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
1 WSV SIR m/z Analyte RT (Min) SIR m/z Cone voltage (V) Ascorbic Acid (C).91 177 2 Thiamine (B1) 1.1 265 5 Nicotinic Acid (B3) 1.27 124 15 Pyridoxal (B6) 1.75 168 5 Nicotinamide (B3) 2.48 123 15 Pyridoxine (B6) 2.5 17 1 Ca_Pantothenate (B5) 5.88 242 15 Cyanocobalamin (B12) 7.17 678 2 Folic Acid (B9) 7.22 442 5 B2-5-Phosphate 7.35 457 5 Biotin (B7) 7.5 245 1 Riboflavin (B2) 7.74 377 15 1. SIR Standard Individual B complex vitamins (mg/l ) Vitamin C (mg/l) 1 1. 5 2.75 37.5 3.5 25. 4.25 12.5 5.1 5. 6 75 3.75 7 5 2.5 8 25 1.25 9 1.5 1 5.25 11 1 5 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 2 11 1 8.5 g 1 ml.2 µm PVDF 25 1 2 3 1.34 g 1 ml 15.2 µm PVDF 1 1 2 2 2.2 µm PVDF 2. B C 2
UPLC LC ACQUITY UPLC H-Class 17.5 ACQUITY UPLC HSS T3 1.8 µm 2.1 1 mm A 1 mm.1 B 1 mm.1 5 µl A B ml/ 1. Initial.45 99 1 2. 3..45 99 1 3. 3.1.45 95 5 4. 5.1.45 8 2 5. 7.1.45 2 98 6. 9..45 2 98 7. 9.1.45 99 1 8 17.5.45 99 1 1 ACQUITY UPLC PDA 21~4 nm 27 nm 1 / 2 ACQUITY QDa ESI+ 8 6.8 kv MS 5~8 m/z SIR 5 Hz 15 V 1 SIR SIR m/z 2 3 3
12 1 8 2 2.5 7.25 MS m/z 2 2 D F m/z=442 m/z =678 2 UV 2A 2H UV 3 MS 2 4 5 6 1- 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 B2 1 3 8 11 1 9 12 7 1. 8 12 SIR 4
Compound name: Biotin (B7) Correlation coefficient: r =.998455, r 2 =.996912 Calibration curve: 145.948 * x + 6.8733 type: External Std, Area A Compound name: Thiamine (B1) Correlation coefficient: r =.99633, r 2 =.992673 Calibration curve: 731.882 * x + -4517.39 type: External Std, Area B 15 6 1 5 4 2 Compound name: Riboflavin (B2) Correlation coefficient: r =.998258, r 2 =.996518 Calibration curve: 87.96 * x + -59.1265 type: External Std, Area C Compound name: Folic Acid (B9) Correlation coefficient: r =.999226, r 2 =.998452 Calibration curve: 1.7885 * x + 1.57451 type: External Std, Area D 8 1 6 4 75 5 2 25 Compound name: Riboflavin 5 phosphate Correlation coefficient: r =.999416, r 2 =.998832 Calibration curve: 9.91594 * x + 22.87 type: External Std, Area E Compound name: Cyanocobalamin ( B12) Correlation coefficient: r =.999745, r 2 =.99949 Calibration curve: 34.1976 * x + 14.9612 type: External Std, Area F 3 6 4 2 2 1 Conc Compound name: Nicotinic Acid (B3) Coefficient of Determination: r 2 =.99938 Calibration curve:.99786 * x 2 + 41.17 * x + -9.2113 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 =.999727, r 2 =.999454 Calibration curve: 58.377 * x + -59.1867 type: External Std, Area H 3 2 1 4 2 2. 5
MS UV 3 5 5 µg/l SIR UV 3A 27 nm 3A UV MS 5.e-4 A: UV at 27 nm..25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 RT 2.46, S/N = 63 B: Pyridoxine SIR m/z 17 5..25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 RT 1.75, S/N = 32 C: Pyridoxal SIR m/z 168 11..25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 RT 1.24, S/N = 19 D: Nicotinic Acid SIR m/z 124 14..25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 RT 2.43, S/N = 33 E: Nicotinamide SIR m/z 123 11 Time..25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 3.25 3.5 3.75 4. 4.25 4.5 3. 5 µg/ml 27 nm UV SIR SIR 4 B C D E 6
4 2 B5 UV UV B1 UV 1 5 B1 C C UV B1 5A ACQUITY QDa SIR B1 A B C 4.5 5. 5.5 6. 6.5 7. 7.5 2. 4.5 5. 5.5 6. 6.5 7. 7.5 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)) 4.5 5. 5.5 6. 6.5 7. 7.5 D 2. PDA Vitamin water 2 14: Diode Array 4.5 5. 5.5 6. 6.5 7. 7.5 Time 4. 2 B5 5.9 MS A C UV B D 1 Thiamine, 1.1 RT 1.1 A: SIR of thiamine (B1).5 1. 1.5 2. 2.5 1 Ascorbic acid,.91 RT.91 B: SIR of ascorbic acid(c).5 1. 1.5 2. 2.5 2.e-1 Ascorbic acid,.9 RT.9 C: UV at 27 nm 1.e-1.5 1. 1.5 2. 2.5 Time 5. 25 A B1 SIR B C SIR C C 27 nm UV B1 UV 7
2 4 UPLC 6 UV 6B B12 ACQUITY QDa LC A: SIR of cyanocobalamin 7.17 13: SIR of 1 Channel ES+ 678 (Cyanocobalamin (B12) 4 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7. 7.1 7.2 7.3 7.4 75. 7.6 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 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7. 7.1 7.2 7.3 7.4 75. 7.6 6. B12 A UV B 8
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 41 68 96 2 A 1 Diluted x 1 7.71 SIR at m/z 377 Riboflavin (B2) 6. 6.5 7. 7.5 8. 8.5 1 B Undiluted 7.71 SIR at m/z 377 Riboflavin (B2) 6. 6.5 7. 7.5 8. 8.5 C 1 Undiluted 7.17 7.55 7.85 SIR at m/z 678 Cyanocobalamin (B12) D 6. 6.5 7. 7.5 8. 8.5 7.7 UV at 27nm 2.e-1 Undiluted 1.e-1 6. 6.5 7. 7.5 8. 8.5 Time 7. 2 1 A B12 C B UV D 9
B 4 5 4 2 1 2 RSD=.6 25 mg/l 1 5 5 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) 3 4. 2.75 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) 11.53 Riboflavin 5 phosphate 14.29 Biotin (B7) 2.77 Riboflavin (B2) 2.28 5. 25 mg/l 1 B 1
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. 33. 2. E Riches. The Rapid Simultaneous Analysis of 12 Water Soluble Vitamin Compounds. Waters Application Note No. 72352en, June, 29. 3. 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. 51. 4. AOAC Method 211.9: Vitamin B12 in Infant Formula and Adult Nutritionals. (212). UV LC-MS Empower 3 Mass Lynx 2 LC ACQUITY QDa www.waters.com 141 1-3-12 5 TEL 3-3471-7191 FAX 3-3471-7118 53211 5-14-1 11F TEL 6-634-8888 FAX 6-63-1734 Waters ACQUITY UPLC UPLC Empower MassLynx QDa T he Science of What s Possible Waters Corporation 214 Waters Corporation. Printed in Japan. 214 5 72496JA PDF