The coupling of thin-layer chromatography with mass spectrometry (TLC-MS) for the analysis of monomeric flavanols and proanthocyanidins in samples presented as complex matrices has been studied. The elution conditions for TLC-MS were optimised and full scans were compared with selected reaction monitoring for the MS detection of compounds. The performance of silica gel and cellulose plates with different developing solvents in TLC-MS was assessed. Cellulose plates provided superior sensitivity while ionisation suppression was encountered with silica plates. The use of a HILIC guard column beyond the elution head was found to facilitate detection of monomer compounds on silica plates. A new comprehensive TLC x MS procedure for screening flavanols in the entire chromatogram was developed as an alternative to the use of 4-dimethylaminocinnamaldehyde to determine the locations of compounds on the plate. This new procedure was applied to detect flavanols in the peel of Punica granatum L. fruits and in seeds of Juniperus communis L. , in which flavanols and proanthocyanidin dimers and trimers were detected for the first time.
COBISS.SI-ID: 36620037
A major factor in the direct determination of lutein in spinach extracts proved to be obtaining reproducible and stable chromatography of lutein. This was achieved on a C30 column with the mobile phase acetone-0.1 M triethylammonium acetate (TEAA) buffer (pH 7) 9:1 (v/v). Extraction of 10 mg of lyophilized spinach with 10 mL of extraction solvent (ethanol, acetone, ethanol-ethyl acetate 1:1 (v/v), methanol-THF 1:1 (v/v)) for 15 min with magnetic stirring under nitrogen resulted in equal yields of lutein. The yields were enhanced by addition of 15% of 1 M TEAA buffer pH 7 to all four extraction solvents. As confirmed by recovery experiments, no loss of lutein occurred during the extraction. The relative standard deviation from triplicate extractions was less than 5%. The addition of 15% TEAA pH 7 to acetone enhanced the extraction yield of lutein also from unlyophilized spinach. The content of lutein in different spinach samples ranged from 5 to 15 mg/100 g of fresh weight. The first separation is reported of all the carotenoids and chlorophylls on a C18 core-shell column and the addition of 15% of 1 M TEAA buffer pH 7 to acetone also enhanced the extraction yield of beta-carotene compared to the yield produced by pure acetone.
COBISS.SI-ID: 5168410
Separation of three triterpenic acids (ursolic, oleanolic and betulinic acid) was achieved on different thin-layer chromatography (TLC) (silica gel 60) and high-performance thin-layer chromatography (HPTLC) sorbents (silica gel 60, C-2 RP and C-18 RP) using several developing solvents, based on the non-polar diluent n-hexane, and ester (methyl acetate, ethyl acetate, ethyl propionate) as selector. Anisaldehyde and molybdophosphoric acid detection reagents were used. Finally, a simple method on a C-18 RP HPTLC plate was developed using n-hexane-ethyl acetate (5:1 v/v) as a developing solvent in a horizontal developing chamber. The method was used for the screening of ursolic, oleanolic and betulinic acids in different vegetable extracts. Other plant triterpenoids (lupeol, alpha-amyrin, beta-amyrin, cycloartenol, lupenone, friedelin, lupeol acetate, cycloartenol acetate) and phytosterols (beta-sitosterol, stigmasterol) did not interfere. TLC-MS was used as a tool for the additional confirmation of the presence of ursolic, oleanolic, and betulinic acids in some of the studied vegetable extracts. Ursolic and oleanolic acids were found in radicchio Leonardo and white-colored radicchio di Castelfranco extracts for the first time, while betulinic acid was not detected in the eggplant extract by MS, although it was suggested at first by TLC analysis. Pre-chromatographic bromination on the HPTLC silica gel 60 plates and subsequent development in toluene-chloroform-diethyl ether-formic acid (20:16:4:0.1, v/v) provided a superior resolution of these compounds.
COBISS.SI-ID: 5206810
An isocratic HPLC method was developed for the determination of eight xanthophylls (lutein, capsanthin, zeaxanthin, canthaxanthin, beta-apo-8'-carotenal, ethyl-8'-apo-beta-carotene-8'-oate, citranaxanthin and beta-cryptoxanthin; registered as additives in poultry feeding) in egg yolks. Optimum separation of all-E isomers of these xanthophylls was achieved in less than 18 min on a ProntoSIL C30 column at 27 degrees C using acetone-methanol-0.5 M triethylammonium acetate buffer pH 7 14:5:1 (v/v) as the mobile phase with a flow rate of 1 mL/min using spectrophotometric detection at 450 nm. Other mobile phases were also found suitable, including acetone-water 93:7 (v/v) and acetone-methanol 1:4 (v/v) and the influences of column temperature on the separation and addition of triethylammonium acetate buffer pH 7 to the mobile phase on enhancement of the peak areas were evaluated. Preparation of test solution from yolks included a short vortexing of 0.5 g of yolk in 10 mL of acetone, followed by 15 min magnetic stirring under nitrogen and centrifugation. The method was validated for 5 analytes. The calibration range was between 0.04 and 2 mu g/mL and the mean recovery of the analytes (95%) and the intra-day precision of the method (RSD less than 5%) on three levels in triplicates were obtained. Analyses of eggs from four husbandry classes showed the presence of up to four xanthophylls (lutein, zeaxanthin, canthaxanthin and ethyl-8'-apo-beta-carotene-8'-oate) and traces of beta-cryptoxanthin.
COBISS.SI-ID: 5334042
During coffee roasting major changes occur in coffee bean composition. Among others dark coloured melanoidins are formed, which are high molecular weight Maillard reaction products. A new approach is presented here to monitor the influence of roasting conditions on the antioxidant capacity of melanoidins and chlorogenic acids (CGAs) in a coffee brew. Validated Folin–Ciocalteu (FC) and ABTS assays were used as on-line antioxidant assays coupled (post-column) with high performance sizeexclusion chromatography (HPSEC). HPSEC enabled the separation of melanoidins from CGAs and the determination of the antioxidant capacity of each fraction, within a total elution time of 25 min. Besides the on-line assay measurements, both assays were also applied off-line with flow injection analysis (FIA). The maximum antioxidant capacity was determined to be at a light-to-medium roast degree, measured with both ABTS-FIA and FC-FIA assays as well as on-line ABTS assay. With FC on-line assay the maximum was found to be at a very light roast degree. Based on the peak areas obtained with the new coupled technique the roasting effects on the variability of melanoidin and CGA contents in coffee brews were studied. The majority of melanoidins are already formed in the early stage of the roasting process and the relative contribution of melanoidins to the total antioxidant capacity increases towards darker roasts, mainly because CGAs degrade during roasting. A new parameter, the ratio of melanoidin to CGA peak area, was introduced as a possible predictor of the roast degree.
COBISS.SI-ID: 36620293