Developed HPTLC Method Research Articles

Combinatorial analysis of quercetin and resveratrol by HPTLC in Sesbania grandiflora/phyto-based nanoformulations

A sensitive HPTLC method was developed for the simultaneous estimation of quercetin (QUR) and resveratrol (RES). The chromatographic separation was achieved using mobile phase toluene:chloroform:ethyl acetate:formic acid (3:2:4.9:0.1% v/v) and densitometric scan performed at 280 nm. The developed method was linear at 2–10 µg/mL with correlation coefficient of 0.9907 (QUR) and 0.9917 (RES). The method was validated for its precision, specificity, detection and quantification limits and % RSD was found to be less than 4.0%. The developed HPTLC method was evaluated in QUR and RES-loaded nanoformulation and Sesbania grandiflora leaf extract. The amount of QUR and RES present in the SG leaf extract was found to be 26.13 ± 0.7 µg/mg and 4.31 ± 0.8 µg/mg, respectively. The pH-dependent stability of RES has checked using the developed method. The above-developed method can be used to check the QUR/RES content in herbal/pharmaceutical formulation with scope towards industries.

Optimization of microwave-assisted extraction technique for isolation of betulinic acid from Dillenia indica Linn. and its quantification using developed HPTLC method

Structure determination of betulinic acid from the leaves of Lophira lanceolata Van Tiegh. Ex Keay (Ochnaceae)Sani Audu Ali, Rafat Ojuolape Abdulraheem, Sikirat Sani Abdulkareem, Emmanuel Taiwo Alemika, Mohammed Ilyas

Stability Indicating TLC Method for Quantification of Brexpiprazole in Bulk and Its Pharmaceutical Dosage Form and Determination of Content Uniformity.

A sensitive, selective and precise high performance thin layer chromatographic method has been developed and validated for the quantification of Brexpiprazole in bulk drug and in pharmaceutical dosage form. The method employed HPTLC aluminum plates (pre-coated with silica gel 60 F254) as stationary phase while n-butanol was used as mobile phase. The Rf value of Brexpiprazole was observed to be 0.38. The densitometric analysis was carried out in absorbance mode at 215 nm. The linear regression analysis data for the calibration plots showed a good linear relationship for Brexpiprazole over a concentration range of 200-1,600 ng band-1. The limit of detection and limit of quantification for Brexpiprazole was found to be 66 and 200 ng band-1. To find out the possible degradation pathway, forced degradation studies were performed. The stock solutions of Brexpiprazole (1,000 μg mL-1) were subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photo degradation. The drug was found to be susceptible to acid and alkali hydrolysis, chemical oxidation, photo degradation and dry heat. The degraded product peaks were well resolved from the pure drug peak with significant difference in their Rf values. Stressed samples were analyzed using developed HPTLC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of Brexpiprazole in marketed formulation and determination of content uniformity of tablet formulation. Statistical analysis showed that the method is repeatable, selective, and precise.

Qualitative and Quantative estimation of phyto-constituents in Hemidismus indicus roots by using advanced analytical tools

The roots of Hemidismus indicus have been used in traditional and indigenous system of medicines thought the humankind for thousands of years. The plant Hemidismus indicus enriched with many phyto-constituents, which are responsible to cure many ailments. The present study was aimed to physiochemical screening and estimation of the bioactive compounds by using advance analytical tools. The study results in the qualitative and quantative estimation of the secondary metabolites; which are industrially important in standardization of the herbal drug products. The heavy metals and residues are the main intimidation in herbal drug formulations. The advance analytical instruments like HPLC and ICPMS are used in the estimation of Aflotoxins and Heavy metals in the study. The validated analytical methods and tools are applied here for the identification and quantifications of the Hemidismus indicus root powder. The same method and assay can be used as quality control parameters in standardization of the plant extracts and herbal drug formulations. The developed HPTLC method is found to be sensitive, specific, precise and accurate for the identification and quantification of isovanillin, rutin and coumarin in Hemidismus indicus. The finger prints derived from the HPTLC can be used as the athentification tool for the Hemidismus indicus roots.

