An approach that allows setting up under predefined ionization conditions a rugged self-consistent quantitative experimental scale of electrospray ionization (ESI) efficiencies of organic compounds is presented. By ESI ionization efficiency (IE) we mean the efficiency of generating gas-phase ions from analyte molecules or ions in the ESI source. The approach is based on measurement of relative ionization efficiency (RIE) of two compounds (B 1 and B 2 ) by infusing a solution containing both compounds at known concentrations (C 1 and C 2 ) and measuring the mass-spectrometric responses of the protonated forms of the compounds (R 1 and R 2 ). The RIE of B 1 and B 2 is expressed asThe relative way of measurement leads to cancellation of many of the factors affecting IE (ESI source design, voltages in the source and ion transport system, solvent composition, flow rates and temperatures of the nebulizing and drying gases). Using this approach an ESI IE scale containing ten compounds (esters and aromatic amines) and spanning over 4 logRIE units has been compiled. The consistency of the scale (the consistency standard deviation of the scale is s ¼ 0.16 logRIE units) was assured by making measurements using different concentration ratios (at least 6-fold concentration ratio range) of the compounds and by making circular validation measurements (the logRIE of any two compounds was checked by measuring both against a third compound). Copyright # 2008 John Wiley & Sons, Ltd.The electrospray ionization (ESI) method 1,2 is one of the most widely used ionization methods in contemporary mass spectrometry (MS). Its compatibility with liquid chromatography (LC), soft nature and virtual absence of restrictions on the molecular mass of the compounds have enabled its successful application to a vast number of different analytes. 3,4 The ESI method has played a key role in the spectacular success of LCMS during the recent years. Besides practical applications research has been carried out on elucidating the ESI mechanism 5-12 and on the principles that govern the ESI ionization efficiency of different analytes. 6,9 By ESI ionization efficiency we mean the efficiency of generating gas-phase ions from analyte molecules or ions in the ESI source. ESI is not an efficient method for generation of gas-phase ions. Only a small fraction of analyte molecules are ionized in the ion source and only a part of the resulting gas-phase ions are successfully transmitted to the mass analyzer and eventually detected. 7 The actual process of generating gas-phase ions in the ESI source is complex. A multitude of processes in the liquid phase as well as in the gas phase and on the gas-liquid boundary must be considered. Furthermore, solvents, additives and impurities may cause ionization suppression or an enhancement effect 8,11 that is frequently observed when introducing a solution containing different compounds into an ESI source (also known as the matrix effect). Several models for relating the response of an analyte ion in the mass spectrum to the a...
The aim of this work was to evaluate the contributions of the main chromophores to the total UV absorbance of the spent dialysate and to assess removal dynamics of these solutes during optical on-line dialysis dose monitoring. High performance chromatography was used to separate and quantify UV-absorbing solutes in the spent dialysate sampled at the start and at the end of dialysis sessions. Chromatograms were monitored at 210, 254 and 280 nm routinely and full absorption spectra were registered between 200 and 400 nm. Nearly 95% of UV absorbance originates from solutes with high removal ratio, such as uric acid. The contributions of different solute groups vary at different wavelengths and there are dynamical changes in contributions during the single dialysis session. However, large standard deviation of the average contribution values within a series of sessions indicates remarkable differences between individual treatments. A noteworthy contribution of Paracetamol and its metabolites to the total UV absorbance was determined at all three wavelengths. Contribution of slowly dialyzed uremic solutes, such as indoxyl sulfate, was negligible.
The present study contributes new information on the removal of uremic retention solutes during hemodialysis and on the origin of the optical dialysis adequacy monitoring signal.
In this study, simultaneous removal assessment of marker molecules from three uremic toxin groups was performed during different hemodialysis treatment modalities using optical characteristics of spent dialysate. Results from optical measurements were compared with the results from chemical laboratory. Ten chronic dialysis patients, mean age 59 ± 15 years, were included in the study during 40 hemodialysis sessions. Low-flux hemodialysis (HD), high-flux hemodialysis (HF), and postdilutional online hemodiafiltration (HDF) with different settings were used. The reduction ratio (RR) and total removed solute (TRS) of three uremic solutes were determined: small molecular weight urea, middle molecular β2-microglobulin (B2M), and protein-bound indoxyl sulfate (IS). Concentrations of these solutes in the spent dialysate were measured by laboratory (lab) and optical (opt) methods, in the serum by laboratory methods, and calculated RR values in percentage were compared accordingly. Total removed solute was obtained from the total dialysate collection (TDC) using lab and opt methods. The highest RR values were found for urea and B2M, and the lowest for IS. The difference between RR of lab and opt results estimated as mean accuracy (BIAS) was ≤8.1% for all three solutes. Good correspondence between TRS lab vs. opt was achieved, resulting in strong linear correlation values R from 0.727 for urea to 0.971 for IS. Accuracy for TRS values as BIAS ± standard error (SE), comparing lab vs. opt, showed no statistical difference for any of the observed uremic solutes (P > 0.05). The accuracy of the optical method was not influenced by the dialysis modality (HD, HF, and HDF).
