3200 J. Sep. Sci. 2013, 36, 3200–3205 Muhammed Alzweiri Ruba Tarawneh Mohammad A. Khanfar Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman, Jordan Received April 21, 2013 Revised June 6, 2013 Accepted June 28, 2013 Research Article Gas chromatography/trace analysis of derivatized azelaic acid as a stability marker Azelaic acid, a naturally occurring saturated dicarboxylic acid, is found in many topical formulations for its various medical benefits or as a byproduct of the oxidative decompo- sition of unsaturated fatty acids. The poor volatility of azelaic acid hinders its applicability in GC analysis. Therefore, azelaic acid was derivatized by methylation and silylation pro- cedures to enhance its volatility for GC analysis. Accordingly, dimethyl azelate (DMA) and di(trimethylsilyl) azelate were synthesized and characterized by GC–MS. Subsequently, a GC with flame ionization detection method was developed and validated to analyze trace amounts of azelaic acid in some marketed skin creams. Unlike DMA, di(trimethylsilyl) azelate was chemically unstable and degraded within few hours. Nonane was used as a stable internal standard. Variability due to derivatization and extraction was controlled by a standard addition procedure. DMA analysis was linear in a wide concentration range (100 ng/mL to 100 mg/mL). Moreover, the method was accurate (96.4–103.4%) and pre- cise with inter- and intraday variability <2.0% and LOQ and LOD of 100 and 10 ng/mL, respectively. Keywords: Azelaic acid / Dimethyl azelate / Di(trimethylsilyl) azelate / GC–FID DOI 10.1002/jssc.201300418  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Azelaic acid (1,7-heptanedicarboxylic acid, Supporting Infor- mation Fig. S1) is a naturally occurring saturated dicarboxylic acid often found as a dietary constituent, occurring in whole grains and some meat products, and can be formed endoge- nously and concurrently with some ailments. It has been used in various formulations to treat rosacea, acne, melasma, lentigo maligna, malignant melanoma, and even perioral dermatitis [1–4]. Azelaic acid is widely used for treatment of acne vulgaris due to its irritant, antimicrobial, and anti- inflammatory effects [5, 6]. It is also used in some whitening preparations for its antihyperpigmentive effect [4]. Neverthe- less, azelaic acid is found in some skin preparations as a de- composition product of unsaturated fatty acids such as oleic acid. Azelaic acid is one of the components found in some skin preparations (both the claimed and unclaimed content of azelaic acid) responsible for burning sensations and transient erythematic reactions in 38% of azelaic acid users [7]. Correspondence: Dr. Mohammad A. Khanfar, Department of Phar- maceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan E-mail: m_khanfar@ju.edu.jo Fax: +962-6530-0250 Abbreviations: BSTFA, bis(trimethylsilyl)trifluoroacetamide; DMA, dimethyl azelate; DSA, di(trimethylsilyl) azelate; FID, flame ionization detection The determination of fatty acid derivatives, such as azelaic acid, is most often carried out by GC [8]. How- ever, they are polar, low-volatility compounds, tend to self- associate, or to adhere to walls of GC columns or other surfaces; thus, the separation of unmodified azelaic acid is rather problematic. Therefore, derivatization procedures are usually required for GC analysis to increase the volatil- ity and improve separation. In the derivatization process, the polar and low-volatile azelaic acid is converted into less polar, relatively volatile, and thermally stable derivatives. However, the analysis of azelaic acid by HPLC is also challenging because it lacks chromophoric features. Conse- quently, several methods of azelaic acid derivatization by using chromophoric reagents have been adopted including derivatives with 2,4-dinitrophenylhydrazine [9], 2-bro- moacetyl-6-methoxynaphthalene [10], l-leucine-4-methyl-7- coumarinylamide [11], phenacyl bromide [12], phenacyl es- ters [13], methylmethoxycoumarin [14], and tosylic acid [15]. GC with flame ionization detection (FID) is a sensitive method for the quantitative analysis of trace analytes [8]. FID provides linearity over a wide dynamic range [8]. Therefore, GC–FID was used as an optimum method for azelaic acid analysis in selected cosmetic preparations to determine ei- ther trace amounts of azelaic acid generated by vegetable oil decomposition or azelaic acid used in preparations for its medical benefits. Derivatization by esterification increases the volatility of carboxylic acids, improves the peak configuration, separation, C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com