Browsing by Subject "validation"

The literature review focused on liquid chromatographic-mass spectrometric (LC-MS) methods used to quantify B12 vitamers in food matrices. Various MS methods have been used for the detection of B12, offering more specificity than other commonly used analysis techniques. This thesis aimed to develop a method for quantifying the native forms of B12 in different food matrices and avoiding the commonly used conversion to cyanocobalamin during extraction. In the experimental study, an ultra-high-performance LC-tandem MS (UHPLC-MS/MS) method was developed and validated for selectivity, specificity, recovery, repeatability, reproducibility, trueness, and measurement uncertainty to determine B12 vitamers in fermented plant-based foods and microbial cell supernatants. The development was initiated by setting up mass spectrometer conditions and selecting transitions for multiple reaction monitoring (MRM) to achieve selective and sensitive detection method for individual B12 vitamers. This was followed by developing the UHPLC method utilizing a reversed-phased C18 column and gradient elution with 0.5% formic acid and 0.5% FA in methanol. The vitamers were ionized using electrospray ionization in a positive ion mode and detected in an MRM mode using hydroxocobalamin, cyanocobalamin, adenosylcobalamin, and methylcobalamin. All B12 vitamers were detected and separated with the developed and optimized UHPLC-MS/MS method. The internal standard calibration method was necessary to overcome matrix effects when analyzing food samples. The calibration curve content range was 0.2–200 pg/µL, and the results showed good linearity. The instrumental method was selective, precise, repeatable, and reproducible with detection and quantitation limits of 0.03–0.4 pg/µL and 0.2–2 pg/µL, respectively. The measurement uncertainty of the instrumental method varied between 10% and 20%. For the entire method, recoveries for the B12 vitamers ranged from 40% to 200%, and measurement uncertainties from 40% to 60%. Results for the total B12 content in food samples deviated from those determined using a conventional UHPLC-PDA method: Recovery for tempeh was over 90%, but for fortified bread only 20%. These results indicate the need for further development of sample pretreatment. The instrumental method was successfully validated and separated matrix compounds from B12 vitamers in food samples to some extent. The developed sample pretreatment method is a good starting point for developing more effective sample pretreatment methods in the future.

In atmospheric sciences, measurements provided by remote-sensing instruments are crucial in observing the state of atmosphere. The associated uncertainties are important in nearly all data analyses. Random uncertainties reported by satellite instruments are typically estimated by inversion algorithms (ex-ante). They can be incomplete due to simplified or incomplete modelling of atmospheric processes used in the retrievals, and thus validating random uncertainties is important. However, such validation of uncertainties (or their estimates from statistical analysis afterwards, i.e. ex-post) is not a trivial task, because atmospheric measurements are obtained from the ever-changing atmosphere. This Thesis aims to explore the structure function method – an important approach in spatial statistics – and apply it to total ozone column measurements provided by the nadir-viewing satellite instrument TROPOMI. This method allows us to simultaneously perform validation of reported ex-ante random uncertainties and to explore of local-scale natural variability of atmospheric parameters. Two-dimensional structure functions of total ozone column have been evaluated based on spatial separations in latitudinal and longitudinal directions over selected months and latitude bands. Our results have indicated that the ex-post random uncertainties estimated agree considerably well with the reported ex-ante random uncertainties, which are within 1-2 DU. Discrepancies between them are very small in general. The morphology of ozone natural variability has also been illustrated: ozone variability is minimal in the tropics throughout the year, whereas in middle latitudes and polar regions they attain maxima in local spring and winter. In every scenario, the ozone structure functions are anisotropic with a stronger variability in the latitudinal direction, except at specific seasons in polar regions where isotropic behaviour is observed. Our analysis has demonstrated that the structure function method is a remarkable and promising tool for validating random uncertainties and exploring natural variability. It has a high potential for applications in other remote sensing measurements and atmospheric model data.