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Summary

Ion exclusion chromatography (IEC) is a chromatographic technique based on the exclusion of dissociated sample molecules. This method has many applications in various analytical separations of weak acids. An alternative technique is the vacancy IEC (v-IEC), where the column is equilibrated with the sample solution flowing as mobile phase through the system, whereas pure water is injected as a sample. In this case, characteristic negative “vacant” peaks are obtained. The aim of this paper is to compare the efficiency of four RP-HPLC columns in the separation of C1–C5 aliphatic acids mixture by use of classical HPLC and very high pressure liquid chromatography. The investigations were conducted in IEC and v-IEC modes.

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A precise and sensitive reversed phase high-performance thin-layer chromatography (RP-HPLC) method was developed for the determination of nilotinib (NTB) in spiked plasma, urine, and pharmaceutical capsule formulation. The method was based on derivatization NTB with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the borax buffer (pH 9). The method employs an isocratic elution using acetonitrile and 10 mM orthophosphoric acid (40:60 v/v) as a mobile phase and an C18 column (4.6 mm × 250 mm, 5 μm, Waters Symmetry), with a fluorescence detector (λ ex: 447 nm, λ em: 530 nm). The method validation was performed with respect to linearity, recovery, accuracy, precision, and stability. The linear ranges were 100–600 ng mL−1 in standard solution, plasma, and urine. Correlation coefficients (r 2) were higher than 0.9997 for all of the analytes, indicating good linear relationship. The percentage recovery was 87.89% for plasma, 95.35% for urine, and 96.07% for capsules.

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Qualitative and quantitative analysis was performed on supercritical-fluid and conventional Soxhlet extracts of Betula pendula Roth., Alnus glutinosa (L.) Gaertn., and Platanus hybrida Brot. bark. The effect of the two extraction methods on extraction yields was compared. Lupeol and β-sitosterol were identified in the bark extracts by TLC and by GC-MS. The main components were betulin and lupeol followed by β-sitosterol; betulinic acid seemed to be a minor constituent. Betulin content was determined by RP-HPLC, with acetonitrile-water 80:20 (v/v) as mobile phase. Comparison of the extraction methods showed that supercritical-fluid (scCO2 + EtOH) and ethanolic Soxhlet extraction resulted in the highest extraction yields. Accumulation of betulin derivatives was higher in supercritical-fluid extracts (scCO2 + EtOH) than in conventional Soxhlet extracts.

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Extraction yield of flavonoids from plant material Sambucus nigra L. inflorescence and Polygonum aviculare herb was determined by use of various methods of liquid-solid extraction — Soxhlet extraction, ultrasonification (USAE), microwave-assisted extraction (MASE), and accelerated solvent extraction (ASE). Methanol was used as extractant. Crude extracts were evaporated to dryness and prepared to achieve fractionation of flavonoids by LLE or SPE. Samples containing the flavonoid fraction were analysed by RP-HPLC. For quantitative purposes the external standard method with a calibration plot for every standard was used. It was found that yield depends first of all on the plant material from which flavonoid fraction was extracted. For Sambucus nigra L. inflorescence the most effective method was exhaustive extraction in a Soxhlet apparatus. For Polygonum aviculare the most effective method was ASE. The best purification method was SPE on C18 adsorbent, which resulted in the highest recoveries and repeatabilities.

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The genus Rhizobium colonizes in leguminous plants by symbiotic relationship and enriches soil nitrogen through the formation of the root nodule. Legumes are the appropriate crops for the recovery of marginal lands and they can easily grow in adverse climatic condition. Rhizobium sp. VMA301 was isolated from the root nodules of V. mungo , grown in arsenic contaminated field. The LC 50 value of arsenite for VMA301 was found to be 1.8 mM. Sixteen differentially expressed proteins were identified using RP-HPLC and MALDI ToF mass spectrometry from arsenite induced whole cell lysate soluble proteins. It is also found that nine proteins were up-regulated and seven proteins were down-regulated in comparison to the control group (cells grown without arsenite). These differential protein expressions mitigate the toxic effect of arsenite and stimulate the detoxification process.

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Organic acids, total phenolic compounds, and antioxidant activity were determined in two different juices of gilaburu fruits ( Viburnum opulus L., belonging to the Caprifoliaceae family). Organic acids, analysed by RP-HPLC-UV visible detection, were individually detected and quantified. The predominant organic acid of samples was L-malic acid. The mean concentration of total phenolic compounds of fresh gilaburu juice (FGJ) and pasteurised gilaburu juice (PGJ) was 351.26 and 330.40 mg gallic acid equivalents (GAE)/100 ml, respectively. Average EC 50 values (in the DPPH• test) were 25.06 μl mg −1 DPPH• for FGJ, and 30.87 μl mg −1 DPPH• for PGJ. Total phenolic compounds of both juices were higher than those of some commonly consumed juices and nectars.

