Developed High-performance Thin-layer Chromatographic Method Research Articles

Quality risk assessment and DoE – Practiced validated stability-indicating chromatographic method for quantification of Rivaroxaban in bulk and tablet dosage form

AbstractA systematic DoE and Analytical Quality by Design (AQbD) approach was utilized for the development and validation of a novel stability indicating high-performance thin–layer chromatographic (HPTLC) method for Rivaroxaban (RBN) estimation in bulk and marketed formulation. A D-optimal design was used to screen the effect of solvents, volume of solvents, time from spotting to development and time for development to scanning. ANOVA results and Pareto chart revealed that toluene, methanol, water and saturation time had an impact on retention time. The critical method and material attributes were further screened by Box-Behnken design (BBD) to achieve optimal chromatographic condition. A stress degradation study was carried out and structure of major alkaline degradant was elaborated. According to the design space, a control strategy was used with toluene: methanol: water (6:2:2) and the saturation time was 15 min. A retention factor (RF) of 0.59 ± 0.05 was achieved for RBN using chromatographic plate precoated with silica gel at detection wavelength 282 nm with optimized conditions. The linear calibration curve was achieved in the concentration range of 200–1,200 ng/band with r2 > 0.998 suggesting good coordination between analyte concentration and peak areas. The quadratic model was demonstrated as the best fit model and no interaction was noted between CMAs. The optimized HPTLC method was validated critically as stated in International Conference on Harmonization (ICH) Q2 (R1) guideline and implemented successfully for stress degradation study of RBN. The developed HPTLC method obtained through AQbD application was potentially able to resolve all degradants of RBN achieved through forced degradation study. The obtained results demonstrate that a scientific AQbD approach implementation in HPTLC method development and stress degradation study drastically minimizes the number of trials in experiments, ultimately time and cost of analysis could be minimized.

Development of a stability indicating HPTLC method for the estimation of olanzapine in pharmaceutical dosage forms

We report herein an accurate, precise, and economical stability indicating high performance thin layer chromatographic (HPTLC) method developed to assess the safety of olanzapine in pharmaceutical formulations. Olanzapine was subjected to forced degradation studies to assess the effect of environmental conditions on its stability. Stress conditions such as hydrolysis under acidic and alkaline environment, degradation and oxidation by heat, light and air were used to study the stability of olanzapine. Mobile phase comprising of toluene: methanol (5:5 v/v) and aluminum plate pre-coated with silica gel 60 F254 as a stationary phase were used for the development of chromatogram by HPTLC technique. Densitometric analysis of olanzapine carried out at 297 ​nm gave sharp symmetrical peak with Rf value of 0.50 and a satisfactory baseline resolution for all components. The drug was found to undergo degradation under acidic, alkaline and oxidative conditions. A single distinct peak in acidic and alkaline media while two peaks obtained as a result of oxidative degradation were well resolved along with the parent drug. The degradation products and parent drug showed significantly different Rf values. The developed HPTLC method gave quick and reproducible results for the olanzapine content in the tablets. The mean recoveries were 100.75% which confirms accuracy of the proposed method. The method was further validated for specificity, ruggedness and robustness. Based on the results, it can be suggested that the developed HPTLC method is quite efficient in separating the olanzapine from its degradation products; hence it can be used by pharmaceutical industries and regulatory bodies for the routine analysis of olanzapine in various pharmaceutical dosage forms.

