Analytische Chemie
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Hepcidin-25 has attracted much attention ever since its discovery in 2001. It is widely recognized that this liver produced peptide hormone plays a major role in the regulation of iron levels in mammals and can reveal important clinical information about several pathological states in patients suffering from iron-related disorders.
With the aim to tackle the current difficulties in hepcidin quantification and improve the status of this promising biomarker in the clinical field, we developed a rapid and robust analytical strategy for the quantification of hepcidin-25 in human samples based on HPLC-MS/MS (QqQ) to be implemented in routine laboratories. The novelty of the method is the use of special HPLC vials to avoid adsorptive losses due to the basic character of the peptide that causes interaction with the silanol groups of the vial’s glass surface. Up to 90% decrease in the MS/MS signal was observed, when commercial HPLC vials were used, while vials treated with 3-(2-aminoethylamino)propylmethyl-dimethoxysilane or 1H,1H,2H,2H-perfluorooctyltriethoxysilane, leading to no significant losses in the dynamic range of physiological hepcidin-25 mean serum levels (10-20 µg/L).
Careful analytical validation was performed for determining the reproducibility, repeatability, limit of quantification (0.5 µg/L) and linearity (0.5-40 µg/L) of the method. Serum samples from 9 healthy volunteers were analyzed with a median hepcidin-25 level of 3.3 µg/L, comparable to results reported in the literature.
Referee Report 06 March 2017
(2017)
A 400 m2 soil test field with gas injection system was built up, which enables an experimental validation of linear membrane-based gas sensors – a sensor technology which was developed based on the selective permeation of gases through membranes. Several soil watering and injection experiments with carbon dioxide at different days with varying boundary conditions were performed showing that the sensor behaves mostly insensitive to the environmental conditions investigated, i.e., barometric pressure fluctuations, soil temperature, air temperature and humidity, sun duration, and wind speed. Furthermore, depending on water infiltration, a gas phase displacement could be observed in-situ based on the changed measurement signal. The results of the validation experiments highlight the potential of the method for rapid leak detection and localization qualifying the sensor particularly for safety applications, e.g., in underground gas storage areas.
Many applications of nanometer- and micrometer-sized particles include their surface functionalization with linkers, sensor molecules, and analyte recognition moieties like (bio)ligands. This requires knowledge of the chemical nature and number of surface groups accessible for subsequent coupling reactions. Particularly attractive for the quantification of these groups are spectrophotometric and fluorometric assays, which can be read out with simple instrumentation. In this respect, we present here a novel Family of cleavable spectrophotometric and multimodal reporters for conjugatable amino and carboxyl surface groups on nano- and microparticles. This allows determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface, as well as the remaining thiol groups on particle, by spectrophotometry and inductively coupled optical emission spectrometry (32S ICP-OES). Comparison of the performance of these cleavable reporters with conductometry and conventional labels, utilizing changes in intensity or Color of absorption or emission, underlines the analytical potential of this versatile concept which elegantly circumvents Signal distortions by scattering and Encoding dyes and enables straightforward validation by method comparison.
Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the sticky character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing an LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend an LC-MS/MS-based quantification method with a dynamic range of 0.5–40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials.
Hepcidin-25 has attracted much attention ever since its discovery in 2001. It is widely recognized that this peptide hormone plays a major role in the regulation of iron levels in mammals and can reveal important clinical information about several iron-related disorders. However, the development of a reliable assay to quantify hepcidin proved to be problematic and serum hepcidin-25 concentrations determined by various assays differ substantially. Challenges arise in the MS analysis of hepcidin due to the “sticky” character of the peptide and the lack of suitable standards.
With the aim to tackle the current difficulties in hepcidin quantification and improve the status of this promising biomarker in the clinical field, we developed a rapid and robust analytical strategy for the quantification of hepcidin-25 in human samples based on HPLC-MS/MS (QqQ) as a reference method candidate to be implemented in routine laboratories. The novelty of the method is the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces. Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions where the use of acidic mobile phases was compared with a novel approach involving solvents at high pH containing 0.1% of ammonia. Both methods were carefully validated and applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with very good correlation of the results.
The determination of chloride is still one of the main tasks for the evaluation of reinforced concrete structures.
The corrosion of the reinforcement induced by the penetrating chlorides is the dominant damage process affecting the lifetime of concrete structures. In the recent years different research groups demonstrated that LIBS can be a fast and reliable method to quantify chlorine in cement-bound materials. Because chlorine in concrete can only occur as solved ions in the pore solution or bound in salts or hydrated cement phases, the detected emission of chlorine can be correlated with the chloride concentration determined e.g. with potentiometric titration. This work inter alia describes the production of reference samples and possible side effects during the production process. Due to transport processes in the porous matrix of the cement a misinterpretation of the concentrations is possible. It is shown how to overcome these effects and higher precisions of the single measurements can be realised. Using the calibration method, blank sample method and noise method, three different ways of calculating the limit of detection (LOD) and limit of quantification (LOQ) are compared. Due to the preparation of the reference samples a precision of the whole calibration model of sx0 = 0.023 wt% is determined.