Analysis of Chromium (III)Picolinate in Capsule dosage form by using Stability indicating HPTLC Method

Chromium (III) picolinate is used as a nutritional supplement and has been valuable effects on carbohydrate and lipid metabolism alleviating symptoms associated with diabetes. A chromium supplement produces beneficial results in reducing insulin sulfonylurea or metformin requirements. Hence, stability indicating HPTLC method was developed for estimation of Chromium picolinate in capsule formulation. The development of HPTLC method optimization on precoated silica gel 60 F254 aluminium plates of 20 cm x 20 cm, 250μm thickness. The mobile phase used was methanol: ethyl acetate6:4 (v/v). The densitometry detection was done at 264 nm. Also, the forced degradation studies were performed and method was validated with as per ICH guidelines. The Rf value obtained was 0.39 ±0.05.Linearity data for the calibration curve gave a good linear relationship over the concentration range of 100-600ng/spot for Chromium picolinate (correlation coefficient R2 =0.9997). The percent recovery of Chromium picolinate in marketed formulation was found in the range of 99.56%.Force degradation and validation of method was done as per ICH guidelines and the results are within the compliance limit.The developed HPTLC method gave good results for force degradation studies show that the method is stability indicating. Thus, this method can be used for routine analysis of Chromium picolinate and can be use for its future usage

Simultaneous HPTLC Densitometric Estimation of KBA and AKBA from Boswellia serrata

Background: Boswellic acids (BAs) are extracted from oleo gum of Boswellia serrata and are utilized as potential anti-inflammatory, hypolipidemic, immunomodulatory and antitumor specialists. The present examination was meant to assess KBA and AKBA in Boswellia serrata separate by High-Performance Thin Layer Chromatography (HPTLC). Methods: The separation of bioactive compounds was performed utilizing mobile phase glacial acetic acid, n-hexane, ethyl acetate and toluene (0.3: 1: 8: 2) (v/v/v/v) and distinguished at wavelength 254 nm. The technique was approved for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and so forth by International Conference on Harmonization guidelines. Results: The calibration range was observed to be 2- 14 μg/band for both the bioactive compounds. KBA was isolated with an Rf estimation of 0.39 ± 0.02 and AKBA with an Rf estimation of 0.42 ± 0.02. The accuracy was seen to be as high as 99.17% and 97.42 for KBA and KBA, respectively. The percentage RSD value for intra-day and between day varieties was under 2%. The system indicated high affectability and specificity. Conclusion: The developed HPTLC method was simple, precise, robust, specific, rapid, and costeffective and could be used for quality control analysis and quantification of KBA and AKBA in different herbal formulations containing the plant species.

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR QUANTITATIVE ESTIMATION OF PALIPERIDONE

A planar chromatography (HPTLC) method has been developed and validated for quantitative estimation of the atypical antipsychotic drug paliperidone, which is weakly basic and poorly water soluble. The Camag HPTLC system, operated by software winCATS (ver.1.4.1.8), was used. Sample application was facilitated by Linomat 5 applicator. After sample application, plates were subjected to ascending development in twin trough chamber of size 10 cm x10 cm, using about 10 mL of mobile phase. The optimized mobile phase was composed of ethyl acetate:chloroform:toulene:methanol (2.5:2.5:2.5:2.5 v/v/v/v). In post development, the plates were air dried and then scanned densitometrically using a UV detector at 278 nm in absorbance mode. In HPTLC densitogram, well defined peak was obtained for paliperidone with starting position at 0.27 Rf, max position at 0.31 Rf and end position at 0.34 Rf. The optimal Rf value for paliperidone was found to be 0.31. Performance characteristics of proposed and newly developed HPTLC method were statistically validated as per recommendations of ICH guidelines of analytical method validation. The HPTLC method was found to be linear across the range from 20 to 140 ng/band. The LOD and LOQ values were found to be 1.288 and 3.905ng/band, respectively. The method was found to be accurate, precise, robust and economical for the determination of paliperidone from bulk and its extended release tablet formulation.

