| dc.description.abstract | Norway is the largest fish industry of Atlantic salmon. With a worth of more than United States Dollar (USD) 7 billion. That ́s why Atlantic salmon production in Norway is 3 times more than the total volume of all other types of meat. Despite large advancements in the fish industry. Fish health is still a major concern because of the high mortality rate of about 27.8%. That results in significant economic loss. For good fish health, vitamin D is an important factor. Because of its role to increase immunity against disease, immunoregulatory effect, phagocytic activity, and deposition of calcium and phosphorous in bones. In Salmonid vitamin D deficiency in feed caused growth retardation. For that vitamin D play its role as a biomarker for farmed fish health.
For vitamin D quantification Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) consider the gold standard.
So, the aim of the study is to develop and validate a high-quality method for monitoring vitamin D in fish plasma by LC-MS/MS.
Method: Sample preparation was optimized by using different concentrations of different salts (NaCl, MgSO4.7H20, (NH4)2SO4) with different water-miscible solvents acetone, methanol, water, and Acetonitrile (ACN). Among different water-immiscible solvents (heptane, ethyl acetate) through Liquid-Liquid Extraction (LLE) for maximum extraction recovery, derivatization, separation, and detection of 25 Hydroxy Vitamin D3 (25-(OH)D3. Signal enhancement was done by using different concentrations of derivatizing agent 4-Phenyl-1-2,4-tiazoline-3,5-dione (PTAD). Separation of derivatized 25-Hydroxy Vitamin D3 (25(OH)D3-PTAD) was maximized by analyzing under different conditions. While using 0.2% ammonium hydroxide and formic acid and ACN in water. Detection of 25(OH)D3-PTAD increased by optimal Multiple Reaction Monitoring (MRM) transitions in Electrospray Ionization Positive Mode (ESI+) under Electrospray Ionization (ESI) of tandem mass spectrometry. The concentration of 25(OH)D3 was determined by using the deuterium label internal standard. After that, the method validation was done.
Results: LLE with MgSO4, acetone, and heptane gave the best results for extraction recovery with low ion suppression. The derivatization step increases the signal response by ESI+. High sensitivity for 25(OH)D3-PTAD was achieved by using 0.2% ammonium hydroxide. The repeatability (CV%) of replicates and individual samples of small, marine, and plant-based feed was 6%, 4%, 5%, 29%, 25%, and 26% respectively. The intermediate precision for first and second quality control was 12 & and 13% over the period of six days. The linear range of 0.4-100 nmol/L with R2 of 0.995-0.999 was found by the linear regression method. The LOD and LOQ were 0.15 and 0.4 nmol/L respectively. Conclusion: Different fish plasma samples were measured from small, marine, and plant-based feed. That gave a much higher value than our Limit of Detection (LOD) and Lower Limit of Quantification (LOQ). For small fishes from 1.33-4.07 nmol/L. For marine feed-based fishes from 7.7-24.3 nmol/L. For plant feed-based fishes from 4.07-20.6 nmol/L. These differences are higher than intermediate precision.
So, it is suggested that the developed method could be useful to monitor the level of vitamin D in fish. | |