Quantitation of Fat Soluble Vitamins in Different Walnut Genotypes and Commercial Cultivars; a Comprehensive and Robust LC-MS/MS Method Validation

Walnut, being both nutritious and delicious, has been regarded as a health food due to its preventive and therapeutic effects. In the present study, a robust LC-MS/MS analytical method was developed to simultaneously quantify five fat soluble vitamins (D2, D3, K2, E, K1) in eleven walnut genotypes and two commercial cultivars. Linearity, accuracy, inter- and intra-day precision, detection limit (LOD), quantification limit (LOQ) and uncertainty (U at confidence level of 95% (k = 2)) were the validation parameters of the developed LC-MS/MS method. The established linearity ranges for the studied vitamins were between 50 and 1200 mg L？1 for vitamins D2, D3, K1, between 100-2400 mgL？1 for vitamin E and between 200-4800 mg L？1 for vitamin K2. The coefficients of determination for the vitamins D2, D3, K2, E, K1 were between 0.9990, 0.9991, 0.9995, 0.9997, 0.9991, respectively. Vitamins D3, K2 and K1 were not present in the analysed walnut samples. On the other hand, vitamins E and D2 were in high quantities in all studied walnut samples. D2 and E were the most abundant fat soluble vitamins in MDE32 (29.1 mg g-1 dry plant) and MDE5 (15.2 mg g-1 dry plant) genotypes. Despite the fact that the walnut was selected as real life sample, the validated LC-MS/MS method is enforceable to a broad range of food species