Densitometric High-performance Thin-layer Chromatographic Method Research Articles

Simultaneous quantification of five marker compounds of Betula utilis stem bark using a validated high‐performance thin‐layer chromatography method

A sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for five marker compounds, namely betulin, lupeol, oleanolic acid, 3-acetyloleanolic acid and β-sitosterol, known for their various therapeutic activities. The marker compounds have been isolated from the stem bark of Betula utilis, well characterized by the spectral analysis, and their simultaneous quantitative determination carried out by high-performance thin-layer chromatography (HPTLC) method. The resolution of marker compounds was carried out on silica-gel 60 plates, using n-hexane:ethyl acetate (8:2 v/v) as the mobile phase. The HPTLC densitometry was performed at 500-nm wavelength after the post chromatographic derivatization with ceric ammonium sulfate reagent. The optimized method provided good linear relation (r>0.9960) for all the investigated analytes. The method is simple, and reproducible, which may be applied for quantitative analysis of the above-mentioned marker compounds.

Effective separation and analysis of E- and Z-guggulsterones in Commiphora mukul resin, guggulipid and their pharmaceutical product by high performance thin-layer chromatography-densitometric method

A high performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of the hypolipidemic agents, E- and Z-isomers of guggulsterone in Commiphora mukul resin, guggulipid (ethyl acetate extract of resin), and its pharmaceutical formulation, was developed. The developed system was efficient enough to separate both isomers from their conger, 17,20-dihydroguggulsterone ( 3). HPTLC glass plates, pre-coated with silica gel 60F-254, were used as a stationary phase. The mobile phase consisted of toluene:acetone (9.3:0.7, v/v) which gave well resolved spots for E- and Z-guggulsterones ( R f: 0.52 ± 0.01, and 0.67 ± 0.01, respectively) following double development of chromatoplate with the same mobile phase under unsaturated conditions. The analyte stability towards the developed chromatographic procedure was also investigated by two-dimensional (2D) HPTLC analysis. 17,20-Dihydroguggulsterone ( 3) was identified by the electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI–QTOF–MS/MS) analysis.

UV densitometric HPTLC method for analysis of nitrazepam in pharmaceutical formulations

A UV densitometric high-performance thin-layer chromatographic (HPTLC) method for analysis of nitrazepam in pharmaceutical formulations is described. Chromatography was performed on silica gel F254 plates with benzene-ethanol 5:1 (v/v) as mobile phase. Densitometric detection was at 196 nm. Under these conditions the RF of nitrazepam was 0.68. The method provided good results, but only when calibration was performed by non-linear regression analysis, with fitting to the equation y = bxm. The method can be used for routine quality-control analysis of nitrazepam in pharmaceutical formulations. Quantification was achieved over the range of 0.25–10.0 μg per spot. Mean recovery was 98.86% and 98.80 % for tablet and pure powder, respectively. Precision and accuracy, expressed as RSD (relative standard deviation) were 1.26% and 1.18%, respectively. The was no interference from tablet excipients in the assay procedure. The method was validated and found to be sufficiently precise and reproducible.

Stability-indicating densitometric HPTLC method for qualitative and quantitative analysis of arbutin in commercial whitening creams

High-performance thin-layer chromatographic (HPTLC) densitometric method for analysis of arbutin in commercial whitening creams was developed and validated. Aluminum-backed silica gel 60 F254 plates were used as stationary phase while methanol-chloroform-acetic acid 3.5:6:0.5 (%, v/v/v) mixture was used as mobile phase. Under these chromatographic conditions, arbutin was well separated from other ingredients. This system was found to give a well defined, sharp, and compact spot of arbutin at retention factor (RF) value of 0.40 ± 0.02. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 42.25 and 112.45 ng per spot respectively. The proposed method with high degree of precision and accuracy was employed for the analysis of arbutin both qualitatively and quantitatively in commercial whitening creams. Due to the efficiency of the method in separating arbutin from other ingredients including its degradation products, it can be applied as a stability-indicating method.

