Browsing by Subject "UHPLC"

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.