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Authors: Mhaveer Singh, Yunus Kamal, Ennus Tamboli, Rabea Parveen, Shahid Ansari and Sayeed Ahmad

A novel HPTLC method has been developed for the estimation of glabridin in Licorice rhizome and its Unani polyherbal formulation (Qurs-e-Gul). Separation was achieved on silica using toluene, dichloromethane, and ethyl acetate in equal ratios. A compact, well resolved peak of glabridin with R F value 0.56 ± 0.02 was observed. Calibration curve revealed a good linear relationship with r 2 value of 0.993 between the peak area and concentration in the range of 25–500 ng spot−1. The proposed method was validated as per the International Conference on Harmonization (ICH) guidelines. The stability assessment was carried out by studying degradation of glabridin stressed by acid, base, oxidation, thermal, and humidity. Photodegradation was also carried out after keeping the drug in sunlight, dark, and in UV lights. The method proposed can be used for routine determination of glabridin in crude drugs and in herbal formulations containing Licorice as one of the ingredients, for quality control as well as for stability testing with high precision, accuracy and a wide range of linearity.

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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 quantification (LOQ) for SPF were found to be ≈13 and ≈40 ng spot−1, respectively, and those for FLB ≈27 ng spot−1 and ≈82 ng spot−1, respectively. The developed method was found to be linear (r 2 = 0.999), precise (% RSD < 1.5% repeatability and <2.55% for intermediate precision), accurate (mean recovery of within the range of 98–102%), specific, and robust. Stress-induced degradation studies revealed the suitability of method for the quantitative determination of drugs in the presence of degradants. The developed method has been successfully applied for the determination of entrapment efficiency, drug loading, in vitro drug release profile and stability assessment of dual drug loaded PLGA nanoparticles (NPs).

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The present research work was undertaken to develop and validate a sensitive, fast, and reproducible high-performance thin-layer chromatography (HPTLC) method for the analysis of trigonelline in fenugreek seeds. Chromatographic analysis was carried out using thin-layer chromatography (TLC) aluminum plates pre-coated with silica gel G60 F254 as the stationary phase, and the samples were spotted with the help of a CAMAG Linomat 5 TLC applicator. For optimization of the method, different combinations of extraction solvents and mobile phases were tested, and methanol was found to be the ideal extraction solvent; the best separation was achieved using n-propanol‒methanol‒water‒ammonia (10:1.5:15:0.25, v/v) as the mobile phase. Densitometric scanning of the plates was used for the quantification of trigonelline. This method gave compact spots with retardation factor (R F) value corresponding to 0.40 ± 0.02 for trigonelline. The method was validated as per the International Conference on Harmonization (ICH) guidelines in terms of limit of detection (LOD), limit of quantification (LOQ), specificity, precision (intra-day and inter-day), accuracy, and robustness. The accuracy of the method was checked by recovery study of 4 different levels with average percentage recoveries of 100.077 ± 0.41. While employing this method, dried fenugreek seed samples were found to contain trigonelline in the range of 0.262% to 0.386% (w/w). This method is being reported for the first time and could be successfully applied for the quantification and routine quality control of this marker compound in several plant samples, herbal extracts, and market preparations.

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A simple, sensitive, specific, rapid, and accurate high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous estimation of quercetin (QCT) and resveratrol (RSV). Chromatographic separation was performed over pre-coated TLC plates (60 F254, 20 × 10 cm, 250 µm thickness, Merck, Darmstadt, Germany) through a linear ascending technique. Among the different combinations of the mobile phases used, the best separation was achieved in the toluene-ethyl acetate-formic acid (6:2.5:1.5, V/V) mixture. Detection and quantification were achieved at 286 nm through the spectrodensitometric analysis. Analytical performance of the proposed HPTLC method was validated according to the International Conference for Harmonization (ICH) guidelines with respect to linearity, accuracy, precision, detection, and quantitation limits, robustness, and specificity. The calibration curves were linear in the range of 50–2500 ng per spot for both QCT and RSV, with a correlation coefficient (R 2) of 0.998 and 0.997 for the QCT and RSV, respectively. The detection limits were 122.33 and 370.7 for QCT and RSV, respectively, and the quantitation limits were 27.35 and 82.93 for QCT and RSV, respectively. Additionally, forced degradation studies of QCT and RSV were established. The validated HPTLC method was successfully applied to the simultaneous determination of QCT and RSV in the nanoformulation.

