, was higher than 98%. The lower limit of detection was found to be 50 ng spot−1.

HPTLC analysis of octachlorodipropyl ether in insecticide formulations

A simple, rapid, and sensitive high-performance thin-layer chromatographic method for analysis of octachlorodipropyl ether (OCDPE) in insecticide formulations has been established and validated. Known amounts of analytical grade OCDPE and its emulsifiable concentrate (EC) or wettable powder (WP) formulations were characterized by HPTLC on silica gel with toluene-acetic acidwater 20:20:1 ( v/v ) as mobile phase; detection was by spraying with silver nitrate-2 m alcoholic potassium hydroxide as chromogenic reagent and exposure to UV light. The plates were evaluated densitometrically at 399 nm. The results indicated that the calibration plot for OCDPE was logarithmic in the range 0.2–5.0 μg per band, and the correlation coefficient for the calibration equation in this range was 0.99. Recoveries from laboratory-prepared test EC and WP formulations using this method were 98.5–103.9% and 95.3–104.3%, respectively, and the respective RSDs were 3.39–4.89% and 2.92–5.33%. The accuracy and precision of the method w...

Determination of fluvoxamine and moclobemide in tablets by densitometric and videodensitometric TLC

New, simple, rapid, and accurate thin-layer chromatographic methods have been established for determination of fluvoxamine and moclobemide in tablets. The drugs were chromatographed on silica gel 60 F 254 plates in horizontal chambers with benzene-acetone-ethanol-25% aqueous ammonia, 9 + 7 + 2 + 1 ( v/v ), as mobile phase. Densitometric detection and quantification were performed at λ = 249 nm and λ = 236 nm, respectively, for fluvoxamine and moclobe-mide; videodensitometric detection was performed at λ = 254 nm for both drugs. The range of linearity was 1–10 μg per spot for fluvoxamine and moclobemide for both methods. The relative standard deviation for determination of these antidepressants in pharmaceuticals was less than 2.5% for densitometry and less than 5.1% for videodensitometry. Recovery of fluvoxamine from tablets was 101.20% by use of densitometry and 98.96% by use of videodensitometry; recovery of moclobemide from tablets was 101.15% and 98.81% respectively.

A rapid thin-layer chromatographic method for comparison of different ethoxylation products in the manufacture of non-ionic surfactants

A rapid thin-layer chromatographic method has been developed for comparing the different ethoxylation products during the manufacture of non-ionic surfactants. The ethoxylation products are separated on silica gel layers with ethyl acetate-acetone-water (12:5:0.5) as mobile phase. The chromatograms were visualized by exposure to iodine vapor for 3 min; densitometric scanning was then performed at 460 nm in reflectance mode and plates were evaluated by use ofRF values and peak-area data. The separation conditions were investigated and optimized. The average numbers of the oxyethyl groups in the molecules of the adducts, and their distribution curves, were determined and used as criteria for monitoring the extent of ethoxylation of the intermediates and the final products. The method is recommended for the purpose because of the advantageous features of TLC.

Thin-layer immunoaffinity chromatography with bar code quantitation of C-reactive protein.

A rapid thin-layer immunoaffinity chromatographic method for quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacteria] infections, is described, where material and reagent costs are minimal. The analysis is based on the "sandwich" assay format using monoclonal antibodies directed against two sites of CRP. One of the antibodies is covalently bound to defined zones on a thin-layer immunoaffinity chromatography membrane, while the other antibody is covalently bound to deeply dyed blue latex particles. After incubation (CRP sample and latex particles), the CRP-latex immunocomplex is allowed to migrate along the immunoaffinity chromatography membrane. In the presence of antigen, a sandwich is formed between the CRP-latex immunocomplex and membrane-bound antibodies, which results in the appearance of blue lines on the membrane. Antibody immobilization on the TLC membrane is made with a redesigned piezoelectric-driven ink-jet printer. The time required for the analysis is less than 10 min. Quantitation is achieved either by counting the lines visually, with scanning reflectometry, or with a modified bar code reader. The limit of detection was estimated in the low femtomolar range using the naked eye as detector.

Rapid thin-layer chromatographic screening method for the detection of five sulfonamides in swine tissues: collaborative study.

A rapid thin-layer chromatographic screening method for five sulfonamides in swine tissues was studied collaboratively in nine Dutch laboratories. All collaborators were sent fifteen swine muscle and fifteen swine kidney samples. These were obtained from treated swine or from swine tissues spiked with sulfonamides at levels ranging from 0 to 0.4 mg/kg. The study indicates that the method is suitable for the screening of animal tissues for these sulfonamides. False scores occasionally occur at the lowest levels.

Rapid Thin Layer Chromatographic Method for Determination of Zearalenone and Zearalenol in Grains and Animal Feeds

A rapid and inexpensive method has been developed for the analysis of zearalenone and zearalenol in grains and animal feeds. The method involves extraction with 75% methanol, precipitation of pigments with lead acetate, and defatting with petroleum ether. The mycotoxins are subsequently partitioned into toluene-ethyl acetate, chromatographed on high performance thin layer chromatographic plates, and detected after treatment with Fast Violet B salt solution. Sensitivity of the method is better than 80 ng/g for zearalenone and 200 ng/g for zearalenol. Ten samples can be completed in less than 2 h. The method is applicable for zearalenone in corn, wheat, barley, millet, and swine feeds.