Silica Gel High-performance Thin-layer Chromatography Research Articles

CHROMATOGRAPHIC AND PHYTOBIOLOGICAL ASSESSMENT OF SOME NATURAL BORON-CONTAINING COMPOUNDS

Boron (B) has many roles in the plant kingdom, being an essential element for fundamental metabolic pathways, with reference to carbohydrates, ribonucleic acid, phenolic compounds, indole acetic acid derivatives, respiration, as well as for the structure of cell wall and integrity of plasma membrane, cell division, differentiation, and elongation at the level of meristematic tissues, growth of the pollen tube, grain maturation. It has also been shown that the low amount of B can cause sterility during herbal reproductive stage.For identification and quantitative determination of some natural B-containing compounds (BCCs), such as boric acid, fructoboric acid and its derivatives (Ca–fructose–B complex and Zn–fructose–B complex), sodium tetraborate, high-performance thin-layer chromatography (HPTLC)–ultraviolet (UV) densitometry method was used, based on the following experimental conditions: stationary phase HPTLC silica gel G 60 F254 precoated glass plates, mobile phase 2-propanol–water 8:2 (v/v), derivatization with chlorogenic acid.The phytobiological assessment of natural BCCs was carried out using Triticum test. A slight stimulation of the mean radicular elongation of wheat-germinated caryopses, compared to the reference, was recorded for fructoboric acid and its derivatives at a concentration of 0.1% expressed in B.

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.

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.

Screening bisphenols in complex samples via a planar Arxula adeninivorans bioluminescence bioassay

The Arxula yeast bisphenol screen (A-YBS) utilizes the bioluminescent Arxula adeninivorans yeast–based reporter cells for tailored analysis of bisphenols, one of the major endocrine-disrupting compound groups. For the first time, this bioreporter has been applied on the high-performance thin-layer chromatography (HPTLC) adsorbent surface to develop a respective planar bioluminescence bioassay (pA-YBS). The goal was to combine the advantages of HPTLC with a more selective bioassay detection for bisphenols. The performance of this pA-YBS bioluminescence bioassay was demonstrated by calculating the half-maximal effective concentration (EC50) of bisphenols compared to references. The EC50 ranged from 267 pg/band for bisphenol Z and 322 pg/band for bisphenol A (BPA) to > 1 ng/band for other bisphenols (BPC, BPE, BPF, and BPS) and references (17β-estradiol and 17α-ethinylestradiol). The EC50 value of BPA was three times more sensitive in signal detection than that of 17β-estradiol. The visual or videodensitometric limit of detection of BPA was about 200 pg/zone. The higher signal intensity and sensitivity for BPA confirmed the tailored bioassay selectivity compared to the existing estrogen screen bioassay. It worked on different types of HPTLC silica gel plates. This HPTLC–UV/Vis/FLD–pA-YBS bioluminescence bioassay method was used to analyze complex mixtures such as six tin can migrates, five thermal papers, and eleven botanicals. The detected estrogenic compound zones in the tin can migrates were successfully verified via the duplex planar yeast antagonist estrogen screen (pYAES) bioassay. The two bisphenols A and S were identified in one out of five thermal papers and confirmed with high-resolution mass spectrometry. No bisphenols were detected in the botanicals investigated via the pA-YBS bioluminescence bioassay. However, the botanicals proved to contain phytoestrogens as detected via the pYAES bioassay, which confirmed the tailored bioassay selectivity. This HPTLC–UV/Vis/FLD–pA-YBS bioluminescence bioassay is suited for cost-efficient analysis of BPA in complex samples, with no need for sterile conditions due to the fast workflow.

