Sample Preparation for Determination of Biological Thiols by Liquid Chromatography and Electromigration Techniques

Wydrukowano z dostarczonych Wydawnictwu UŁ gotowych materiałówMajority of the bioanalytical or environmental methods do not use just one chromatografie or electrophoretic step, but rather involve several sample pretreatment steps which simplfy the matrix, and often preconcentrate and chemically modify the analytes. This work surveys typical procedures for sample preparation for most commonly analyzed biofluids with particular emphasis placed on chemical derivatization of sulfur amino acids for their determination by liquid phase separation techniques. Recent author's laboratory contribution to the development of sample preparation procedures is merked.Zadanie pt. Digitalizacja i udostępnienie w Cyfrowym Repozytorium Uniwersytetu Łódzkiego kolekcji czasopism naukowych wydawanych przez Uniwersytet Łódzki nr 885/P-DUN/2014 zostało dofinansowane ze środków MNiSW w ramach działalności upowszechniającej naukę

Reinforcement of natural rubber by precipitated silica: the influence of processing temperature

The thermal history and in particular the mixing dump temperature is a parameter of paramount importance in mixing rubber and silica with a silane coupling agent in order to achieve proper silanization of silica and to avoid premature scorch reactions. In this work, the influence of mixing dump temperature on the performance of silica reinforced Natural Rubber (NR) is investigated. The investigation also includes the effect of non-rubber constituents, primarily proteins in NR, by using deproteinized Natural Rubber (DPNR) and synthetic polyisoprene (IR). The vulcanization properties and rubber-to-filler interactions of silica reinforced NR in presence and absence of a silane coupling agent are highlighted. With increasing mixing dump temperature, the silanization reaction between silica and silane coupling agent proceeds further. At sufficiently high dump temperature, filler-filler interactions in the NR-silica compounds are reduced and silica-rubber interaction improved as evidenced by a drop in the Payne effect and increment in chemically bound rubber. It is demonstrated that NR and IR compounds mixed till above the optimum dump temperature exhibit cure reversion and reduction in tensile properties. On the other hand, DPNR-silica vulcanizates show slightly more constant physical properties

Quantification of sirolimus by liquid chromatography-tandem mass spectrometry using on-line solid-phase extraction

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune((R))) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC)-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 mul) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB(R) material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray((R)) source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6-50 mug/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 mug/l in routine use; the instrumentation proved to be reliable and required minimal maintenance

The fracture toughness of small animal cortical bone measured using arc-shaped tension specimens: Effects of bisphosphonate and deproteinization treatments

Small animal models, and especially transgenic models, have become widespread in the study of bone mechanobiology and metabolic bone disease, but test methods for measuring fracture toughness on multiple replicates or at multiple locations within a single small animal bone are lacking. Therefore, the objective of this study was to develop a method to measure cortical bone fracture toughness in multiple specimens and locations along the diaphysis of small animal bones. Arc-shaped tension specimens were prepared from the mid-diaphysis of rabbit ulnae and loaded to failure to measure the radial fracture toughness in multiple replicates per bone. The test specimen dimensions, crack length, and maximum load met requirements for measuring the plane strain fracture toughness. Experimental groups included a control group, bisphosphonate treatment group, and an ex vivo deproteinization treatment following bisphosphonate treatment (5 rabbits/group and 15 specimens/group). The fracture toughness of ulnar cortical bone from rabbits treated with zoledronic acid for six months exhibited no difference compared with the control group. Partially deproteinized specimens exhibited significantly lower fracture toughness compared with both the control and bisphosphonate treatment groups. The deproteinization treatment increased tissue mineral density (TMD) and resulted in a negative linear correlation between the measured fracture toughness and TMD. Fracture toughness measurements were repeatable with a coefficient of variation of 12–16% within experimental groups. Retrospective power analysis of the control and deproteinization treatment groups indicated a minimum detectable difference of 0.1 MPa·m1/2. Therefore, the overall results of this study suggest that arc-shaped tension specimens offer an advantageous new method for measuring the fracture toughness in small animal bones

Measurement of adenosine triphosphate and some other metabolites in blood cells by isotachophoresis. I. Preparative technique and enzymatic confirmation.

This study investigated the optimal conditions for detection of nucleotides in blood using an IP-1B capillary isotachophoretic apparatus. The system used 10 mM HCl-beta-alanine (pH 4.2) as the leading electrolyte and n-caproic acid as the terminal electrolyte. Direct application of lysed red blood cells was shown to be inaccurate, and a method of deproteinization based on heat in a microwave oven was developed. The zones for 2,3-diphosphoglycerate, ATP, inorganic phosphate, and lactate were identified enzymatically by withdrawal of pure samples of each zone via a special withdrawal cell. The quantitative values obtained by isotachophoresis were also confirmed enzymatically. The technique is now available for convenient and accurate identification of these metabolites simultaneously.</p

Effect of deproteinization and tubular occlusion on microtensile bond strength and marginal microleakage of resin composite restorations

Dentin adhesion procedure presents limitations, especially regarding to lifetime stability of formed hybrid layer. Alternative procedures have been studied in order to improve adhesion to dentin. OBJECTIVE: The aim of this study was to evaluate in vitro the influence of deproteinization or dentin tubular occlusion, as well as the combination of both techniques, on microtensile bond strength (µTBS) and marginal microleakage of composite resin restorations. MATERIAL AND METHODS: Extracted erupted human third molars were randomly divided into 4 groups. Dentin surfaces were treated with one of the following procedures: (A) 35% phosphoric acid gel (PA) + adhesive system (AS); (B) PA + 10% NaOCl + AS; (C) PA + oxalate + AS and (D) PA + oxalate + 10% NaOCl + AS. Bond strength data were analyzed statistically by two-way ANOVA and Tukey's test. The microleakage scores were analyzed using Kruskal-Wallis and Mann-Whitney non-parametric tests. Significance level was set at 0.05 for all analyses. RESULTS: µTBS data presented statistically lower values for groups D and B, ranking data as A>C>B>D. The use of oxalic acid resulted in microleakage reduction along the tooth/restoration interface, being significant when used alone. On the other hand, the use of 10% NaOCl alone or in combination with oxalic acid, resulted in increased microleakage. CONCLUSIONS: Dentin deproteinization with 10% NaOCl or in combination with oxalate significantly compromised both the adhesive bond strength and the microleakage at interface. Tubular occlusion prior to adhesive system application seems to be a useful technique to reduce marginal microleakage

Comparison of four different algorithms for the calculation of radioimmunoassay standard curves

Peer Reviewe

Development and application of an UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (Pregnenolone, 17-OH-Pregnenolone, Progesterone, 17-OH Progesterone, Androsterone, Androstenedione, DHEA, DHEAS, Testosterone, Cortisol, Corticosterone, Aldosterone, 11-Deoxycortisol, 11-Deoxycorticosterone, Dihydrotestosterone, Estrone, Estradiol) has been developed in equine serum using the UHPLC-MS/MS technique. 400 μL of sample were deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in UHPLC-MS/MS triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60 % and with the percentage of coefficient of variation (CV) lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922–0.9986, and the limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.002–2 ng ml-1 and 0.0055-5.5 ng ml-1, respectively. The detected LOQ for testosterone (i.e. 50 pg ml-1) is two-fold lower with respect to its threshold admitted in geldings plasma (100 pg ml-1 free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25-51.26 ng ml-1) and cortisol (range 32.57-52.24 ng ml-1), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone