Authors:Mitja Križman, Jernej Jakše, Mirko Prošek, Dea Baričevič, and Branka Javornik
Agarose gel electrophoresis is a basic separation tool used in molecular biology, mostly for qualitative DNA analysis. There are constraints limiting its use in quantitative analysis, namely low repeatability and a narrow linear range. However, by using an internal standard or internal normalization, repeatability and linear range could be significantly improved. In the work discussed in this paper it was shown that an approximately fivefold improvement in repeatability and an over threefold wider linear range could be achieved by applying internal normalization. Using the proposed approach, genetic markers, for example RAPD and PCR-RFLP, or even microsatellite markers, could be conveniently quantitatively assessed using agarose gel electrophoresis.
Authors:Mirko Prosek, Luka Milivojevic, Mitja Krizman, and Maja Fir
A new on-line TLC-MS interface, with computer-controlled extraction of substances from selected spots on a TLC or HPTLC plate, has been constructed. The controlled collection of the sample and its programmed injection into the mass spectrometer is the advantage of this type of interface. The interface has been tested and validated with a standard solution of caffeine as test substance. The results were compared with those from a previously established and now routinely used off-line TLC-SPE-APCI-MS extraction procedure.
Authors:Petra Jazbec, Andrej Šmidovnik, Mateja Puklavec, Mitja Križman, Jernej Šribar, Luka Milivojević, and Mirko Prošek
Coenzyme Q10, an essential factor of oxidative phosphorylation and an important antioxidant, is used as food additive in industrial production of poultry meat. In this study chickens were fed with water-soluble coenzyme Q10-β-cyclodextrin complex for 40 days and the distribution of CoQ10 and cholesterol in the chicken-breast cells was then measured. Cell fractions were analyzed by HPTLC and results were confirmed with a new HPLC-MS method. Both methods are suitable for quantitative evaluation, but HPTLC is particularly suitable for screening purposes. Validation revealed HPTLC is a reliable, sensitive, and flexible analytical tool in comparison with HPLC-MS, despite its inherently lower sensitivity and selectivity.