Analytical method development and validation for the simultaneous estimation of quercetin,berberine, rutin and curcumin in a polyherbal formulation using high-performance thin-layer chromatography

JPC – Journal of Planar Chromatography – Modern TLC - People all over the world are affected by the chronic, disabling autoimmune illness known as rheumatoid arthritis (RA). Herbal...     

Development and validation of a high-performance thin-layer chromatography densitometric method for the simultaneous determination of novel 1-acridinyl-1,2,3-triazole derivatives

JPC – Journal of Planar Chromatography – Modern TLC - High-performance thin-layer chromatographic (HPTLC) method provides a simple, sensitive, and accurate analytical method for the...     

Development of a validated high-performance thin-layer chromatography method for the standardization of an Ayurvedic formulation using berberine and ursolic acid

Giloy Tulsi tablet is an Ayurvedic preparation containing Tinospora cordifolia (Giloy) and Ocimum sanctum (Tulsi) and it is recommended for boosting the body’s immune response. This research work is about the marker-based standardization of this Ayurvedic preparation using high-performance thin-layer chromatography method. Standardization is based on the determination and quantification of the phytoconstituents berberine and ursolic acid present in Giloy Tulsi tablets. Separation was performed on pre-coated silica gel 60 F₂₅₄ plate as the stationary phase, with chloroform‒acetone‒formic acid (6:3.5:0.5, V/V) as the mobile phase. Identification and quantification were conducted densitometrically at 330 nm. The developed method resulted in good quality peak shape and enabled high-quality resolution of biomarkers. The R_F value for berberine (0.46 ± 0.02) and for ursolic acid (0.68 ± 0.02) in both reference standard and formulation were found to be comparable. The method was validated for specificity, linearity, limit of detection, limit of quantification, intra-day and inter-day precisions, accuracy, and robustness. The limit of detection values were 91 and 153 ng/band for berberine and ursolic acid, respectively. The limit of quantification values were 175 and 465 ng/band for berberine and ursolic acid, respectively. Regression analysis of the calibration data revealed a good linear relationship between peak area response and concentration in the range 200‒1000 ng/band for berberine (r² = 0.995) and 500‒2500 ng/band for ursolic acid (r² = 0.9968). The accuracy of the method, determined by measurement of recovery at three different levels, was in the range 98‒102% for both markers. These results are indication of reliability, reproducibility, accuracy, and precision of the method.

     

Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide—a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non‐validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A–B–C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 m ) and MeCN (60:40, v/v), B corresponds to NaClO₄·H₂O (pH 6.0; 0.2 m ) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser‐porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of thin-layer chromatography and high-performance thin-layer chromatography methods for the simultaneous determination of linagliptin and empagliflozin in their co-formulated dosage form

JPC – Journal of Planar Chromatography – Modern TLC - A sensitive, simple and validated thin-layer chromatography (TLC) method (method A) and a high-performance thin-layer...     

Development and validation of a high-performance thin-layer chromatography method for the estimation of bromfenac in ophthalmic solution

A novel, simple, precise, specific, accurate high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of bromfenac in ophthalmic solution. Diclofenac sodium was used as an internal standard (IS) because of its structural resemblance with bromfenac to develop a more accurate and precise method. Silica gel 60 F₂₅₄ HPTLC plates were used to separate bromfenac from the formulation with a mobile phase consisting of toluene-ethyl acetate-glacial acetic acid (65:35:0.2, V/V). Densitometric scanning was performed at 274 nm after the HPTLC plates were air-dried. Well-resolved bands and good peak shapes were obtained for both bromfenac and diclofenac sodium, with retention factor (R_F) values of 0.28 and 0.44, respectively. The proposed method was validated as per International Council for Harmonisation Q2 (R1) guidelines for specificity, precision, robustness, accuracy, and recovery. The drug shows linearity in the concentration range of 60‒270 ng/band and the correlation coefficient was found to be 0.999. The mean percent recovery of bromfenac was found to be 100.7%. The limit of detection and limit of quantification values for bromfenac were found to be 7.4 ng/band and 22.5 ng/band, respectively. The method was found to be novel since no HPTLC methods have yet been reported for the estimation of bromfenac. The developed method was successfully applied for the quantitative analysis of the drug in the ophthalmic formulation.

     

A validated quantification of gallic acid and ellagic acid in Triphala using a high-performance thin-layer chromatography method

JPC – Journal of Planar Chromatography – Modern TLC - A well-known Ayurvedic preparation, Triphala, consists of Amla (Emblica officinalis Gaertn.), Baheda (Terminalia bellirica Roxb.)...     

