High-performance Thin-layer Chromatographic Method Research Articles

Effects of Hydrolysis Condition and Detection Method on the Monosaccharide Composition Analysis of Polysaccharides from Natural Sources

Monosaccharide composition analysis is essential to the structural characterization and research into the biological activity of polysaccharides. In this study, a systematic comparison was performed among commonly used monosaccharide composition analysis methods, including colorimetric and chromatographic methods. These were tested on 16 aldoses, ketoses, alditols, amino sugars, and uronic acids. Furthermore, the effect of hydrolysis methods was also investigated. The results showed that the phenol sulfuric acid method is greatly affected by the type of monosaccharide that is used as the reference substance. The determination of uronic acid using sulfuric acid carbazole is less affected by neutral sugars than that method using m-hydroxybiphenyl. The high-performance thin-layer chromatography (HPTLC) method can simultaneously analyze multiple samples and accurately determine the type of uronic acid. High-performance liquid chromatography (HPLC) can provide a good qualitative and quantitative analysis of aldose, amino sugars, and uronic acids, while gas chromatography–mass spectrometry (GC-MS) can detect aldose, ketose, and alditols. Fructose was detected in a large amount in inulin and Codonopsis pilosula after one-step hydrolysis, while it was totally destroyed in two-step hydrolysis. The release of galacturonic acid significantly increased after two-step hydrolysis in pectin and Lycium barbarum, which indicated that one-step hydrolysis is not enough for acidic polysaccharides. The results of this study are beneficial for selecting appropriate hydrolysis and analysis methods in order to accurately analyze the monosaccharide compositions of natural polysaccharides.

Comprehensive high-performance thin-layer chromatography analysis of Monteverdia ilicifolia leaf and its adulterants

The leaves of Monteverdia ilicifolia (syn. Maytenus ilicifolia) are widely used in traditional South American medicine to treat gastrointestinal problems such as gastritis and ulcers. Several herbal products containing the leaves of M. ilicifolia can be found in the market. However, other species with similar leaf morphology are confounding materials, e.g. Monteverdia aquifolia (Celastraceae), Citronella gongonha (Cardiopteridaceae), Jodina rhombifolia (Santalaceae), Sorocea bonplandii (Moraceae) and Zollernia ilicifolia (Fabaceae). This study aimed to identify M. ilicifolia and distinguish it from its potential adulterants using high-performance thin-layer chromatography (HPTLC) technique. Comprehensive HPTLC analysis revealed specific fingerprints that can be used to assess the minimum content of epicatechin and the quality of commercial espinheira-santa samples. The results of the study demonstrated that the HPTLC method is capable of detecting adulterations and distinguishing M. ilicifolia from all confounding materials in commercial products available on the market, showing that most of the products are of poor quality due to adulterations.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC METHOD FOR BUDESONIDE

Objective: A stability-indicating high-performance thin-layer chromatographic method has been developed for budesonide. Methods: Chromatographic separation was achieved on aluminum plates pre-coated with silica gel 60 F254 as the stationary phase using ethyl acetate: toluene (7:3) v/v as the mobile phase. The densitometric evaluation was carried out at 246 nm. The developed method of stability-indicating was validated as per the ICH Q2 (R1) guidelines. Stress degradation studies such as hydrolysis under different pH conditions, photolytic degradation, thermal degradation, and oxidative degradation were performed as per ICH Q1A (R2) and Q1B guidelines. Results: The Rf value of budesonide was found to be 0.48±0.03. The response in terms of peak area was linear over the concentration range of 500–2500 ng/band, with the regression coefficient value greater than 0.99. The LOD and LOQ were 28.04 ng/band and 84.96 ng/band, respectively. Conclusion: This method can conveniently be used for quantitative analysis of budesonide on a routine basis.

