Liquid Chromatography\nMass Spectrometric Method and\na Fluorometric 96 Well Plate Assay for Determination of Thiamine in\nSalmonid Eggs

Thiamine deficiency is a large contributor to reduced\nreproduction\nsuccess among salmonids throughout the northern hemisphere. In Scandinavia,\nthis reproduction disorder is known as M74; while in North America,\nit is known as early mortality syndrome (EMS). The disorder fluctuates\nin magnitude from year to year. During years with high prevalence\nof the disorder, salmonid hatcheries that stock various aquatic systems\nto maintain the population size experience difficulties filling their\nquotas without thiamine treatment of alevins. The disorder is monitored\nboth by observing the survival rate and by measuring the thiamine\ncontent of prefertilized eggs in the hatcheries. Here, a simple extraction\nprocedure is presented, which allows for quantitative determination\nof the various phosphorylated forms of thiamine using liquid chromatography\nmass spectrometry but also allows for extraction in 96 deep-well plates\nand measurement of the total thiamine content using fluorescence monitoring\nwith a plate reader, following oxidation of thiamine to thiochrome.\nThe latter procedure could also be integrated into a highly portal\nsystem where the thiochrome is determined using the DeNovix QFX analyzer.\nThe newly developed extraction procedure and cleanup method for fluorescence\nmeasurement represent the most versatile and simple methods to date\nfor monitoring of thiamine in salmonid eggs. The methods produced\naccurate and precise data with quantification limits below the limit\nwhere the deficiency causes 100% lethality.