An eco-friendly multi-analyte high-performance thin layer chromatographic densitometric determination of amoxicillin,metronidazole, and famotidine in their ternary mixtures and simulated gastric juice: A promising protocol for eradicating Helicobacter pylori

Gastrointestinal tract disorders constitute a heavy burden to healthcare providers. To eradicate Helicobacter pylori infection, different triple therapy protocols have been proposed. Among which are combinations of proton pump inhibitors (e.g., omeprazole), histamine-2 receptor antagonists (e.g., famotidine), along with antibiotics (e.g., amoxicillin). In this work, a sensitive and accurate high-performance thin-layer chromatographic method was developed for the simultaneous determination of amoxicillin, metronidazole, and famotidine in bulk powder and laboratory-prepared combined-tablet mixtures. Complete separation of the cited compounds was achieved using pre-coated silica gel plates with a mixture of methanol:chloroform:toluene:water:glacial acetic acid (5:2:1.5:0.5:0.1 v/v/v/v/v) as the mobile phase. The method was fully validated as per the international conference of harmonization guidelines. Good linearity, a correlation coefficient of 0.9991, was obtained in the concentration ranges 0.1–1.6 μg/band (amoxicillin), 0.1–0.9 μg/band (metronidazole), and 0.1–0.9 μg/band (famotidine). Since the method allowed the determination of the three compounds in combined tablets with a high degree of selectivity, accuracy, precision, with cost-effectiveness, it could be used for regular quality control. Moreover, the applicability of the proposed method was extended to the determination of the ternary mixture in simulated gastric juice. Method greenness was assessed using different green metrics.     

Elution–extrusion counter-current chromatographic separation and theoretical mechanism of antioxidant from Robinia pseudoacacia flower

Robinia pseudoacacia flowers have attracted much attention because of numerous bioactivities. In this study, its extract showed the potential scavenging ability for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) and 1,1-diphenyl-2-picrylhydrazyl free radicals. Under the guidance of antioxidant activity, the antioxidant extract was enriched by liquid-liquid extraction. The partition coefficients of the two main components in antioxidant extracts differed greatly, so in this study, elution-extrusion counter-current chromatography with the solvent system of n-hexane-ethyl acetate-methanol-water (2.5:5:2.5:5, v/v) was used to enhance the separation efficiency, and the two main components were successfully obtained. Among them, kaempferol showed strong antioxidant activity, which can be responsible for the activity of the extract. In order to deeply understand the antioxidant mechanism of kaempferol, the thermodynamics, frontier molecular orbital, and kinetics of scavenging free radicals were investigated by density functional theory. The results showed that 4′-OH in kaempferol was the most active group, which can scavenge free radicals by hydrogen atom transfer in non-polar solvents and activate 3-OH to generate double hydrogen atom transfer in the gas phase. But in polar solvents, it was more inclined to clear radicals through single electron transfer and proton transfer. The kinetic result showed that kaempferol needed 9.17 kcal/mol of activation energy to scavenge free radicals.     

Validated high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in fruits of Terminalia chebula,Phyllanthus emblica,and Quercus infectoria

A simple and rapid high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in dried fruits of Terminalia chebula, Phyllanthus emblica, and Quercus infectoria has been developed. The chromatographic development was carried out on precoated silica gel 60 F₂₅₄ plates in a mixture of toluene:ethyl acetate:chloroform:formic acid (4:8:1:3 v/v/v/v). The plate was scanned densitometrically at a wavelength of 280 nm. The retention factor value of gallic acid and ellagic acid was found to be 0.63 ± 0.2 and 0.53 ± 0.1, respectively. The developed method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, specificity and stability as per the international conference of harmonization guidelines. The method showed good linear relationship over a range of 100–600 ng/band (gallic acid) and 100–500 ng/band (ellagic acid) with a regression coefficient (r²) of 0.997 (gallic acid) and 0.996 (ellagic acid). The method showed high accuracy (99.65%–100.85%). The percentage relative standard deviation of intra-day and inter-day precision studies was not more than 2%. The method is highly robust and has displayed high specificity. The developed method is new, simple, and accurate and can be successfully employed in routine analysis of raw materials and formulations containing gallic acid and ellagic acid.     