Development of a highly sensitive and eco-friendly high-performance thin-layer chromatography approach for the determination of empagliflozin, pioglitazone, and rosuvastatin simultaneously in pharmaceutical preparations and different biological fluids

A simple, accurate, green and selective high-performance thin-layer chromatography (HPTLC) method has been developed and validated for the simultaneous estimation of empagliflozin, pioglitazone, and rosuvastatin in their synthetic ternary mixture and different biological fluids. These three drugs are used for the treatment of type 2 diabetes mellitus and dyslipidemia and have shown synergistic effects on cardiovascular outcomes. The ternary combination was separated on silica gel TLC plates G60 F254, utilizing a mixture of n-hexane‒ethyl acetate‒methanol‒glacial acetic acid in ratio (4.2:4:1.75:0.05, V/V) as a developing system using ultraviolet (UV) detection at 230 nm. All experimental parameters were optimized with a linearity range of 5‒250 ng per band for each drug, with good sensitivity and low limit of detection values reached, namely 1.72, 1.79, and 1.52 ng per band for empagliflozin, pioglitazone, and rosuvastatin, respectively. The developed method was applied for separation of the studied drugs in their synthetic ternary mixture and different biological fluids, with good recovery results ensuring high efficiency of the proposed approach. Eco scale, green analytical procedure index, and AGREE metric tools were used to evaluate the greenness of the proposed method.

A validated high-performance thin-layer chromatography method for the simultaneous quantification of amino acids and sugars in Eulophia nuda Lindl. corm

A validated high-performance thin-layer chromatography method for the simultaneous quantification of amino acids and sugars in Eulophia nuda Lindl. corm

Novel chromatographic methods for fingerprinting the bioactive constituents of herbal medicines used as hepatoprotective and adaptogenic in herbo-mineral regimen

Shilajit, along with Triphala, has been one of the most widely used herbal drug combinations for the symptomatic treatments of hyperthyroidism. However, the high polarity difference among active constituents proposes difficulties while analyzing its quality. In this study, two high-performance thin-layer chromatography methods were developed for chromatographic fingerprinting of the proposed herbal mixture of Triphala and Shilajit. A reliable HPTLC method effectively analyzed bioactive phytoconstituents such as quercetin, myricetin, gallic acid, ellagic acid, tannic acid, ascorbic acid, and humic acid. Method 1 showed good separation of quercetin, myricetin, and tannic acid, but separating gallic and ellagic acid proved difficult. To address this issue, the 2D-TLC approach involves a second mobile phase. Although humic acid and ascorbic acid were not detected due to their highly polar and non-polar nature, so a different HPTLC method 2 was developed. The successful method of development using the Quality by Design approach and validated. The developed method successfully analyzed the bioactive constituents of Shilajit, in combination with Triphala, and validation parameters such as linearity, accuracy, precision, range, specificity, robustness, the limit of detection (LOD), and limit of quantification (LOQ) agreed with given values.

Development and validation of a high-performance thin layer chromatography method for the simultaneous determination of amoxicillin and clavulanic acid combinations in tablet dosage forms

A simple and sensitive high-performance thin layer chromatography (HPTLC) method was developed and validated as per the International Council for Harmonization (ICH) guidelines for the simultaneous determination of amoxicillin (AMX) and clavulanic acid (CLA) combinations in tablet formulations. Chromatography was performed on precoated glass plates with normal phase silica gel 60 F254. The mobile phase was acetone:ethyl acetate:glacial acetic acid:water (11:9:4:2 (v/v)). The plate was scanned at a wavelength of 428 nm after derivatization with ninhydrin. The validation of the method revealed that the linearity range lies between 400 and 1200 ng/band for AMX and 100–300 ng/band for CLA, with coefficients of determination of 0.9997 and 0.9966, respectively. Recoveries in standard addition accuracy studies were 100.3 % for AMX and 96.75 % for CLA. The limit of detection (LOD) and limit of quantitation (LOQ) of the developed method are 20.3 ng/band and 61.6 ng/band for AMX and 18.5 ng/band and 56.2 ng/band for CLA, respectively. The new, novel high-performance thin layer chromatography (HPTLC) method that was successfully developed in this study was applied for the simultaneous determination of AMX and CLA in their fixed-dose tablet dosage forms obtained from retail pharmacies and offered comparable results with the official British Pharmacopoeial high-performance liquid chromatography (HPLC) method.

STABILITY INDICATING HPTLC METHOD DEVELOPMENT AND VALIDATION FOR CERITINIB IN BULK AND FORMULATION

Ceritinib is an anti-cancer of drug used in treatment of non-small cell lung cancer. A high-performance thin layer chromatography (HPTLC) method has been developed for ceritinib, which is stability indicating and simple, precise, and discriminating. The stationary phase used was aluminum-backed silica gel60 F254 plates with chloroform: methanol: glacial acetic acid as the mobile phase in the ratio of 8.5:1.5:0.5 (V/V/V). The retention factor was found to be 0.34 ± 0.02. The densitometric scanning was performed at 277 nm. The linear range for analysis was 100–600 ng band-1, which gave good linear relationship with regression coefficient of 0.998. Method accuracy was proved by the recovery studies. The detection limit and quantification limit were 7.38 and 10.03 ng band-1, respectively. Stress degradation studies like hydrolysis under different pH conditions, photolytic, thermal, and oxidative degradation were carried out as per ICH Q1A (R2) and Q1B guidelines. The method established was found to be robust, and thus it can be used as a stability-indicating method.