Stability-Indicating HPTLC Method for Estimation of Benazepril in Bulk and Tablet Dosage Form

A stability-indicating high-performance thin-layer chromatography method for determination of benazepril both in bulk and pharmaceutical formulation has been developed and validated as per ICH guidelines. The benazepril was analyzed on pre-coated silica gel GF254 plates using Carbon tetrachloride: Methanol: Triethylamine (7.6: 2.4: 0.1 v/v) as the mobile phase. The concentration of benazepril was determined by densitometry at 240 nm. The method was found to be linear, with r2 = 0.998±0.3347 in the concentration range 200 - 1200 ng per spot. The LOD and LOQ were 19.48 ng and 59.03 ng per spot respectively. Benazepril was subjected to forced degradation like acid and alkali hydrolysis, oxidation, photo and thermal degradation. The drug was found to degrade under acidic, basic, oxidative and thermal conditions. The degraded product was well resolved from the pure drug with significantly different Rf value. In validation, it was found that the method is repeatable, selective and accurate for the estimation of benazepril. The proposed developed stability-indicating HPTLC method can be applied for identification and quantitative determination of benazepril in bulk drug and pharmaceutical formulation.

A Validated High-Performance Thin-Layer Chromatographic Method for the Simultaneous Determination of Zofenopril Calcium and Hydrochlorothiazide in the Presence of the Hydrochlorothiazide Impurities: Chlorothiazide and Salamide.

The chromatographic analysis of either process-related impurities or degradation products is very important in the pharmaceutical industry. In this work, a simple, selective, and sensitive HPTLC method was developed and validated for the simultaneous determination of zofenopril calcium (ZOF) and hydrochlorothiazide (HCT) in the presence of the HCT impurities: A) chlorothiazide (CT) and B) salamide, in raw materials and in pharmaceutical formulation. The separation was carried out on HPTLC silica gel 60 F254 using ethyl acetate-glacial acetic acid-triethylamine (10 + 0.1 + 0.1, v/v/v) as a developing system. The separated bands were scanned densitometrically at 270 nm. Polynomial equations were used for the regression. Calibration curves were constructed for ZOF, HCT, CT, and salamide in the ranges of 0.5-10, 0.2-4, 0.05-1.4, and 0.05-1.0 μg/band, respectively. Different parameters affecting the suggested method, including developing systems of varying composition/ratios and different detection wavelengths, were studied to achieve the best resolution and precision with good sensitivity. System suitability parameters were also tested. The proposed method was validated as per the International Conference on Harmonization guidelines and was successfully applied for the quantification of the studied drugs in their pharmaceutical formulation, with no interference from excipients observed. The results obtained by the developed HPTLC method were compared statistically with those obtained by the reported HPLC method using Student's t and F ratio tests, and no significant difference was obtained, indicating the ability of the proposed method to be used for routine analysis of drug product.

To study the Photodynamic antimicrobial activity of Henna extract and preparation of topical Gel formulation

Henna, known to possess antibacterial & antifungal activities is reported to contain naphthoquinone derivatives which have potential as photodynamic agents. Current study comprises of evaluation of Photodynamic antimicrobial activity of alcoholic extract of henna, followed by preparation & evaluation of a topical gel formulation. The alcoholic extract prepared by maceration was analyzed for phytochemicals. Lawsone (2- Hydroxy-1,4 naphthoquinone) was isolated from Henna and was characterized by UV, IR & MASS to confirm its structure. The HPTLC fingerprinting of extract and quantification of lawsone in extract and fraction was carried out. The effect of light was tested on extract and fraction to assess its photosensitizing property. The antimicrobial activity of the extract was tested where one set was exposed to light and other kept in dark and zone of inhibition calculated for both sets. The yield of fraction was found to be 0.5±0.1% w/w. The quantity of lawsone in alcoholic extract & fraction was found to be 1.3 ± 0.02 % w/w and 59.57 ± 1.01 % w/w respectively. The extract is nearly 75% as active as standard Gentamicin and the activity gets 20% potentiated on exposure to light for all test samples indicating Photodynamic antimicrobial activity. The gel was evaluated for its stability with developed HPTLC method and also for physicochemical parameters. The gel containing henna extract, exhibiting good Photodynamic antifungal & antibacterial properties, was found to be stable. The current study thus has resulted in developing a potentially effective herbal topical formulation for management of skin, scalp & hair infections