Validated stability-indicating thin layer chromatographic determination of nadifloxacin in microemulsion and bulk drug formulations

A stability-indicating high performance thin layer chromatographic (HPTLC) method for the densitometric analysis of nadifloxacin in microemulsions was developed and validated according to the guidelines of the International Conference on Harmonization (ICH). The compact spot for nadifloxacin was found at an Rf value of 0.39±0.02 at an absorption wavelength of 288 nm using a mobile phase consisting of chloroform : methanol : formic acid (7.5 : 2.0 : 0.5 v/v). The linear regression data for the calibration plots (r 2= 0.9981) was found with respect to peak area in the concentration range of 50-600 ng/spot. The limit of detection (LOD) and limit of quantification (LOQ) were 9.4 and 20.5 ng respectively. The drug was subjected to acid and alkaline hydrolysis, oxidation, photo degradation and dry heat treatment. The peaks of degradation products were well-resolved from the peak of the standard drug with significantly different Rf values. Statistical analysis revealed that the developed HPTLC method is reproducible, selective and accurate for the determination of nadifloxacin in its formulations. The method can effectively separate the drug from its degradation products and be used for stability-indicating assay.

Development and validation of a simple HPTLC method for the determination of new hepatitis C subtype 4 antiviral agents in their tablet dosage form

A new, simple, precise, accurate and selective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of ledipasvir and sofosbuvir in their tablet dosage form. Chromatographic separation was carried out on Merck TLC aluminum sheets of silica gel 60 F254 using ethyl acetate:hexane:methanol in the ratio of 8:1.25:0.75 (% v/v) as the mobile phase followed by densitometric measurement at 256 nm. The method was validated in terms of linearity, accuracy, precision, limit of detection, limit of quantification and specificity in accordance with the International Conference on Harmonization (ICH) guidelines. The calibration curve was found to be linear between 60 to 1980 and 45 to 3600 ng/band for ledipasvir and sofosbuvir, respectively, with significantly high value of regression coefficient (r2 > 0.9999) with linear and homoscedastic residuals. The limits of detection and quantification were found to be 16.5 and 50 ng/band, respectively, for ledipasvir and 13 and 39.5 ng/band, respectively, for sofosbuvir. Comparative study was performed between the developed HPTLC method and the reported high-performance liquid chromatography (HPLC) method. The quantitative results of the two analytical methods did not show statistically significant difference, whereas the developed HPTLC method is both time- and cost-effective.

Quality by Design-Based Optimization and Validation of a High-Performance Thin-Layer Chromatography Method for the Estimation of Rivaroxaban in Bulk and Its Pharmaceutical Dosage Form

The objective of this study was to develop a new rapid and robust high-performance thin-layer chromatographic (HPTLC) method for the estimation of rivaroxaban (RRB) in tablet dosage form using a quality by design approach. Chromatography was performed using a pre-coated silica gel aluminum plate 60 F254 (10 cm × 10 cm) as the stationary phase and toluene-methanol (7:3, V/V) as the mobile phase. Detection was carried out at 250 nm. The linear regression analysis data for the calibration plots showed r2 > 0.99 with a concentration range from 100–600 ng per band. A Box–Behnken experimental design with a response surface methodology was applied to study the effects of chamber saturation time, band length, and solvent front on the RF value and area of RRB. The RF value was predicted to be 0.63 ± 0.05 for RRB to optimize the chromatographic conditions based on the preliminary trials. The optimized HPTLC method was validated according to the International Conference on Harmonization (ICH) guideline Q2 (R1). The results of this study indicate that the quality by design (QbD) concept could be effectively applied to optimize a HPTLC method with a minimum number of experimental runs. The developed HPTLC method was successfully applied for routine analysis of RRB in tablet dosage form.

Development and Validation of a High-Performance Thin-Layer Chromatographic (HPTLC) Method for Simultaneous Quantification of Reserpine, Atropine, and Piperine in Sarpagandha Ghanvati, a Classical Ayurvedic Preparation.