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The alternative system of medicines like Unani and Ayurveda is preferred worldwide nowadays due to its therapeutic efficacy, lower side effects, holistic approach, psychological dimensions, and qualitative action of weather and seasonal requirement. A simple procedure is described for the simultaneous extraction and estimation of piperlongumine and piperine in a well-known Unani polyherbal formulation using reversed-phase high-performance liquid chromatography (HPLC). The chromatography was carried out on reversed-phase C18 (250 × 4.6 mm) column with a mobile phase containing acetonitrile—water (50:50 v/v). Detection was accomplished with ultraviolet (UV) detection at λ = 325 nm. The flow rate was kept as 1.0 mL−1. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines for accuracy (94.4–105.0%), precision (0.37–2.17% RSD), and robustness (0.14–2.11% RSD). The limit of detection (LOD) values were found as 30 and 10 ng mL−1, while limit of quantification (LOQ) was 100 and 30 ng mL−1 for piperlongumine and piperine, respectively, which proved the sensitivity of the method satisfactory enough for accurate analysis of the both piperlongumine and piperine.

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Bacoside A, a triterpenoid saponin, is a major constituent isolated from Bacopa monnieri (L.) Wettst. (Scrophulariaceae), used as a memory enhancer. Bacoside A and B are active ingredients in Bacopa herb and have antioxidant and hepatoprotective activities


A new rapid, simple, and economical high-performance thin-layer chromatographic (HPTLC) method was developed and validated for densitometric quantitative analysis of bacoside A in powdered leaves from different geographical regions of India.

Materials and methods

An amount of 10 mg mL−1 methanol extract of powdered leaves from different geographic regions was used for sample application on precoated silica gel 60 F254 aluminum sheets. Standard bacoside A (1 mg mL−1) was used for calibration curve. HPTLC separation was performed on percolated silica gel aluminum plate 60 F254 (20 cm × 10 cm with 0.2 mm thickness) as a stationary phase using ethyl acetate–methanol–water (4:1:1) as the mobile phase. Quantification was achieved by densitometric analysis at 598 nm over the concentration range of 500–4000 ng band−1.


Compact and well-resolved bands for bacoside A from powdered leaves of different geographic regions were found at retardation factor (R f) 0.53 ± 0.02. The linear regression analysis data for calibration curve showed good linear relationship with regression coefficient r 2 = 0.9996 and r 2 = 0.99810 with respect to peak area and peak height. The method was validated for precision, recovery, and robustness as per the International Conference on Harmonization (ICH) guidelines. Variation in quantitative analysis of bacoside A in powdered leaves sample from different geographic regions was found by HPTLC method.

Discussion and conclusion

The highest and lowest content of bacoside A in powdered leaves sample from Jammu and Kerala regions, respectively. The variety of B. monnieri in Jammu is superior to other regions of India. The proposed developed HPTLC method can be applied for the quantitative determination of bacoside A in powdered leaves of plant and its formulation.

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Authors: Y.T. Kamal, Sayeed Ahmad, Nanjaian Mahadevan, Prawez Alam, Shahana Salam, Yahya I Asiri, Abdullatif Bin Muhsinah and Abdulrhman Alsayari


A new High Performance Liquid Chromatography–Photodiode Array Detector (HPLC–PDA) method has been developed for the chromatographic separation and simultaneous quantitative determination of nine bioactive compounds, i.e. four phenolic (gallic acid, ellagic acid, chebulinic acid, and tannic acid), two flavanoids (rutin and quercetin), two anthraquinones (sennoside A and B) and one oxygenated hydrocarbon (vitamin C) in a well-known Unani polyherbal formulation namely Itrifal's. Separation was accomplished on a C18 LiChrospher 100 column (5 µm, 250 × 4.6 mm) with a gradient elution and recorded at 254 nm. The results demonstrated that the proposed method is reproducible, accurate, economic, and suitable for the quality control of traditional polyherbal Unani formulations containing complex compounds with different structures such as Itrifals.

Open access