A need for dialysate-based, on-line, continuous monitoring systems for the control of dialysis efficiency and the prevention of dialysis-associated complications is arisen due to increasing number of dialysis patients and related treatment quality requirements. The aim of this study was to investigate the wavelength dependence between the the ultra-violet (UV) absorbance in the spent dialysate and the retained solutes removed during the hemodialysis in order to explain possibilities to estimate removal of the solutes by the optical dialysis adequacy sensor. Ten uremic patients, during 30 hemodialysis treatments, were followed at the Department of Dialysis and Nephrology, North-Estonian Regional Hospital. The dialysate samples were taken and analyzed with spectrophotometer to get absorbance spectra. The results confirm previous studies considering similarity for the UV-spectrum on the spent dialysate samples during a single dialysis session indicating presence of the same type of chromophores in the spent dialysate removed from the patient's blood for different patients groups. At the same time the highest correlation in the spent dialysate for urea, creatinine, potassium, and phosphate was obtained at the wavelength 237 nm that is a new finding compared to earlier results. The highest correlation between the UV-absorbance and uric acid in the spent dialysate was obtained at the wavelength 294 nm. Presence of at least two different wavelength ranges may add selectivity for monitoring several compounds. Our study indicates that the technique has a potential to estimate the removal of retained substances.
In this paper, we present a new method for structure determination of flexible "random-coil" peptides. A numerical method is described, where the experimentally measured 3J(H(alpha)Nalpha) and [3J(H(alpha)Nalpha+1 couplings, which depend on the phi and psi dihedral angles, are analyzed jointly with the information from a coil-library through a maximum entropy approach. The coil-library is the distribution of dihedral angles found outside the elements of the secondary structure in the high-resolution protein structures. The method results in residue specific joint phi,psi-distribution functions, which are in agreement with the experimental J-couplings and minimally committal to the information in the coil-library. The 22-residue human peptide hormone motilin, uniformly 15N-labeled was studied. The 3J(H(alpha)-N(i+1)) were measured from the E.COSY pattern in the sequential NOESY cross-peaks. By employing homodecoupling and an in-phase/anti-phase filter, sharp H(alpha)-resonances (about 5 Hz) were obtained enabling accurate determination of the coupling with minimal spectral overlap. Clear trends in the resulting phi,psi-distribution functions along the sequence are observed, with a nascent helical structure in the central part of the peptide and more extended conformations of the receptor binding N-terminus as the most prominent characteristics. From the phi,psi-distribution functions, the contribution from each residue to the thermodynamic entropy, i.e., the segmental entropies, are calculated and compared to segmental entropies estimated from 15N-relaxation data. Remarkable agreement between the relaxation and J-couplings based methods is found. Residues belonging to the nascent helix and the C-terminus show segmental entropies, of approximately -20 J K(-1) mol(-1) and -12 J K(-1) mol(-1), respectively, in both series. The agreement between the two estimates of the segmental entropy, the agreement with the observed J-couplings, the agreement with the CD experiments, and the assignment of population to sterically allowed conformations show that the phi,psi-distribution functions are indeed meaningful and useful descriptions of the conformational preferences for each residue in this flexible peptide.
The aim of this study was to evaluate the contribution and removal dynamics of the main fluorophores during dialysis by analyzing the spent dialysate samples to prove the hypothesis whether the fluorescence of spent dialysate can be utilized for monitoring removal of any of the protein bound uremic solute. A high performance liquid chromatography system was used to separate and quantify fluorophoric solutes in the spent dialysate sampled at the start and the end of 99 dialysis sessions, including 57 hemodialysis and 42 hemodiafiltration treatments. Fluorescence was acquired at excitation 280 nm and emission 360 nm. The main fluorophores found in samples were identified as indole derivatives: tryptophan, indoxyl glucuronide, indoxyl sulfate, 5-hydroxy-indoleacetic acid, indoleacetyl glutamine, and indoleacetic acid. The highest contribution (35 ± 11%) was found to arise from indoxyl sulfate. Strong correlation between contribution values at the start and end of dialysis (R2 = 0.90) indicated to the stable contribution during the course of the dialysis. The reduction ratio of indoxyl sulfate was very close to the decrease of the total fluorescence signal of the spent dialysate (49 ± 14% vs 51 ± 13% respectively, P = 0.30, N = 99) and there was strong correlation between these reduction ratio values (R2 = 0.86). On-line fluorescence measurements were carried out to illustrate the technological possibility for real-time dialysis fluorescence monitoring reflecting the removal of the main fluorophores from blood into spent dialysate.In summary, since a predominant part of the fluorescence signal at excitation 280 nm and emission 360 nm in the spent dialysate originates from protein bound derivatives of indoles, metabolites of tryptophan and indole, the fluorescence signal at this wavelength region has high potential to be utilized for monitoring the removal of slowly dialyzed uremic toxin indoxyl sulfate.
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