OPTIMIZATION OF CONDITION OF METHOD DEVELOPMENT DETERMINATION OF SWEETENERS, PRESERVATIVES AND DYES IN ESSENCES SYRUP SIMULTANEOUSLY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Aceh International Pharmacy Conference 1 Suprianto, 2 Effendy De Lux Putra, and 3 Siti Morin Sinaga 1 College of Health Sciences Helvetia, Medan, Indonesia ,2,3 University of Sumatera Utara, Medan, Indonesia Coorresponding Author:

BACKGROUND SYRUP (BSN,1994) Composition: Sugar Preservatives Dyes (BSN,1994) EE ssence Syrup (Satuhu, S, 1994) No Sweeteners

BACKGROUND OPTIMIZATION OF CONDITION OF METHOD DEVELOPMENT DETERMINATION OF SWEETENERS, PRESERVATIVES AND DYES IN ESSENCES SYRUP SIMULTANEOUSLY USING HPLC Saccharin and Cyclamate (Qualitative: BSN, 1992) Benzoic and Sorbic Acids ( HPLC: BSN, 1992 ) Tartrazine and Sunset Yellow (TLC: BSN, 1992) Saccharin (Hayun, et al., 2004) Cyclamate (Novelina, et al.,2009) Benzoic and Sorbic Acids ( BSN, 1992 ) Tartrazine and Sunset Yellow (Veni, et al., 2011) HPLC METHOD ANALYSIS METHOD (BSN)

OPTIMUM CONDITION PROPERTIESS:  SOLUBLE IN WATER  Maximum Wavelength: 202 nm (Saccharin) 194 nm (Cyclamate) 225 nm (Benzoate) 255 nm (Sorbate) 427 nm (Tartrazine) 481 nm (SYellow) FOOD ADDITIVES LITERATURE S O O Na H N O S N O O O C-O-Na O C H H3CH3C C H C - O - K O C H C H SO 3 Na N N NaO 3 S OH NaOOC N N N N OH NaO 3 S SO 3 Na SACCHARIN CYCLAMATE TARTRAZINE SUNSET YELLOW BENZOATE SORBATE HPLC D AD REVERSE PHASE  RETENSI TIME  CAPACITY FACTOR  TAILING FACTOR  RESOLUTION  SELECTIVITY  PLATES  VOID VOLUME  WAVELENGTHS  pH of MOBILE PHASE  MOBILE PHASE COMPOSITION.  FLOW RATE  COLUMN TEMPERATURE

Materials and instruments MATERIAL Methanol (E. Merck) Sodium Dihydrogen phospate (E Merck) Ortohydrogen phospate (E. Merck) Saccharin (Sigma-Aldrich) Cyclamate (Sigma-Aldrich) Sodium Benzoate (Sigma- Aldrich) Potasium Sorbate (Sigma- Aldrich) Tartrazine (Sigma-Aldrich) Sunset Yellow (Sigma-Aldrich) Instrument Probe UV 1800 spectrophotometer (Shimadzu) UFLC system Agilent Series 1290 DAD. ZORBAX Eclipse Plus C18 Column (100 mm x 4.6 mm, 3.5 μm) pen type digital pH meter (ATC) Sonicator (Bransonic) Analytical Balance (Boeco) Vacuum Pump (Bust ) Cellulose Nitrate Filter 0.45 µm Syringe Filter Polytetrafluoroethylene (PTFE) 0.45 µm Volumetric Flask (Oberoi) Volume Pipette (Oberoi) Beaker Measuring Cylinder METHODE

1,36 gram NaH 2 PO 4 0,78 ml H 3 PO 4 Mr = 98 g/mol  = 1,685 g/ml % = 85% a. preparation 1. Buffer phospate solution SOLUTION A 10 mM SOLUTION B 10 mM Ph 4,7± 0, ml Dropwise pH 4,5 ± 0,1 pH 4,3 ± 0,1 pH 4,0 ± 0,1