Anticancer activity and concurrent analysis of ursolic acid, β-sitosterol and lupeol in three different Hibiscus species (aerial parts) by validated HPTLC method

The genus Hibiscus contains about 275 species of flowering plants widely grown in the tropics and sub-tropics. The available literature revealed that several Hibiscus species exhibited excellent anticancer activity against several cancer cells like lung, breast, and liver. This motivated the authors to explore the anticancer property of other Hibiscus species (Hibiscus calyphyllus, H. deflersii and H. micranthus) along with development of a validated HPTLC method for the concurrent analysis of three anticancer biomarkers (ursolic acid, β-sitosterol and lupeol) in different Hibiscus species. The anticancer activity of various fractions (petroleum ether, toluene, dichloromethane, ethyl acetate and n-butanol) of all the Hibiscus species (aerial parts) were evaluated in vitro against HepG2 and MCF-7 cell lines using MTT assay. The HPTLC analysis was carried out using chloroform and methanol as mobile phase (97:3; v/v) on 20 × 10 cm glass-backed silica gel 60F254 plates and analyzed different phytoconstituents present in all fractions at λ = 575 nm wavelength. Of the tested fractions of H. calyphyllus, H. deflersii and H. micranthus, HdP (H. deflersii petroleum ether fraction) exhibited the most potent cytotoxic effect on HepG2 and MCF-7 (IC50: 14.4 and 11.1 μg/mL, respectively) cell lines. Using the developed HPTLC method a compact and intense peak of ursolic acid, β-sitosterol and lupeol were obtained at Rf = 0.22, 0.39 and 0.51, respectively. The LOD/LOQ (ng) for ursolic acid, β-sitosterol and lupeol were found as 42.30/128.20, 13.20/40.01 and 31.57/95.68, respectively in the linearity range 100–1200 ng/spot. The obtained result showed maximum presence of ursolic acid, β-sitosterol and lupeol (5.50, 11.85 and 7.47 μg/mg, respectively) in HdP which also supported its strong anticancer effect. Our data suggest that H. deflersii petroleum ether fraction (HdP) can be further subjected to the isolation of active cytotoxic phytoconstituents and establishment of their mechanism of action. The maiden developed HPTLC method for concurrent analysis of anticancer biomarkers may be further employed in the in process quality control of herbal formulation containing the said biomarkers.

Back Cover: Two chromatographic methods for the quantitative determination of some pesticides applied for cucumber pests in Egypt

Sep. Sci. plus 2018, 1, 334–342 DOI: 10.1002/sscp.201800012 The cover picture shows two novel sensitive and accurate thin layer chromatography and HPLC methods that were used for monitoring the residues of four pesticides; mandipropamid, metalaxyl, thiophenate methyl and carbendazim, which are widely used to eliminate cucumber pests in Egypt; the first method is a HPTLC method, using silica gel plates as a stationary phase and hexane, methanol, acetone, and glacial acetic acid as a developing system; the second method is HPLC using isocratic elution and a mobile phase consisting of acetonitrile and potassium dihydrogen phosphate buffer with ultra violet detection. The proposed HPTLC and RP-HPLC methods are precise, accurate and validated analytical procedures. The advantage of the developed HPTLC method is that many samples can be run simultaneously using a small quantity of developing system, it shows a high selectivity in short analysis time. The suggested chromatographic methods can easily be used for quality analysis of cucumber to make sure the pesticides dose is within acceptable limit. Application of these methods to commercial formulations showed that excipients do not interfere with the determination of the studied pesticides and hence it can be used for their determination either in bulk powder or in commercial formulations.

