Talanta 71 (2007) 639–643 Validation of HPLC stability-indicating method for Vitamin C in semisolid pharmaceutical/cosmetic preparations with glutathione and sodium metabisulfite, as antioxidants Adriana M. Maia a , Andr´ e Rolim Baby a,∗ , Wilson J. Yasaka b , Eunice Suenaga b , Telma M. Kaneko a , Maria Val´ eria R. Velasco a a Department of Pharmacy, School of Pharmaceutical Sciences, University of S˜ ao Paulo, 580 Prof. Lineu Prestes Av., bl. 13, Conjunto das Qu´ ımicas, Cidade Universit´ aria, 05508-900 S ˜ ao Paulo, SP, Brazil b Department of Pharmacology, Institute of Biomedical Sciences, University of S˜ ao Paulo, 1374 Prof. Lineu Prestes Av., ICB II, Cidade Universit´ aria, 05508-900 S ˜ ao Paulo, SP, Brazil Received 1 April 2006; received in revised form 7 May 2006; accepted 7 May 2006 Available online 12 June 2006 Abstract HPLC stability-indicating method was validated for Vitamin C (ascorbic acid) in semisolid pharmaceutical/cosmetic formulations containing glutathione and sodium metabisulfite, as antioxidants. The described procedure included a reliable, precise, accurate and specific method deter- mination employing a 250 mm × 4.6 mm C 18 column, 0.2% metaphosphoric acid/methanol/acetonitrile (90:8:2, v/v/v) as the mobile phase and detection at 254 nm. Nicotinic and ascorbic acids were employed as standards, both presenting purity of 99.0%. Linearity was established for the ascorbic acid concentrations ranging form 1.0 to 12 g mL -1 , accuracy/recovery percentage was 95.46–101.54%, precision values were 0.38 (intra-day) and 1.22% (inter-days), and LOD and LOQ were found to be 0.05 and 0.17 g mL -1 , respectively. The working mobile phase elevated the ascorbic acid retention time to ≈3.5 min at a flow rate of 1.0 mL min -1 and provided resolution of the active from the nicotinic acid (internal standard), degradation product (oxalic acid) and other excipients from the pharmaceutical/cosmetic preparations. © 2006 Elsevier B.V. All rights reserved. Keywords: Ascorbic acid; Pharmaceutical preparations; HPLC stability-indicating method; Oxalic acid; Cosmetic formulations 1. Introduction Vitamin C, also known as ascorbic acid, has been widely employed in pharmaceutical and cosmetic preparations, includ- ing semisolid topical dosage forms, for the formulation pro- tection against oxidation and to exert physiological/biological activities. The Vitamin C major biologic activity is related to the maintenance of the organism oxidation–reduction poten- tial. Besides its antioxidant property, the ascorbic acid acts on the free-radical inactivation, enzymatic co-factor, inhibi- tion of the nitrosamines formation, participation on the syn- ∗ Corresponding author at: Laboratory of Pharmaceutical Technology, Depart- ment of Pharmacy, School of Pharmaceutical Sciences, University of S˜ ao Paulo, FCF-USP, 580 Prof. Lineu Prestes Av., bl. 13, Conjunto das Qu´ ımicas, Cidade Universit´ aria, 05508-900 S˜ ao Paulo, SP, Brazil. Tel.: +55 11 3091 3623; fax: +55 11 3815 4418. E-mail addresses: andrerb@usp.br, andre rolim@uol.com.br (A.R. Baby). thesis of collagen, carnitine, tyrosine and reactions dependent of the cytocrome P 450 , intervention on the iron and histamine metabolism, skin depigmentant, and immunological reactions [1–7]. The ascorbic acid presents reasonable stability when the storage conditions protect the active substance from humidity, heat and luminosity. In aqueous solutions, it is decomposed by the environmental oxygen and oxidant agents, particularly in the presence of alkalis and heavy metals, obtaining the dehy- droascorbic acid and, lately, the diketogluconic acid. This last stage of oxidation is irreversible and biological activities were already compromised. As the final degradation product of the ascorbic acid, the oxalic acid is formed [8,9]. An assortment of techniques has been described to the quan- titation of Vitamin C in pharmaceutical dosage forms, as: titri- metric, spectrophotometric, electrochemical, fluorimetric, enzy- matic and chromatographic [10–13]. Since the matrix of a pharmaceutical/cosmetic dosage form, developed as an emul- 0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.05.006