50 mg 2. Stock solution  Saccharin  Cyclamate  Sodium Benzoate  Potasium Sorbate  Tartrazine  Sunset Yellow 3. Single solution  Saccharin: 5,24 ppm  Cyclamate: 80,48 ppm  Sodium Benzoate: 8,40 ppm  Potasium Sorbate: 2,30 ppm  Tartrazine: 84,48 ppm  Sunset Yellow: 4,16 ppm 4. Mixed solution MS 10 ml STOCK 50 ml  Saccharin: 1 ppm  Cyclamate: 75 ppm  Sodium Benzoate: 3 ppm  Potasium Sorbate: 6 ppm  Tartrazine: 10 ppm  Sunset Yellow: 5 ppm SS 10 ml METHODE

b. Maximum wavelength determination 1. WAVELENGTH  : 200 nm nm Maximum Wavelength SINGLE SOLUTION  Saccharin: 5,24 ppm  Cyclamate: 80,48 ppm  Sodium Benzoate: 8,40 ppm  Potasium Sorbate: 2,30 ppm  Tartrazine: 84,48 ppm  Sunset Yellow: 4,16 ppm 2. VOID VOLUME  Voids Volume: 20%; 30%; 40%  : 200 nm  Mobile Phase pH: 4,5  Mobile Phase Composition: 75 : 25  Flow Rate: 0,8 ml/min  Column Temperature: 30 o C 3. WAVELENGTH  Void Volume: Optimum  : nm, 440 – 470 nm  Mobile Phase pH: 4,5  Mobile Phase Composition: 75 : 25  Flow Rate: 0,8 ml/min  Column Temperature: 30 o C  Rretensi times  Capacity Factor  Tailing Facttor  Resolution  Selectivity  Plates MIXED SOLUTION  Saccharin: 1 ppm  Cyclamate: 75 ppm  Sodium Benzoate: 3 ppm  Potasium Sorbate: 6 ppm  Tartrazine: 10 ppm  Sunset Yellow: 5 ppm MIXED SOLUTION  Saccharin: 1 ppm  Cyclamate: 75 ppm  Sodium Benzoate: 3 ppm  Potasium Sorbate: 6 ppm  Tartrazine: 10 ppm  Sunset Yellow: 5 ppm METHODE C. metHode HPLC OPTIMIZATION

4. Mobile Phase pH  Void Volume: Optimum  : Optimum  Mobile Phase pH: 4,0; 4,3; 4,5; 4,7  Phosphate buffer-methanol Composition: 75 : 25  Flow rate: 0,8 ml/menit  Column Temperature: 30 o C 5. MOBILE PHASE COMPOSITION  Void Volume: Optimum  : Optimum  Mobile Phase: Optimum  Phosphate buffer-methanol Composition: 73:27; 75:25; 77:23  Flow Rate: 0,8 ml/min  Column Temperature 25 o C 6. FLOW RATES  Void Volume: Optimum  : Optimum  Mobile Phase pH: Optimum  Mobile Phase Composition: Optimum  Flow Rates: 0,8; 1,0 ; 1,2 ml/min  Column Temperature: 30 o C 7. COLUMN TEMPERATURE  Void Volume: Optimum  : Optimum  Mobile Phase: Optimum  Mobile Phase Composition: Optimum  Flow Rate: Optimum  Column Temperature: 25, 30, 35 o C  Rretensi times  Capacity Factor  Tailing Facttor  Resolution  Selectivity  Plates c. metHode HPLC OPTIMizATION MIXED SOLUTION  Saccharin: 1 ppm  Cyclamate: 75 ppm  Sodium Benzoate: 3 ppm  Potasium Sorbate: 6 ppm  Tartrazine: 10 ppm  Sunset Yellow: 5 ppm MIXED SOLUTION  Saccharin: 1 ppm  Sunset Yellow: 5 ppm  Sodium Benzoate: 3 ppm  Potasium Sorbate: 6 ppm  Tartrazine: 10 ppm  Cyclamate: 75 ppm METHODE

results 1. Determination of Maximum Wavelength 201 nm 224 nm 254 nm 197 nm 427 nm 482 nm

results Table 1. Effect of Void Volume on the Optimization Parameters 2. Optimation of Void volume

results 2. Optimation of wavelengths Table 2. The data of Tailings Factor on the Wavelength Optimization Ree and Stoa, 2011 Novelina, et al., 2009 Jurcovan, et al., 2012

