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A new multivitamin supplement containing fat (A and D 3 ) and water soluble (C, B 2 , B 3 , B 6  and B 12 ) vitamins together with nutrients for young athletes was formulated and analyzed. Due to the complexity of the matrix (excipients: Aegina peanut butter, pomegranate juice, royal jelly and chocolate), a purification process was developed using a freezing and liquid extraction technique and optimized with central composite design (CCD) methodology. For the analysis of the samples, an efficient liquid chromatography (LC) gradient elution method was developed and validated, focusing on the stability of sensitive vitamins. After investigation, a diode array detector was chosen instead of electrospray ionization mass spectrometry (ESI-Q/MS) to determine the analytes. The most suitable chromatographic conditions were selected based on a D-optimal process where a mixture component (acetonitrile and phosphate buffer 20 mM) was cross-correlated with three factors: pH, flow rate, and column temperature. The analysis was performed on a cyano column (250 × 4.6 mm, 5 µm) and validated (recovery from 98.7 to 100.4; relative standard deviation &lt;1.6%; limits of detection from 0.02 to 0.10 µg mL −1 ; limits of quantitation from 0.06 to 0.30 µg mL −1 ) according to International Conference on Harmonization (ICH) guidelines. The robustness was characterized based upon a Box-Behnken design process. Stability experiments indicated that the vitamins were stable for at least 1 year (recovery &gt;97.1%).

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Response Surface and Freezing-Out Methodologies for the Extraction, Separation, and Validation of Seven Vitamins in a Novel Supplement with Determination by High-Performance Liquid Chromatography