International Journal of Applied Pharmaceutics ISSN - 0975 - 7058 Vol 12, Special Issue 1, 2020 DEVELOPMENT OF A DIRECT METHOD OF ANALYZING TRANEXAMIC ACID LEVELS IN WHITENING CREAM USING REVERSED PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY BAITHA PALANGGATAN MAGGADANI, JIHAN YASMINA, HARMITA HARMITA* Department of Pharmacy, Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia. Email: igakadeharmita@gmail.com Received: 26 September 2019, Revised and Accepted: 17 December 2019 ABSTRACT Objective: Whitening cream is a cosmetic that contains ingredients that can alleviate hyperpigmentation. Tranexamic acid (TA) is one of the potential anti-pigmentation agents that work through inhibiting plasmin. TA is used in cosmetic formulations at a concentration of 2.5% as a whitening and moisturizing agent. To date, research on TA in both cosmetics and other pharmaceutical products using high-performance liquid chromatography (HPLC) has not been done directly (without derivatization). Therefore, this study aimed to develop a simple and rapid analytical method for TA (without derivatization) in cosmetic cream samples using reverse-phase HPLC and water as a solvent. Methods: Optimization was conducted by evaluating several parameters that affect sample extraction, as well as composition and mobile phase types. The optimal method must fulfill suitability and validation requirements. The optimal method should be able to detect and quantify TA in cream samples without derivatization. Results: The optimal analysis condition used a ultraviolet detector at a wavelength of 210 nm, acetonitrile: double-distilled water: phosphoric acid (64:34:2) as the mobile phase and a flow rate of 0.8 mL/min. The retention time of the analyte occurred in the 2 nd min. Conclusion: The analytical method that met the validation requirements was characterized using parameters such as accuracy, precision, linearity, selectivity, limit, of detection, and limit of quantitation. This method is applicable for analyzing TA content in samples with a concentration of 1.02%. Keywords: Reverse-phase high-performance liquid chromatography, Optimization and validation, Tranexamic acid, Whitening cream. INTRODUCTION Tranexamic acid (TA) (Fig. 1) is an antifibrinolytic agent used to treat menorrhagia. In addition, TA also has a whitening effect against hyperpigmentation caused by melasma and ultraviolet (UV) radiation [1]. TA has been studied for its anti-melasma potential compared with standard therapy [2]. These reports revealed that oral or topical TA is similarly effective as standard therapy in patients with melasma [3]. Some studies additionally stated that TA has greater efficacy with fewer side effects. TA has emerged as a promising treatment for melasma both alone and in combination with other treatments [4-6]. TA is used as a whitening and moisturizing agent in cosmetic formulations at a concentration of 2.5% [7]. According to Japanese regulations regarding products containing TA, a cosmetic product is considered safe as a whitening agent if its TA concentration does not exceed 1.5–2% [8]. TA can also cause severe irritation and allergies under skin conditions that are sensitive to the agent [9-11]. TA does not have a high number of chromophore groups, and thus it is difficult to detect through UV spectroscopy. Analyses of TA in pharmaceutical products through high-performance liquid chromatography (HPLC) always involve derivatization to obtain a higher number of chromophore groups. The previous studies on the derivatization of TA used derivative agents such as 0.2% ninhydrin in methanol [12], phenyl isothiocyanate [13], 2-hydroxynaphthaldehyde in ethanol [14], sodium picryl sulfonate [15], benzenesulfonyl chloride [16], and 2,4-dinitrofluorobenzene [17]. None of these studies reported direct analysis using UV-HPLC. Therefore, this study analyzed TA content in a cosmetic sample in the form of a cream without derivatization using reverse-phase HPLC. The method of sample preparation and HPLC analysis was optimized to increase its sensitivity and selectivity to permit TA analysis without derivatization through a simpler method. METHODS Instrumentation An LC 20AT HPLC system (Shimadzu, Japan) was equipped with a pump, SunFire™ C 18 column, SPD-10A UV-Vis detector (Shimadzu), manual injector, and data processor (LC-Solution). A UV-Vis spectrophotometer (Jasco V-530), HPLC syringe (SGE, Australia), centrifuge (Labofuge 5100), vortex (Thermo Scientific), micropipette (Eppendorf), Ultrasonic Sonicator, hotplate (IKA ® C-MAG HS 7), pH meters (Eutech Instruments pH 510), and 0.45-µm Whatman filter membrane were also utilized. Chemicals and reagents TA (Hunan Dongting Pharmaceutical Co., Ltd.), HPLC grade acetonitrile (Merck), glacial acetic acid (Merck), ammonium acetate (Merck), double-distilled water (Ikapharmindo), potassium dihydrogen phosphate (Merck), methanol (Merck), and cream whitening samples were obtained from commercial suppliers. Chromatographic conditions Chromatographic separation was conducted using a C 18 column as the stationary phase and acetonitrile: double-distilled water: phosphoric acid (64:34:2) v/v/v as the mobile phase at a flow rate of 0.8 mL/min. Chromatographic detection was performed using a UV-Vis detector at a wavelength of 210 nm. Standard and working solution preparation The standard stock solution of TA (1000 µg/mL) was prepared by dissolving 100 mg of TA in 70 mL of distilled water in a 100-mL volumetric flask. The solution was saturated for 15 min and solvent was added up to a volume of 100 mL. The working solution was prepared by diluting the stock solution with solvent to obtain 200 µg/mL TA. Research Article © 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ijap.2020.v12s1.FF015 The 4 th International Conference on Global Health 2019