Deuterated vitamin D standards are used commonly as internal standards in LC-MS/MS analysis of vitamin D3 and 25-hydroxyvitamin D3 in food. However, the use of various eluent additives, such as methylamine, formic acid and ammonium formate, also contributes to matrix effects and the performance of analysis by affecting accuracy and robustness. For the first time, continuous post-column infusion experiments of isotopically labelled vitamin D3-[d6] were performed to evaluate ion-suppression in a wide variety of food (salmon, cheese, pork fat, pork meat, and egg yolk). Furthermore, results collected using five analytical methods, employing DAD/UV and MS/MS-detectors, were evaluated with in-house and standardised reference materials. The matrix effect was significant when analysing vitamin D3 in most food matrices using the deuterium labelled internal standard. Even though the use of the 13C5-labelled internal standard reduced matrix effects, a standardised method is needed to agree on the true value of vitamin D in food.