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Summary

The objective of the work was continuation of our study on identification of target coumarins in extracts from Peucedanum alsaticum L. and Peucedanum cervaria (L.) Lap., by use of ChromSword, on the basis of relationships between retention data and descriptors such as partial molecular volume of structural fragments in water and energies of electrostatic interactions of bond dipoles with water (QSRR). The coumarins were mainly identified by comparing UV spectra from gradient runs with spectra from the literature. The presence of columbianadin, ostruthin, and 8-methoxypeucedanin in the fruits of P. cervaria (L.) Lap. and oxypeucedanin and isoimperatorin in the fruits of P. alsaticum was confirmed. The optimum conditions obtained from DryLab simulation were used for both RP-HPLC and RP-UPLC.

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Acta Alimentaria
Authors: D. Giuffrida, M. Ziino, A. Verzera, C. Condurso, and V. Romeo

The biogenic amine content of “Provola dei Nebrodi”, a typical “pasta filata” Sicilian cheese, was studied at different ripening stages. Reversed-phase high performance liquid chromatography (RP-HPLC) with fluorimetric detection was employed for the identification and quantification of eight different amines: ethanolamine, histamine, serotonine, tyramine, tryptamine, 2-phenylethylamine, putrescine and cadaverine. Histamine was the most representative amine in all the analysed cheese samples, followed by serotonin and tyramine. The total amount of biogenic amines was lower than 0.1 ppm in fresh samples, whereas it reached 33.57 ppm in samples with the longest ripening time. The biogenic amine contents were correlated with the proteolytic maturation coefficient, defined as the water-soluble nitrogen/total nitrogen (WSN/TN %) percent ratio. The results showed amine contents lower than the toxic level even for samples with the longest ripening time.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Mihaela Vlassa, Virginia Coman, Miuţa Filip, Florina Copaciu, Aurora Mocanu, and Maria Tomoaia-Cotişel

The basic of all proteins is constituted by approximately 20 common amino acids that differ only in the structure of their side chain. The purpose of our work was to use the overpressured layer chromatography (OPLC) technique for the separation and identification of the essential and non-essential amino acids from some most common proteins, like hen egg yolk protein, bovine serum albumin (BSA), and Type I Collagen from bovine achilles tendon. In this order, the target proteins were acid-hydrolyzed and derivatized. For the separation of essential amino acids from egg yolk, two successive OPLC developments (normal and overrunning) were performed, followed by direct derivatization with 4-hydroxybenzaldehyde and ninhydrin on the chromatoplate for identification. In the hydrolyzed egg yolk, nine essential amino acids were identified. Between lysine and histidine, a poor separation was obtained. The amino acids from BSA and Type I Collagen were separated and identified as phenylthiohydantoin (PTH) derivatives by OPLC. The supposed identity of the amino acids as PTH derivatives was confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC). Seven essential amino acids (arginine, histidine, threonine, valine, methionine, isoleucine, and leucine) were found in bovine serum albumin (BSA), and six (histidine, threonine, valine, methionine, leucine, and lysine) in Type I Collagen. The same eight non-essential amino acids (cysteine, asparagine, glutamine, glycine, alanine, serine, tyrosine, and proline) were identified in these two proteins. By RP-HPLC, other two non-essential amino acids (aspartic acid and glutamic acid) were found both BSA and Type I Collagen. Cysteic acid was also found in BSA. In our samples, tryptophan was missing due to its destruction during hydrolysis.

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Simple, sensitive, selective, and precise stability-indicating thin-layer chromatography (TLC)—densitometric and reversed-phase high-performance liquid chromatography (RP-HPLC) methods were developed and validated for the determination of citicoline sodium (CT) in the presence of its alkaline degradation products (CT Deg.) and in pharmaceutical oral solution. TLC—densitometry employs aluminum TLC plates precoated with silica gel 60 F254 as the stationary phase and ammonia—ethyl acetate—triethylamine (6:3.5:0.5, v/v) as the mobile phase to give compact spots for citicoline sodium (R F = 0.35) and its degradation product (R F = 0.1); the chromatogram was scanned at 272 nm. RP-HPLC utilizes a C18 column and a mobile phase consisting of methanol-water-acetic acid (60:40:0.1, v/v); the pH level was adjusted to 4 using 0.1% orthophosphoric acid, at a fow rate of 1 mL min−1 for the separation of citicoline sodium (t R = 1.801) and its degradation product (t R = 3.422). Quantitation was achieved by ultraviolet (UV) detection at 272 nm. Citicoline sodium was exposed to alkaline hydrolysis, and a comparative study was carried out to show the advantages of the proposed chromatographic methods in determination of citicoline sodium in the presence of its alkaline degradation products. The chromatographic methods were developed and validated as per the International Conference on Harmonization guidelines. As the methods could effectively separate the drug from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients.

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