Simultaneous quantification of pharmacological markers quercetin, berberine HCl and curcumin using High-performance thin-layer chromatography (HPTLC) from polyherbal formulation

Quercetin, curcumin and berberine HCl have anti-acne, antimicrobial, anti-inflammatory, antioxidant properties. 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 quercetin, curcumin and berberine HCl from polyherbal gel prepared for the treatment of Acne. The method was developed using HPTLC silica gel GF254 precoated aluminium plate as a stationary phase. The solvent system consisted of Toluene, Ethyl acetate, Methanol, Formic acid. Densitometric analysis was carried out in the absorbance mode at 254nm. This system was found to give compact spots for quercetin, curcumin and berberine HCl at specific Rf values. The linearity of quercetin, curcumin and berberine HCl were obtained between 200 and 1000 ng/spot with good correlation coefficient values. The % of quercetin, curcumin and berberine HCl from polyherbal gel was found to be 99.42±0.98, 98.67±0.76% and 99.6±1.06% respectively, which was well within the limit. It was concluded that the developed HPTLC method would be an important tool in the quality control of polyherbal formulations.

Ecofriendly chromatographic estimation of spasmolytic pharmaceutical mixture along with official toxic impurity, 3,5-dichloroaniline: Complete green profile appraisal.

Nowadays, Green Analytical Chemistry is widely applied to provide various analytical methods with eco-friendly procedures employing the least toxic, harmful reagents on humans and the environment without affecting the efficacy of the determination. Accordingly, two eco-friendly, accurate, and reliable high-performance thin-layer chromatography-densitometry and high-performance liquid chromatographic methods were established for the determination and separation of two antispasmodic drugs, namely phloroglucinol and trimethylphloroglucinol in their pure, combined dosage form along with phloroglucinol toxic impurity, 3,5-dichloroaniline. For high-performance thin-layer chromatography-densitometry, efficient separation was developed via utilizing the stationary phase of high-performance thin-layer chromatography silica gel 60 F254 plates and developing a system comprising of ethyl acetate-butanol-ammonia in the ratio of 8.0:2.0:0.2, by volume and scanning of the developed bands at 210.0nm. The subsequent method is isocratic high-performance liquid chromatography with diode array detection in which separation was successively attained using XTerra RP-C18 (250 × 4.6mm, 5μm) column as stationary phase and methanol-10.0mM phosphate buffer, pH 3.7±0.1 as mobile phase in the ratio of 75.0:25.0, v/v at flow rate 1.0ml/min and scanning at 220.0nm. The developed liquid chromatography methods were validated according to the International Council for Harmonization guidelines, and all results acknowledged their efficacy. Additionally, the proposed methods worked well for assessing the cited drugs in binary combined commercially available pharmaceutical formulation. The greenness profile of the present methods was assessed and estimated using various assessment tools, namely; Green Analytical Procedure Index, analytical eco-scale method, National Environmental Method Index in addition to Analytical GREEnness tool to evaluate the greenness of the provided methods with a statistical comparison between them to assess the more green ones.

Research of <I>Rhodiola rosea</I> Rhizomes and Roots Using High Performance Thin Layer Chromatography

Introduction. The research results obtained through the analysis of different varieties of Rhodiola rosea rhizomes and roots using high-performance thin layer chromatography (HPTLC), grown in the Saint-Petersburg State Chemical-Pharmaceutical University (SPCPU) medicinal plants nursery garden, are represented.Aim. To carry out a comparative analysis of Rhodiola rosea rhizomes and roots samples, grown in the SPCPU medicinal plants nursery using HPTLC.Materials and methods. HPTLC analysis was performed on a CAMAG device (Switzerland), using MERCK HPTLC silica gel 60 F 254, 20 × 10 cm plates. Extracts were obtained from raw materials using an ultrasonic bath "Sapphire-4.0 TTC" (Russia). The Rhodiola rosea rhizomes and roots were harvested in the SPCPU medicinal plants nursery garden (Leningrad Region, Vsevolozhsk district, Priozerskoe Highway, 38 km) in August 2019.Results and discussion. In the course of the research, extracts from Rhodiola rosea rhizomes and roots were obtained using 70 % ethyl alcohol and methyl alcohol as extractants. The extracts were investigated by HPTLC in ethyl acetate – methanol – water – formic acid (77 : 13 : 10 : 2) solvent system. After scanning densitometric analysis at 254 nm, the much better separation of methanol extracts in this solvent system was found than ethanol extracts. The densitograms of individual tracks were compared with each other in order to identify samples of Rhodiola rosea rhizomes and roots with a higher biologically active compounds content. As a result of the comparison of 18 samples tracks of the Rhodiola rosea rhizomes and roots (the extractant was ethyl alcohol 70 %), samples № 6 and 17 have been proposed as promising for further research and cultivation.Conclusion. As a result of the analysis of alcohol extracts from Rhodiola rosea rhizomes and roots by HPTLC, the samples 6 (Tomsk, Russia) and 17 (Valla Di Aposta/Hirvos varieties, country of origin – Italy/Finland) were found to have a higher content of biologically active compounds.

