Spectrofluorimetric Method for the Determination of Rhodamine B in Syrup Asmiyenti Djaliasrin Djalil 1 , Bondan Bagus Wijaksono 2 , Pri Iswati Utami 3 1,2 Vice Dean, Department of Pharmacy, Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Jl. Raya Dukuhwaluh, PO. Box 202 Purwokerto 53182, Indonesia 3 Student of Department of Pharmacy, Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Jl. Raya Dukuhwaluh, PO. Box 202 Purwokerto 53182, Indonesia 1 asmiyentidjaliasrindjalil@ump.ac.id 2 bondanbagusw@gmail.com 3 priiswatiutami@ump.ac.id Abstract— The rhodamine B has been used as a textile colorant. Its use in food is strictly prohibited because of its ability to form carcinogenic compounds. This study describes a method for determining rhodamine B in syrup by a fluorescence spectrophotometry. The method was validated by determining linearity, range, limit of detection, limit of quantitation, precision, and recovery. Five red syrup samples were collected from markets in Purwokerto city, Indonesia. A fluorescent compound was measured fluorometrically at 570 nm (λ ex , 550 nm). Linear relationship with good correlation coefficients (0.9975) has been obtained in the range of 0.5 - 2.5 μg/ml, with a detection limit of 0.1436 μg/ml and a quantitation limit of 0.4787 μg/ml. The precision of the method was satisfactory; the relative standard deviation was 0.29%. The method was successfully applied to the analysis of rhodamine B in syrup with good accuracy; the recovery was 102.8%. The result showed that five syrup samples did not have rhodamine B. Keywords— Analysis method, rhodamine B, spectrofluorometric, syrup, validation I. INTRODUCTION Synthetic dyes have been widely used in textile, food, pharmaceuticals, and cosmetic. On the other hand, toxicity issues caused by synthetic colorants have initiated an intense research especially in food colorants. The color of a food and beverage play essential criteria in a food's acceptability. 1 Rhodamine B (Fig 1.) is a derivative of the xanthene dyes class. It may cause ovarian toxicity and cervical epithelial cell proliferation in the uterus. 2-3 Due to the toxic effects of rhodamine B, FDA and many countries have banned it from using in food samples. In accordance with the Regulation of the Ministry of Health Republic of Indonesia and the National Agency of Drug and Food Control, Indonesia currently prohibits its use as food additive. 4-6 The food fraud is the act of deceiving consumers with the aim of increasing company profits. FDA has been reported a number of imported food containing rhodamine B, in 2012. There were approximately 82 food products from 11 developing countries. 7 In 2018, Saeeds’s group research has successfully identified the presence of rhodamine B in syrup samples sold in Faisalabad, Punjab, Pakistan. Of 75 samples studied, there were 40% syrup containing rhodamine B. 8 Therefore, a continuous monitoring of prohibited colorants in food is needed to ensure food safety. Many liquid chromatography methods were developed for the determination of rhodamine in food, cosmetic, or wastewater samples. 9-11 Tatebe’s group research has developed an HPLC method for the simultaneous determination of rhodamine B, pararosaniline, and auramine in processed foods. 9 Furthermore, Chiang’s group research has determined that rhodamine B and rhodamine 6G could be separated by HPLC combined with fluorescence detection. 10 Although HPLC method provides good sensitivity for determination of rhodamine B, it has its limitation concerning the high price of the instrument. Thus far, some spectrophotometric method was reported for the detection of rhodamine B. 11-15 Even though UV-visible spectrophotometer is a simple and low cost apparatus than other instrument, the quantitative detection of rhodamine B in several matrixes needs a sample preparation technique. It step takes a long time and a lot of chemicals or solvents. Rhodamine B is a fluorescent dye. In this research, based on its native fluorescence, a direct quantitative analysis method of rhodamine B in syrup was developed and investigated using spectrofluorometer. The objective of the present study was to detect the presence of rhodamine B quantitatively in syrup samples collected from local market in Purwokerto City, Central Java, Indonesia by spectrofluorometric method.