Intra- and interlaboratory reproducibility of ultra performance liquid chromatography-time-of-flight mass spectrometry for urinary metabolic profiling.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a major platform in metabolic profiling but has not yet been comprehensively assessed as to its repeatability and reproducibility across multiple spectrometers and laboratories. Here we report results of a large interlaboratory reproducibility study of ultra performance (UP) LC-MS of human urine. A total of 14 stable isotope labeled standard compounds were spiked into a pooled human urine sample, which was subject to a 2- to 16-fold dilution series and run by UPLC coupled to time-of-flight MS at three different laboratories all using the same platform. In each lab, identical samples were run in two phases, separated by at least 1 week, to assess between-day reproducibility. Overall, platform reproducibility was good with median mass accuracies below 12 ppm, median retention time drifts of less than 0.73 s and coefficients of variation of intensity of less than 18% across laboratories and ionization modes. We found that the intensity response was highly linear within each run, with a median R(2) of 0.95 and 0.93 in positive and negative ionization modes. Between-day reproducibility was also high with a mean R(2) of 0.93 for a linear relationship between the intensities of ions recorded in the two phases across the laboratories and modes. Most importantly, between-lab reproducibility was excellent with median R(2) values of 0.96 and 0.98 for positive and negative ionization modes, respectively, across all pairs of laboratories. Interestingly, the three laboratories observed different amounts of adduct formation, but this did not appear to be related to reproducibility observed in each laboratory. These studies show that UPLC-MS is fit for the purpose of targeted urinary metabolite analysis but that care must be taken to optimize laboratory systems for quantitative detection due to variable adduct formation over many compound classes.

Drop deformation dynamics and gel kinetics in a co-flowing water-in-oil system.

Drop deformation and superimposed gel kinetics were studied in a fast continuous-flow process for a water-in-oil system. Highly monodisperse drops were generated in a double capillary and then deformed passing through a narrowing rectangular channel geometry. Nongelling deformation experiments were used to establish the process and compare it with existing theories. Thereafter, temperature induced drop gelation was included to study its effect on deformation and gel kinetics on short timescales and at high temperature gradients. The disperse phase was a kappa-carrageenan solution with additional sodium and potassium ions for gelation experiments. Sunflower oil was used for the continuous phases. Nongelling experiments showed that shear forces are able to deform drops into ellipsoids. A comparison with the small deformation theory by Taylor was surprisingly good even when drop deformation and flow conditions were not in steady state. Superimposed gelation on the deformation process showed clearly the impact of the altered rheological properties of the dispersed and continuous phase. Deformation first increased on cooling the continuous phase until the onset of gel formation, where a pronounced decrease in deformation due to increasing droplet viscosity/viscoelasticity was observed. Drop deformation analyses were then used to detect differences in gelation kinetics at high cooling rate within process times as short as 1.8 s.

Formation of shaped drops in a fast continuous flow process.

Drop shaping, i.e., flow-induced deformation and fixation by gel formation, was studied under dynamic conditions in a fast continuous process for a water-in-oil system. The system consisted of sunflower oil with different surfactant concentrations (0.1-2% Admul Wol) and a 1.5% kappa-carrageenan solution with different Na(+) and K(+) concentrations. The continuous phase flowed in a 10-mm-wide straight channel into which the dispersed phase was injected via a thin needle. A subsequent shaping channel with a width of 1 or 2 mm deformed the drops. Gel formation was induced by a temperature gradient between the continuous and dispersed phase. Drop sizes in the range 220-roughly 1000 microm were produced at the needle tip by varying the ratio between the oil and carrageenan flow rate. A diffusion zone before the narrow channel allowed the surfactant to adsorb at the interface. In the elongation flow at the entrance of the shaping geometry, drops underwent initial elongation. In the narrow channel, the drops developed a parabolic shape within a residence time of 0.03-0.15 s. Choosing the correct parameter combinations made it possible to fix the deformation by gel formation within this time period. Shaped drops were shown to be functional. At a concentration of 25% in an emulsion, they increased the viscosity by about 15-20% compared to spherical drops even though 45% of the shaped drops had an aspect ratio of less than 1.2.

