Relative quantification of albumin and fibrinogen modifications by liquid chromatography tandem mass spectrometry in the diagnosis and monitoring of acute pancreatitis.

The increasing availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories provides the opportunity to replace or complement present underperforming immuno- and chemometric assays. Amylase and lipase show limited specificity and sensitivity for pancreatic inflammation and lack the capacity of monitoring the disease due to their short half-lives. Previous findings suggested that cleavage products of the pancreatic enzyme carboxypeptidase A could be a more suitable indicator for defining and classifying pancreatic inflammation. The plasma proteins albumin and ß-fibrinogen were digested with trypsin and truncated forms (des-Leu-albumin, and des-Gln-ß-fibrinogen) quantified against their non-truncated forms by LC-MS/MS. Four hundred fifty eight samples from 83 patients were used to evaluate the novel method and affirm its suitability for detecting acute pancreatitis. A robust, selective, precise and accurate LC-MS/MS method was set up to measure the proportion of truncated proteins. Reference ranges for the proportion of the truncated albumin and ß-fibrinogen were from 2% to 9% and 3% to 25%, respectively. Acute pancreatitis patients had values above these ranges and were distinctly separated from reference control individuals. The longer circulating half-lives of albumin and fibrinogen compared to pancreatic enzymes themselves provide the potential to diagnose pancreatitis more specifically over a longer time period, to monitor the course of the disease, and to track recurrent complications. The wide range of the proportion and the differential half-life of both truncated proteins could also be used for assessing the severity of pancreatitis.

Differential extraction of endogenous and exogenous 25-OH-vitamin D from serum makes the accurate quantification in liquid chromatography-tandem mass spectrometry assays challenging.

BACKGROUND: Extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is the method of choice when it comes to the accurate quantification of 25-OH-vitamin D in blood samples. It is generally assumed that the addition of exogenous internal standard allows for the determination of the endogenous analyte concentration. In this study we investigated the extraction properties of endogenous and exogenous 25-OH-vitamin D. METHODS: Eight samples were used for the evaluation of the extraction procedure and 59 patients' samples for a method comparison. The methanol-to-sample ratio (v/v) and the sample-to-hexane ratio (v/v) were varied and the LC-MS/MS signals of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and internal standard of the extracts recorded. The optimized 'in-house' LC-MS/MS assay was compared to two automated chemiluminescence immunoassays from DiaSorin and Abbott. RESULTS: Mathematical analysis of the data revealed a differential extraction of endogenous 25-OH-vitamin D3, spiked 25-OH-vitamin D2 and non-equilibrated internal standard. Exogenous 25-OH-vitamin D can be measured accurately if a definite methanol-to-sample ratio is used. Endogenous 25-OH-vitamin D is affected by critical quantification issues due to a differential slope in the extraction profile. The actual 25-OH-vitamin D concentration can be one-third above the measured extractable concentration. Results confirm that the 'in-house' LC-MS/MS assay provides reproducible 25-OH-vitamin D results. CONCLUSIONS: Discordant concentrations of 25-OH-vitamin D from LC-MS/MS assays can be caused by selection of suboptimal extraction conditions. Furthermore, a different sample pretreatment or solvent extraction system may result in a different dissociation and extraction yield of endogenous 25-OH-vitamin D and therefore contribute to variations of LC-MS/MS results.

Hb Ashburton [ß12(A9)Thr → Pro; HBB: c.37A > C], a novel, mildly unstable variant and the first substitution identified at codon 12.

We report the identification of a novel, slightly unstable hemoglobin (Hb) variant [ß12(A9)Thr → Pro; HBB: c.37A > C] that came to our attention during Hb A1C ion exchange chromatography where it migrated as a trailing shoulder on the Hb A0 peak. On electrospray ionization mass spectrometry (ESI MS), this electrophoretically silent variant was detected as an unresolved ß component with a 2 Da decrease in average ß chain mass. Reversed phase high performance liquid chromatography (HPLC) confirmed the presence of a more hydrophilic ß chain with a mass 4 Da less than normal ß(A) and showed it represented 40.0% of the total ß-globin. Tryptic mapping revealed the [M + 1H] ion of peptide ßT-2 had shifted from 932.5 to 928.5 m/z, suggesting a point mutation of Thr → Pro at position ß12(A9). This substitution was confirmed by fragmentation analysis of the [M + 2H] ion (464.8 m/z) of the new ßT-2 peptide and it represents a novel mutation which we have named Hb Ashburton.