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

High-performance thin-layer chromatographic (HPTLC) method provides a simple, sensitive, and accurate analytical method for the simultaneous determination of certain synthesized 1-acridinyl-1,2,3-triazole derivatives without interference from starting materials and intermediates. Separation was carried out on Merck HPTLC silica gel 60F254 plates, using chloroform‒methanol (9:1, V/V) and hexane‒ethyl acetate (3:2, V/V) as mobile phases. Validation of the method was performed based on the basis of the International Council for Harmonisation (ICH) guidelines in terms of linearity, sensitivity, limit of detection, limit of quantification, precision, selectivity, and specificity. Least-square equations were calculated for the studied compounds in the ranges of 25–500 and 10–500 ng/spot for ultraviolet (UV) and fluorescence measurements, respectively. Correlation coefficients (r) values were found ranging from 0.9913 to 0.9992 for analytes. The method provides selectivity and specificity which ensure that synthesized compounds are in the pure form without the interference of starting materials and intermediates. The detection limits for the studied compounds ranged from 11.02 to 51.09 ng/spot and 3.84 to 31.95 ng/spot and quantification limits were 33.39–154.82 ng/spot and 11.63–73.67 ng/spot for both spectrophotometric and spectrofluorimetric methods, respectively, indicating applicability for good qualitative and quantitative determination of members of this series at the nanogram concentration levels in biological fluids.

Thin-layer chromatography of DNS amino acids derivatives in systems with silica gel and silanized silica gel plates

Abstract Investigations into separation selectivity of High-Performance Thin-layer chromatography (HPTLC) of dansyl (DNS) derivatives of amino acids in silica gel (Silica gel 60 F254s plates) and silanized silica gel (RP-18 W plates) systems are presented. The results have been obtained for mobile phases containing different concentrations of acetonitrile (ACN) in formic acid (FA) water solution (final concentration of FA in the mobile phase was equal to 265 mmol/dm3). The data obtained show differences in separation selectivity of the solutes between employment of HPTLC silica gel and RP-18 W systems.

HPTLC determination of ergosterol in wheat and structure elucidation by NMR: Toward confirming method selectivity

Ergosterol (ERG) is considered as an excellent fungal biomarker, therefore it was utilized for the determination of the degree of fungi infection in wheat grains. A novel, fast, cost-effective and selective high performance thin layer chromatography (HPTLC) with reflectance/absorbance densitometry method was developed and validated for ERG determination. Separation was attained on pre-coated silica gel HPTLC plate with ethyl acetate: petroleum ether (6: 4 v/v) as a mobile phase. This system gave compact bands at retardation factor (Rf) of 0.45 ± 0.002 and quantification was performed at 282 nm over concentration range of 40–600 ng/band. The lower detectable limit was 10.79 ng per band which is three times more sensitive than the previously reported TLC method. The developed method could be utilized efficiently for the detection of ERG in pure powders and in fungi infected wheat samples. Resolution of ERG from other compounds in wheat grain extract allowed utilizing preparative TLC for separating and purifying ERG in a sufficient quantity for structure elucidation by nuclear magnetic resonance spectroscopy (NMR) for the first time. H1 and C13 NMR spectra indicated the purity of ERG extracted from the scrapped silica of preparative TLC plate at the Rf of the standard ERG verifying the method selectivity.

