Rapid High-performance Thin-layer Chromatographic Method Research Articles

HPTLC METHOD FOR ESTIMATION OF ISOTRETINOIN IN TOPICAL FORMULATIONS, EQUILIBRIUM SOLUBILITY SCREENING, AND IN VITRO PERMEATION STUDY

A new, simple, and rapid high-performance thin-layer chromatographic method was developed and validated for quantitative determination of Isotretinoin. Isotretinoin was chromatographed on silica gel 60 F254 TLC plate using toluene - methanol (9.0 + 1.0 v/v) as the mobile phase. Isotretinoin was quantified by densitometric analysis at 340 nm. The method was found to give compact spots for the drug (RF = 0.43 ± 0.01). The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9995 in the concentration range 100–500 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 13.72 ng/spot, whereas the limit of quantitation was found to be 41.58 ng/spot. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of Isotretinoin. The method was successfully employed for the estimation of equilibrium solubility, quantification of Isotretinoin as a bulk drug, in commercially available preparation and in-house micro-emulsion based gel formulations.

HPTLc method development and validation for analysis of risperidone in formulations, and in-vitro release study

Summary 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 (RF 0.34 ± 0.01). Linear regression analysis of calibration data revealed a good linear relationship (r2 = 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...

Development and Validation of HPTLC Method for Estimation of Carbamazepine in Formulations and Its In Vitro Release Study

A new, simple, and rapid high-performance thin-layer chromatographic method was developed and validated for quantitative determination of Carbamazepine. Carbamazepine was chromatographed on silica gel 60 F254 TLC plate using ethyl acetate-toluene-methanol (5.0 + 4.0 + 1.0 v/v/v) as mobile phase. Carbamazepine was quantified by densitometric analysis at 285 nm. The method was found to give compact spots for the drug (Rf=0.47 ± 0.01). The linear regression analysis data for the calibration plots showed good linear relationship with r2=.9995 in the concentration range 100–600 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 16.7 ng/spot, whereas the limit of quantitation was found to be 50.44 ng/spot. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of Carbamazepine. The method was successfully employed for the estimation of equilibrium solubility, quantification of Carbamazepine as a bulk drug, in commercially available preparation, and in-house developed mucoadhesive microemulsion formulations and solution.

HPTLC method for estimation of tazarotene in topical gel formulations and in vitro study

A new, eco friendly, simple, and rapid high-performance thin-layer chromatographic method was developed and validated for quantitative determination of tazarotene. The HPTLC separation was achieved on an aluminium-backed layer of silica gel 60F254 using toluene-methanol (9.0 + 1.0 v/v) as mobile phase. Quantitation was achieved by densitometric analysis at 327 nm over the concentration range of 100–500 ng/mL. The method was found to give a compact spot for the drug (Rf = 0.75 ± 0.01). The linear regression analysis data for the calibration plots showed a good linear relationship with r2 = 0.9995. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 18.59 ng/spot, whereas the limit of quantitation was found to be 56.34 ng/spot. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of tazarotene. The method was successfully employed for the estimation of equilibrium solubility, quantification of tazarotene as a bulk drug, in commercially available gel preparation and in-house developed microemulsion based gel formulations.

Development and Validation of HPTLC Method for Estimation of Tacrolimus in Formulations

A new, simple, and rapid high-performance thin-layer chromatographic method with a derivatization procedure was developed and validated for quantitative determination of tacrolimus. Tacrolimus was chromatographed on silica gel 60 F254 TLC plate using toluene–acetonitrile–glacial acetic acid (6:4:0.1, by volume) as mobile phase. Tacrolimus was visualized using a derivatization reagent containing anisaldehyde–sulfuric acid in absolute alcohol and quantified by densitometric analysis in the reflectance mode at 675 nm. The method was found to give compact spots for the drug (Rf = 0.40 ± 0.03). The linear regression analysis data for the calibration plots showed good linear relationship with r2 = .9989 in the concentration range 100–800 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 28.90 ng, whereas the limit of quantitation was found to be 97.04 ng. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the analysis of tacrolimus. The method was successfully employed for the estimation of equilibrium solubility and quantification of tacrolimus as a bulk drug and in commercially available capsules and in-house developed self-microemulsifying formulations.

