Three TLC methods were used for an initial screening of some common plant triterpenoids and phytosterols in cuticular wax extracts of different vegetables (zucchini, eggplant, tomato, red pepper, mangold, spinach, lettuce, white-colored radicchio di Castelfranco, raddichio Leonardo, white cabbage, red cabbage and savoy cabbage). The preliminary experiments showed that the studied vegetables are potential sources of triterpenoids and phytosterols. To identify the compounds present in the extracts with high certainty, the first TLC–MS/MS method was developed for the analysis of eight triterpenoids (lupeol, α-amyrin, β-amyrin, cycloartenol, cycloartenol acetate, lupeol acetate, lupenone and friedelin) and two phytosterols (β-sitosterol and stigmasterol). This method takes the advantages of: (1) a satisfactory separation of the target compounds; (2) their differentiation according to the band colors; and (3) the potential of their discrimination by the acquired first-order mass (MS) and product ion (MS/MS) spectra. Since the closely eluting compounds have complex and similar MS/MS spectra, distinguishing between them was possible by the proposed characteristic ions. Using a custom-built mass spectral library, the head to tail MS/MS spectra comparison of sample test solution zones and standard aided the compound identification. In addition to the molecular mass information, the developed atmospheric pressure chemical ionization method (APCI) in positive ion mode provided structural information, regarding the presence of functional group in the molecule. This approach resulted in many positively assigned compounds in the investigated vegetable waxes, from which more than a half are reported for the first time. First developed TLC-MS/MS method will enable identification of triterpenoids and phytosterols with anti-cancer and anti-inflammatory activities in other vegetables and fruits and consequently will support other studies dealing with daily intake of these important compounds with biological activity.
COBISS.SI-ID: 5635354
The recently developed comprehensive TLC × GC technique using headspace sampling is presented. The main advantage of this approach, as demonstrated in lipid analysis, is the possibility to include a transesterification step of glycerides into fatty acid methyl ester derivatives (FAME) because no particular constraints in terms of operational time between TLC and GC are present. Besides being a relatively low-cost solution, TLC × GC by means of headspace sampling provides many benefits in terms of flexibility of separation conditions and modulation sampling width. The technique provides over two orders of magnitude of linear range with TLC sample loads of about 1 mg with good reproducibility and accuracy, as demonstrated by multiple headspace extraction (MHE) tests. The technique is a viable alternative to the established but more expensive HPLC × GC technique. The useful range of TLC × GC in terms of analyte volatility can be further extended with a future development of devices based on thermal desorption. Since the implementation of the method in relatively simple and quick, we can expect a wider use of comprehensive two-dimensional analysis and evaluation of lipid samples in many analytical laboratories.
COBISS.SI-ID: 5728538
Since Chinese lantern (Physalis alkekengi L.) represents a rich source of various bioactive secondary metabolites, there is an urge for its detailed characterization. Non-polar flavonoid aglycones represent one of the few bioactive species found in plant’s cuticular waxes. The separation of flavonoids is already extensively covered in the literature, but methods dedicated to separation and identification of methylated flavonoids are rather scarce. In the present study a nontargeted approach for the separation, isolation and identification of methylated flavonoids present in P. alkekengi L. var. franchetii cuticular waxes was established. A rapid and simple separation on HPTLC silica gel was developed for preliminary screening of flavonoids. Fast HPLC-UV-MSn and HPLC-UV methods using a C6-Phenyl and a C18 stationary phase were also developed, respectively. In both cases, the right combination of temperature and tetrahydrofuran, as a mobile phase modifier, were shown to be crucial for a baseline separation of all studied compounds. By employing a semi-preparative analog of the C18 column, a simultaneous isolation of pure unknown analytes was achieved. Using these developed methods in combination with NMR, four 3-O-methylated flavonols were detected and identified in P. alkekengi L. var. franchetii cuticular waxes: myricetin 3,7,3’-trimethyl ether, quercetin 3,7-dimethyl ether, myricetin 3,7,3’,5’-tetramethyl ether and quercetin 3,7,3’-trimethyl ether. Developed isocratic HPLC-UV-MSn method is simple and fast (under 8 min) and therefore useful in quality control of P. alkekengi L. var. franchetii by enabling chromatographic fingerprinting of external methylated flavonols. A rationale for the mechanism of separation of these metabolites by HPLC establishes a foundation for future development of chromatographic methods for methylated flavonols and related compounds.
COBISS.SI-ID: 5867290
On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol–formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280 nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6’-O-malonyl)-hexoside). Developed methodology is suitable not only for identification of flavan-3-ols and proanthocyanidins, but also for many other groups of pharmacologically important compounds (flavonoids, physalines), that are in the world the subject of numerous studies (novel compounds – biological activities, new raw materials for medicines, food supplements).
COBISS.SI-ID: 6075930
We performed comparative study of different approaches for multivariate image analysis on plant resins chromatograms as a representative samples with rich chemical profiles. We confirmed that by the use of chemometric classification methods, application of powerful scanning and image capturing and processing devices and algorithms, advancement in development of novel stationary phases, as well as various separation modalities, high-performance thin-layer chromatography (HPTLC) fingerprinting is becoming attractive and fruitful field of separation science.
COBISS.SI-ID: 6048282