Authors:Irena Vovk, Nadja Gerčar, Breda Simonovska, and Mihael Sok
Cholesterol is an essential component of mammalian cells, but its role in cancer is unclear. We have determined the total cholesterol content in the healthy and the cancerous lung tissues of the same patient. Tissues of 13 patients (7 women and 6 men, different histological type, different stages of the disease) were obtained during surgical intervention. The samples (0.056–2.004 g) were hydrolyzed in alkaline medium, and the total cholesterol was extracted into n-hexane and simultaneously determined by thin-layer chromatography (TLC) on silica gel high-performance thin-layer chromatography (HPTLC) plates and nonaqueous reversed-phase high-performance liquid chromatography (HPLC), which both proved to be suitable for the determination of cholesterol in lung tissues and gave comparable results. This is the first report about the comparison of the total cholesterol content in the healthy and the cancerous tissue of the same patient. The difference in the results for the total cholesterol from both types of tissues was remarkable. For 13 patients, the mean contents and standard deviations determined by TLC were 4.01 ± 0.75 mg g−1 in the healthy lung tissue and 7.75 ± 1.93 mg g−1 in the cancerous lung tissue. Comparable results were obtained by HPLC analyses of the same samples: 3.91 ± 0.73 mg g−1 in the healthy and 6.95 ± 1.83 mg g−1 in the cancerous lung tissue. The range of total cholesterol content determined by TLC was between 3.20 mg g−1 and 5.83 mg g−1 in healthy lung tissues, and between 4.64 mg g−1 and 12.01 mg g−1 in lung cancer tissues, while the ranges obtained by HPLC were between 2.80 mg g−1 and 5.49 mg g−1 for healthy lung tissue, and between 4.38 mg g−1 and 11.24 mg g−1 for lung cancer tissue. The cancerous lung tissue of each of the thirteen patients contained higher amounts of total cholesterol compared to their healthy lung tissue. In 8 of the patients, the total cholesterol in the cancer tissue was more than 60% higher than in the healthy tissue; furthermore, in 5 patients, it was more than 100% higher.
Authors:Vesna Glavnik, Breda Simonovska, Alen Albreht, and Irena Vovk
A thin-layer chromatographic (TLC) method for fast screening of trans-resveratrol, pterostilbene, and p-coumaric acid in samples of recombinant bacterial cultures, food supplements, and wine was developed. The separation was performed on high-performance thin-layer chromatography (HPTLC) silica gel 60 plates using n-hexane-ethyl acetate-formic acid (20:19:1, v/v) as developing solvent in tank configuration of horizontal developing chamber, in which better resolution between trans-resveratrol and p-coumaric acid than in sandwich configuration of the same chamber or in automatic developing chamber (ADC) was obtained. Compounds were detected before and after post-chromatographic derivatization (three detection reagents) by image analyzing system (at 366 nm or white light) and by densitometer (absorption-reflectance and fluorescence mode). The lowest densitometric limits of detection (LOD) 2 ng for trans-resveratrol (303 nm), 5 ng for pterostilbene (303 nm), and 15 ng for p-coumaric acid (286 nm) were found before derivatization in absorption-reflectance mode. Post-chromatographic derivatization with anisaldehyde-sulfuric acid detection reagent resulted in higher LOD in the same mode: 13 ng for trans-resveratrol and pterostilbene at 500 nm and 30 ng for p-coumaric acid at 566 nm. Natural fluorescence of both stilbenes was less sensitive than UV absorption and less selective than post-chromatographic derivatization with anisaldehyde reagent at densitometric screening of trans-resveratrol in the samples. A complementary high-performance liquid chromatography (HPLC) method was developed for screening and quantification of the three compounds in recombinant bacterial cultures. Adequate separation of the analytes was performed in 35 min by a gradient elution from a stainless-steel column Hypersil ODS (150 × 4.6 mm I.D., particle size: 5 μm) with the mobile phase consisting of 50 mM sodium acetate buffer pH 5.6 (solvent A) and acetonitrile (solvent B) at the flow rate of 1.5 mLmin−1.
Authors:Katerina Naumoska, Breda Simonovska, Alen Albreht, and Irena Vovk
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, C2 RP and C18 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 C18 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, α-amyrin, β-amyrin, cycloartenol, lupenone, friedelin, lupeol acetate, cycloartenol acetate) and phytosterols (β-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.
Authors:Tero Wennberg, Irena Vovk, Pia Vuorela, Breda Simonovska, and Heikki Vuorela
The computer-assisted simulation program DryLab has been used to simulate TLC separations. The simulations were based on data from preliminary TLC separations. For DryLab data entry
values from TLC were converted to retention times, the development distance on the plate was used as column length, and the plate thickness was used as the column diameter. To achieve reasonably accurate simulations it was found necessary to run three preliminary runs in which differences between organic modifier concentration in two adjacent runs was more than 5%. The possibility of predicting HPLC separation on the basis of TLC separations was also studied. It was found that the method can be transferred from TLC to HPLC and that DryLab can be used to predict HPLC separation on the basis of the information obtained from TLC experiments. To produce a reasonably accurate HPLC simulation on the basis of TLC data, however, a relatively large number of preliminary experiments is required.
