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A new, simple, and rapid normal-phase high-performance thin-layer chromatography method was developed for the simultaneous separation of retinoic acid isomers (tretinoin and isotretinoin). The isomers were baseline-resolved on aluminum-backed silica gel 60 F254 layer using toluene-ethyl acetate-methanol (8:1:0.5, v/v) as the mobile phase. Quantification was achieved with ultraviolet (UV) detection at 334 nm. The developed analytical method was validated according to International Conference on Harmonization guidelines in terms of parameters like accuracy, precision, linearity, and specificity. Good linearity was observed in the concentrations ranging from 30 to 70 ng band−1 for both the isomers. Recovery study results ranged from 95 to 102% for both tretinoin and isotretinoin. The final optimized methodology was successfully applied to separate the isomers and was proven to be reproducible and accurate for its quantitative determination.

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This study was designed to develop a simple, sensitive, selective, and precise high-performance thin-layer chromatography (HPTLC) fingerprint and quantitative estimation method for the analysis of three phenolic compounds from nine Leucas species. The developed HPTLC method was validated according to the International Conference on Harmonization guidelines. The method permits reliable quantification of one important phenolic acid (gallic acid) and two hydroxycinnamic acids (caffeic and ferulic acids) in 50% hydroethanolic extracts on silica gel with toluene—ethyl acetate—formic acid 8:2:1 (v/v) as the mobile phase. The system showed good resolution and separation of caffeic, ferulic, and gallic acids (at R f values 0.48, 0.60, and 0.30, respectively) from the other constituents of the extract. Densitometric scanning was done at 300 nm in absorbance mode. All the results obtained by the developed method were statistically compared for validation parameters, for accuracy and good precision. Caffeic, ferulic, and gallic acids were estimated in all the aforesaid nine Leucas species, but the quantity varied from species to species. Further, these phenolics were reported from the aforesaid Leucas species for the first time, except for Leucas lanata and Leucas urticifolia. However, Leucas biflora, Leucas decemdentata, and Leucas stricta were documented for their chemical constituents for the first time. The proposed HPTLC method can be used for routine quality testing of Leucas species.

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Lupenone was isolated for the first time from the stem bark of Diospyros melanoxylon and characterized with the help of physical and spectroscopic data. A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the quantification of lupenone in D. melanoxylon stem bark. HPTLC analysis was performed on HPTLC plates by using a binary mobile phase of n-hexane–ethyl acetate (8.2:1.8, v/v). It was quantified at 395 nm after derivatization with methanol—sulfuric acid reagent. The limits of detection and quantification were found to be 40 ng and 100 ng per spot, respectively. The linear regression analysis data for the calibration plot in the range of 100–500 ng spot−1 showed a good linear relationship between peak area and concentration (r 2 = 0.9997). The instrumental precision was 1.01% (coefficient of variation [CV]) and the repeatability of the method was 2.17% (CV). The method was validated for precision, recovery, and repeatability as per the International Conference on Harmonization guidelines. The developed HPTLC method is very accurate and precise and has been successfully used for the estimation of lupenone in D. melanoxylon stem bark.

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This paper describes a validated high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of ivermectin (IVM) and albendazole (ALB) in combined tablet dosage forms. The HPTLC separation was carried out on aluminum-backed silica gel 60 F254 layers using toluene-ethyl acetateglacial acetic acid, 6:4:0.5 (ν/ν/ν), as the mobile phase. Quantification was achieved with UV detection at 247 nm. The developed analytical method was validated according to International Conference on Harmonization guidelines in terms of parameters such as accuracy, precision, linearity, and specificity. Good linearity was observed in the concentrations ranging from 0.12 to 0.54 and 8 to 36 μg per band for IVM and ALB, respectively. The recovery study results ranged from 98% to 101% for IVM and ALB, showing the accuracy of the method. The method was successfully applied for the analysis of the drugs in laboratory-prepared mixtures and commercial tablets. The developed method is simple, precise, and sensitive, and applicable for the simultaneous determination of IVM and ALB in pure powder and tablets.

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A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the quantification of two bioactive lupane triterpenoids, namely, lupeol and betulin from Diospyros melanoxyon stem bark. Chromatographic separation was achieved on aluminium foil-backed HPTLC plates using ethyl acetate-hexane (1.8:8.2, v/v) as mobile phase. The compounds were quantified at their wave length of maximum absorbance in the range of 100–500 ng per spot. The instrumental precision was 0.82% and 1.07% (CV) and the repeatability of the method was 1.33% and 1.17% (CV), respectively, for lupeol and betulin. The minimum detectable amount was found to be 40 and 50 ng per spot for lupeol and betulin, respectively. The linear regression analysis data for the calibration plots showed a good linear relationship with r 2 = 0.9996 for lupeol and 0.9997 for betulin. The method was validated for precision, recovery, and repeatability as per the International Conference on Harmonization guidelines. The developed HPTLC method is very accurate and precise, and has been successfully applied for the assay of these bioactive molecules in D. melanoxylon stem bark.