Development and validation of a high-performance thin-layer chromatography method for the quantification of α-mangostin in three lesser-known Garcinia species of Assam

Development and validation of a high-performance thin-layer chromatography method for the quantification of α-mangostin in three lesser-known Garcinia species of Assam

Chromatographic Development and Validation of High-performance Thin Layer Chromatography Method for Concurrent Estimation of Azelnidipine and S (-) Metoprolol Succinate in Bulk and Tablets

Chromatographic Development and Validation of High-performance Thin Layer Chromatography Method for Concurrent Estimation of Azelnidipine and S (-) Metoprolol Succinate in Bulk and Tablets

A simple, easy, and efficient HPTLC method for simultaneous determination of polyamines (putrescine, spermidine, and spermine) in plant tissues

Polyamine analysis has received a lot of attention in recent years because they are important in several physiological processes, including cell proliferation, differentiation, and growth. This study presents the development and validation of a user-friendly and convenient high-performance thin-layer chromatography (HPTLC) method, using benzoyl chloride as a derivatization agent for the analysis of three abundant polyamines. A comparative study with HPLC-MS/MS was performed to evaluate the putrescine, spermidine, and spermine content of plant tissues. The linear ranges for the determination of putrescine and spermine by HPTLC were lower than those of HPLC-MS/MS. Lower LODs were found for polyamine analysis using HPLC-MS/MS except for spermidine. The Pearson correlation coefficients of the two methods for putrescine, spermidine, and spermine were 0.982, 0.998, and 0.980, respectively, which confirms the applicability of both HPTLC-UV and LC-MS/MS methods. According to these results, the optimized HPTLC-UV method may be used as a practical analytical tool for screening polyamine levels in the horticultural, food, and pharmaceutical industries for quality control purposes.

A validated bioanalytical method for the simultaneous estimation of telmisartan and gallic acid in rat plasma samples by high-performance thin-layer chromatography-mass spectrometry: Application to an oral pharmacokinetic study.

A novel and cost-effective high-performance thin-layer chromatography (HPTLC) method, combined with densitometric quantification, was developed for the biomedical analysis of telmisartan (TEL) and gallic acid (GA). Recent research indicates that when used in combination, these compounds offer improved therapeutic efficacy for the treatment of cardiovascular diseases with reduced side effects. The study focused on the simultaneous quantification and pharmacokinetic analysis of drugs in rat plasma. The separation was conducted using HPTLC silica gel 60 F254 plates with dimensions of 20 × 10 cm and a thickness of 0.2 mm. The mobile phase used for separation consisted of a mixture of ethyl acetate, methanol, chloroform, and acetic acid in the ratio of 4:2:2:0.2 (v/v). GA and TEL were analyzed using ultraviolet detection at specific wavelengths, with GA at 280 nm and TEL at 296 nm. Peak purity was assessed through spectral correlation analysis using Vision CATS software. The method underwent validation following the guidelines of the US Food and Drug Administration (US FDA). Calibration plots demonstrated linearity in the concentration range of 200-1200 ng/spot, with high correlation coefficients (R2 ). The retention factors (Rf ) were 0.67 for TEL and 0.60 for GA. The identity of the separated compounds was further confirmed using MS, with GA having a mass-to-charge ratio (m/z) of 168.9 in negative mode and TEL with m/z 515.2 in positive mode. In the pharmacokinetic study, the maximum peak plasma concentration (Cmax ) for GA was 899.7 ng/mL, and for TEL, it was 1013 ng/mL. The time to reach maximum concentration (Tmax ) was 2 h for GA and 6 h for TEL. This simultaneous qualitative and quantitative determination of the drugs in an oral pharmacokinetic study involving Wistar rats can serve as a valuable tool for future investigations into pharmacokinetic interactions, quality control, and routine analysis of these drugs, both in their pure forms and in novel formulations.