Development and validation of a densitometric HPTLC method for simultaneous analysis of wedelolactone and asiaticoside in a polyherbal formulation

Summary A sensitive, accurate, and robust high-performance thin-layer chromatographic (HPTLC) method has been established for simultaneous analysis of wedelolactone (WED) and asiaticoside (ASI) in Eclipta alba and Centella asiatica Linn., respectively. Chromatography was performed on silica gel with toluene-acetone-methanol-formic acid 3.0:2.0:2.0:0.05 (υ/υ) as mobile phase. Densitometric scanning at 317 nm for WED and at 530 nm, after derivatisation with 10% methanolic sulphuric acid, for ASI was used. The method was validated in accordance with the guidelines of the International Conference on Harmonization (ICH). RF values of 0.26 and 0.75 were obtained for ASI and WED, respectively. The linear ranges were 50–250 and 150–550 ng per band for WED and ASI, respectively, with good correlation coefficients (r2 = 0.999 and 0.9989, respectively). Accuracy was 99.29% and 99.45% for WED and ASI, respectively. The method was found to be precise, robust, and suitable for routine quality-control analysis of plant ...

Development and Validation of a Reversed-Phase High-Performance Thin-Layer ChromatographyDensitometric Method for Determination of Atorvastatin Calcium in Bulk Drug and Tablets

Atorvastatin calcium is a synthetic HMG-CoA reductase inhibitor that is used as a cholesterol-lowering agent. A simple, sensitive, selective, and precise RP-HPTLC-densitometric determination of atorvastatin calcium both as bulk drug and from pharmaceutical formulation was developed and validated according to International Conference on Harmonization guidelines. The method used aluminum sheets precoated with silica gel 60 RP18F254S as the stationary phase, and the mobile phase consisted of methanol-water (3.5 + 1.5, v/v). The system gave a compact band for atorvastatin calcium with an Rf value of 0.62 +/- 0.02. Densitometric quantification was carried out at 246 nm. The linear regression analysis data for the calibration plots showed a good linear relationship with r = 0.9992 in the working concentration range of 100-800 ng/band. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, LOD, and LOQ. The LOD and LOQ were 6 and 18 ng, respectively. The drug underwent hydrolysis when subjected to acidic conditions and was found to be stable under alkali, oxidation, dry heat, and photodegradation conditions. Statistical analysis proved that the developed RP-HPTLC-densitometry method is reproducible and selective and that it can be applied for identification and quantitative determination of atorvastatin calcium in bulk drug and tablet formulation.

A validated high-performance thin-layer chromatographic method for quantification of sertraline in tablets

A quantitative densitometric high-performance thin-layer chromatographic method for analysis of sertraline hydrochloride, estimated as sertraline, in pharmaceutical preparations has been established and validated. Sertraline was separated and identified on silica gel 60 F 254 HPTLC plateswith chloroform-ethyl acetate-triethylamine 5:3:0.2 ( v/v ) as mobile phase. The plates were developed to a distance of 7 cm. Densitometric quantification was performed at 279 nm by reflectance scanning. Well resolved bands were obtained for sertraline. The method was validated for precision, recovery, specificity, and ruggedness. The calibration plot for sertraline was linear with r = 0.9988. The method is selective and specific with potential application in pharmaceutical analysis.

High-Performance Thin-Layer Chromatography Densitometric Method for the Quantification of Harmine, Harmaline, Vasicine, and Vasicinone in Peganum harmala

Peganum harmala Linn. seed is a reputed drug of the Indian systems of medicine. We report a simple high-performance thin-layer chromatography (HPTLC) densitometric method for the quantification of 4 alkaloids, viz., harmine, harmaline, vasicine, and vasicinone from P. harmala seed. The 4 compounds were resolved in ethyl acetate-methanol-ammonia (7 + 1 + 0.3, v/v/v) mobile phase. The method was validated for precision, repeatability, and accuracy. Instrumental precision was 0.27, 1.53, 0.39, and 1.15% [relative standard deviation (RSD)] and repeatability of the method was 1.01, 0.79, 0.13, and 1.62% RSD for harmine, harmaline, vasicine, and vasicinone, respectively. Accuracy of the method was checked by a recovery study conducted at 3 different levels, and the average recovery was 97.88% for harmine, 97.69% for harmaline, 98.38% for vasicine, and 98.28% for vasicinone. The 4 compounds were quantified in P. harmala seed using the method, and it was found to contain 0.44, 0.096, 0.25, and 0.0007% (w/w) of each, respectively. The proposed HPTLC densitometric method for the quantification of the 4 compounds was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of P. harmala seed and has the ability to quantify the 4 marker compounds in the formulations containing P. harmala. It also can be used to quantify any of these marker compounds in other herbal drugs.

