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Summary

A simple, precise, rapid, and accurate liquid chromatography-mass spectrometry (LC-MS) compatible reversed phase high-performance liquid chromatography-photodiode array detection (RP-HPLC-PDA) method has been developed and validated for the estimation of oxcarbazepine (OXC) in bulk and tablet formulations. The chromatographic separation was achieved on Phenomenex C18 column (150 mm · 4.6 mm, 5.0 μm particle size) using the mobile phase comprising methanol-formic acid (0.02% v/v in water) in the ratio of 50:50 (v/v) at a flow rate of 1 mL min−1, and OXC was eluted at 6.4 min. Quantification and linearity were achieved at 229 nm over the concentration range of 10–50 μg mL−1, and the mean percentage of assay was found to be 100.03. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), stability, and robustness as per the International Conference on Harmonisation (ICH) guidelines and it is suitable to be employed in quality control.

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A new, simple, and rapid high-performance thin-layer chromatographic method has been established for quantitative analysis of risperidone. Chromatography was performed on silica gel 60 F254 plates with methanol-ethyl acetate 8.0:2.0 (v/v) as mobile phase. Risperidone was quantified by densitometric analysis at 285 nm. The method gave compact bands for the drug (R F 0.34 ± 0.01). Linear regression analysis of calibration data revealed a good linear relationship (r 2 = 0.9996) between response and amount of risperidone in the range 100–600 ng per band. The method was validated for precision, recovery, repeatability, linearity, specificity, and robustness in accordance with ICH guidelines. The minimum detectable amount was 22.44 ng per band and the limit of quantification was 68.01 ng per band. Statistical analysis of the results showed the method enabled precise, accurate, reproducible, and selective analysis of risperidone. The method was successfully used for estimation of the equilibrium solubility of risperidone, and for quantification of risperidone as the bulk drug in a commercially available preparation, in in-house-developed mucoadhesive microemulsion formulations, and in solution.

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The γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter system in the brain. Either imbalance in the extreme can result in several mental diseases like schizophrenia, cerebral stroke, temporal lobe epilepsy (TLE), Parkinson’s disease (PD), Huntington’s disease (HD), and anxiety disorders even death. Measurement of GABA in tissue will help to elucidate the metabolic role and diagnostic value. Various analytical techniques are employed to estimate GABA in biological samples but the experimental procedure and tedious techniques are required. The present study demonstrates a simple, feasible, and cost-effective high-performance thinlayer chromatography (HPTLC) method for estimation of GABA in mice brain tissue. The method was validated in terms of specificity, linearity, and detection limit. The precision was done for inter-day, intra-day, instrumental, and reproducibility. Accuracy was checked by recovery study. The results indicate that this method is fast, sensitive, and suitable for quantitative assessment.

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A sensitive, selective, precise, and robust densitometric method for the analysis of nicotine (drug component of Nicotiana tabacum) in various tobacco products (TPS), including smoked (cigarette and beedi), sniffing (sniffing niswar), dipping (niswar), and chewing (gutka) tobacco brands, was developed and validated as per the ICH guideline. TLC glass plates, precoated with silica gel 60F-254, were used for the analysis. Densitometric analysis of nicotine was carried out in the absorbance mode at 262 nm. The mobile phase consisted of petroleum ether-acetone-diethylamine (7.6:2:0.4) v/v, and gave a sharp and symmetrical nicotine peak (R F = 0.57 ± 0.005) under saturated environment. Linear regression data (LRD) for the calibration curve showed a good linearity with r = 0.997 ± 0.0013. The method was validated for precision, recovery, and robustness. The limits of detection (LOD) and quantitation (LOQ) were found to be 3.15 and 9.54 ng per spot, respectively. Statistical analysis indicated that the method is reproducible and selective for the estimation of nicotine in various TPS.

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Summary

A high-performance liquid chromatographic (HPLC) method coupled with photodiode array (PDA) detection has been developed and validated for simultaneous analysis of six active components (syringin, hyperoside, baicalin, quercetin, baicalein, and farrerol) of the Chinese medicinal preparation Qin-Bao-Hong antitussive tablet. The optimum conditions for separation were achieved on a 3.9 mm × 150 mm i.d., 5-μm particle, C18 column with a linear mobile phase gradient prepared from acetonitrile and 1% acetic acid at a flow rate of 1.0 mL min−1. Because of the different UV characteristics of these compounds, four detection wavelengths were used for the quantitative analysis (265 nm for syringin, 256 nm for hyperoside and quercetin, 277 nm for baicalin and baicalein, and 296 nm for farrerol). For all the analytes a good linear regression relationship (r > 0.999) was obtained between peak area and concentration over a relatively wide range. The method was validated for repeatability, precision, stability, accuracy, selectivity, and robustness. The validated method was successfully applied to simultaneous analysis of these active components in Qin-Bao-Hong antitussive tablet from different production batches.