Estimation of apigenin from Abrus precatorius Linn. leaves extract by validated HPTLC densitometric method coupled with mass spectrometry

Abrus precatorius Linn. is recognised as Indian liquorice, valued for its medicinal properties, containing apigenin, a promising therapeutic scaffold, however, its quantification is lacking. Hence, this study established and validated the high-performance thin layer chromatography method for quantitation of apigenin from methanolic extract of A. precatorius leaves (APM). The optimised mobile phase was toluene: ethyl acetate: formic acid (6:3:1, v/v/v) for separation of apigenin. Apigenin was also confirmed by tandem mass spectrometry. The developed method was sensitive with correlation coefficient (r2 = 0.998) with limit of detection and quantification of 12.66 and 38.38 ng/band respectively. APM extract contained 0.559% w/w apigenin. Spectroscopic analysis confirmed apigenin with m/z value of 271.3. This method has been reported for the first time and proven accurate, precise and specific. As apigenin is a promising therapeutic agent, the established method can be used for quality control and be a template for drug formulation.

Identification of adulteration in the market samples of saffron using morphology, HPLC, HPTLC, and DNA barcoding methods.

Saffron, the stigma of Crocus sativus L., is the most expensive spice used for culinary, medicinal, dye, and cosmetics purposes. It is highly adulterated because of its limited production and high commercial value. In this study, 104 saffron market samples collected from 16 countries were tested using morphology, high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), and deoxyribonucleic acid (DNA) barcoding. Overall, 45 samples (43%) were adulterated. DNA barcoding identified the highest number of adulterated saffron (44 samples), followed by HPTLC (39 samples), HPLC (38 samples), and morphology (32 samples). Only DNA barcoding identified the adulterated samples containing saffron and other plants' parts as bulking agents. In addition, DNA barcoding identified 20 adulterant plant species, which will help develop quality control methods and market surveillance. Some of the adulterant plants are unsafe for human consumption. The HPLC method helped identify the saffron samples adulterated with synthetic safranal. HPLC and HPTLC methods will help identify the samples adulterated with other parts of the saffron plant (auto-adulteration).

Antioxidant activity and simultaneous estimation of four polyphenolics in different parts of Carica papaya L. by a validated high-performance thin-layer chromatography method

Antioxidant activity and simultaneous estimation of four polyphenolics in different parts of Carica papaya L. by a validated high-performance thin-layer chromatography method

Quantification of Suvorexant in Human Urine Using a Validated HPTLC Bioanalytical Method.

Suvorexant (SUV) is a new sedative/hypnotic medicine that is recommended to treat insomnia. It is an important medicine from a forensic point of view due to its sedative/hypnotic and depressant effects. To the best of our knowledge, high-performance thin-layer chromatography (HPTLC) bioanalytical methods have not been published to measure SUV in human urine and pharmaceutical samples. Accordingly, this study was designed and validated a sensitive and rapid bioanalytical HPTLC method to determine SUV in human urine samples for the very first time. The densitometric measurement of SUV and the internal standard (IS; sildenafil) was performed on glass-coated silica gel normal-phase-60F254S TLC plates using a mixture of chloroform and methanol (97.5:2.5 v/v) as the eluent system. Both the SUV and IS were detected at a wavelength of 254 nm. Both analytes were extracted using the protein precipitation technique utilizing methanol as the solvent. For the IS and SUV, the Rf values were 0.09 and 0.45, respectively. The proposed bioanalytical method for SUV was linear in the 50-1600 ng/band range. The current bioanalytical technique was linear, precise (% RSD = 3.28-4.20), accurate (% recovery = 97.58-103.80), robust (% recovery = 95.31-102.34 and % RSD = 2.81-3.15), rapid, and sensitive (LOD = 3.73 ng/band and LOQ = 11.20 ng/band). These findings suggested that the current bioanalytical method can be regularly used to determine SUV in wide varieties of urine samples.

Eco-Friendly High-Performance Thin-Layer Chromatography Method for the Determination of Tenoxicam in Commercial Formulations.