HPTLC METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF CINNAMALDEHYDE IN POLYHERBAL FORMULATION WITH ANTI-DIABETIC PROPERTY

In this research article HPTLC method for the estimation of cinnamaldehyde content in cinnamomum zeylanicum, laboratory batches (MC-I, MC-II, MC-III) and marketed formulation (MCM) was developed. In this method, precoated Silica Gel F254 plates were used as stationary phase and Toluene: ethyl acetate: formic acid (8:1:1v/v) as mobile phase. Developed chromatogram was scanned at 295 nm, the wavelength of maximum absorption for cinnamaldehyde. The aptness of developed HPTLC method for estimation of cinnamaldehyde was established by validating it as per the ICH guidelines. The developed method has been successfully used for the estimation of cinnamaldehyde in polyherbal anti-diabetic formulation. Keywords: Polyherbal formulation, Cinnamaldehyde, Cinnamomum zeylanicum, HPTLC.

DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR ESTIMATION OF ELLAGIC ACID IN ANTIDIABETIC HERBAL FORMULATION

A simple, sensitive, precise, rapid and reliable HPTLC method for the estimation of ellagic acid in marketed herbal formulation, Glucomap tablet was developed. In this method, precoated Silica Gel F254 Plates were used as stationary phase and Toluene: ethyle acetate: formic acid: methanol (3:3:8:2 v/v) as mobile phase. Developed chromatogram was scanned at 280 nm, the wavelength of maximum absorption for ellagic acid. The aptness of developed HPTLC method for estimation of ellagic acid was established by validating it as per the ICH guidelines. The content of ellagic acid in crude drug Terminalia arjuna and polyherbal formulation was also studied. The developed method has been successfully applied for the determination of ellagic acid in polyherbal formulation.

Concurrent analysis of bioactive triterpenes oleanolic acid and β-amyrin in antioxidant active fractions of Hibiscus calyphyllus, Hibiscus deflersii and Hibiscus micranthus grown in Saudi Arabia by applying validated HPTLC method

In this study, we developed a validated HPTLC method for concurrent analysis of two natural antioxidant triterpenes, oleanolic acid (OA) and β-amyrin (BA) in the biologically active fractions (petroleum ether, toluene, chloroform, ethyl acetate and n-butanol) of aerial parts of three Hibiscus species (H. calyphyllus, H. deflersii and H. micranthus). The chromatography was conducted on normal HPTLC (ready to use glass-plate coated with silica gel 60 F254) plate with chloroform and methanol (97:3, V/V) used as mobile phase. The derivatization of the developed plate was done with p-anisaldehyde and scanned at λmax = 575 nm. Well resolved and intense peaks of OA and BA were obtained at Rf = 0.36 and 0.57, respectively. The linear regression equation/correlation coefficient (r2) for OA and BA were Y = 6.65x + 553.35/0.994 and Y = 9.177x + 637.23/0.998, respectively in the linearity range of 100–1200 ng/spot indicated good linear relationship. The low values of %RSD for intra-day/inter-day precision of OA (1.45–1.61/1.38–1.59) and BA (1.52–1.57/1.50–1.53) suggested that the method was precise. The recovery/RSD (%) values for OA and BA were found to be 99.21–99.62/1.39–1.95 and 98.75–99.70/1.56–1.80, respectively assures the reasonably good accuracy of the proposed method. Fifteen samples were analyzed to check the content of OA and BA by using the developed HPTLC methods. The content of OA in different samples were found to be 3.87 (HmP) > 1.212 (HcP) > 0.673 (HdC) > 0.493 (HdP) > 0.168 (HdE) > 0.059 (HcC) > 0.015 (HcE) > 0.008 (HmT) µg/mg of the dried weight of extract. However the content of BA was found as: 2.293 (HmP) > 1.852 (HdT) > 0.345 (HdC) > 0.172 (HmT) > 0.041 (HdE) > 0.008 (HcC) µg/mg of the dried weight of extract. Some Hibiscus species fractions exhibited good antioxidant potential like: HcE (IC50 = 17.6 ± 1.8) > HdB (IC50 = 32.16 ± 0.9) > HmP (IC50 = 80.4 ± 4.5) > HmT (IC50 = 99.7 ± 8.2) when compared with ascorbic acid (IC50 = 14.2 ± 0.5), while other fractions exhibited only mild antioxidant capability. The developed HPTLC method can be further exploited for analysis of these markers in the quality assessment of raw material as well as herbal formulations available in the market.