Background: Anxiety disorders are the most common of emotional disorders, affecting more than 20 million people annually. Sarpagandha Ghanvati is a classical Ayurvedic polyherbal formulation prescribed in conditions of insomnia, hysteria, and is used as an anxiolytic agent. Standardization and quality control are the two major issues that need to be addressed for herbal formulations, especially those containing multiple herbal ingredients. Objective: An HPTLC method was developed for the simultaneous quantification of reserpine, atropine, and piperine from Sarpagandha Ghanvati containing Rauwolfia serpentine (root), Hyoscyamus niger (seed), and Piper longum (root and stem). Methods: The marker compounds were effectively resolved on a silica gel G TLC plate using toluene-ethyl acetate-diethyl amine (7+2+1, v/v) as the mobile phase. The detected wavelengths for reserpine, atropine, and piperine were 269, 220, and 254 nm, respectively. The method was validated as per the International Conference on Harmonization guidelines. Results: R. serpentine roots contained 0.82% w/w of reserpine. Atropine content in the seeds of H. niger was found to be 0.004% w/w, whereas P. longum roots were found to contain 0.508% of piperine. The method was found to be accurate, which was evident from 98.93, 99.46, and 99.10% recovery of reserpine, atropine, and piperine, respectively, when the respective herbs were spiked with them. By the developed HPTLC method, 1.0 g of Sarpagandha Ghanvati was found to contain 4.94, 0.049, and 0.318 mg of reserpine, atropine, and piperine, respectively. The recoveries of these three markers from the formulation were found to be 90.32, 92.45, and 89.97%, respectively. Conclusions: The developed method can be successfully used for simultaneous estimation of these marker compounds and for the quality control of the classical Ayurvedic formulation Sarpagandha Ghanvati. Highlights: This works describes effects of extraction solvents on the quantities of marker compounds in the formulations. It also suggests a simple and reliable HPTLC method for simultaneous quantification of three different marker compounds from a poly-herbal formulation.

HPTLC METHOD FOR THE DETERMINATION OF LUPEOL FROM ANDROGRAPHIS ECHIOIDES LEAVES

Objective: A new simple, precise, rapid and selective high‐performance thin‐layer chromatographic (HPTLC) method has been developed for the determination of lupeol in methanolic leaves extract of Andrographis echioides.Methods: As per International Conference on Harmonization (ICH) guidelines we have applied different concentrations of lupeol as standard on HPTLC plates for the quantification of lupeol from the Andrographis echioides leaves. The concentration of standard lupeol was 1 mg/ml.Results: The retention factor of lupeol was found to be 0.55. Linearity was obtained in the range of 5000 ng‐10000 ng for lupeol. The developed and validated HPTLC method was employed for lupeol in methanolic leaves extract of Andrographis echioides for standardization of the content of the marker. The linear regression data for the calibration plots showed a good linear relationship with r=0.99917 for lupeol. Satisfactory recoveries of 99.80 % were obtained for lupeol.Conclusion: The results obtained in validation assays indicate the accuracy and reliability of the developed HPTLC method for the quantification of lupeol in methanolic leaves extract of Andrographis echioides.

Development of Simultaneous HPTLC Method for estimation of Boswellic acid, Curcumin and Diosgenin in Polyherbal Transdermal Gel

Background: Boswellic acid, curcumin and diosgenin are important phytoconstituents present in Boswellia serrata, Curcuma longa and Trigonella foenum-graecum, respectively. Traditionally, these plants are used in the treatment of arthritis. In the present study an attempt has been made to develop a simple, precise, rapid, selective and cost-effective high-performance thin-layer chromatographic (HPTLC) method for simultaneous estimation of boswellic acid, curcumin and diosgenin from polyherbal transdermal gel prepared for the treatment of arthritis. Material and method: The method employed TLC aluminium plates precoated with silica gel 60F254 as the stationary phase. The solvent system consisted of hexane and acetone. Densitometric analysis was carried out in the absorbance mode at 423nm for curcumin whereas for boswellic acid and diosgenin, plates were scanned at 540nm after derivatization. Result: This system was found to give compact spots for boswellic acid (Rf value of 0.54), curcumin (Rf value of 0.30), and diosgenin (Rf value of 0.64). Response was a linear function of the amount applied to the plate in the ranges 1–6 μg for boswellic acid, curcumin and diosgenin. The % of boswellic acid, curcumin and diosgenin from transdermal gel was found to be 99.46%, 99.47% and 98.85% respectively, which was well within the limit. Conclusion: The developed HPTLC method would be an important tool in the quality control method for polyherbal formulations.