There is a dearth of information in the literature regarding environmentally benign high-performance thin-layer chromatography (HPTLC) methods to determine tenoxicam (TNX). Therefore, designing and validating an HPTLC method to detect TNX in commercial tablets and capsules was the goal of this investigation. The green mobile phase utilized was the combination of ethanol/water/ammonia solution (50:45:5 v/v/v). The TNX was quantified at a wavelength of 375 nm. The proposed method's greenness profile was established using the Analytical GREEnness (AGREE) approach. The proposed methodology for determining TNX was linear in the range of 25-1400 ng/band. The proposed methodology for measuring TNX was accurate (% recoveries = 98.24-101.48), precise (% RSD = 0.87-1.02), robust (% RSD = 0.87-0.94), sensitive (LOD = 0.98 ng/band and LOQ = 2.94 ng/band), and environmentally friendly. The AGREE scale for the present methodology was derived to be 0.75, indicating an outstanding greenness profile. TNX was found to be highly stable under acidic, base, and thermal stress conditions. However, it completely decomposed under oxidative stress conditions. Commercial tablets and capsules were found to have 98.46 and 101.24% TNX, respectively. This finding supports the validity of the current methodology for measuring TNX in commercial formulations. The outcomes of this work showed that the proposed eco-friendly HPTLC methodology can be used for the routine analysis of TNX in commercial formulations.

Novel green HPTLC and organic solvent-free micellar LC methods for the simultaneous determination of Ozenoxacin and benzoic acid; greenness assessment and applications

Ozenoxacin (OZ) is a novel synthetic quinolone antimicrobial agent that has been recently approved by US-FDA for the treatment of impetigo in combination with its preservative, benzoic acid (BA). Meanwhile, implementing greener chromatography has been a recent goal of a great ecological importance. In the proposed research, two novel green methods have been validated for the simultaneous determination of OZ and BA in skin creams for the first time. An organic-solvent free mixed-micellar HPLC technique was developed using RP-C8 core-shell column with a mobile phase consisting of 0.01 M Brij-35, 0.15 M sodium dodecyl sulfate, and 0.02 M ammonium acetate in water, adjusted to pH 4.5. The mobile phase flow rate was 1 mL min−1 and detecting analytes in short retention times (3.4 and 4.7 min for BA and OZ, respectively) at wavelength 235 nm. The working concentration ranges were 1–10 and 10–100 μg mL−1 for BA and OZ, respectively. Another eco-friendly high-performance thin-layer chromatography (HPTLC) method was designed using HPTLC silica-gel 60 F254 plates as a stationary phase and ethyl acetate–ethanol–ammonia (6:4:2, v/v/v) as mobile phase. The detection was also performed at λmax = 235 nm. The method was found linear across concentration range of 0.5–1.8 and 0.05–0.18 μg/band for OZ and BA, respectively which gave symmetric, compact band with Rf values of 0.21 and 0.69 for OZ and BA, respectively. The proposed methods were applied successfully in their combined marketed pharmaceutical cream. The proposed methods were typically based on safe biodegradable reagents, and their ecological superiority was proven using recent greenness metrics.

Development of High-performance Thin-layer Chromatography (HPTLC) Method for Quality Control of Actinidiae Fructus Vermicultus

Development of High-performance Thin-layer Chromatography (HPTLC) Method for Quality Control of Actinidiae Fructus Vermicultus

Development of Green HPTLC method for simultaneous determination of a promising combination Tamsulosin and Mirabegron: stability-indicating assay was examined

Recently, mirabegron has been added to tamsulosin to treat overactive bladder in men with benign prostatic hypertrophy. A Rapid, selective, sensitive, and green high-performance thin-layer chromatography (HPTLC) approach was developed for the simultaneous determination of tamsulosin (TAM) and mirabegron (MIR) in pure and laboratory-prepared mixture. Complete separation was obtained on silica gel F254 using the solvent system methanol-ethyl acetate-ammonia (3:7:0.1, v/v). Short-wave ultraviolet light at 270 nm was used to view the chromatographic bands. For MIR and TAM, the suggested technique revealed compact spots with retention factor Rf values of 0.42 and 0.63, respectively. Within concentration ranges of 0.15–7.5 µg/band and 0.05–2.5 µg/band, good linearity was observed, with mean percentage recoveries of 100.04 ± 0.56 and 99.98% ± 0.95 for MIR and TAM, respectively. Green assessment of the developed HPTLC technique was estimated using different green analytical chemistry metrics such as Analytical eco-scale Analytical GREEness (AGREE), and Green Analytical Procedure Index (GAPI) metrics. The proposed method was effectively used as a stability-indicating assay to assess the presence of MIR and TAM in the pharmaceutical dosage form in the presence of their degradation product. The statistical analysis showed high precision and accuracy.