GNAS1 mutation analysis in gastrointestinal tumors.

GNAS1 codes for a part of the a-stimulatory subunit (Gsa) of the G protein. Mutation of GNAS1 has been frequently found in myxoid soft tissues, however, in gastrointestinal tumors, little is known about the mutation status of GNAS1. The aim of the study was to analyze the occurrence of GNAS1 mutations indifferent gastrointestinal, gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and colorectal tumors. Mutation status of GNAS1 exon 8 was analyzed in one hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) gastrointestinal tumor samples including 45 tubular-villous adenomas, 11 tubular adenomas, 6 villousadenomas, 10 hyperplastic gastric polyps, 31 GEP-NETs and 32 colorectal adenocarcinomas by using polymerase chain reaction (PCR) and direct sequencing. Five GNAS1 mutations were found in 2 tubular-villous adenomas, 2 villous adenomas and 1 colorectal adenocarcinoma. No mutations were detected in the tubular adenomas, the hyperplastic gastric polyps or GEP-NETs. GNAS1 mutation is not a frequent molecular event in GEP-NETs or hyperplastic gastric polyps. The study confirms the presence of GNAS1 mutations in colon tumors with villous differentiation.

Analysis of GNAS1 mutations in myxoid soft tissue and bone tumors.

The aim of this study was to characterize the prevalence of GNAS1 mutations in various tumor types, including intramuscular myxomas, fibrous dysplasias, and other myxoid tumors and implications for malignant transformation. PCR and direct sequencing were applied to analyze GNAS1 mutation status using genomic DNA isolated from 97 formalin-fixed and paraffin-embedded samples, including 63 intramuscular myxomas, 19 various myxoid lesions, 8 cases of sporadically occurring fibrous dysplasia, and 7 cases of atrial myxoma. Mutations of GNAS1 were detected in 23 out of 63 (36.5%) intramuscular myxoma patients, with mutational hotspots R201H and R201C being equally affected. GNAS1 mutations in codon 201 were found in 5 out of 8 fibrous dysplasias (62.5%), while no mutations of GNAS1 were detected in the other studied entities, including atrial myxomas. GNAS1 mutation analysis has diagnostic value in screening patients with intramuscular myxoma and patients with fibrous dysplasia.

Influence of kappa-carrageenan gel structures on the diffusion of probe molecules determined by transmission electron microscopy and NMR diffusometry.

The influence of the microstructures of different kappa-carrageenan gels on the self-diffusion behavior of poly(ethylene glycol) (PEG) has been determined by nuclear magnetic resonance (NMR) diffusometry and transmission electron microscopy (TEM). It was found that the diffusion behavior was determined mainly by the void size, which in turn was defined by the state of aggregation of the kappa-carrageenan. The kappa-carrageenan concentration was held constant at 1 w/w%, and the aggregation was controlled by the amount of potassium and/or sodium chloride and, for samples containing potassium, also by the cooling rate. Gels containing potassium formed microstructures where kappa-carrageenan strands are rather evenly distributed over the image size, while sodium gels formed dense biopolymer clusters interspersed with large openings. In a gel with small void sizes, relatively slow diffusion was found for all PEG sizes investigated. Extended studies of the self-diffusion behavior of the 634 g mol(-)(1) PEG showed that there is a strong time dependence in the measured PEG diffusion. An asymptotic lower time limit of the diffusion coefficient was found in all gels when the diffusion observation time was increased. According to the ratio, D/D(0), where D(0) is the diffusion coefficient in D(2)O and D is the diffusion coefficient in the gels, the gels could be divided into three classes: small, medium, and large voids. For quenched kappa-carrageenan solutions with salt concentrations of 20 mM K(+), 100 mM K(+), or 20 mM K(+)/200 mM Na(+) as well as slowly cooled solutions with only 20 mM K(+), D/D(0) ratios between 0.18 and 0.29 were obtained. By quenching a kappa-carrageenan solution with 100 mM K(+), the D/D(0) was 0.5, while D/D(0) ratios between 0.9 and 1 were obtained in a quenched solution with 250 mM Na(+) and slowly cooled samples with 20 mM K(+)/200 mM Na(+) or 250 mM Na(+).