Effect of hydrolysis on the yield of hederagenin and High-performance thin-layer chromatography densitometric quantification of hederagenin in fruit pericarp of Sapindus spp

Fruit pericarp of Sapindus species are reported to contain glycosides with hederagenin as an aglycone. To free the aglycone from the glycosides, they need to be hydrolyzed, and the commonly used method is hydrolysis with either hydrochloric or sulfuric acid. In the present work, we studied the effect of hydrolysis on the yield of hederagenin from the fruit pericarp of 3 species of Sapindus, viz., S. mukorossi, S. laurifolius, and S. emarginatus. A high-performance thin-layer chromatography densitometric method for the quantification of hederagenin was developed and validated. It involved automated application of samples as bands onto silica gel 60F254 plates, development with toluene-ethyl acetate-formic acid (7 + 3 + 5, v/v/v) mobile phase, detection with anisaldehyde-sulfuric acid reagent, and scanning at 595 nm. The yield of hederagenin ranged from 0.035 to 1.29% (w/w) with different methods of hydrolysis. Hydrolysis with 3.5 M aqueous sulfuric acid under reflux for 6 h gave the maximum yield of hederagenin in all 3 species, with the highest amount in S. emarginatus (1.29%, w/w).

A Validated and Densitometric HPTLC Method for the Quantification of Withaferin-A and Withanolide-A in Different Plant Parts of Two Morphotypes of Withania somnifera

A simple, sensitive and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the estimation of withaferin-A and withanolide-A in different plant parts such as, leaf, root, stem and fruit of two morphotypes of Withania somnifera. HPTLC of W. somnifera methanolic extract was performed on Si 60 F254 (20 cm × 20 cm) plates with toluene:ethyl acetate:formic acid (5:5:1), as mobile phase. Quantitative evaluation of the plate was performed in the absorption-reflection mode at 530 nm. The method was validated for precision, repeatability, and accuracy. The average recovery of withaferin-A and withanolide-A in two levels were 96.0 and 96.7%, showing the excellent reproducibility of the method. The calibration curves were linear for both in the range of 200–3,200 ng. The technique has been applied, for the first time, for the estimation of withaferin-A and withanolide-A in different parts of the two morphotypes of Withania somnifera. The method is simple, precise, specific, sensitive and accurate and can be used for routine analysis as well as for quality control of raw materials and herbal formulations.

Validation of a high-performance thin-layer chromatographic method, with densitometric detection, for quantitative analysis of nebivolol hydrochloride in tablet formulations

A quantitative densitometric high-performance thin-layer chromatographic method for determination of nebivolol hydrochloride in pharmaceutical preparations has been established and validated. Nebivolol hydrochloride from the formulations was separated and identified on silica gel 60 F 254 HPTLC plates with toluene-ethyl acetate-methanol-formic acid, 8 + 6 + 4 + 1 ( v / v ), as mobile phase. The plates were developed to a distance of 8 cm. Densitometric quantification was performed at λ = 285 nm by reflectance scanning. Well resolved bands were obtained for nebivolol hydrochloride. The method was validated for precision, recovery, robustness, specificity, and ruggedness. The calibration plot for nebivolol hydrochloride standard was linear with r = 0.9991, slope = 3.291, and intercept = 864.50. The limits of detection and quantification were 18.65 and 62.18 ng per band, respectively. The method is selective and specific with potential application in pharmaceutical analysis.