Development,method validation and simultaneous quantification of eleven bioactive natural products from high-altitude medicinal plant by high-performance liquid chromatography

Herein, a novel, rapid, reliable, simple method validation and simultaneous quantification of 11 bioactive compounds (mostly xanthones) have been described. International Conference on Harmonization guidelines were used for the analytical method validation. Good linearity, repeatability, intra- and inter-day precision, accuracy and reliability were well-illuminated in the method validation procedure. The calibration curves showed a good linear relationship (r > 0.999) within test range. Precision was evaluated by intra- and inter-day tests with relative standard deviation <2.79% and accuracy validation recovery of 74.16%–91.84%. On quantification study, the validated method described the high content of bioactive xanthone derivatives, including 1-hydroxy-3, 5-dimethoxyxanthone ( 7 ), 2-(allyloxy)-8-hydroxy-1, 6-dimethoxyxanthone ( 6 ) 1, 7, 8-trihydroxy-3-methoxyxanthone ( 9 ) and Coxanthone E ( 5 ) in Codonopsis ovata, which is advantageous given the numerous pharmacological and biological effects associated with these compounds, which mostly exhibit anti-cancerous, antioxidant, anti-inflammatory, anti-mutagenic and anti-obesity effects. The bulk abundance of these compounds can also be used for further modification to produce better lead molecules for drug discovery with low toxicity and high potency. The proposed method makes it possible to simultaneously determine all bioactive compounds in one run and can be extended to marker-based standardization of herbal formulations in medicinal and pharmaceutical industries.     

A validated high-performance thin-layer chromatography method for the simultaneous quantification of 6-gingerol,guggulsterone E and guggulsterone Z in coded formulation AYUSH SG-5 prepared for rheumatoid arthritis

JPC – Journal of Planar Chromatography – Modern TLC - A new, simple, selective, sensitive, precise, and robust high-performance thin-layer chromatography (HPTLC) method for the...     

Development and validation of a high-performance thin-layer chromatography assay for the analysis of tacrolimus ointments

JPC – Journal of Planar Chromatography – Modern TLC - A simple and rapid high-performance thin-layer chromatography (HPTLC) assay was developed and validated for the quantification of...     

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as 'estoraque do campo' or 'cuia de brejo', has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal tissues

A method of high-performance liquid chromatography with UV detection has been established for simultaneous quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residues for carbadox (CBX) and olaquindox (OLA), respectively, in the muscles and livers of porcine and chicken and in the muscle of fish. Tissue samples were subject to acid hydrolysis followed by liquid-liquid extraction and Oasis MAX solid-phase extraction clean-up. The method was validated according to the EU Commission Decision 2002/657/EC. The decision limits (CCalpha) were 0.7-2.6microg/kg and the detection capabilities (CCbeta) were 1.3-5.6microg/kg for QCA and MQCA in tissues. The recoveries of QCA and MQCA, spiked at levels of 2-100microg/kg, were from 70 to 110%; the relative standard deviation values were <20%. This simple, fast and economic method could be applied to the monitoring for the possible misuse of CBX and OLA in animal edible tissue samples.     

Development and validation of high-performance thin-layer chromatographic method for determination of amygdalin

ABSTRACT

A new method for the extraction and quantitative determination of amygdalin has been proposed. Accelerated solvent extraction was applied for the extraction, and reversed-phase high-performance thin-layer chromatography method was developed, validated, and applied for the determination of amygdalin in the extracts of apricot, plum, almond, and peach kernels. The chromatographic system used was RP-18 silica, as stationary phase and acetonitrile/water (50:50, v/v), as mobile phase. Densitometric scanning was performed at 210 nm. The method was validated with respect to specificity, linearity, precision, and accuracy. The results showed that the peak area responses were linear within the concentration range of 2.5–50.0 µg/spot (R² = 0.9984). The limit of quantification was 4.28 µg/spot, and the detection limit 1.28 µg/spot. The intra-day and inter-day reproducibility, in terms of %RSD, were in the range of 0.81–1.15 and 1.32–1.89, respectively. The accuracy data were in the range from 99.98 to 100.56%. The method is linear, quantitative and reproducible, and could be used as an efficient and economical green chromatographic procedure for the determination of amygdalin in the fruit kernel.     

