Current Chromatography - Volume 2, Issue 1, 2015

The search for new pharmacologically active compounds, whose source can be the natural products, influences the development of techniques with higher separation efficient and identification capacity. From these techniques, chromatography is the most useful and versatile one, since it may aggregate separation, isolation and identification, depending on the detection system used. For identification and structural characterization, the spectroscopic methods as nuclear magnetic resonance, mass spectrometry and infrared spectrometry are essential in the research of biologically active natural products. Nowadays, several modern chromatographic techniques, automated and hyphenated with spectroscopic and spectrometric methods, are disposable to be used on investigation of natural products from different matrixes, whose components can be investigated in a rapid, sensitive and reliable determination. This review is focused on the brief discussion and application about the chromatographic and spectroscopic methods that are currently available for analysis of compounds from natural sources.

Natural products have been used throughout human history for many applications. The matrices of natural products show a myriad of biological activities that can result from a specific compound or from the synergistic effect of several or even hundreds of different components. Moreover, because natural products are derived from living organisms and hence are affected by biotic and abiotic factors during their production, it is extremely difficult to ensure a constant level of activity for these substances among different batches. In order to overcome such obstacles and improve the quality control of these products, which is necessary to obtain reproducible biological activities and approve new commercial uses, the development and application of analytical methods are fundamental. The development of applicable analytical methods along with the qualitative and quantitative characterization of active ingredients and possible interferents need to be extensively studied. However, guidelines or general protocols that can be applied to all samples do not exist, and there is no simple analytical validation method. Every matrix has specific demands and different markers, and quality control laboratories possess different instrumental configurations. Considering these technical difficulties, this review article presents examples, experimental strategies, and arguments demonstrating the importance of qualitative and quantitative knowledge of natural products, analytical validation, and the influence of these matrices over biological results. The major criteria that should be addressed in the development of analytical methods are also discussed. Among several validation parameters, the study of extraction efficiency is highlighted.

For the first time, the study describes the potentialities of a powerful preconcentration technique called fieldenhanced sample injection (FESI) combined with water-in-oil microemulsion electrokinetic chromatography (W/O MEEKC) for the quantification of inorganic anions. Water-in-oil microemulsions as background electrolytes show unique selectivities of W/O MEEKC compared to the standard capillary-electrophoresis analysis of anions. The effect of a set of microemulsion parameters such as the concentration and type of the buffer solution, oil, and surfactant was attained using the separation of the test mixture. The combination of the FESI technique followed by optimized W/O MEEKC provided up to a 930-fold improvement in the detection sensitivity compared to the conventional sample injection. The potential of the developed method was demonstrated by the determination of iodide anions in hen eggs.

Four alkynes having a range of polarities and functional groups are bonded to a silica hydride surface via hydrosilation. The success of the reaction and characterization of the bonded material is determined by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and solid state nuclear magnetic resonance spectroscopy. The retention properties of each of these stationary phases are evaluated by using both hydrophobic and hydrophilic analytes. Plots of retention time vs. mobile phase composition are used to determine whether the materials have reversed-phase, aqueous normal phase (ANP) or both types of chromatographic modes.

A study comparing two GC/MS analytical techniques: High Resolution (HRGC/HRMS) and Low Resolution (HRGC/LRMS) Mass Spectrometry was reported on a sample series during an investigation made for assessing PCB contamination levels on a particularly contaminated site. HRGC/HRMS is confirmed to be necessary for a correct quantification especially for Dioxin-like PCBs whereas total PCBs can be determined, with acceptable accuracy, also with HRGC/LRMS. Only few DL-PCB congeners are interfered in HRGC/LRMS mostly due to their low concentration with respect to the other congeners present. Concentrations from 30 to 25000 µg/kg for total PCBs and from 2 to 1800 µg/kg for total Dioxin-like PCBs were found. Levels from 7 to 7000 µg/kg for PCB 209 (decachlorobiphenyl) were found as well. Lo-res/Hi-res response ratios calculated in the three cases ranged from 85% to 155%, from 75 to 195% and from 80% to 160% respectively. The high resolution/low resolution response ratios calculated for each investigated congener were generally close to 1 except for dioxin-like congener where ratios up to 20 were observed.

Rapid reverse phase TLC-densitometry and HPLC-UV detection methods were developed and validated for simultaneous determination of fenofibrate (FBT), its metabolite fenofibric acid (FFA) and co-formulated/ co-administered statins; rosuvastatin (RST) or atorvastatin (AST). The first method was based on reversed phase (C18) thin layer chromatographic separation of the studied drugs followed by densitometric measurements. The second method was based on isocratic reverse-phase liquid chromatographic separation of the selected compounds on C18 column (250 x 4.6 mm, 10 µ) maintained at controlled temperature (25°C). The mobile phase consisted of acetonitrile-water (82:18, v/v) and quantification was made at 254 nm for both methods. The flow rate was maintained at 1.5 mL/ min. The calibration graphs were linear in the concentration ranges of 0.25 - 4 µg/ spot and 5.0 - 50 µg mL-1 for C18 TLC-densitometry and HPLC-UV methods respectively. The developed methods were validated for specificity, linearity, precision, accuracy, and robustness. All criterions were within the acceptable range for the studied compounds. Statistical analyses proved that the investigated assays were incomparable. Moreover HPLC method was applied for determination of FBT and its metabolite and co-administered statins in human plasma. The assay procedure involved a simple one-step extraction of FBT, FFA, RST and AST from human plasma by salting out with sodium chloride and 0.1% orthophosphoric acid. The chromatographic separation was obtained within 5.4 min.

A molecular-kinetic model of a multicomponent separation process has been suggested. The expression for multicomponent separation potential (the value function) has been obtained based on the “fundamental principles”. The feature of the separation process, which leads to an infinite value of the potential for pure components, has been discussed.