Combination of ultracentrifugation and solid-phase extraction with subsequent chromatographic analysis of α-tocopherol in erythrocyte membranes.

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.

New approach for the clinical monitoring of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by ultra high performance liquid chromatography with MS/MS based on the standard reference material 972.

Biomarkers, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , are important indicators of the vitamin D general status and are monitored in several pathophysiological disorders, such as osteoporosis, diabetes, heart disease, etc. A novel ultra-HPLC with MS/MS methodology for the analysis of 25-hydroxyvitamin D derivatives coupled with a very simple and highly rapid sample preparation step was developed. Analytical parameters obtained showed linearity (R(2) ) above 0.999 for both vitamins with accuracies between 95.8 and 102%. The LODs were as low as 0.22 and 0.67 nmol/L for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , respectively. Intra-assay precision (%RSD) was lower than 4.5%, and inter-assay precision (%RSD) was lower than 6.5%. The feasibility of the developed methodology to be applied in clinical routine analysis has been proved by its application in blood samples from non-agenarian patients, patients with familial hypercholesterolemia and patients suffering from age-related macular degeneration.

Determination of fluoroquinolone antibiotics in wastewater using ultra high-performance liquid chromatography with mass spectrometry and fluorescence detection.

A new ultra HPLC (UHPLC) method using both MS and fluorescence detection (FD) was developed for the determination of five fluoroquinolones in wastewaters. Systematic method development approach was compared with a conventional one. During the systematic approach, a possibility of automatic switching among four independent analytical columns of different chemistries has been used. Acidic as well as basic pH using ACN and methanol as organic modifiers was tested. The best separation of fluoroquinolones was obtained on phenyl analytical column at pH 10.5, which is a completely novel approach for separation of fluoroquinolones. Further, a new SPE procedure was developed for the sample preparation using basic pH as well. The sensitivity and selectivity of FD and MS detection were compared. FD at basic pH 10.5 demonstrated lower sensitivity than at acidic pH, which is conventionally performed. At basic pH, UHPLC-MS/MS was found about two orders of magnitude more sensitive than FD. Both methods were validated and subsequently UHPLC-FD method was used for the evaluation of stability of fluoroquinolones. UHPLC-MS/MS method was used for the analysis of wastewater samples. Norfloxacin and ciprofloxacin were detected in samples of influent and effluent from wastewater treatment plant. Ofloxacin was detected only in influent from wastewater treatment plant.

Recent trends in the analysis of vitamin D and its metabolites in milk--a review.

Vitamin D plays an important role in calcium metabolism and affects other metabolic pathways. Despite the intense interest in vitamin D, no comprehensive overview addressing the analysis of vitamin D in milk has been published. Historically, immunoassay techniques have been mainly used for the routine quantification of vitamin D and its metabolites. However, the greater accuracy and precision of chromatography makes it one of the most important methods in the analysis of vitamin D. The determination of vitamin D and its metabolites by LC-MS is the gold standard for its assessment. LC-MS has unique advantages for vitamin D determination and quantification due to its high sensitivity and specificity. In this review, the current status of vitamin D and its metabolites analysis in milk, human and bovine, including sample pre-treatment and chromatography analysis, are critically discussed and summarised.

The Effect of LDL-Apheresis and Rheohaemapheresis Treatment on Vitamin E.

Lipid apheresis (extracorporeal lipoprotein elimination) is administered to patients with familial hypercholesterolemia who fail to respond to standard therapy. The nature of the treatment process raises the suspicion that it decreases not only cholesterol but also antioxidants. A group of 12 patients (average age 47±17 y, 4 homozygous and 8 heterozygous individuals) with familial hypercholesterolemia treated by LDL-apheresis or rheohaemapheresis for 3-12 y was included in the study. In addition to cholesterol and triacylglycerol levels, vitamin E and vitamin A and also other markers of antioxidant activity were investigated. Nevertheless, the most important determined parameter was the vitamin E/cholesterol ratio in serum and lipoproteins. The results indicate that both extracorporeal elimination methods are effective and suitable ways to treat severe familial hypercholesterolemia, as the LDL fraction of cholesterol decreased by approximately 77% and 66% following LDL-apheresis and rheohaemapheresis, respectively. In addition, the serum vitamin E decreased by 54% and 57% and the decrease of the serum vitamin A was approximately 20%. However, the main marker of antioxidant capacity, vitamin E/cholesterol ratio, in the serum, VLDL and LDL significantly increased. The increase of vitamin E levels in the erythrocyte membranes of 2% following LDL-apheresis and a significant increase of 4% following rheohaemapheresis were confirmed. The presented results indicate that LDL-apheresis and rheohaemapheresis can be considered to be safe procedures according to the antioxidant capacity of the serum, VLDL and LDL lipoprotein fractions and the erythrocyte membrane.

Blood levels of antioxidants during age-related macular degeneration treatment by rheohaemapheresis.