IDENTIFICATION, QUANTIFICATION AND VALIDATION OF STIGMASTEROL FROM ALPINIA CALCARATA USING HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY METHOD

Objective: The present study is designed to develop a new simple, precise, rapid and selective high‐performance thin‐layer chromatographic (HPTLC) method for the determination of stigmasterol in methanolic rhizomes extract of Alpinia calcarata.Methods: As per International Conference on Harmonization (ICH) guidelines we have applied different concentrations of stigmasterol as standard on HPTLC plates for the quantification of stigmasterol from the Alpinia calcarata rhizomes. The concentration of standard stigmasterol is 1 mg/ml.Results: The retention factor of stigmasterol was 0.58. Linearity was obtained in the range of 50 ng‐250 ng for stigmasterol. The developed and validated HPTLC method was employed for stigmasterol in methanolic rhizomes extract of Alpinia calcarata for standardization of the content of the marker. The linear regression data for the calibration plots showed a good linear relationship with r=0.99977 for stigmasterol, respectively Satisfactory recoveries of 99.77 % were obtained for stigmasterol.Conclusion: The results obtained in validation assays indicate the accuracy and reliability of the developed HPTLC method for the quantification of stigmasterol in methanolic rhizomes extract of Alpinia calcarata

Development of validated chemical fingerprint for an Ayurvedic formulation Kantakaryaavaleha

Kantakaryaavaleha is a most trusted Ayurvedic formulation among the Ayurvedic medicines. Kantakaryaavaleha is mentioned throughout the ancient literature of Ayurvedic medicine and used for svasa (asthma), kassa (cough), sula (colic). A simple, sensitive, selective, precise and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the determination and routine analysis of plumbagin in a Traditional Indian formulation (Kantakaryaavaleha) and its crude drug extracts. The validation parameter of developed HPTLC method was found to be reproducible. Analysis was performed on TLC aluminum plates pre-coated with silica gel 60F-254 as the stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of toluene: ethyl acetate: MeOH (8:1:1). Camag TLC scanner III was used for spectro densitometric scanning and analysis in absorbance mode at 265nm. The system was found to give compact spots for plumbagin (Rf value of 0.49 ± 0.02). The HPTLC linear regression analysis data for the calibration plots showed good linear relationship (r2= 0.9998 ± 0.0003) in the concentration range 200 – 600 ng spot–1 with respect to peak area, moreover the method was validated for precision, recovery, robustness and ruggedness according to the International Conference on Harmonization (ICH) guidelines. The plumbagin content was quantified and estimated from the formulation and the plumbago zeylanica plant part. Statistical analysis of the data showed that the method is reproducible and selective for the quantitative determination of plumbagin.

Quantification of the two pharmacologically important triterpenes in various plant parts ofDiospyros sylvaticausing validated high-performance thin-layer chromatography

In view of high pharmacological significance and urgent need to identify high-yielding plant sources of betulin (1) and betulinic acid (2), quantitative high-performance thin-layer chromatographic (HPTLC) analysis has been done on various parts of Diospyros sylvatica. For separation of both compounds, HPTLC silica gel 60 F254 plates were used with chloroform-methanol-formic acid (98:2:2) as the mobile phase. The method was validated for selectivity, sensitivity, accuracy and precision. While betulin was found in maximum amount in stems (0.08%), betulinic acid accumulates maximally in roots (0.29%). The developed HPTLC method is simple, rapid, and highly suitable for the quantification of betulin and betulinic acid in various related species and genera.