The validation of the model is based on different test sets, which are varying in their composition
(different Cl-salts, water-to-cement ratios and additives). The determined mean error of the validation is
0.595 ± 0.063 wt%, which is comparable to standardised methods like potentiometric titration, direct potentiometry
or photometry (0.40 ± 0.06 wt%) [1].
The process of ensuring reliability of NDT applications contains various aspects, such as determining the performance and probability of success, the uncertainty in measurement, the provision of clear and functional procedures and ensuring the correct application accordingly. Test specimens have become powerful elements in supporting many of these aspects. Within the committee for NDT in Civil Engineering (NDT-CE) of the German Society for Nondestructive Testing (DGZfP), the subcommittee on Quality Assurance (UA-QS) therefore addresses the design and the integration of test specimens in the quality assurance process. Depending on the specific purpose, the requirements on test specimens can vary significantly based on the defined simulated scenario. The most prominent purposes of test specimens might be seen in providing references for inspection systems in regard to function control, calibration and validation. Further aspects can be parametric studies, basic investigation of physical principles related to NDT or a simplified and therefore comprehensive demonstration of inspection concepts (e.g. for teaching purposes). The specific purpose of a test specimen dictates the requirements regarding its conception, including the exact design, the material or the fabrication accuracy and the conditioning. In the development of a general guideline by the UA-QS for application-specific procedures and their validation, the use of test specimens is addressed and specific concepts for the design of test specimens are made. This includes the analysis of the measurement process regarding any given application, deriving an adequate calibration approach for it and designing test specimens (calibration specimens) accordingly. Furthermore, it includes the validation of the procedure taking into account all conditions related to the specific application in the field. The validation requires a statistically sufficient number of trials. Thorough evaluation of each trial can only be established if the ground-truth is known. Therefore, test specimens providing a realistic but controlled simulation of the inspection problem are valuable and indispensable elements in the validation process. The requirement of being fully realistic will often not be possible to fulfill due to practical restrictions. Any aspect that cannot be included in the simulation realistically needs to be simulated conservatively. This again, requires a sufficient understanding of the inspection principle and technique to ensure conservativeness. Among other quality-assurance-related aspects, the UA-QS establishes concepts and guidelines regarding sound and efficient approaches for the specific purposes of test specimens. This subcommittee brings together representatives of different Groups along the entire value chain of NDT-CE, including researchers, practitioners, manufacturers and clients. They all work together in establishing a common understanding and level of quality assurance in the industry.
The field of non-destructive testing of civil structures (NDT-CE) has been continuously growing. Due to the complexity and diversity of civil constructions as well as the heterogeneity of concrete, specific standards or guidelines for the application of modern NDT-CE are still missing. The development of individual solutions is the current approach, which is just as challenging as it is common for NDT-CE.
With the increasing development and commercialization of NDT-CE technology, the group of practitioners is growing. To ensure a good level of quality in the industry, it appears necessary to establish adequate means.
Naturally, the performance of NDT-CE methods regarding a specific application is strongly dependent on choosing the most suitable inspection technique and applying it correctly, generally referred to as the inspection procedure in the field of NDT. There are well-defined guidelines regarding procedure documentation and handling in many fields of NDT (e.g. nuclear, aerospace or automotive) according to the high importance of procedures in assuring a successful and reliable application. For a long time, this has not always been the case with NDT-CE, which is still considered a unique discipline of NDT. Part of the reason for that might be the young development state of NDTCE, the heterogeneity of building materials like concrete, timber or masonry as a material and the diversity of civil structures. In consequence, NDT-CE procedure development is considered challenging.
Among other aspects, addressed in the subcommittee on Quality Assurance (UA-QS) within the committee for NDT-CE of the German Society for Nondestructive Testing (DGZfP), part of its work aims at establishing an adequate basis for NDT-CE procedure development. While some of the highly developed approaches from other industries are taken into consideration, they need to be analyzed regarding their suitability for NDT-CE and adapted accordingly. For a procedure to be as defined as possible, it needs to contain sufficient information, such as the scope and limitations regarding material, geometry and condition of the test object, inspection parameters, calibration, data acquisition, analysis criteria as well as requirements regarding the inspection personnel.
For a successful implementation in the field, it is important to define the specific procedure as precisely as possible. Despite the necessity of a great amount of information to be included, the procedure needs to be suitable for efficient field application.
The UA-QS is developing a guideline for NDT-CE procedures suitable for application in this field of NDT to ensure correct and reproducible application. To demonstrate and evaluate this concept, specific examples of procedures are also produced. In particular, the UA-QS has developed a procedure for the detection and positioning of tendon ducts using Ground Penetrating Radar (GPR). This procedure is tested regarding the practical applicability in a roundrobin on a defined type of reference test block.