New solvent systems to separate some estrogenically active compounds by high-performance thin-layer chromatography (HPTLC)

Two solvent mixtures for high-performance thin-layer chromatographic (HPTLC) separation of some compounds showing estrogenic activity in the yeast estrogen screen (YES) assay are presented. The new method, planar yeast estrogen screen (pYES) combines the YES assay and a chromatographic separation on silica gel HPTLC plates with the performance of the YES assay. For separation, the analytes were applied bandwise to HPTLC plates (10 × 20 cm) with fluorescent dye (Merck, Germany). The plates were developed in a vertical developing chamber after 30 min of chamber saturation over a separation distance of 70 mm, using cyclohexane‒methyl-ethyl ketone (2:1, V/V) or cyclohexane‒CPME (3:2, V/V) as solvents. Both solvents allow separation of estriol, daidzein, genistein, 17β-estradiol, 17α-ethinyl estradiol, estrone, 4-nonylphenol and bis(2-ethylhexyl) phthalate.

Identification and quantitative determination of arbutin in the herb of Orthilia secunda

Introduction. Orthilia secunda (L.) House is a perennial herb that grows in Europe, Siberia, Asia Minor and Central Asia. The herb of Orthilia secunda is actively used in folk medicine as a diuretic, wound-healing and anti-inflammatory agent. From literary sources it is known that this medicinal plant raw material (PRM) contains flavonoids, tannins, organic acids, vitamins, as well as simple phenols and their derivatives (arbutin and hydroquinone). The presence of arbutin is responsible for the plant's high antioxidant and anti-inflammatory properties. But the use of Orthilia secunda in official medicine is limited due to the lack of complete information on the chemical composition and criteria for standardization of this type of medicinal product.Aim. Identification and quantification of arbutin by chromatographic methods in Orthilia secunda (L.) House, harvested in various phytocenotic zones.Materials and methods. The investigated medicinal plant material – the herb of Orthilia secunda – was harvested in various phytocenotic zones: in July 2018, harvesting was carried out in the northern part of Kazakhstan (Kokshetau district), in July-August 2019 in the Perm Territory and in the Tyumen Region. Preliminary identification of arbutin and related phenols – gallic acid and hydroquinone – was carried out by high performance thin layer chromatography (HPTLC) on a CAMAG instrument with a UV cabinet (Merck HPTLC silica gel 60 F154 plates, 20 × 10), semi-automatic Linomat 5 applicator (sample application). Elution of the plates was performed in a CAMAG Automatic Developing Chamber (ADC2). Image fixation was performed on a CAMAG Scanner 3 spectrodensitometer. The quantitative determination of arbutin was carried out by the method of highperformance liquid chromatography, which was carried out on a Prominence LC-20 device (Shimadzu, Japan) according to the validated method described in the European Pharmacopoeia 10.0. Diode array detector SPD-M20A, column Intersil C18 column (250–4.6 mm, 5 μm) (Phenomenex, USA). The results were processed using the LabSolution software. The identification and quantification of arbutin was carried out in comparison with a standard solution containing a reference sample (RS) of arbutin (C = 0,025 mg/ml) and RS of hydroquinone (C = 0,0125 mg/ml).Results and discussion. HPTLC analysis made it possible to detect arbutin and gallic acid – the main product of hydrolytic degradation/ precursor of the biosynthesis of tannins of the hydrolysable group – in the herb of Orthilia secunda from different places of growth. HPLC analysis demonstrates a different chromatographic profile of Orthilia herb harvested in different phytocenotic zones. However, in all studied objects, the absence of hydroquinone and the presence of substances that can presumably be attributed to its derivatives were confirmed, which is confirmed by the visual similarity of the spectra of these compounds and the proximity of the extrema. It was found that arbutin does not belong to the marker (majority) compounds of Orthilia. Its content is low and reaches a maximum (about 0,021 %) in the herb of Orthilia secunda growing on the territory of Kazakhstan, while in the herb of Orthilia harvested in the Perm Territory arbutin was not identified. From the data obtained, it follows that the greatest accumulation of arbutin occurs in areas with a warmer and drier climate (northern part of Kazakhstan).Conclusion. HPTLC analysis of the herb Orthilia secunda allowed the identification of arbutin and gallic acid (the main precursor of tannins of the hydrolysable group). The results of HPLC analysis of Orthilia herb harvested in various phytocenotic zones suggest quantitative differences in the content of arbutin depending on the region of growth. From the experimental data, it follows that Orthilia growing in the northern part of Kazakhstan accumulates the maximum (0,021%) amount of arbutin, in comparison with the samples harvested in the Tyumen region and the Perm region. At the same time, Orthilia harvested in the Perm Territory does not accumulate arbutin. The presence of hydroquinone has not been confirmed (by HPTLC and HPLC methods); therefore, it is not justified to talk about the hydrolytic cleavage of arbutin in the process of biosynthesis or drying. However, in all studied objects there are peaks of substances with spectral characteristics like hydroquinone, which makes it possible to assume the presence of its derivatives. Therefore, it is not advisable to position arbutin as a marker compound of Orthilia secunda harvested on the territory of the Russian Federation, and to standardize raw materials for this compound.