DEVELOPMENT OF A NEW HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD FOR QUANTITATIVE ESTIMATION OF LAMIVUDINE AND ZIDOVUDINE IN COMBINED TABLET DOSAGE FORM USING QUALITY BY DESIGN APPROACH

The purpose of this study was to develop a new rapid and robust high-performance thin layer chromatographic (HPTLC) method for separation and quantitation of lamivudine and zidovudine in combined tablet dosage form using a quality by design approach. A Box–Behnken experimental design with response surface methodology was utilized to study the effects of chromatographic chamber saturation time, mobile phase migration distance, and mobile phase composition on R f value. The R f value was predicted for lamivudine and zidovudine in between 0.2 and 0.8 to optimize the chromatographic conditions based on the preliminary trials. The optimized chromatographic conditions were 21 min saturation time, 50 mm migration distance, and ethyl acetate:hexane:methanol:acetic acid (4:4:2:0.1 v/v/v/v) as a mobile phase. The optimized HPTLC method was validated according to International Conference on Harmonization (ICH) guideline Q2 (R1). The results of study clearly indicate that quality by design concept could be effectively applied to optimize HPTLC method with the minimum number of experimental runs. Developed HPTLC method was successfully applied for routine analysis of lamivudine and zidovudine in combine tablet dosage form.

Development and Validation of a Stability-Indicating High-Performance Thin-Layer Chromatographic Method for the Simultaneous Quantification of Sparfloxacin and Flurbiprofen in Nanoparticulate Formulation

A simple, sensitive, precise, rapid, and reliable high-performance thin-layer chromatographic (HPTLC) method for the simultaneous estimation of sparfloxacin (SPF) and flurbiprofen (FLB) in bulk drug as well as in dual drug loaded poly-(lactic-co-glycolic acid) (PLGA) nanoparticles was developed. In this method, aluminumbacked silica gel 60 F254 plates (20 × 10 cm: 200 μm thickness) were used as stationary phase and chloroform-methanol-formic acid (7.5:1:1, v/v) as an optimized mobile phase. Developed chromatogram was scanned at 258 nm, the wavelength of maximum absorption SPF and FLB. Regression analysis of the calibration data showed an excellent linear relationship between peak area versus drug concentration. Linearity was found to be in the range of 100–600 ng spot−1 and 40–800 ng spot−1 for SPF and FLB, respectively. The suitability of the developed HPTLC method for simultaneous estimation of SPF and FLB was established by validating it as per the ICH guidelines. The limits of detection (LOD) and quan...

Development and validation of a high performance thin layer chromatographic method for determination of 1, 8-Cineole in Callistemon Citrinus.

A new, simple, precise, rapid, and selective high performance thin layer chromatographic (HPTLC) method has been developed and validated for the estimation of 1, 8-cineole in volatile oil of leaves of Callistemon Citrinus obtained by hydro distillation. The method was validated as per ICH guidelines and can be utilized for routine analysis. The retention factor for 1, 8-cineole was found to be 0.52. The linearity was found to be in the range of 3 μg-12 μg. The recovery obtained for 1, 8-cineole was 98%, which is satisfactory. The result obtained in validation indicate the accuracy, reproducibility, and reliability of the developed HPTLC method for determination of 1, 8-cineole.

Development of Pharmacognostical Parameters and Estimation of β-sitosterol using HPTLC in Roots of Gmelina arborea Roxb

