1 WSV SIR m/z Analyte RT (Min) SIR m/z Cone voltage (V) Ascorbic Acid (C) Thiamine (B1) Nicotinic Acid (B3) Pyridoxal (

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1 ACQUITY UPLC H-Class ACQUITY QDa Mark E. Benvenuti, Dimple Shah, and Jennifer A. Burgess Waters Corporation, Milford, MA, USA MS ACQUITY QDa UPLC HPLC UV ACQUITY QDa LC EC 1925/26 CFR 21 HPLC 1 LC-MS ACQUITY QDa 12 WSV ACQUITY UPLC H-Class ACQUITY UPLC QDa ACQUITY UPLC H-Class ACQUITY QDa WSV QDa 1

2 1 WSV SIR m/z Analyte RT (Min) SIR m/z Cone voltage (V) Ascorbic Acid (C) Thiamine (B1) Nicotinic Acid (B3) Pyridoxal (B6) Nicotinamide (B3) Pyridoxine (B6) Ca_Pantothenate (B5) Cyanocobalamin (B12) Folic Acid (B9) B2-5-Phosphate Biotin (B7) Riboflavin (B2) SIR Standard Individual B complex vitamins (mg/l ) Vitamin C (mg/l) WSV 1 mg/ml B2 B7 B9 98 µl 1N NaOH 2 µl C ph3.2 C 1.25 ml 25 ml 25 ml C 5 ppm 1 ppm g 1 ml.2 µm PVDF g 1 ml 15.2 µm PVDF µm PVDF 2. B C 2

3 UPLC LC ACQUITY UPLC H-Class 17.5 ACQUITY UPLC HSS T3 1.8 µm mm A 1 mm.1 B 1 mm.1 5 µl A B ml/ 1. Initial ACQUITY UPLC PDA 21~4 nm 27 nm 1 / 2 ACQUITY QDa ESI kv MS 5~8 m/z SIR 5 Hz 15 V 1 SIR SIR m/z 2 3 3

4 MS m/z 2 2 D F m/z=442 m/z =678 2 UV 2A 2H UV 3 MS Ascorbic Acid C 2- Thiamine B1 3- Nicotinic acid B3 4- Pyridoxal B6 5- Nicotinamide B3 6- Pyridoxine B6 7- Ca_Pantothenate B5 8- Cyanocobalamin B12 9- Folic acid B9 1- Riboflavin 5 phosphate 11- Biotin B7 12- Riboflavin B SIR 4

5 Compound name: Biotin (B7) Correlation coefficient: r = , r 2 = Calibration curve: * x type: External Std, Area A Compound name: Thiamine (B1) Correlation coefficient: r =.99633, r 2 = Calibration curve: * x type: External Std, Area B Compound name: Riboflavin (B2) Correlation coefficient: r = , r 2 = Calibration curve: * x type: External Std, Area C Compound name: Folic Acid (B9) Correlation coefficient: r = , r 2 = Calibration curve: * x type: External Std, Area D Compound name: Riboflavin 5 phosphate Correlation coefficient: r = , r 2 = Calibration curve: * x type: External Std, Area E Compound name: Cyanocobalamin ( B12) Correlation coefficient: r = , r 2 = Calibration curve: * x type: External Std, Area F Conc Compound name: Nicotinic Acid (B3) Coefficient of Determination: r 2 = Calibration curve: * x * x type: External Std, Area Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans: None G Compound name: Ca_Pantothenate (B5) Correlation coefficient: r = , r 2 = Calibration curve: * x type: External Std, Area H

6 MS UV µg/l SIR UV 3A 27 nm 3A UV MS 5.e-4 A: UV at 27 nm RT 2.46, S/N = 63 B: Pyridoxine SIR m/z RT 1.75, S/N = 32 C: Pyridoxal SIR m/z RT 1.24, S/N = 19 D: Nicotinic Acid SIR m/z RT 2.43, S/N = 33 E: Nicotinamide SIR m/z Time µg/ml 27 nm UV SIR SIR 4 B C D E 6

7 4 2 B5 UV UV B1 UV 1 5 B1 C C UV B1 5A ACQUITY QDa SIR B1 A B C MS Vitamin water 1 PDA Vitamin water 1 MS Vitamin water 2 7: SIR of 1 Channel ES+ 242 (Ca_Pantothenate (B5)) 14: Diode Array 7: SIR of 1 Channel ES+ 242 (Ca_Pantothenate (B5)) D 2. PDA Vitamin water 2 14: Diode Array Time 4. 2 B5 5.9 MS A C UV B D 1 Thiamine, 1.1 RT 1.1 A: SIR of thiamine (B1) Ascorbic acid,.91 RT.91 B: SIR of ascorbic acid(c) e-1 Ascorbic acid,.9 RT.9 C: UV at 27 nm 1.e Time A B1 SIR B C SIR C C 27 nm UV B1 UV 7

8 2 4 UPLC 6 UV 6B B12 ACQUITY QDa LC A: SIR of cyanocobalamin : SIR of 1 Channel ES+ 678 (Cyanocobalamin (B12) B: UV at 27 nm (1) PDA Ch1 27 nm at 1.2 nm 1.e-2 8.e-3 6.e-3 4.e-3 2.e-3 Time B12 A UV B 8

9 1 6 µg B12 16 mg B3 LC-MS 7 7A 7B B2 1 SIR 7C B12 SIR 27 nm UV 7D 12.5 ppm A 1 Diluted x SIR at m/z 377 Riboflavin (B2) B Undiluted 7.71 SIR at m/z 377 Riboflavin (B2) C 1 Undiluted SIR at m/z 678 Cyanocobalamin (B12) D UV at 27nm 2.e-1 Undiluted 1.e Time A B12 C B UV D 9

10 B RSD=.6 25 mg/l RSD=1 Analyte RSD for retention time Thiamine (B1).6 Nicotinic Acid (B3).19 Pyridoxal (B6).23 Nicotinamide (B3).22 Pyridoxine (B6).26 Ca_Pantothenate (B5) 4 Cyanocobalamin ( B12) 3 Folic Acid (B9) 3 Riboflavin 5 phosphate 3 Biotin (B7) 2 Riboflavin (B2) mg/l 1 25 mg/l 1 Analyte RSD for area Thiamine (B1) 6.78 Nicotinic Acid (B3) 2.35 Pyridoxal (B6) 2.62 Nicotinamide (B3) 2.24 Pyridoxine (B6) 2.65 Ca_Pantothenate (B5) 4.6 Cyanocobalamin ( B12) 7. Folic Acid (B9) Riboflavin 5 phosphate Biotin (B7) 2.77 Riboflavin (B2) mg/l 1 B 1

11 UV ACQUITY QDa SIR ACQUITY QDa 1. G F M Ball (Editor). Bioavailabilty and Analysis of Vitamins in Foods. Chapman and Hall. (1998). Ch2, pp E Riches. The Rapid Simultaneous Analysis of 12 Water Soluble Vitamin Compounds. Waters Application Note No en, June, Heudi et al. Separation of water-soluble vitamins by reversed-phase high performance liquid chromatography with ultra-violet detection: Application to polyvitaminated premixes. J Chrom A. (25) 17: 49 56, pp AOAC Method 211.9: Vitamin B12 in Infant Formula and Adult Nutritionals. (212). UV LC-MS Empower 3 Mass Lynx 2 LC ACQUITY QDa TEL FAX F TEL FAX Waters ACQUITY UPLC UPLC Empower MassLynx QDa T he Science of What s Possible Waters Corporation 214 Waters Corporation. Printed in Japan JA PDF

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