Simultaneous determination of aliskiren hemifumarate, amlodipine besylate and hydrochlorothiazide in their triple mixture dosage form by capillary zone electrophoresis Mohamed Salim a,b*, Walid M. Ebeid Gabor Patonay a a,c , Nahed El-Enany b, Fathalla Belal a b , Mohamed Walash b , Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, Georgia 30302-4098, USA. b Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, P.O. Box 35516, Mansoura, Egypt c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt. Page S-1 Table OF Contents. Title Page Figure S-1. Effect of buffer pH on separation of the studied drugs. S-3 Figure S-2. Effect of buffer conc. on separation of the studied drugs. S-3 Figure S-3. Effect of organic modifiers on separation of the studied drugs. (A) methanol (B) ethanol (C) acetonitrile. S-4 Figure S-4. Effect of applied voltage (kV) on separation of the studied drugs. S-5 Figure S-5. Effect of injection time (sec) on separation of the studied drugs. S-5 Figure S-6. Effect of capillary cartridge temperature (◦C) on separation of the studied drugs. S-6 Figure S-7. Selection of the detection wavelength. S-6 Figure S-8. Application of the proposed method for the simultaneous determination of the studied drugs in Amturnide® tablets (7.5 g/ml of AML, 179.25 g/mL ALS, 13.5 g/mL HCZ and 25 g/mL MXP) under the optimized conditions. S-7 Figure S-9. Application of the proposed method for the simultaneous determination of synthetic mixture of 8 g/ml of AML, 240 g/mL ALS, 20 g/mL HCZ and 25 g/mL MXP under the optimized conditions. S-7 Tables S-1. Performance data for the determination of the studied drugs by the proposed CZE method. S-8 Tables S-2. Robustness data for the determination the studied drugs by the proposed CZE method. S-9 Tables S-3. Assay results for the determination of the studied drugs in their synthetic mixture and comparison methods. S-10 Page S-2 160000 140000 Absorbance (mAU) 120000 100000 pH 7.0 80000 pH 6.5 60000 pH 6.0 40000 pH 5.5 20000 pH 5.0 0 -20000 0 2 4 6 8 10 Time (min) 12 14 16 18 20 Figure S1. 160000 140000 Absorbance (mAU) 120000 100000 50 mM, 53 µA 80000 40 mM, 41 µA 60000 30 mM, 31 µA 40000 20 mM, 22 µA 20000 10 mM, 12 µA 0 -20000 0 1 2 3 4 5 6 7 8 Time (min) Figure S2. Page S-3 9 10 11 12 13 14 Absorbance (mAU) 180000 (A) 160000 140000 120000 100000 80000 60000 15 % 40000 10 % 20000 5% 0 -20000 0 2 4 6 8 10 12 14 16 18 20 25000 (B) 20000 15000 10000 5000 0 -5000 0 2 4 6 8 10 12 14 16 18 20 250000 200000 (C) 150000 100000 50000 0 0 -50000 2 4 6 8 10 Time (min) Figure S3. Page S-4 12 14 16 18 20 300000 200000 150000 20 kV 17 kV 15 kV 13 kV 10 kV 5 kV 100000 50000 0 0 2 4 6 8 10 12 14 16 18 20 Time (min) -50000 Figure S4. 200000 Absorbance (mAU) Absorbance (mAU) 250000 150000 100000 5sec 4sec 3sec 2sec 1 sec 50000 0 0 -50000 5 10 Time (min) Figure S5. Page S-5 15 20 Absorbance (mAU) 200000 150000 100000 30 ◦C 25◦C 20 ◦C 50000 0 0 5 10 15 20 Time (min) -50000 Figure S6. Absorbance (mAU) 240000 190000 275 nm 260 nm 140000 250 nm 245 nm 90000 240 nm 230 nm 220 nm 40000 210 nm 203 nm -10000 0 5 10 Time (min) Figure S7. Page S-6 15 20 12000 11000 ALS 10000 Absorbance (mAU) 9000 8000 7000 6000 AML 5000 4000 3000 IS HCZ 2000 1000 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 Time (min) 6 6.5 7 7.5 8 8.5 9 7.5 8 8.5 9 9.5 10 Figure S8. 20000 ALS 18000 Absorbance (mAU) 16000 14000 12000 10000 AML 8000 HCZ 6000 IS 4000 2000 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 Time (min) Figure S9. Page S-7 6 6.5 7 9.5 10 Table S1. Performance data for the determination of the studied drugs by the proposed CZE method AML HCZ ALS 1-10 2.5-25 30-300 Intercept (a) -0.0009 0.1298 0.0846 Slope (b) 0.0110 0.0203 0.0122 Correlation coefficient (r) 0.9999 0.9999 0.9998 S.D. of residuals (Sy/x) 0.0005 0.0026 0.0277 S.D. of intercept (Sa) 0.0004 0.0021 0.0215 S.D. of slope (Sb) 0.0001 0.0002 0.0001 Percentage relative standard deviation, % RSD 0.79 0.67 0.80 Percentage relative error, % Error 0.32 0.27 0.33 Limit of detection, LOD (µg/mL) 0.11 0.33 5.83 Limit of quantitation, LOQ (µg/mL) 0.33 1.01 17.65 Parameter Linearity range (µg/mL) Page S-8 Table S2. Robustness data for the determination the studied drugs by the proposed CZE method Migration times (min.) Parameters AML ALS HCZ AML ALS HCZ 4.20 4.40 5.40 0.066 2.976 0.538 5.82 4.33 4.51 5.59 0.065 2.961 0.534 6.21 4.05 4.25 5.15 0.067 2.979 0.538 35 4.10 4.25 5.20 0.064 2.969 0.537 45 4.25 4.80 5.55 0.069 2.994 0.528 15 4.76 4.98 6.13 0.076 2.606 0.524 20 3.46 3.62 4.43 0.076 2.645 0.561 23 4.40 4.65 5.83 0.068 2.805 0.537 27 4.08 4.25 4.95 0.066 2.928 0.585 Standard Buffer pH Buffer Peak area ratios concocentration (mM) Applied voltage (kV) Capillary temp. (oC) N.B. Each result is the average of three separate determinations. Page S-9 Table S3. Assay results for the determination of the studied drugs in their synthetic mixture and reference methods Ratio Compound Amount taken (g/mL) Amount found (g/mL) % Found % Found 3.14 3.15 100.29 100.52 7.50 7.50 99.97 99.35 10.00 10.01 100.06 100.24 Mean ± S.D. 100.11± 0.17 100.04± 0.61 % RSD 0.17 % Error 0.10 t 0.22 (2.36) F 1.68 (19.29) AML 5.65 5.61 99.26 99.18 13.50 13.71 101.58 100.84 18.00 18.05 100.27 99.71 Mean ± S.D. 100.37± 1.17 99.91± 0.85 % RSD % Error 1.16 1.0 : 1.8: 23.9 m/m/m HCZ AML: HCZ: ALS Comparison method [46] Proposed method t 0.67 0.18(2.36) F 1.62 (5.79) 75.00 73.94 98.59 98.63 179.25 177.94 99.27 101.05 239.00 238.07 99.61 99.82 Mean ± S.D. 99.16± 0.52 99.83± 2.21 % RSD % Error 0.52 t 0.65(2.36) F 2.27 (5.79) ALS 0.30 N.B. Each result is the average of three separate determinations. The values between parentheses are the tabulated t and F values at P = 0.05 [58]. Page S-10