Simple, accurate and rapid HPTLC method for analysis of theophylline in post-mortem blood and validation of the method

Theophylline (1,3-dimethylxanthine) is used both in the prophylaxis of chronic asthma and chronic obstructive airways disease (COAD), and to treat emergencies in acute severe asthma. Administration of theophylline to patients in respiratory distress can be fatal and, therefore, it is essential to know whether the person has achieved a theophylline blood concentration expected to produce therapeutic effects or otherwise. A method using sodium fluoride-preserved post-mortem blood samples has been established to enable evaluation of theophylline levels in the blood at the time of death. The simple, rapid, and accurate high-performance thin-layer chromatography (HPTLC) method with UV-densitometric detection at 277 nm was validated for analysis of theophylline in postmortem blood. Theophylline was extracted at pH 8.5 with chloroform-isopropanol 8:2 from post-mortem blood after acid hydrolysis. Recovery ranged from 89.1 to 93.4% at a concentration of 10 μg mL −1 in the pH range 8.3 to 8.6. An average analytical...

Standardization of the Ayurvedic Formulation Haridra Khanda Using High-Performance Thin-Layer Chromatography-Densitometry

The present study aimed to standardize the Ayurvedic preparation Haridra Khanda containing Curcuma longa as a major ingredient. Various physicochemical parameters such as alcohol-soluble extractive, water-soluble extractive, total ash, and acid-insoluble ash were determined according to the Ayurvedic Pharmacopoeia of India. Microscopic evaluation of the formulation revealed the presence of various diagnostic cell structures of C. longa. Trace metal analysis indicated the absence of toxic metals such as As, Cd, Hg, and Pb. High-performance thin-layer chromatographic (HPTLC) fingerprint patterns at multiple wavelengths (254, 366, and 430 nm) identified the number of components present at each wavelength. The bioactive markers curcumin (C1), demethoxycurcumin (C2), and bisdemethoxycurcumin (C3) were quantified by using a simple, rapid, and efficient HPTLC method using plates precoated with silica gel 60F254 stationary phase. The instrumental precision [coefficient of variation (CV)] was 0.51, 0.64, and 0.79% and the repeatability of the method (CV) was 0.89, 1.11, and 0.95%, respectively, for C1 to C3. Limits of detection and quantitation for compounds C1 to C3 were 20, 20, and 15 ng and 50, 40, and 50 ng, respectively. Response was a linear function in the ranges of 50-350, 40-240, and 50-300 ng with correlation coefficient (r) = 0.9998, 0.9995, and 0.9992, respectively, for C1 to C3. The mean recovery values of 99.63 (C1), 98.65 (C2), and 98.97% (C3) indicated the excellent accuracy of the method. It is shown that HPTLC can be applied successfully for the marker evaluation of the formulation containing C. longa.

Rapid and sensitive HPTLC method for determination of epalrestat in human plasma

Epalrestat is a new drug for diabetic neuropathy. A new, simple, rapid very sensitive, and accurate high performance thin-layer chromatographic (HPTLC) method has been developed and validated for estimation of epalrestat in plasma. HPTLC was performed on silica gel 60F 254 plates with ethyl acetate-toluene-acetic acid, 60 + 40 + 2 ( v / v ), as mobile phase. Nitrofurantoin was used as internal standard. Densitometric scanning was performed in absorbance mode at λ = 390 nm. The R F values of the internal standard and drug were 0.4 and 0.6, respectively. The response was a linear function of concentration over the range 0.1–6.0 μg mL −1 ( r 2 = 0.9974). Mean extraction recovery was > 63%. Intra-day and inter-day precision (% CV) of the assay were in the range 1.62–4.68% and accuracy was > 95%. This method can be applied to pharmacokinetic and bioequivalence studies.