Development of a general separation strategy by countercurrent chromatography using sanshools from Zanthoxylum bungeanum oleoresin as a case study

Three kinds of sanshools were separated from Zanthoxylum bungeanum oleoresin by high-speed countercurrent chromatography. Sanshools are a series of amide compounds extracted from the Zanthoxylum bungeanum. Due to similar structures, polarities, and dissociation constants, it was challenging to select an appropriate solvent system for their complete separation by countercurrent chromatography. To address this challenge, a solvent-system-selection strategy was proposed to identify a relatively suitable solvent system. Additionally, a separation procedure incorporating multi-elution modes selection was established to separate similar compounds in a logical order. Ultimately, a solvent system comprising n-hexane:ethyl acetate:methanol:water in a ratio of 19:1:1:5.67 was selected. Three amide compounds with high purity were obtained through the use of recycling elution mode to improve separation resolution: hydroxy-ε-sanshool (8.4 mg; purity: 90.64%), hydroxy-α-sanshool (326.4 mg; purity: 98.96%), and hydroxy-β-sanshool (71.8 mg; purity: 98.26%) were obtained from 600 mg sanshool crude extract. The summarized solvent-system-selection strategy and separation procedure incorporating multi-elution modes may instruct countercurrent chromatography users, particularly novices, seeking to separate compounds with highly similar chemical properties.     

Green micellar stability-indicating high-performance liquid chromatography method for determination of rupatadine fumarate in the presence of its main impurity desloratadine: Oxidative degradation kinetics study

A green micellar stability-indicating high-performance liquid chromatography method was developed for rupatadine fumarate determination in existence with its main impurity desloratadine. Separation was attained using Hypersil ODS column (150 × 4.6 mm, 5 μm), the micellar mobile phase consisted of 0.13 M sodium dodecyl sulfate, 0.1 M disodium hydrogen phosphate adjusted by phosphoric acid to pH 2.8 and 10% n-butanol. The column was maintained at 45^◦C and detection was carried out at 267 nm. A linear response was achieved over the range of 2–160 μg/ml for rupatadine and 0.4–8 μg/ml for desloratadine. The method was applied for rupatadine determination in alergoliber tablets and alergoliber syrup without the interference of methyl paraben and propyl paraben present as main excipients. Rupatadine fumarate revealed pronounced susceptibility to oxidation; further study of oxidative degradation kinetics was carried out. Rupatadine was found to follow pseudo-first-order kinetics when exposed to 10% H₂O₂ at 60 and 80°C and the activation energy was found to be 15.69 Kcal/mol. At a lower temperature (40°C), degradation kinetics regression was best fitted as a polynomial quadratic relationship, thus rupatadine oxidation at a lower temperature tends to adopt a second-order kinetics rate. Oxidative degradation product structure was revealed using infrared and found to be rupatadine N-oxide at all temperature values.     

An efficient high-speed counter-current chromatography method for the preparative separation of sesquiterpenoids from the rhizomes of Cyperus rotundus L. combined with evaluation of the anti-inflammation activity in vitro and molecular docking

Cyperus rotundus L. has been extensively used in ancient medication for the treatment of different disorders worldwide, in which sesquiterpenes are the most representative components. In this study, sesquiterpenes were effectively purified by two-dimensional counter-current chromatography in combination with continuous injection and inner-recycling mode with a solvent system of n-hexane/ethyl acetate/methanol/water (1:0.2:1:0.2, v/v/v/v). For one-dimension separation, continuous injection mode was used with three times injection and the inner-recycling mode was adopted for the separation of two mixtures for two-dimensional separation. Finally, four sesquiterpenoids, including scariodione ( 1 ), cyperenoic acid ( 2 ), scariodione ( 3 ), and α-cyperone ( 4 ), were obtained with purities over 98%. Mass spectrometry and nuclear magnetic resonance were applied to identify their structures. The results from the anti-inflammation effect with zebrafish demonstrated that cyperenoic acid exhibited stronger anti-inflammation activity. Molecular docking results suggested that cyperenoic acid possessed lower binding energies –9.4545 kcal/mol with 1CX2 to form formed hydrogen bond interaction with ARG120. In general, all the obtained findings proved that the strong anti-inflammation capacity of cyperenoic acid can have the potential of being adopted for treating diseases resulting from inflammation.     