Stability-indicating high-performance thin-layer chromatography method for the simultaneous estimation of emtricitabine and tenofovir alafenamide fumarate

A simple, precise, rapid and accurate high-performance thin-layer chromatographic method was developed and validated for the simultaneous estimation of emtricitabine (EMT) and tenofovir alafenamide fumarate (TAF) in combined dosage forms and forced degradation studies were carried out as per the International Council for Harmonisation (ICH) guidelines. The stationary phase used was Merck TLC silica gel 60 F₂₅₄ aluminum plates. The mobile phase used was ethyl acetate‒n-hexane‒methanol‒ammonia solution (4:4:2:0.2, V/V). Densitometric evaluation was carried out at 260 nm, EMT and TAF. The concentration ranges used for the study are 400–2000 ng/band for EMT, 50–250 ng/band for TAF. EMT and TAF gave well defined and sharp peaks at retardation factor 0.43 and 0.56, respectively. The stability study showed that samples degraded with acid, base, hydrogen peroxide and light gave well separated peaks of EMT and TAF as well as some additional peaks at different retardation factor values. The proposed method is simple, suitable, accurate and stable in accordance with the ICH guidelines.

     

A validated high-performance thin-layer chromatography method for the quantification of chlorogenic acid in the hydroalcoholic extract of Gynura cusimbua leaves

JPC – Journal of Planar Chromatography – Modern TLC - A simple, reliable, and reproducible high-performance thin-layer chromatography (HPTLC) method was developed for the quantitative...     

A validated high‑performance thin-layer chromatography method for the simultaneous determination of quercetin and gallic acid in Annona reticulata L.

JPC – Journal of Planar Chromatography – Modern TLC - A sensitive and reliable high-performance thin-layer chromatography (HPTLC) method has been developed to simultaneously estimate...     

Development and validation of a novel high-performance thin-layer chromatography method for the quantitative estimation of neohesperidin from Citrus aurantium peel extract

JPC – Journal of Planar Chromatography – Modern TLC - A high-performance thin-layer chromatography method was developed for the estimation of neohesperidin from Citrus aurantium peel....     

A validated high-performance thin-layer chromatography method for quantification of echioidin from Andrographis echioides plant

JPC – Journal of Planar Chromatography – Modern TLC - A simple, reliable and reproducible high-performance thin-layer chromatography (HPTLC) method was established for determining...     

An image analysis system for thin-layer chromatography quantification and its validation

Quantitation of thin-layer chromatography (TLC) using image analysis is attractive for its low cost and convenience. The image analysis is investigated by designing a digital imaging system with simple equipment, developing an image analysis software based on our algorithm, and validated the system in the TLC quantitative assay of cichoric acid present in Echinacea purpurea (L.) Moench. TLC used a polyamide thin-layer plate with chloroform-methanol formic acid-water (3:6:1:1) as the mobile phase and 3% (m/v) aqueous aluminum chloride solution as the visualization reagent. Images are acquired with a standard digital camera under a UV viewing lamp (365 nm) in a dark room. The three-dimensional gray scale digital image dataset (x, y, gray) is reduced to two-dimensional dataset (distance, accumulative gray) and then plotted as a curve. The area under the peak corresponding to the cichoric acid spot is integrated and used for quantitation. The whole method was validated by the assay tests of detection limit, calibration curve, repeatability, reproducibility, and recovery. The results showed that our digital imaging method and image analysis algorithm were applicable for the quantification of TLC. The whole method is convenient, efficient, and moderately accurate for the quantitative assay of cichoric acid present in Echinacea purpurea (L.) Moench.     

Modification and validation of a high-performance liquid chromatography method for quantification of Huperzine A in Huperzia crispata

The present study describes a rapid and sensitive HPLC method for the quantification of huperzine A (HupA) in Huperzia crispata (Huperziaceae). The sample extraction and preparation involved a simple, time-saving, single-solvent extraction, with each sample being analyzed within 12 min. The mobile phase was ammonium acetate (0.1 M, pH 6.0)--methanol (64 + 36, v/v) at a flow rate of 1.0 mL/min. Detection was at 308 nm. The calibration curve was linear from 0.049 to 7.84 microg (R2 = 0.9997), with intraday and interday precision RSD of less than 2%. The extraction recovery rate was over 98.49%. Quantification of HupA was performed using this modified method, and the content of HupA was 1.86 times higher in the whole plant of H. crispata (218.17 +/- 1.55 microg/g) than in that of H. serrata (117.03 +/- 2.97 microg/g). In the whole plant of H. crispata, HupA mainly accumulated in the actively growing shoot tips, the apical bud, and the 10 youngest leaves, reaching 455.23 +/- 2.97 microg/g. The content of HupA in the samples from sunshine-sheltered sites was 3.45 times higher than in that from sunshine-abundant sites. The satisfactory results indicate that this modified method can be applied in the quality control of large-scale Huperziaceae plant extracts and that changes should be made in the cultivation of H. crispata so as to maximize the production of HupA.