DEVELOPMENT AND VALIDATION OF A HPTLC METHOD FOR SIMULTANEOUS DENSITOMETRIC ANALYSIS OF GLYCYRRHETIC ACID AND SOLASODINE IN HERBAL DRUG FORMULATION

Objective: To develop and validate a simple, precise, selective, and accurate high-performance thin layer chromatographic method for simultaneous densitometric analysis of glycyrrhetic acid and solasodine in the polyherbal formulation.Methods: The method was developed using HPTLC silica gel GF254precoated aluminium plate as the stationary phase and Chloroform: Methanol (9:1 v/v) as the mobile phase. Quantization of glycyrrhetic acid was achieved by determining the area under the curve at 267 nm using CAMAG TLC Scanner and CATS 3 software. Since the structure of solasodine lacks conjugated double bond, it does not give any fluorescence either in the absorbance mode or reflectance mode hence solasodine was derivatized using 0.5%anisaldehyde sulphuric acids which gave a bluish spot as seen on TLC plate. These spots were scanned at 546 nm wavelength using CAMAG TLC Scanner and CATS 3 software.Results: The retention factor for glycyrrhetic acid and solasodine were found to be 0.52±0.01, 0.40±0.01% w/w respectively. The developed HPTLC method was validated using parameters described in International Conference on Harmonization (ICH) guideline. The proposed method showed good linearity in the range of 400-2000 ng spot-1 for glycyrrhetic acid as well as for solasodine. The content of glycyrrhetic acid and solasodine in marketed polyherbal formulation were found to be 0.67%±0.8 and 0.10±0.35%w/w respectively.Conclusion: The developed method can be used as quality control tool for the routine analysis of glycyrrhetic acid and solasodine in polyherbal formulation.

HPTLC: A Tool for Determination of Curcumin in Mammalian Samples

Curcumin, an important phytoconstituent obtained from Curcuma longa L. (turmeric) is used traditionally in the treatment of various inflammatory diseases like arthritis, stroke and bowel diseases, etc. Despite its many health benefits, instability of curcumin in plasma is a major issue. The retention of curcumin in plasma must be properly evaluated in order to establish its stability in biological systems. The current study presents an HPTLC method undertaken for detection of curcumin and determination of its stability in plasma and different tissues of rats. The plasma and tissue samples were appropriately processed to render them suitable for HPTLC analysis. The method employed HPTLC glass plates precoated with silica gel 60F254 as the stationary phase. The mobile phase developed consisted of chloroform, methanol and glacial acetic acid which successfully gave distinct bands for curcumin with a Rf value of 0.95. This newly developed HPTLC method was found to be reproducible and accurate in quantifying curcumin in mammalian samples. This method was further utilized to efficiently estimate the time for which curcumin is retained in its native form in mammalian tissues and plasma alike.