AIMS: Rheohaemapheresis treatment influences rheological markers and most likely improves metabolism in affected retinal areas, resulting not only in absorption of soft drusen but also reduction or complete disappearance of drusenoid retinal pigment epithelium detachments. However, the character of the treatment process has raised suspicion that there is a decrease not only in cholesterol but also in antioxidants, such as vitamin E and vitamin A. METHODS: Twenty-three patients with the progressive dry form of age-related macular degeneration were each treated with 8 procedures of rheohaemapheresis. We measured levels of vitamin E (α-tocopherol), the vitamin E/cholesterol ratio in serum and lipoproteins (VLDL, LDL, HDL). Vitamin E in erythrocyte membrane and serum vitamin A (retinol) were also measured. These parameters were determined before and after rheohaemapheresis. Erythrocyte superoxide dismutase, erythrocyte glutathione peroxidase and serum malondialdehyde were analysed as markers of antioxidant activity and lipid peroxidation, respectively. RESULTS: In serum, the VLDL and LDL fraction ratios of vitamin E/cholesterol increased significantly. Additionally, the HDL fraction ratio showed an increase but this was not statistically significant. The patients showed no clinical signs of vitamin E deficiency, and their serum concentrations of vitamin E did not differ from normal values. The results show that rheohaemapheresis in addition to causing a significant reduction in atherogenic LDL cholesterol, may have favourable additive anti-atherogenic effects due to a relative increase in the content of vitamin E in the lipoprotein fractions.

Miniaturised medium pressure capillary liquid chromatography system with flexible open platform design using off-the-shelf microfluidic components.

Trends towards portable analytical instrumentation of the last decades have not been equally reflected in developments of portable liquid chromatography (LC) instrumentation for rapid on-site measurements. A miniaturised medium pressure capillary LC (MPLC) system with gradient elution capability has been designed based on a flexible modular microfluidic system using primarily off-the-shelf low cost components to ensure wide accessibility to other analysts. The microfluidic platform was assembled on a breadboard and contained microsyringe pumps and switch valves, complemented with an injection valve and on-capillary detectors, all controlled by a PC. Four miniaturised microsyringe pumps, with 5, 20 and 100 µL syringe volume options, formed the basis of the pumping system. Two pairs of pumps were used for each mobile phase to create gradient elution capability. The two microsyringe pumps in each pairs were linked by two electrically operated microfluidic switching valves and both pairs of pumps were connected through a zero void volume cross-connector, thus providing a low hold-up volume for gradient formation. Sample was injected by a 20 nL nano-LC sampling valve, directly connected to a 18 cm long 100 µm i.d. Chromolith CapRod RP-18 monolithic capillary column. On-capillary LED-based UV-vis photometric detection was conducted through a piece of equal diameter fused silica capillary connected after the column. The performance of the portable LC system was evaluated theoretically and experimentally, including the maximum operating pressure, gradient mixing performance, and the performance of the detectors. The 5 µL microsyringe pump offered the best performance, with typical maximum operating pressures up to 11.4 ± 0.4 MPa (water) and gradient pumping repeatability of between 4 and 9% for gradients between 0.10% s(-1) and 0.33% s(-1). Test analytes of charged and uncharged dyes and pharmaceuticals of varying hydrophobicity showed typical RSD values of 0.7-1.4% and 3.3-4.8% in isocratic mode and 1.2-4.6% and 3.2-6.4% in gradient mode, respectively for retention time and peak area repeatability.

Determination of steroid hormones in biological and environmental samples using green microextraction techniques: an overview.

Residues of steroid hormones have become a cause for concern because they can affect the biological activity of non-target organisms. Steroid hormones are a potential risk for wildlife and humans through the consumption of contaminated food or water. Their determination requires extraction and clean-up steps, prior to detection, to reach low concentration levels. In recent years, a great effort has been made to develop new analytical methodologies, such as microextraction techniques, that reduce environmental pollution. Researchers have modified old methods to incorporate procedures that use less-hazardous chemicals or that use smaller amounts of them. They are able to do direct analysis using miniaturised equipment and reduced amounts of solvents and wastes. These accomplishments are the main objectives of green analytical chemistry. In this overview, we focus on microextraction techniques for the determination of steroid hormones in biological (e.g., human urine, human serum, fish, shrimp and prawn tissue and milk) and environmental (e.g., wastewaters, surface waters, tap waters, river waters, sewage sludges, marine sediments and river sediments) samples. We comment on the most recent applications in sorptive-microextraction modes, such as solid phase microextraction (SPME) with molecularly imprinted polymers (MIPs), in-tube solid-phase microextraction (IT-SPME), stir-bar sorptive extraction (SBSE) and microextraction in packed sorbent (MEPS). We also describe liquid-phase microextraction (LPME) approaches reported in the literature that are applied to the determination of steroid hormones.