results 3. Optimization of Mobile phase pH Table 3. Effect of Buffer pH 4.0 and 4.3 on the Optimization Parameters Table 4. Effect of Buffer pH 4.5 and 4.7 on the Optimization Parameters

results 4. Optimization of mobile phase composition Table 5. Effect of Mobile Phase Composition on the Optimization Parameters

results 5. Flow rate Optimization Table 6. Effect of Flow Rate on the Optimization Parameters

results 6. Optimization of Column Temperature Table 7. Effect of Column Temperature on the Optimization Parameters

results Optimum codition of HPLC method Fuigure 2. Chromatograms of mixed standard solution Ft = Ft = Ft = Ft = Ft = Ft = Compounds k'NRsα TAR SAC CYC SUN BEN SOR  Rs ≥ 2  0,5 ≤ k ≤ 20  0,9 ≤ Ft < 2,0   > 1  N >>> Snyder, et al, 2010 Snyder, et al, 1997

Conclusions The results showed that the optimum conditions for the test were the wavelength of 200 nm for the detection of cyclamate, a wavelength of 220 nm for the detection of saccharin, benzoate and sorbate and a wavelength of 450 nm for the detection of tartrazine and sunset yellow, void volume of 30%, mobile phase pH 4.5 phosphate buffer : methanol (75 : 25, v/v), flow rate of 1,0 ml/min, column temperature of 30 o C.

References 1.Badan Standarisasi Nasional (2004). Bahan Tambahan Pangan Pemanis Buatan – Persyaratan Penggunaan dalam Produk Pangan. SNI Jakarta: Badan Standarisasi Nasional. pp Hartono, E. (2007). Pengaruh pH pada Penetapan Kadar Natrium Benzoat dalam Sirup Melalui Isolasi dengan Pelarut Eter Secara KCKT. Pharmacon 8(1): 28–33. 3.Jurcovan, M.M., Atudosiei, N.L., Daniela Mihaila, D., (2012). A simple HPLC Method for Determination of Tartrazine and Sunset Yellow in Soft Drinks Samples. Bulletin UASVM Agriculture, 69(2): 267 – Novelina, Y.M., Sutanto dan Fatimah, A. (2009). Validasi Metode Analisis Penetapan Kadar Senyawa Siklamat dalam Minuman Ringan. Prosiding PPI Standarisasi; 2009; Nov 9; Jakarta: Badan Standarisasi Nasional. 5.Pylypiw, H.M., dan Grether, M.T. (2000). Rapid High Performance Liquid Chromatography Method for the Analysis of Sodium Benzoate and Potassium Sorbate in Foods. Journal of Chromatography A. 888(1): 299– Ree, M., dan Stoa, E. (2011). Simultaneous Determination of Aspartame, Benzoic Acid, Caffeine, and Saccharin in Sugar-Free Beverages Using HPLC. Concordia College Journal of Analytical Chemistry 2(1): Satuhu, S. (1994). Penanganan dan Pengolahan Buah. Jakarta: PT Penebar Swadaya. Hal Serdar, M., dan Knezevic, Z. (2011). Determination of Artificial Sweeteners in Beverages and Special Nutritional Products Using High Performance Liquid Chromatography. Arh. Hig. Rada Toksikol. 62(1): Snyder, L.R., Kirkland, J.J., dan Dolan, J.W. (2010). Introduction to Modern Liquid Chromatography. Edisi 3. New York: A John Willey & Sons, Inc. pp 20-83, , Veni, N.K., Menyyanathan, S.N., Babu, B.N., Sharma, A.K., Srikanth, B.A., Satyam A.B., dan Sureh, B. (2011). Simultaneous Estimation of Colorants Sunset Yellow and Tartrazine in Food Products by RP HPLC, International Journal of Research in Pharmaceutical Sciences 2(4): Xiao-tong ， J., Guo-song ， C., Ling-ling ， J.dan Yan-li., Z. (2011). Simultaneous HPLC Determination of 6 Sweeteners. Analysis Detected Food Science. 32(6): Zatar, N.A. (2007). Simultan Determination of Seven Synthetic Water Soluble Food Colorants by Ion Pair Reversed Phase HPLC. Journal Food and Technology 5(3):