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Eschborn, Germany

Kuhn J.,Ruhr University Bochum | Vollmer T.,Ruhr University Bochum | Martin C.,Waters GmbH | Hendig D.,Ruhr University Bochum | Knabbe C.,Ruhr University Bochum
Journal of Pharmaceutical and Biomedical Analysis | Year: 2012

We developed a stable isotope dilution ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay to measure nicotine and cotinine, the major oxidative and pharmacologically less active metabolite of nicotine, in human urine. A simple dilution step was used as sample preparation and the measurement of nicotine and cotinine was performed during a 1.5-min run-time using nicotine-d 4 and cotinine-d 4 as internal standards. Multiple calibration curves for the analysis of both nicotine and cotinine exhibited a consistent excellent linearity and reproducibility in the range of 5-35,000μg/L (r>0.999). Limits of Detection were 0.7μg/L for nicotine and 0.4μg/L for cotinine, and Lower Limits of Quantification were 1.7μg/L for nicotine and 1.1μg/L for cotinine. The intraassay coefficients of variation (CVs) for nicotine and cotinine were <4% and <2%, respectively, the interassay CVs were <6% for nicotine and <4% for cotinine. The inaccuracy was <6% for both substances. The mean recovery was 103.2% (range 96.8-105.1%) for nicotine and 97.4% (range 94.3-99.2%) for cotinine. A method comparison showed that the values of nicotine metabolites in human urine samples (n=98) measured by a commercially available chemiluminescent immunoassay tested on analyzer IMMULITE 2000 were much higher than the cotinine concentration in the same urine samples measured by our UPLC-MS/MS assay. The Passing-Bablok regression line was: immunoassay=4.62 (UPLC-MS/MS)+3.64 [μg/L]; r=0.75. This robust, sensitive and interference-free UPLC-MS/MS assay permits rapid and accurate determination of nicotine and cotinine in human urine. © 2012 Elsevier B.V. Source

Lange S.,Max Planck Institute for Infection Biology | Lange S.,Waters GmbH | Rosenkrands I.,Statens Serum Institute | Stein R.,I and B | And 3 more authors.
Journal of Proteomics | Year: 2014

Secreted proteins of bacteria are preferentially capable of interacting with host cells and are therefore of special biological and medical interest. Narrow pH range 2-DE and MALDI-TOFTOF-MS combine high-resolution protein separation with highly sensitive identification of proteins. Secreted proteins of Mycobacterium tuberculosis were separated at the protein species level, distinguishing different protein species of one protein. We focused on the pI range 4.0-4.7 and the Mr range 6-20. kDa of the 2-DE pattern. Out of 128 analyzed spots, 121 were identified resulting in 33 different proteins with 277 different protein species, accumulating in a mean of 8.4 protein species per protein. Overrepresentation was found for the protein classes "virulence, detoxification, adaption", "information pathways", "cell wall and cell processes" and "intermediary metabolism and respiration". Thus far, 15 protein species of the ESX-1 family are characterized with 100% sequence coverage. More automated 2-DE procedures and more sensitive identification techniques are required for complete characterization of all of the protein species even in highly enriched samples, such as culture filtrates. Only then the functional level of proteomics will be achieved and potential biomarkers can be postulated at the molecular level. Biological significance: Proteomics is dominated by bottom-up approaches largely ignoring protein speciation. A prerequisite to reach the protein species level is to obtain 100% sequence coverage, which is a major challenge in proteomics. Here we show complete sequence information with a 2-DE-MS approach for 15 protein species. Acetylation of the N-terminus of ESAT-6 inhibits interaction with CFP-10, with direct consequences for pathogen-host interaction.This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 Elsevier B.V. Source

Schmidt A.H.,Chromicent GmbH | Schmidt A.H.,Free University of Berlin | Wess C.,Waters GmbH
Journal of Liquid Chromatography and Related Technologies | Year: 2014

A state-of-the-art ultra-performance liquid chromatographic (UPLC) method has been developed for purity testing of carbamazepine. Successful chromatographic separation of the active pharmaceutical ingredient (API) from its impurities was achieved on a WATERS ACQUITY UPLC CSH C18 column with the dimensions 2.1 mm × 100 mm and 1.7 m particle size with gradient elution of 0.2% phosphoric acid and acetonitrile in only 5 min. Incorporating Quality by Design (QbD) principles to the method development approach by using the statistical software package Fusion AE allows the study of the relationship between chromatographic parameters (factors) and the resolution (response) between the peaks of interest. In a screening phase, the factors known to have a major effect on column selectivity (stationary phase, pH of the aqueous eluent, organic eluent type, gradient time, and slope) were studied. In the second phase, the chromatographic parameters that were identified as affecting the resolution were studied with additional instrument settings. In both phases, statistical concepts with experimental design plans (Design-of-Experiments) are used as an efficient and fast tool to simultaneously gain knowledge regarding the influencing factors and interactions. An operating space within the design space was established and a verification study confirmed the robustness of the final method. Total analysis time was only 5 min, which is an impressive 22-fold increase in productivity in comparison to the method published in the European Pharmacopeia. © 2014 Taylor and Francis Group, LLC. Source

Frenkel J.,Friedrich - Schiller University of Jena | Wess C.,Waters GmbH | Vyverman W.,Ghent University | Pohnert G.,Friedrich - Schiller University of Jena
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2014

The proline derived diketopiperazine has been identified in plants, insects and fungi with unknown function and was recently also reported as the first pheromone from a diatom. Nevertheless the stereochemistry and enantiomeric excess of this natural product remained inaccessible using direct analytical methods. Here we introduce a chiral separation of this metabolite using supercritical fluid chromatography/mass spectrometry. Several chromatographic methods for chiral analysis of the diketopiperazine from the diatom Seminavis robusta and synthetic enantiomers have been evaluated but neither gas chromatography nor high performance liquid chromatography on different chiral cyclodextrin phases were successful in separating the enantiomers. In contrast, supercritical fluid chromatography achieved baseline separation within four minutes of run time using amylose tris(3,5-dimethylphenylcarbamate) as stationary phase and 2-propanol/CO2 as mobile phase. This very rapid chromatographic method in combination with ESI mass spectrometry allowed the direct analysis of the cyclic dipeptide out of the complex sea water matrix after SPE enrichment. The method could be used to determine the enantiomeric excess of freshly released pheromone and to follow the rapid degradation observed in diatom cultures. Initially only trace amounts of c(d-Pro-d-Pro) were found besides the dominant c(l-Pro-l-Pro) in the medium. However the enantiomeric excess decreased upon pheromone degradation within few hours indicating that a preferential conversion and thus inactivation of the l-proline derived natural product takes place. © 2014 Elsevier B.V. Source

Today's mass spectrometry plays a key role for the analysis of complex protein mixtures. Nevertheless, it reaches technical limits already, which can be overcome via combination with ion mobility spectrometry. Source

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