Quantification of (−)-Epicatechin and Procyanidin B2 in Chocolates

JPC – Journal of Planar Chromatography – Modern TLC - Validation of a method for the quantification of (−)-epicatechin and procyanidin B2 in chocolates is reported. After a simple...     

Ultra-thin-layer chromatography mass spectrometry and thin-layer chromatography mass spectrometry of single peptides of angiotensin-converting enzyme inhibitors

The separation of structurally related angiotensin-converting enzyme (ACE) inhibitors lisinopril, cilazapril, ramipril and quinapril and their corresponding active diacid forms (prilates) by conventional TLC silica gel 60 plates was contrasted with that afforded by monolithic ultra-thin-layer chromatographic (UTLC) plates. For the use of UTLC plates technical modifications of the commercially available equipments for the sample application, development and detection were made. Plates were developed in modified horizontal developing chamber using ethyl acetate-acetone-acetic acid-water (4:1:0.25:0.5, v/v). Detection of the separated compounds was performed densitometrically in absorption/reflectance mode at 220 nm and after exposure to iodine also by image analysis. The obtained results showed that monolithic layer is more efficient for the separation of structurally similar polar compounds, such as prilates than conventional silica layers. Identification of the compounds was confirmed by ESI-MS after their on-line extraction from the UTLC and TLC plates by means of Camag TLC-MS interface.     

TLC determination of mebendazol and pentoxifylline as residues on pharmaceutical equipment surfaces

This paper presents the applicability of thin-layer chromatographic methods with a subsequent densitometric or video densitometric quantitation for determination of residues in controlling pharmaceutical equipment cleanliness. Analytical methods were developed for monitoring residues of pentoxifylline at 10 mg/M2 and mebendazol at 1 mg/M2 on stainless steel surfaces. Simulated samples were prepared by addition of a calculated amount of pharmaceutical (as a solution) on a 35 x 35 cm stainless steel surface. After evaporation of solvent, the residues were wiped with wetted cotton. The cotton was extracted with dichloromethan-methanol (1 + 1). Filtered extract was concentrated by vacuum evaporation and an aliquot applied to the plate, where standards were also applied. In the narrow concentration range near the acceptable residue limits, linear calibration curve could be obtained for both substances. The mean recovery (n = 4) obtained by densitometric quantitation was 93.4% for pentoxifylline and 85.6% for mebendazol, with coefficients of variation of 3.5 and 8.3%, respectively. Results of video densitometric quantitation did not differ significantly. However, data acquisition and evaluation is faster compared with densitometry and allows better archiving possibilities as required by the regulatory authorities. Both quantitation modes can be applied to routine control of pharmaceutical equipment cleanliness.     

High-performance thin-layer chromatography method for monitoring norfloxacin residues on pharmaceutical equipment surfaces

This paper presents a high-performance thin-layer chromatography (HPTLC) method with direct fluorescence measurement for the determination of norfloxacin. The method was validated for the monitoring of norfloxacin residues on stainless steel surfaces at the allowed limit of 10 mg of norfloxacin per square meter. However, it can be adapted for lower amounts of residues owing to the low detection limit of norfloxacin (about 5 ng) and can also be used for other surface materials. Test solutions were analyzed by the new HPTLC method and the known HPLC method for comparison. Accuracy and precision of the new HPTLC method, with a subsequent quantification by densitometer or video system, are comparable with those of the HPLC method.     

Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.     

Determination of inulin in foods

A method was developed for determining fructan inulin in various foods (yogurts, honey cakes, chocolates). Warm water was applied for extraction of samples, and mono- and dissacharides were determined by a thin-layer chromatographic densitometric method. A portion of the test solution was hydrolyzed 30 min with 1% oxalic acid in a boiling water bath. Fructose was determined in the hydrolysate. The amount of inulin in a sample was calculated as the difference between the amount of fructose in the sample before and after hydrolysis. The fructose from sucrose formed during the hydrolysis was also considered. The mean recovery from yogurt fortified with 4% inulin was 95.5 +/- 4.5% (mean +/- standard deviation); from honey cakes extract fortified with 10% inulin, 97.3 +/- 5.5%; and from chocolate extract fortified with 30% inulin, 98.6 +/- 6.6% (6 replicates in all cases). Determination of glucose is not necessary for analyzing fructans with the composition expressed shortened to GFn-1 (G, glucose; F, fructosyl) with the average degree of polymerization 8 < or = n < or = 15.     

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate-formic acid-water (85:10:15, v/v/v) and n-hexane-ethyl acetate-formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis. Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr = 562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).     

Separation of eight selected flavan-3-ols on cellulose thin-layer chromatographic plates

The potential of microcristaline cellulose as sorbent in the separation of eight compounds: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), procyanidin B1 and procyanidin B2 was studied. Cellulose HPTLC plates prewashed in water (not necessary, when water was used as developing solvent) and dried with a hair dryer, bandwise application and development in horizontal developing chamber (sandwich configuration) gave the best results. Detection was performed using vanillin-H3PO4 reagent. Four new developing solvent systems were proposed: water, 1-propanol-water (20:80, v/v), 1-propanol-water-acetic acid (4:2:1, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v), and at least two of them were needed for the differentiation between all eight compounds. Surprisingly, water enabled the separation of epimers C from EC and GC from EGC, as well as the dimers procianidin B1 and B2. Additionally, C, EGC, B1 and B2 were separated from all the other compounds. The best choice for developing solvent is given for each of the studied compounds. The best separation of the five main catechins (EC, GC, EGC, ECg, EGCg) present in green tea extract was achieved using 1-propanol-water-acetic acid (20:80:1, v/v). The chromatograms of oak bark extract developed in solvents with higher water content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v)) showed less bands than chromatograms developed in solvents with higher organic modifier content (e.g. 1-propanol-water-acetic acid (4:2:1, v/v)). It was proved that such behavior was due to the presence of procyanidins beside the main component catechin.     

Determination of three major triterpenoids in epicuticular wax of cabbage (Brassica oleracea L.) by high-performance liquid chromatography with UV and mass spectrometric detection

Lupeol, together with alpha- and beta-amyrins in smaller quantities, has been found for the first time in the epicuticular wax of white cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC) leaf surface extract. The three triterpenoids were identified by a new high-performance liquid chromatographic (HPLC) method with UV and mass spectrometric (MS) detection using atmospheric pressure chemical ionization (APCI). All three isomeric compounds gave a parent ion peak at m/z 409 [M+H-18](+) and the relative intensities of some characteristic fragment ion peaks in tandem mass spectrometric (MS-MS) spectra of this parent ion enabled differentiation between the isomers. An additional peak at m/z 439 [M+H](+), which could be oleanonic or ursonic aldehyde, was detected by HPLC-APCI-MS. Saponification of cabbage leaf surface extract with 20% NaOH in methanol at 65 degrees C for 2h had no influence on lupeol, or alpha- or beta-amyrins, but lead to the formation of three additional compounds, which were not identified.