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A thin-layer chromatographic (TLC) method is developed for the analysis of cefixime using precoated silica gel 60F254 TLC aluminum sheets as stationary phase. The mobile phase was optimized to ensure that no other drug from cephalosporin class and degradation products (formed under stress condition) of the drug interfered with the estimation. By using toluene-ethyl acetate-formic acid-water (10:58:22:10, ν/ν) as mobile phase, a sharp, well-defined, and symmetrical peak was obtained in the chromatogram with an R F value 0.54 ± 0.02. Densitometric measurement was made in the reflectance/absorbance mode at 293 nm. The calibration curve was established as dependence of peak area on amount 500–1500 ng. The second-order polynomial fit was deduced to be suitable for the quantitative analysis. The method was validated as per International Conference on Harmonization guidelines. The method is simple, sensitive, selective, and precise. It can be applied for the quantitative analysis of cefixime in raw materials, marketed tablets, and counterfeit samples.

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Valsartan is a potent and specific competitive antagonist of the angiotensin-II AT 1 -receptor and is used orally for treatment of hypertension. A sensitive, selective, precise, and stability-indicating high-performance thin-layer chromatographic method for analysis of valsartan both as the bulk drug and in formulations has been developed and validated. The method uses aluminum-backed silica gel 60F 254 plates with toluene-ethyl acetate-methanol-formic acid 60.0:20.0:20.0:1.0 ( v/v ) as mobile phase. The system gave compact bands for valsartan ( R F 0.44 ± 0.05). Densitometric analysis of valsartan was performed in absorbance mode at 250 nm. Linear regression analysis data for the calibration plots revealed a good linear relationship with r 2 = 0.9946 ± 0.0013 in the working concentration range 200 to 1600 ng per band. The method was validated for precision, robustness, and recovery. The limits of detection and quantification were 25 and 150 ng per band, respectively. Valsartan was subjected to different stress conditions — acid and alkaline hydrolysis, oxidation, photo degradation, and dry and wet heat treatment — as prescribed by International Conference on Harmonization guidelines. The degradation products were well resolved from the pure drug with significantly different R F values. Since the method could effectively separate the drug from its degradation products, it could be used as a stability-indicating method for analysis of valsartan.

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Transfer of two rapid thin-layer chromatography (TLC) screening methods used to detect markedly substandard and fake pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods is demonstrated using a model procedure that was published earlier. These TLC methods for diazepam and amodiaquine are contained in a Compendium of methods by Kenyon and Layloff and a Minilab method manual from Global Pharma Health Fund E.V., respectively, for use in countries with limited resources. Merck HPTLC Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 were used for detection, identification, and quantification. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software. Accuracy was estimated by the standard addition approach with overspotting standard and sample solutions. HPTLC gives better efficiency, selectivity, and resolution than TLC, and the new methods overcome the deficiencies in technology related to manual application and visual zone comparison that do not allow the Compendium and Minilab TLC procedures to support regulatory compliance actions. These new methods can be fully validated according to the International Conference on Harmonization guidelines or by interlaboratory studies if required by their applications.

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Summary

A simple, rapid, and specific reversed-phase HPLC method has been developed for simultaneous analysis of withaferin-A and 6-gingerol in a polyherbal formulation containing Withania somnifera and Zingiber officinalis extracts. HPLC analysis was performed on a C18 column using a 40:60 (v/v) mixture of acetonitrile and water as isocratic mobile phase at a flow rate of 1.5 mL min−1. UV detection was at 227 nm for withaferin-A and 278 nm for 6-gingerol. The method was validated for accuracy, precision, linearity, specificity, and sensitivity in accordance with International Conference on Harmonization guidelines. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r 2 > 0.9996) were obtained for calibration plots in the ranges tested. Limits of detection were 0.2 and 0.4 μg and limits of quantification were 0.5 and 1.0 μg for withaferin-A and 6-gingerol, respectively. Intra and inter-day RSD of retention times and peak areas were less than 2.1%. Recovery was between 94.5 and 98.8% for withaferin-A and 94.2 and 102.4% for 6-gingerol. The established HPLC method is appropriate and the two markers are well resolved, enabling efficient quantitative analysis of withaferin-A and 6-gingerol. The method was successfully used for quantitative analysis of these two marker constituents in a marketed polyherbal formulation.

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Transfer of four thin-layer chromatography (TLC) Global Pharma Health Fund Minilab mobile kit protocols for detecting fake pharmaceutical products to quantitative high-performance TLC (HPTLC)—densitometry methods was carried out using a model process published earlier. The developed and validated methods were for the drugs quinine sulfate, mefloquine, dihydroartemisinin, and piperaquine phosphate. EMD Millipore Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification were used. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software, and accuracy was estimated by the standard addition approach. HPTLC gives better efficiency, selectivity, and resolution than TLC, and the new methods overcome the deficiencies in technology related to manual application and visual zone comparison that do not allow the Minilab TLC procedures to support regulatory compliance actions. These new methods should be fully validated according to International Conference on Harmonization guidelines or by interlaboratory studies if required by their applications. In addition, a previously reported transferred simultaneous HPTLC–densitometry method for lumefantrine and artemether was used to analyze a new combination tablet to demonstrate its applicability.

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