This paper deals with the detailed pharmacognostical evaluation of roots of Gmelina arborea Roxb. (Verbenaceae), a highly valued plant in Ayurveda as a bitter tonic, stomachic, laxative and useful in fever, indigestion, anasarca and skin problems. It has been used as an ingredient in many preparations like Dashmoola, Panchmoola di kwatha and Chyawanprasha. Chemical constituents of G. arborea roots have been studied extensively which include lignans, flavonoids, alkaloids and various phenyl propanoid glycosides but pharmacognosy of roots has not been reported. The main objective of the present work is to develop detailed pharmacognostical and physicochemical parameters recommended by World Health Organization (WHO) and various pharmacopoeias for the roots, as this has not been reported so far for the roots of the plant. Preliminary analysis showed the presence of phenolics and flavonoids, which led us to estimate total phenolics in roots. The present study also focuses on development of high-performance thin-layer chromatographic (HPTLC ) method for the quantitative analysis of roots using β-sitosterol as a chemical marker. Major microscopic features of roots are lignified cork, presence of oil globules, scattered stone cell islets in secondary cortex and pitted wood elements. Phytochemical screening reveals the presence of flavonoids, lignans, sterols, tannins, carbohydrate, coumarins and alkaloids. Total phenolics were found 1.89% w/w in roots and β-sitosterol was found 0.120 ± 0.018% in methanolic extract of the roots by the developed HPTLC method. These findings will aid in the standardization of one of the major roots used in a well known Ayurvedic formulation, Dashmoola. Validated HPTLC method developed can be used as a tool for standardization of roots in different formulations using β-sitosterol as a marker.

Stability-Indicating TLC-Densitometric Determination of Nebivolol Hydrochloride in Bulk and Pharmaceutical Dosage Form

A simple, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for densitometric determination of nebivolol hydrochloride both as a bulk drug and in formulation was developed and validated as per the international conference on harmonization guidelines (ICH). The method employed TLC aluminium plates precoated with silica gel 60F254 as the stationary phase. The solvent system consisted of toluene-methanol-triethylamine (3.8:1.2:0.2 v/v/v). Densitometry analysis of nebivolol hydrochloride was carried out in the absorbance mode at 281 nm. The system was found to give compact spot for nebivolol hydrochloride (R(f) value of 0.33 +/- 0.02). The linear regression analysis data for the calibration plots showed good relationship with r(2)= 0.9994 +/- 0.0002 in the concentration range 500-3000 ng/spot. The mean value +/- SD of slope and intercept were 3.761 +/- 0.017 and 127.39 +/- 19.53 with respect to peak area. The limits of detection (LOD) and limit of quantitation (LOQ) were 63.10 ng/spot and 191.23 ng/ spot, respectively. Nebivolol hydrochloride was subjected to acid and alkali hydrolysis, oxidation, thermal degradation, and photodegradation. All the peaks of degradation products were well-resolved from the standard drug with significantly different R(f) values. Statistical analysis proves that the method is repeatable, selective and accurate for the estimation of said drug. The proposed developed HPTLC method can be applied for identification and quantitative determination of nebivolol hydrochloride in the bulk and pharmaceutical dosage form.

Stability-Indicating HPTLC Determination of Ambroxol Hydrochloride in Bulk Drug and Pharmaceutical Dosage Form

A simple, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of ambroxol hydrochloride both as a bulk drug and in formulations was developed and validated. The method employed HPTLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of methanol-triethylamine (4:6 v/v). The system was found to give a compact spot for ambroxol hydrochloride (R(f) value of 0.53 +/- 0.02). Densitometric analysis of ambroxol hydrochloride was carried out in the absorbance mode at 254 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r(2) = 0.9966 +/- 0.0013 with respect to peak area in the concentration range 100-1000 ng/spot. The mean value +/- standard deviation of slope and intercept were 164.85 +/- 0.72 and 1168.3 +/- 8.26 with respect to peak area. The method was validated for precision, recovery, and robustness. The limits of detection and quantitation were 10 and 30 ng/spot, respectively. Ambroxol hydrochloride was subjected to oxidation and thermal degradation. The drug undergoes degradation under oxidation and heat conditions. This indicates that the drug is susceptible to oxidation and heat. Statistical analysis proves that the method is repeatable, selective, and accurate for the estimation of said drug. Stability indicating of new chemical entities is an important part for the drug development of ambroxol hydrochloride and for its estimation in plasma and other biological fluids; the novel Statistical analysis proves that the method is repeatable and selective for the analysis of ambroxol hydrochloride as bulk drug and in pharmaceutical formulations. The proposed developed HPTLC method can be applied for identification and quantitative determination of ambroxol hydrochloride in bulk drug and dosage forms. This work is to determine the purity of the drug available from the various sources by detecting the related impurities.