Using high performance thin layer chromatography for the detection of pharmacologically active secondary metabolites in Empetrum nigrum L.

Introduction. The article presents the results of the detection of pharmacologically active secondary metabolites in black crowberry Empetrum nigrum L. using the method of high performance thin layer chromatography (HPTLC).Aim. To show the efficiency of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites in promising medicinal plant species.Materials and methods. HPTLC analysis was carried out on a CAMAG device (Switzerland), using MERCK HPTLC silica gel 60 F154, 20 × 10 cm plates. For the evaporation of the samples, a Heidolph vacuum rotary evaporator (Germany) was used. The aerial parts (shoots) of Empetrum nigrum were harvested next to St. Petersburg State Chemical and Pharmaceutical University (SPCPU) nursery garden of medicinal plants (Leningrad Region, Vsevolozhsky District, Priozerskoe Highway, 38 km) in August 2019.Results and discussion. In the course of the research, four fractions from the aerial parts of Empetrum nigrum were obtained: hexane, dichloromethane, butanol, and water. Then, these fractions were investigated by HPTLC in two solvent systems – n-butanol : acetic acid : water (BAW) (4 : 1 : 2) and hexane : dichloromethane : methanol (HDM) (1 : 2 : 0.5). After scanning densitometric analysis of the plates eluted in the HDM system, it was revealed, that the hexane and dichloromethane fractions have a similar composition and contain the greatest amount of compounds, compared to the butanol and water fractions, and in the BAW system, it was found, that the butanol fraction contains the greatest variety of metabolites. As a result of UV spectroscopy, it was found, that the main groups of compounds contained in the hexane and dichloromethane fractions are derivatives of chalcones, dihydrochalcones, bibenzyls and 9,10-dihydrophenanthrenes. While in the butanol fraction, the main groups of secondary metabolites were derivatives of flavonoids and tanninsConclusion. The data obtained allow us to note the efficiency, speed and simplicity of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites of promising medicinal plant species.

Identification of type B trichothecenes and zearalenone in Chilean cereals by planar chromatography coupled to mass spectroscopy

ABSTRACT High-performance thin-layer chromatography (HPTLC) and HPTLC coupled with mass spectrometry (MS) methods were described for the simultaneous determination of zearalenone (ZEA); type B trichothecenes (TCT-B); nivalenol (NIV) and deoxynivalenol (DON) along with its acetylated derivatives: 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON). The extract samples were cleaned-up with Bond Elut Mycotoxin® solid-phase extraction cartridges. Then, separation was performed on HPTLC silica gel 60 F254 plates using toluene, ethyl acetate and formic acid (1:8:1 v/v/v) as mobile phase. Derivatisation was then performed with 10% aluminium trichloride in 50% methanol. Mycotoxin standards and spiked cereals grains were identified by UV spots at 366 nm, with retention factors (R F) of 0.20 (NIV), 0.39 (DON), 0.45 (15-ADON), 0.50 (3-ADON) and 0.60 (ZEA). Some parameters of validation were determined. Calibration data (n = 5) fitted a linear regression model with determination coefficients, R2 > 0.99. The recovery was determined in triplicate at two levels, ranging from 84.3 ± 2.2% to 114.2 ± 11.7%. Detection limits ranged from 80 to 120 μg kg−1 and quantification limits ranged from 120.0 to 200 μg kg−1. The analysis by HPTLC/electrospray (ESI)-MS in negative mode confirmed the presence of TCT-B and ZEA standards in Chilean cereals with mass signals at m/z 355, 371, 337, and 317 for DON, NIV, 3-ADON and 15-ADON, and ZEA, respectively.