Authors:Irena Vovk, Breda Simonovska, Pia Vuorela, and Heikki Vuorela
The separation of (+)-catechin and (−)-epicatechin on cellulose TLC plates with 1-butanol-water-acetic acid, 4 + 2 + 1 (
), has been studied under different development conditions on prewashed and untreated TLC plates. Plates were developed in horizontal chambers (tank and sandwich configuration) and in twin trough (unsaturated and saturated) developing chambers. Tank configurations were used with and without preconditioning.Prewashing of the TLC plates, preconditioning, and the type of development chamber used had a large effect on the chromatographic results. Without prewashing of the TLC plate unsatisfactory or misleading results were obtained.Anisaldehyde-sulfuric acid dipping reagent proved to be more selective than vanillin-phosphoric acid as detection reagent for the detection of flavan-3-ols in oak bark extract; the latter was, however, more sensitive.
Authors:Irena Vovk, Breda Simonovska, Lidija Kompan, and Mirko Prošek
The ratio of lactulose/mannitol excretion in urine after their administration is of great importance for evaluation of malabsorption and intestinal permeability disruption in some diseases. An analytical method has been developed for determination of lactulose and mannitol in urine on the same amino HPTLC plate. The method enables densitometric quantification of lactulose by use of fluorescence mode, and mannitol by use of absorption mode after detection with AgNO
Authors:Vesna Glavnik, Breda Simonovska, Irena Vovk, Dragana Pavlović, Danijela Ašperger, and Sandra Babić
Validation of a method for the quantification of (−)-epicatechin and procyanidin B2 in chocolates is reported. After a simple preparation of the chocolate test solution, thin-layer chromatography (TLC) on high-performance thin-layer chromatographic (HPTLC) cellulose plates with n-propanol-water-acetic acid (20:80:1, ν/ν) and derivatization with 4-dimethylaminocinnamaldehyde (DMACA) were performed. Densitometry at 655 nm enabled accurate quantification of both analytes in chocolates. The milk chocolate sample which served for precision and recovery experiments contained 13.0 mg/100 g of (−)-epicatechin (relative standard deviation (RSD)) 5.8%, n = 6) and 13.2 mg/100 g of procyanidin B2 (RSD 4.2%, n = 6). Usable polynomial curves were 2–30 ng for (−)-epicatechin and 4–60 ng for procyanidin B2. The validated TLC method can be applied for routine quality control of the two major polyphenolic compounds in different chocolate samples.
Authors:Mirko Prošek, Breda Simonovska, Alenka Golc-Wondra, Irena Vovk, Samo Andrenšek, Elizabeta Mičović, and Terezija Golob
HPTLC and HPLC-MS methods have been developed for quantitative determination of inulin in food products. Samples were applied to silica gel HPTLC plates and developed three times, twice with
-propanol-acetone-water, 45 + 30 + 25 (
), and the third time with
-propanol-acetone-water, 50 + 40 + 10 (
). Total development time was 150 min. Dried plates were dipped into DAP reagent for 10 s. After heating colored spots of the saccharides appeared. Quantitative evaluation of the colored spots was performed in transmission mode by means of a flatbed scanner and densitometer. The precision of measurements for the main inulin fractions was ±6.0%; the limit of quantitation (
) varied from 0.1 to 1 µg per spot; and the ‘linear’ working range was between 0.5 and 4.0 µg per spot. The method was tested on real samples.
Authors:Irena Vovk, Breda Simonovska, Samo Andrenšek, Teijo Yrjönen, Pia Vuorela, and Heikki Vuorela
Extraction of onion (
L.) with 80:20 (
) methanol-water in water by rotation planar extraction (RPE) and medium-pressure solid-liquid extraction (MPSLE) has been studied systematically. Rotation planar extraction was studied by use of an ExtraChrom separation instrument prototye. Only rotation planar extraction performed by use of the ExtraChrom enabled efficient extraction of dried onion. The equilibration time proved to be the most important variable in the extraction. The particle size of the sample had a variable effect on extraction efficiency, mainly because of the carbohydrates predominating in the extracts. It was possible to extract oligofructans with a degree of polymerization of up to 12, and these were the main components of the extract. Fructose, glucose, and saccharose, in total, accounted for 10% of the mass of the extract; their mass ratio in the extract was 3:1:6. After mild hydrolysis of the extract with oxalic acid only fructose and glucose were obtained. Although TLC screening of the extract before acid hydrolysis detected no flavonoids, one phenolic acid was observed. After acid hydrolysis with hydrochloric acid, quercetin could be detected.