Separation and purification of quinolyridine alkaloids from seeds of Thermopsis lanceolata R. Br. by conventional and pH-zone-refining counter-current chromatography

In this work, the preparative separation of quinolyridine alkaloids from seeds of T. lanceolata by conventional and pH-zone-refining counter-current chromatography. Traditional counter-current chromatography separation was performed by a flow-rate changing strategy with a solvent system of ethyl acetate-n-butanol-water (1:9:10, v/v) and 200 mg sample loading. Meanwhile, the pH-zone-refining mode was adopted for separating 2.0 g crude alkaloid extracts with the chloroform-methanol-water (4:3:3, v/v) solvent system using the stationary and mobile phases of 40 mM hydrochloric acid and 10 mM triethylamine. Finally, six compounds, including N-formylcytisine (two conformers) ( 1 ), N-acetycytisine (two conformers) ( 2 ), (-)-cytisine ( 3 ), 13-β-hydroxylthermopsine ( 4 ), N-methylcytisine ( 5 ), and thermopsine ( 6 ) were successfully obtained in the two counter-current chromatography modes with the purities over 96.5%. Moreover, we adopted nuclear magnetic resonance and mass spectrometry for structural characterization. Based on the obtained findings, the pH-zone-refining mode was the efficient method to separate quinolyridine alkaloids relative to the traditional mode.     

Dilute-and-shoot approach for determination of several biomarkers and pharmaceuticals in wastewater using nanoflow liquid chromatography – Orbitrap mass spectrometry

A new method for quantitative analysis of several biomarkers and pharmaceutical compounds in wastewater has been developed employing nanoflow liquid chromatography with Orbitrap mass spectrometry. An easy dilute-and-shoot approach has been used for sample preparation with a dilution factor of 5. Improved retention of ionic and highly polar compounds has been achieved by the addition of tetrabutylammonium bromide as an ion pair reagent into the final diluted sample. The new nanoflow liquid chromatography method has demonstrated low matrix effects (70%–111%), high sensitivity in terms of limits of quantification (0.005 to 0.3 μg/L), low injection volume (70 nl) and solvent consumption, and the ability to analyze diverse polar and ionic analytes within one run using a single reversed-phase nanoflow liquid chromatography column. Wastewater samples (n = 116) from the wastewater treatment plants of different cities in Latvia were analyzed using the developed method. The observed concentrations of biomarkers were in line with the literature data.     

Biphasic alcoholysis coupled with high-speed countercurrent chromatography for high performance on separating phorbol from Croton tiglium Linn extracts

Phorbol is a tetracyclic diterpenoid found in Euphorbia tirucalli, Croton tiglium, and Rehmannia glutinosa, and is nuclear of various phorbol esters. The rapid obtaining of phorbol with high purity highly contributes to its application, such as synthesizing phorbol esters with designable side chains and particular therapeutic efficacy. This study introduced a biphasic alcoholysis method for obtaining phorbol from croton oil by using polarity imparity organic solvents in both phases and established a high-speed countercurrent chromatography method for simultaneous separation and purification of phorbol. The optimized operation conditions of biphasic alcoholysis were a reaction time of 91 min, a temperature of 14°C, and a croton oil-methanol ratio of 1:30 (g:ml). The phorbol during the biphasic alcoholysis was 3.2-fold higher in content than that obtained in conventional monophasic alcoholysis. The optimized high-speed countercurrent chromatography method was using the ethyl acetate/n-butyl alcohol/water at 4.7:0.3:5 (v:v:v) with Na₂SO₄ at 0.36 g/10 ml as the solvent system, using the mobile phase flow rate of 2 ml/min, the revolution of 800 r/min, under which the retention of the stationary phase was achieved at 72.83%. The crystallized phorbol following high-speed countercurrent chromatography was obtained as high purity of 94%.     

Simple high-performance liquid chromatography-ultraviolet method for simultaneous separation and detection of 14 bisphenol pollutants in building materials

A high-performance liquid chromatography-ultraviolet method was developed for rapidly and simultaneously analyzing novel and typical bisphenols in building materials, including bisphenol S, diphenolic acid, bisphenol F, bisphenol E, bisphenol A, bisphenol B, bisphenol AF, bisphenol AP, bisphenol C, bisphenol FL, bisphenol Z, bisphenol BP, bisphenol M, and bisphenol P. By using a Kromasil 100–5 C18 column, these bisphenols were completely separated in 40 min via gradually increasing the concentration of methanol in the mobile phase from 45 to 80% during the elution process. In particular, this method achieved the synchronous analysis of bisphenol S, diphenolic acid, bisphenol FL, bisphenol BP, and bisphenol M through HPLC, which were difficult to separate and had to be identified and detected through mass spectrometry. The limits of detection of the method ranged from 0.002 to 0.040 mg/L for these 14 bisphenols, with a precision of less than 4.9% (n = 7, c = 0.05 mg/L). The analytical results for five types of building materials (phenolic, epoxy, polycarbonate, polyester, and polysulfone resins) indicated that the proposed method is appropriated for the rapid measurement of bisphenols in real samples.