Greenness profile assessment of selective liquid chromatographic methods for determination of a quaternary antimigraine combination along with three of their related official impurities

Two selective, sensitive and environmentally safe LC methods were developed and validated for determination of paracetamol, caffeine, ergotamine tartrate and metoclopramide in coformulated antimigraine tablets along with p-aminophenol, p-nitrophenol and theophylline as officially specified impurities. The first is based on high-performance thin-layer chromatography (HPTLC) coupled with densitometric quantitation. Separation was achieved on HPTLC silica gel 60 F254 plates as stationary phase using ethyl acetate:aqueous ammonium hydroxide solution:glacial acetic acid (10.0:0.4:0.1, by volume) as a developing system followed by scanning of the separated bands at 210.0 nm. The subsequent method depends on HPLC with diode array detection. The LC separation was accomplished on a Scharlau C18 (250 × 4.6 mm, 5 μm) column using a mixture of 20.0 mm sodium dihydrogen phosphate, pH 3.0, adjusted with o-phosphoric acid and methanol, at a flow rate of 1.3 mL/min in a gradient elution program. The separated peaks were detected at 210.0 nm. The proposed methods have been validated and proven to meet the requirements outlined in the International Council for Harmonisation (ICH) guidelines. The greenness profile evaluation was carried out using three tools, namely, the National Environmental Method Index, the Analytical EcoScale and the Green Analytical Procedure Index tool, and a comparative study was then conducted. Successful application of the developed methods for determination of the cited quaternary mixture in Metograine tablets confirms their suitability regarding the analytical performance and ecological impact in quality control assay and impurity profiling purposes.

Green HPLC-DAD and HPTLC Methods for Quantitative Determination of Binary Mixture of Pregabalin and Amitriptyline Used for Neuropathic Pain Management.

First analytical methods were herein developed for determination of pregabalin (PGB) and amitriptyline (AMT) as an active binary mixture used for management of neuropathic pain whether in pure forms or in human biological fluids (plasma/urine). First method is green high-performance liquid chromatography-diode array detector (HPLC-DAD) after derivatization of PGB with ninhydrin (NIN) on a reversed-phase C18 column using a mobile phase consisting of ethanol:water (97:3%, v/v) pumped isocratically at 0.8mL/min; AMT were scanned at 215nm, whereas PGB-NIN was scanned at 580nm. Second method is High-performance thin-layer chromatography (HPTLC), where PGB and AMT were separated on silica gel HPTLC F254 plates, using ethanol:ethyl acetate:acetone:ammonia solution (8:2:1:0.05, by volume) as a developing system. AMT peaks were scanned at 220nm, whereas PGB peaks were visualized by spraying 3% (w/v) ethanolic NIN solution and scanning at 550nm. Linear calibration curves were obtained for human plasma and urine spiked with PGB and AMT over the ranges of 5-100μg/mL and 0.2-2.5μg/band for PGB, and 1-100μg/mL and 0.1-2.0μg/band for AMT for HPLC-DAD and HPTLC methods, respectively. The suggested methods were validated according to Food and Drug Administration guidelines for bioanalytical methods validation and they can be applied for routine therapeutic drug monitoring for the concerned drugs.

Extraction of Anthraquinones from Japanese Knotweed Rhizomes and Their Analyses by High Performance Thin-Layer Chromatography and Mass Spectrometry.

Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol(aq) (20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol(aq), methanol, 70% acetone(aq), acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC–MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone(aq). In STS prepared with 70% acetone(aq), the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene–acetone–formic acid (6:6:1, 3:6:1 and 3:3:1 v/v) and dichloromethane–acetone–formic acid (1:1:0.1, v/v). Non-glycosylated anthraquinones were separated only with toluene–acetone–formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (v/v). This solvent and dichloromethane–acetone–formic acid (1:1:0.1, v/v) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8-O-hexoside, emodin-O-acetyl-hexoside and emodin-O-malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1, v/v) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.

High performance thin-layer chromatography (HPTLC) analysis of cannabinoids in cannabis extracts

Ten different TLC mobile phase systems were evaluated in triplicate to determine the most effective mobile phase for the analysis of cannabinoids in Cannabis sativa L. products using HPTLC. Retardation factors (RF) were recorded and the resolution was calculated for three major cannabinoids, Δ9-THC, CBD and CBN on all ten systems. HPTLC Silica gel 60 F 254 20×10 cm plates were used for nine systems and a RP-18 WF 254 10×10 cm plate was used for one additional system. Two systems, xylene-hexane-diethylamine (25:10:1) and 6% diethylamine in toluene, gave the best results in separating between the three major cannabinoids and from other phytocannabinoids. Both systems were validated according to SWGDRUG validation requirements and showed excellent precision. The results of the analysis of various cannabis products from casework are presented. Using the proper mobile phase system with HPTLC is a superior method when compared to traditional TLC systems for qualitative identification of the common cannabinoids in cannabis products. This method has the potential to provide better resolution and to generate reports for more convenient documentation for peer review of casework in crime labs.

High-performance thin-layer chromatography with atmospheric solids analysis probe mass spectrometry for analysis of gasoline polymeric additives.

The offline coupling of high-performance thin-layer chromatography (HPTLC) with atmospheric solids analysis probe mass spectrometry (ASAP-MS) was evaluated for the characterization of polymeric additives in gasoline. A protocol was developed to optimize the ion signal. A glass capillary was moistened with deionized water, and then dipped into silica gel scratched from an HPTLC plate. The capillary tube was fixed to the ASAP holder and introduced into the ionization source for analysis by MS. Silica gel, reversed-phase C18 and cellulose stationary phases were evaluated. The effect of the stationary phase and the nature of analyte were evaluated using polypropylene glycol and polyisobutylene succinimide polyamine as analyte molecules. The optimal ionization conditions are significantly different between ASAP and HPTLC/ASAP-MS analyses. In particular, a higher desorption gas temperature was required to produce ions from the silica gel HPTLC plate. The presence of the stationary phase reduces the internal energy of the ions and limits the fragmentation. HPTLC/ASAP-MS is a very fast and efficient technique for the analysis of polymers in formulated fuels. Good ionization efficiency was obtained with all investigated stationary phases.

Green validated HPTLC and HPLC methods for determination of ephedrine hydrochloride and naphazoline nitrate in the presence of methylparaben, in their pure forms and pharmaceutical formulation

Green validated high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) methods were established for resolving ephedrine hydrochloride (EPH), naphazoline nitrate (NAP), and methylparaben (MET) mixture. Method A is HPTLC–densitometric one that depends on their separation and quantitation on HPTLC silica gel 60 F254 plates, using ethyl acetate:ethanol:triethylamine (8:2:0.2, v/v) as a developing system followed by densitometric measurement at 220 nm. Method B includes HPLC separation and quantification of the introduced mixture on C18 column using a mobile phase consisting of water:ethanol:acetonitrile (80:20:2, v/v) flowing at 1 mL/min. The separated peaks were detected at 220 nm. International Conference on Harmonization (ICH) guidance for method validation was obeyed; EPH and NAP can be determined in the concentration ranges of 2–16 μg/band and 0.4–1.8 μg/band, respectively, using HPTLC method and 5–30 μg/mL and 2–30 μg/mL, respectively, using HPLC method with correlation coefficients > 0.9995. The methods were proven to be accurate, selective, and repeatable for the determination of the two drugs, and can be used for their estimation to check their purity in powder forms and quantifying them in pharmaceutical formulation. The greenness of the procedures was assessed to make sure that they have no adverse environmental effects.