The concentration of polysaccharides and proteins in EPS of Pseudomonas putida and Aureobasidum pullulans as revealed by 13C CPMAS NMR spectroscopy.

Extracellular polymeric substances were extracted from the bacterial strain Pseudomonas putida and the fungal species Aureobasidium pullulans using three different methods (formaldehyde-NaOH, ethylenediaminetetraacetic acid (EDTA) and cation-exchange-resin). The composition of the extracellular polymeric substances (EPS) was analysed by biochemical and high-resolution solid state 13C nuclear magnetic resonance (NMR) spectroscopic methods. The EPS yield was strongly dependent on the extraction method, with the formaldehyde-NaOH method showing the best extraction efficiency. The NMR method revealed that when using the EDTA extraction method, about 40% of the EDTA accumulated in the EPS and that was responsible for the apparent high extraction yields. EPS protein content determined by the NMR method was up to 30% higher than the protein content determined using the biochemical (Lowry) method for P. putida and for A. pullulans. The average protein carbon content determined by the NMR method was approximately 70% of the total carbon content. NMR results could be supported by elemental analysis, which showed a high nitrogen content (approximately 10%) in the EPS. The carbohydrate carbon content detected with both methods in the cell aggregates and the EPS was approximately 20% in each. In this study, quantitative 13C cross-polarisation magic angle spinning NMR spectroscopy was conducted on unlabeled cell strains, and EPS and could be used to quantify protein and carbohydrate of different samples.

Reconsidering the quantitative analysis of organic carbon concentrations in size exclusion chromatography.

The evaluation of the molecular size distribution of natural organic matter (NOM) in aquatic environments via size exclusion chromatography (SEC) is important for the understanding of environmental processes such as nutrient cycling and pollutant transport as well as of technical water treatment processes. The use of organic carbon (OC) detectors has become popular in recent studies due to improved availability and quantification possibilities, which supposedly are superior to those of ultraviolet (UV) detectors. A set of 12 NOM samples was used to demonstrate the limitations of online OC detection (OCD) when analyzing complex aquatic organic matter. A novel evaluation approach for SEC data is introduced by combining the information from UV absorbance (UVA) and OCD chromatograms as well as offline total OC (t-OC) and dissolved OC-specific UVA (SUVA) measurements. It could be shown that about 70% of certain OC components were not detected with the OCD system used in this study. For the investigated samples, these types of carbon accounted for up to 72% of the t-OC, i.e. for such NOM samples quantification by OCD is not possible or at least highly questionable. The addition of an oxidant improved the overall oxidation efficiency only slightly. Most likely NOM that predominantly consists of polysaccharides and features a nominal molecular weight of 150kg/mol or more was responsible for low OCD yields. For future applications, a further improvement of the OCD system would be worthwhile so that quantitative analytical data on the molecular size distribution of NOM and its structural characteristics such as the SUVA distribution can be obtained.

Search for basic relationships between "molecular size" and "chemical structure" of aquatic natural organic matter--answers from 13C and 15N CPMAS NMR spectroscopy.

To investigate the structural composition of natural organic matter (NOM), a 3-step micro- and ultrafiltration procedure was applied to 3 surface waters from southern Germany, and fractions from all filtration steps were collected. The NOM was characterized using solid-state 13C and 15N nuclear magnetic resonance (NMR) techniques. Routine integration of the 13C NMR spectra and extended data analysis procedures were carried out for a quantitative comparison of the structural components as well as for the elucidation of structural fractionation patterns. A common feature of the large molecular size fractions was the predominance of polysaccharide material, with the dissolved high molecular weight organics being mostly enriched in N-acetylated polysaccharides derived from microbial leftovers. Aromatic structures like lignin and tannin derivatives were most abundant in the intermediate size fraction. All membranes were found to be highly permeable for branched aliphatics, i.e. isoprenoids. Fouling layers of the ultrafiltration membrane were significantly enriched in long-chain aliphatics (lipids). Biofouling was not observed on any of the membranes. Overall, a strong interdependence between the chemical structural characteristics of NOM components and their size, shape, or interaction characteristics could be shown. The results provide the basis for a better understanding of water process technologies as treatment effectiveness is strongly dependent on the chemical composition and the "size" distribution of NOM.