Development and validation of an HPTLC method for analysis of zerumbone, the anticancer marker fromZingiber zerumbet

Summary A simple, accurate, and rapid high-performance thin-layer chromatographic (HPTLC) method for quantification of the anticancer marker zerumbone in different parts of Zingiber zerumbet Smith has been established and validated. Chromatography was performed on aluminium foil-backed silica gel 60 F254 HPTLC plates with the binary mobile phase ethyl acetate-hexane 1.5:8.5. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 250 nm. Regression analysis data for the calibration plot showed there was a linear relationship between response and concentration in the range 60–260 ng with a regression coefficient of 0.9997. The limits of detection and quantification were 20 and 60 ng, respectively. The instrumental precision and repeatability of the method were found to be 0.80 and 1.08%, respectively. Recovery ranging from 97.85 to 100.12% indicated the excellent accuracy of the method. The developed HPTLC method was successfully used for analysis of the marker in differ...

Estimation of Berberine in Ayurvedic Formulations Containing Berberis aristata

A sensitive, simple, rapid, and efficient high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the analysis of berberine in marketed Ayurvedic formulations containing Berberis aristata DC for regulatory purposes. Chromatography of methanolic extracts of these formulations was performed on silica gel 60 F254 aluminum-backed TLC plates of 0.2 mm layer thickness. The plate was developed up to 66 mm with the ternary-mobile phase butanol-acetic acid-water (8 + 1 + 1, v/v/v) at 33 +/- 5 degrees C with 5 min of tank saturation. The marker, berberine, was quantified at its maximum absorbance of 350 nm. The limit of detection and limit of quantitation values were found to be 5 and 10 ng/spot. The linear regression analysis data for the calibration plot showed a good linear relationship with correlation coefficient = 0.9994 in the concentration range of 10 to 50 ng/spot for berberine with respect to peak area. The instrumental precision was found to be 0.49% coefficient of variation (CV), and repeatability of the method was 0.73% CV. Recovery values from 98.27 to 99.11% indicate excellent accuracy of the method. The developed HPTLC method is very accurate, precise, and cost-effective, and it has been successfully applied to the assay of marketed formulations containing B. aristata for determination of berberine.

HPTLC Method for the Quantitative Determination of ar-Turmerone and Turmerone in Lipid Soluble Fraction from Curcuma longa

A simple, sensitive and precise high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the analysis of ar-turmerone and turmerone, the major constituents of the lipid soluble fraction of the herbal medicament (HM) obtained from the rhizomes of Curcuma longa (turmeric). The separation was carried out on HPTLC aluminum plates precoated with silica gel 60F-254, with n-hexane:ethyl acetate (9.8:0.2 v/v) as mobile phase. Densitometric analysis of ar-turmerone and turmerone was carried out in the absorbance mode at 254 nm. This system was found to give compact spots for ar-turmerone and turmerone (Rf values 0.5 ± 0.05; 0.6 ± 0.04, respectively). A good linear regression relationship between peak areas and the concentrations was obtained over the range of 100–600 ng/spot, with correlation coefficients of 0.997 and 0.998 for ar-turmerone and turmerone, respectively. The limit of detection and quantification was found to be 20 and 40 ng/spot for ar-turmerone and turmerone, respectively. The method was further validated for precision and recovery. The RSD values of the precision were in the range 0.49–1.33% and spike recoveries were 99.9 and 100.0% for ar-turmerone and turmerone, respectively. Analysis of different batches of HM using the above method gave ar-turmerone and turmerone contents in the range of 25–30% and 30–38%. The developed HPTLC method can be applied for identification and quantitative determination of ar-turmerone and turmerone in the lipid soluble fractions of turmeric.