Purpose: Magnesium ascorbyl phosphate (MAP) is a water-soluble, stable derivative of Vitamin C, used in several cosmetic preparations. MAP properties include whitening, UV-Protection, acne treatment, and wound/burn care. MAP mechanism of action in dermis is related to its conversion to ascorbate via hydrolysis catalyzed by dermal phosphatase. In order to characterize the in-vitro permeability of MAP through the skin from various formulations and eventually assess its in-vivo kinetic in dermis using intradermal microdialysis, it is necessary to quantify MAP in isotonic solutions. Analysis of MAP in isotonic solutions is challenging, given its ionization reactivity at the most acidic proton, which is the enolic C3-hydrogen (pKa = 4.25). The 2,3-enediol moiety enables compounds to form dimers, resulting in multiple broad peaks along with the solvent front. The goal of this study was to develop and validate a sensitive and selective HPLC-UV method for the quantification of MAP in normal saline.
Methods: Analysis was performed on an Agilent 1100 series (Agilent technologies, Santa Clara, CA) using Waters, Symmetry C18 column (3.9 x 150 mm, 5 µm, 100 A). In order to achieve the desired separation of MAP in saline solution, a volatile ion pairing agent (N-butyl amine) was used to retain MAP for a longer time on the column. The total run time was 6 minutes. The mobile phase consisted of sodium phosphate buffer solution: acetonitrile (99:1) at pH 2.5, added with 0.1 % of N-butyl amine. Flow rate of the pump was 0.5 ml/min at column temperature of 25°C. The detection wavelength of the compound was 246 nm and reference was 360 nm. The method was tested for: accuracy, precision, selectivity, sensitivity, detection and quantitation limits, and calibration range.
Results: MAP peak was well separated from the solvent front with a retention time of 3.9 minutes. The peak was narrow and sharp. Calibration curves were linear in the concentration range of 0.05 µg/ml to 20 µg/ml (R2 > 0.99). LOD was 0.02 µg/ml. LLOQ was 0.05 µg/ml; ULOQ was 20 µg/ml. MAP was stable in normal saline after 72 hr freeze-thaw cycles at -20°C.
Conclusion: An HPLC method for the quantification of MAP in normal saline was developed and validated. The method was successful and applied to perfusion studies of MAP dialysate using in-vitro Franz diffusion studies.
Candace Norton– M.S student, Long Island University, Brooklyn, New York
Benjamin Kuzma– Ph.D student, Long Island University, Brooklyn, New York
Grazia Stagni– Associate professor, Long Island University, Brooklyn