Monitoring the formation of an Aureobasidium pullulans biofilm in a bead-packed reactor via flow-weighted magnetic resonance imaging.

The biofilm-forming fungus, Aureobasidium pullulans DSM 2404, was grown in a bead-packed reactor. Alterations within the reactor were analysed in several cross-sectional slices by magnetic resonance imaging (MRI) with flow contrast. For the first time, biofilm accumulation could be continuously elucidated without using any contrast agents, and the non-stationary flow through the fixed-bed reactor could be visualized. The results indicate that the non-stationary flow through the biofilm reactor changes significantly due to the changing reactor morphology. Preferential flow lines arise during biofilm formation. The accumulation of the biomass was determined and compared to gravimetrical biomass data. The described technique can be used to monitor hydrodynamic transport, and to combine flow-field characteristics with morphological data for the prediction of undesirable reactor processes, e.g. clogging.

Analysis of carbon and nitrogen forms in soil fractions after the addition of 15N-compost by 13C and 15N nuclear magnetic resonance.

A quantitative laboratory assessment of the different C and N forms in soil humus fractions was carried out by incubation of a mineral substrate after the addition of (15)N-labeled compost. The experimental design included (i) preparation of the (15)N-labeled organic matter (city refuse compost, 640 g kg(-1) wheat straw and K(15)NO(3) composted for 80 days), (ii) a further 80 day incubation of a mixture of the labeled compost with a mineral soil (32 g kg(-1)), (iii) measurement of stable isotope ratios, and (iv) isolation and structural comparison by (13)C and (15)N cross-polarization, magic-angle spinning nuclear magnetic resonance (NMR) of different organic fractions, i.e., soluble, colloidal (humic and fulvic type), and particulate (free organic matter and humin), from both the compost and the compost-treated soil. The results showed that the amide forms dominated in all of the newly formed N compounds, but an increased amount of alkali insoluble organic fractions was observed after incubation of the soil. The analysis of the insoluble, particulate fractions shows that nonextractable amides constitute the major pool of newly formed N compounds. The particulate soil fraction isolated by flotation in CHBr(3)-MeOH contained 16.8% of the total soil N and 26% of the (15)N. The (13)C NMR spectra showed that the fulvic acid-like fraction (7.6% of the soil N, 8.8% of (15)N) consisted almost completely of a C=O-containing carbohydrate material, whereas the humic acid-like fraction (20.3% of the total soil N, 8.6% of (15)N) resembled an oxidized lignoproteic fraction containing the most significant aromatic domain. The water soluble fraction was, in both soil and compost, the one with the highest isotopic abundance of (15)N (96%), but the (15)N NMR spectrum revealed minor amounts of soluble mineral N in this fraction and the remainder consisting of amide compounds.

Ultrafiltration of nonionic surfactants and dissolved organic matter.

A brownwater sample with a high content of humic substances (HS) was fractionated by multistage ultrafiltration (mst-UF) into five fractions with nominal molecular weights ranging from >30 to <1 kDa. Fractions were characterized with respect to molecular size distribution and structure. Size exclusion chromatography with online DOC detection revealed that mst-UF yielded fractions with decreasing Mp (molecular weight at peak maximum) and polydispersities from nominally large to small mst-UF fractions. 13C MAS NMR analysis showed that the content of carbohydrate structures decreased from the original sample toward smaller molecular weight (MW) fractions, which in turn contained more carboxylic groups and branched aliphatic structures. Specific UV absorbances (SUVA254) were highest in the >30 kDa fraction and decreased with decreasing MW. To evaluate whether separation mechanisms other than size exclusion were of importance during the fractionation, the behavior of low molecular weight model compounds (MC) with a range of polarities was studied. Recoveries decreased with increasing hydrophobicity of the MC. For selected nonylphenol ethoxylates and 4-nonylphenol the recovery correlated well with the hydrophile-lipophile balance value. The presence of dissolved organic matter (DOM) caused an additional loss of hydrophobic MC, possibly because of sorption of the compounds onto DOM fouling layers. The hydrophilic MC caffeine was recovered almost completely (85-86%) regardless of the DOM content of the model solution. It was concluded that size exclusion was the dominant fractionation mechanism for caffeine, whereas hydrophobic interactions played a major role during the mst-UF fractionation of nonpolar contaminants. For a better understanding of the behavior of polyfunctional molecules such as HS, the effect of other physicochemical properties needs to be investigated in further studies.