Pharmacokinetic analysis of ursolic acid inAlstonia scholarisR. Br. by high-performance thin-layer chromatography

A simple and rapid high-performance thin-layer chromatographic method has been established and validated for determination of ursolic acid in rabbit plasma after ingestion of an aqueous slurry of Alstonia scholaris R. Br. leaves. Chromatography was performed on silica gel HPTLC plates with toluene-ethyl acetate-triethyl-amine-methanol, 7 + 2 + 1 + 1 ( v / v ), as mobile phase. The plates were scanned densitometrically at λ = 366 nm. The response to ursolic acid in spiked plasma was a linear function of concentration in the range 10 to 80 μg mL −1 . The detection and quantification limits were 3.0 and 10 μg mL −1 , respectively. The method has been successfully used to study the pharmacokinetics of Alstonia scholaris R. Br. using ursolic acid as a marker.

Rapid HPTLC method for identification and quantification of curcumin, piperine and thymol in an ayurvedic formulation

A simple, precise, rapid, selective, and cost-effective high-performance thin-layer chromatographic (HPTLC) method has been established for simultaneous estimation of curcumin, piperine, and thymol in an ayurvedic formulation. Chromatography was performed on silica gel 60 F 254 plates with toluene-ethyl acetate-methanol, 9 + 1 + 0.5 ( v/v ), as mobile phase. Plates were developed to a distance of 8 cm at room temperature, without chamber saturation. The plates were scanned and the compounds were quantified at their wavelengths of maximum absorption, 420, 333, and 277 nm for curcumin, piperine, and thymol, respectively. The respective R F values of curcumin, piperine, and thymol were 0.23, 0.30, and 0.64. Response was a linear function of the amount applied to the plate in the ranges 50–250 ng, 10–60 ng, and 100–700 ng for curcumin, piperine, and thymol, respectively. LOD for curcumin, piperine, and thymol were 25, 5, and 50 ng, respectively. The mean results from assay of curcumin, piperine, and thymol in...

Quantitative determination of phyllanthin and hypophyllanthin inPhyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.

Development and validation of a HPTLC method for the simultaneous estimation of atorvastatin calcium and ezetimibe

A simple, precise, accurate and rapid high-performance thin-layer chromatographic method has been developed and validated for the estimation of atorvastatin calcium and ezetimibe simultaneously in combined dosage forms. The stationary phase used was precoated silica gel 60F254. The mobile phase used was a mixture of chloroform: benzene: methanol: acetic acid (6.0:3.0:1.0:0.1 v/v/v/v). The detection of spots was carried out at 250 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 0.8 and 4.0 µg/spot for atorvastatin calcium and 0.1 and 1.0 µg/spot for ezetimibe. The limit of detection and the limit of quantification for atorvastatin calcium were found to be 170 ng/spot and 570 ng/spot respectively, and for ezetimibe, 20 ng/spot and 70 ng/spot respectively. The proposed method can be successfully used to determine the drug content of marketed formulation.

Determination of 1,3-Benzodioxanes in Piper mullesua by High-Performance Thin-Layer Chromatography

A new, simple, precise, and rapid high-performance thin-layer chromatographic method was developed for the determination of 6 benzodioxanes in Piper mullesua extract: 1',3'-benzodioxole-5'-(2,4,8-triene-isobutyl nonanoate), 1',3'-benzodioxole-5'-(2,4,12-triene-isobutyl tridecanoate), fargesin, sesamin, asarinin, 1',3'-benzodioxole-5'-(2,4,8-triene-methyl nonanoate). The ingredients were separated on a precoated Silica Gel 60 F254 plate with a solvent system of toluene-acetone (92 + 8). The 6 benzodioxanes were well separated and easily identified in this chromatographic system. The separated benzodioxanes were visualized by color development with a spray reagent consisting of 1 g vanillin dissolved in 100 mL H2SO4-ethanol (5 + 95, v/v). Quantitation was performed by scanning the spots and comparing the integrated areas of compounds in samples with those of standards. Recoveries from samples spiked with known amounts of the benzodioxanes were excellent. The results were comparable with those estimated by liquid chromatography.