Simultaneous determination of levonorgestrel and ethinyl estradiol in combined dosage form utilizing spectrophotometric methods and high performance thin layer chromatographic method on nanosilica gel plates

Simultaneous quantification of levonorgestrel (LEV) and ethinyl estradiol (EE) was performed utilizing five different spectrophotometric methods and a high performance thin layer chromatographic method (HPTLC). The applied spectrophotometric methods were based on either ratio spectra namely; ratio difference, ratio subtraction and derivative ratio or the presence of isosbestic point specifically; absorbance subtraction and amplitude modulation. The proposed methods had the ability to resolve the overlapped spectra of the drugs with a linear relationship in the concentration range 1-65 µg/mL and 1-95 µg/mL for LEV and EE, respectively. The developed HPTLC method has revealed a good separation of the drugs upon utilizing Nano Silica Gel on TLC plates with fluorescent indicator 254 nm glass plates as the stationary phase and chloroform: methanol (99:1, v : v ) as the mobile phase. The proposed HPTLC method has shown high sensitivity, where the linearity range was 0.02-3.00 µg/band and 0.5-20.0 µg/band, for LEV and EE, respectively. The proposed methods were successfully applied for the analysis of laboratory prepared mixtures as well as combined dosage form. Validation for all methods was conducted in compliance with the ICH guidelines proving the methods to be selective, linear, precise and accurate. The proposed methods were statistically compared with the pharmacopoeial method, where the obtained results showed no significant difference regarding accuracy and precision.

DEVELOPMENT OF HPTLC METHOD FOR THE ESTIMATION OF PIOGLITAZONE AND ALOGLIPTIN IN SYNTHETIC MIXTURE

A precise and accurate HPTLC method for simultaneous estimation of pioglitazone HCl (PIO) and alogliptin benzoate (ALO) in synthetic mixture has been developed. The stationary phase selected was Pre-coated TLC plates (silica gel G60 – F254 aluminium sheet) and the mobile phase selected was n-butanol : water : acetic acid (6 : 3 :1 V/V/V). The separated spots were densitometrically analyzed in absorbance mode at 270 nm. The Rf value of PIO and ALO were found to be 0.70 and 0.39, respectively. The calibration curve of PIO and ALO were plotted in the range of 400 – 1600 ng/band, which showed the linear correction coefficient (r2) 0.9971 for PIO and 0.998 for ALO, respectively. The stock solutions of both the drugs were subjected to acid and alkali hydrolysis, chemical oxidation, dry heat degradation and photo degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their Rf values. Stressed samples were assayed using developed HPTLC method. The method was validated as per ICH guideline Q2 (R1) and applied successfully for determination of both drugs in synthetic mixture.

Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita

Context: Extensive research on Rhus (Anacardiaceae) shows their antioxidant potential, which warrants further evaluation of its other species. Objective: To perform a comparative antioxidant assay on extracts of R. retinorrhoea and R. tripartita, including sakuranetin quantification by a validated HPTLC method. Materials and methods: In vitro antioxidant assay was performed on chloroform and ethanol extracts of R. retinorrhoea Steud. ex Oliv. (RRCE and RREE) and R. tripartita (Ucria) Grande (RTCE and RTEE) by DPPH radical scavenging (at 31.25, 62.5, 125, 250 and 500 μg/mL concentrations) and β-carotene-linoleic acid bleaching methods at 500 μg/mL concentration. Densitometric HPTLC method was developed and validated using toluene: ethyl acetate: methanol (8:2:0.2; v/v/v) as mobile phase, executed on glass-backed silica gel F254 plate and scanned at 292 nm. Results: Antioxidant activity of Rhus extracts tested by the two methods (DPPH/BCB) was found in order of RTEE > RREE > RTCE > RRCE with IC50 118.67/256.26, 315.75/82.35, 827.92/380.0 and 443.69/292.75, respectively. Scanning of the HPTLC plate provided an intense peak of sakuranetin at Rf = 0.59. The estimated sakuranetin content in the dry weight of the extracts was highest in RREE (27.95 μg/mg) followed by RRCE (25.22 μg/mg), RTEE (0.487 μg/mg) and RTCE (0.0 μg/mg). Presence of sakuranetin in RREE, RRCE and RTEE supported the highest antioxidant property of the two Rhus species. Nonetheless, low sakuratenin in R. tripartita indicated the presence of other bioactive constituents responsible for synergistic antioxidant activity. Conclusion: The developed HPTLC method therefore guarantees its application in quality control of commercialized herbal drugs and formulations containing sakuranetin.