Quantitative Analysis of the Vitamin D3 Content in Dietary Supplements Marketed in Hungary Using High-Performance Liquid Chromatography

<b>Background/Objectives:</b> The use of over-the-counter vitamin D₃ supplements has increased substantially in recent years. Compared with pharmaceuticals, dietary supplements are subject to less stringent regulatory oversight, raising concerns regarding labeling accuracy, consumer knowledge, and patient safety. This study aimed to assess public knowledge and preferences related to vitamin D₃ supplementation and to evaluate the content accuracy and short-term stability of commonly used products. <b>Methods:</b> A cross-sectional online survey containing 39 questions was conducted in Hungary between 1 May and 30 June 2024. Based on survey responses, the most frequently used vitamin D₃ supplements (five soft gel capsules and four tablets) were selected for laboratory analysis. Vitamin D₃ content was quantified using a validated high-performance liquid chromatography (HPLC) method with UV detection. Soft gel capsules were additionally exposed to natural daylight for one month to assess short-term photostability. <b>Results:</b> In total, 367 participants (mean age 31.0 ± 12.5 years) completed the survey, and only 3.5% answered correctly all knowledge-based questions. Six commonly reported supplement brands accounted for approximately 90% of responses. Measured vitamin D₃ content remained within the tolerance limit (-20% to +50%). Following sunlight exposure, three of four capsule products showed no substantial vitamin D₃ loss, while one exhibited a 14.7% decrease. <b>Conclusions:</b> Most analyzed vitamin D₃ supplements complied with labeled content claims, but substantial knowledge gaps were identified that may affect patient safety. The validated HPLC method supports pharmacovigilance-oriented quality monitoring of vitamin D₃ supplements and underscores the need for improved professional counseling.