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A reliable and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection method was developed and validated for the quantification of hopantenic acid in human plasma. Hopantenic acid, with protocatechuic acid as the internal standard (IS), was extracted from plasma samples using a liquid-liquid extraction with methanol. A chromatographic separation was achieved on a Luna C18 column (4.6 mm × 150 mm, 5-μm particle size) and precolumn of the same sorbent (2.0 mm). An isocratic elution, at a flow rate of 1.0 mL min−1, was used with a mobile phase consisting of acetonitrile, water, and 0.03% trifluoroacetic acid. The UV detector was set to 205 nm. The elution times for hopantenic acid and IS were ∼4.3 and 5.4 min, respectively. The calibration curve of hopantenic acid was linear (r > 0.9994) over the range of 0.5–100 μg mL−1 in human plasma. The limit of detection and limit of quantification for hopantenic acid were 0.034 and 0.103 μg mL−1, respectively. The present method was successfully applied for the estimation of pharmacokinetic parameters of hopantenic acid following single oral administration of tablets containing 250 mg hopantenic acid to healthy volunteers. For hopantenic acid, the data showed a mean maximum plasma concentration (C max) of 2.32 μg mL−1, with a time to reach peak plasma concentration (t max) of 1.56 h.

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A simple, sensitive, precise, rapid, and reliable HPTLC method for quantitative analysis of artemether and lumefantrine in tablets has been established and validated. The method uses aluminum foil plates precoated with silica gel 60 F 254 as the stationary phase and nhexaneethyl acetate 8:2 ( v/v ) as mobile phase. Bands were scanned at 357 nm, the wavelength of maximum absorption. The method is linear ( r 2 > 0.995), precise (RSD < 2%), accurate (average recovery of 100.5% for artemether and 99.5% for lumefantrine), specific, and robust. The artemether content of the tablets varied from 98.50 to 102.45% and that of lumefantrine from 97.80 to 100.64%. The limits of detection and quantification for artemether were 50 and 150 ng per band, respectively, and those for lumefantrine were 300 and 900 ng per band, respectively. The suitability of this HPTLC method for quantitative analysis of artemether and lumefantrine was proved by validation in accordance with the requirements of pharmaceutical regulatory standards. The method was successfully applied to the analysis of a commercial pharmaceutical tablet dosage form. The method is simple, rapid, reproducible, and accurate and is a more effective option than other chromatographic techniques used for routine quality control.

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Piperine and gallic acid are of different chemical natures — piperine is an alkaloid, while gallic acid a phenolic compound. They are used as marker compounds in many plant-based formulations. A highperformance thin-layer chromatographic (HPTLC)-densitometric method has been developed and validated for the simultaneous quantification of piperine and gallic acid as such and in pharmaceutical dosage forms. Toluene-ethyl acetate (3:7) was used as mobile phase and scanning was done at 254 and 340 nm. The method was validated with respect to linearity, reproducibility, specificity, accuracy, precision, robustness, and ruggedness. Both compounds showed good linearities in the range of 250–1750 ng. LOD and LOQ for piperine were 9.98 and 33.29 ng, while for gallic acid 25 and 83.33 ng. Average % RSD values of precision for piperine and gallic acid were 0.46% and 0.72%, respectively. % Recovery was 96–103%. The method is accurate, reproducible, cost-effective, and can be used in routine analysis.

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Various chromatographic techniques have been designed to estimate soy isoflavones in finished products as well from Glycine max (L.) Merrill. As an attempt to develop a simple, accurate, and validated method for routine analysis of daidzein and genistein, the major isoflavones as an established standardization technique, a high-performance thin-layer chromatographic (HPTLC) method has been developed. A comparative analysis of individual isoflavone (daidzein and genistein) contents in different varieties of G. max CO-2, CO (soy) 3, and local white was also carried out. Extraction efficiency of the targeted isoflavone from the seed matrix with organic solvents using ultrasonication technique was studied. The methanolic extract of the soybean seeds was taken for method development and validation procedures by HPTLC in which separation was achieved on silica gel 60 F254 HPTLC plates using chloroform-methanol (20:1 v/v) as the mobile phase. The individual isoflavones and protein contents were estimated in three different varieties of soybean in which CO-2 variety has shown the highest content.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Maria Gadzikowska, Anna Petruczynik, Monika Waksmundzka-Hajnos, Mirosław Hawrył, and Grzegorz Jóźwiak

M.E. Swartz and I.S. Krull , Analytical Method Development and Validation, Marcel Dekker, New York, 1997. Krull I.S. Analytical Method

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