Ultron ES-VM : HPLC HPLC 28,000, 1-120Å 5-µm Ultron ES-VM [1-] Ultron ES-VM HPLC ph pi 3.8 4.3 Ultron ES-VM ph pi ph pka = 4.4 ph 3.0 6.0 pka = 4.0 9.2 k' 1 αph 1 Ibuprofen H3 C CHCH 2 CH CH H 3 C Chlorpheniramine pka = 4.4 H3 C H 3 C CHCH 2 CH C _ Cl + H C H + CH 2 CH 2 H pka = 4.0 Cl C H + CH 2 CH 2 H pka = 9.2 Cl C H CH 2 CH 2 Hexobarbital _ H 3 C H pka = 8.2 H 3 C 1.
20 mm 0/5 ph 3.0 6.0 k' 1 α= k' 2/k' 1 2 3 ph 3.0 ph 4.0 4.6pH 5.5 2 ph 3.0 6.0 k' 1 2 phk' 1 ph 3.0 phpi 3α ph α ph 2 α 60 50 40 k 30 20 Enantiomeric samples Ibuprofen Chlorpheniramine Hexobarbital ph 2 ph 0 2 3 4 5 ph 6 7 3 2. : Column: Ultron Ultron ES-VM ES-VM : Column size: 6.0 150 mm 150 mm : Mobile phase: 20 mm 20 mm KH 2P KH 4 2 (ph P 4 4.6)/C (ph 4.6)/C 2H 5H 2 H(0/20 5 H (0/20 v/v) v/v) : Pressure: 25 ºC1 kg/cm 2 : Temperature: UV-220 25 C nm ph k' 2 ph 4.0 4.6 k' 1pKa 4.4 ph ph 5.0 6.0 k' 1 ph 5.0 6.0 k' 1 ph 3.0 k' 1 ph 3. α 2 1 0 2 3 4 5 ph Enantiomeric samples Ibuprofen Chlorpheniramine Hexobarbital 6 7 : Column: Ultron Ultron ES-VM ES-VM : Column size: 6.0 150 mm 150 mm : Mobile phase: 20 mm 20 mm KH 2P KH 4 2 (ph P 4 4.6)/C (ph 4.6)/C 2H 5H 2 H(0/20 5 H (0/20 v/v) v/v) : Pressure: 25 ºC1 kg/cm 2 : Temperature: UV-220 25 C nm ph α 2
pka = 3.9pKa = 4.0 9.2 4 3 ph 5.55 20 70 mm / 0/ 20 70 mmk' 5 mm k' 20 mm Ultron ES-VM k' α ph 4.6 4 pka = 8.95 v/v > > > Acetonitrile Ethanol 30 20 Enantiomeric samples First 1 chlorpheniramine Second 2 chlorpheniramine First 1 ketoprofen Second 2 ketoprofen Isopropanol Methanol k 0 0 20 40 60 Phosphate (mm) buffer 80 Time (min) Time (min) Time (min) Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 6.0 150 mm 150 mm : Mobile phase: 20 mm (ph 20 phosphate buffer 4.6)/ (0/ (ph 5.5)/ethanol v/v) : 1.0 ml/min (0/ v/v); buffer concentration = 5,, 70 : Flow rate: 25 ºC1.0 ml/min : Pressure: UV-220 1 nm kg/cm 2 Temperature: 25 C : Column: Ultron Ultron ES-VM ES-VM : Column size: 6.0 150 mm 150 mm : Mobile phase: 20 mm (ph 20 phosphate buffer 4.6)/ (0/ (ph 4.6)/organic modifier v/v) : 1.0 ml/min (0/ v/v) : Flow rate: 25 ºC1.0 ml/min : Pressure: UV-220 1 nm kg/cm 2 Temperature: 25 C 4. 5. 3
1 5 v/v 0/5 0/ v/v 20 mm ph 4.6 k' 2 k' [5] 1. Ultron ES-VM 5 5 k 1 α k 1 α k 1 α k 1 α a 24.76 1.45 12.38 1.40 24.29 1.25 3. 1.06 a 4. 2.60 1.60 1.93 3.25 2.00 2.00 1.00 a 1.70 1.52 1.15 1.23 a 20 mmph 4.6 v/v 2. k 1 α k 1 α a 12.40 1.40 13.20 1.16 a 8.00 2.14 7.80 1.46 a 2.19 1.59 1.85 1.42 a 20 mmph 4.60/ v/v0/7 v/v b 20 mmph 5.50/ v/v0/8 v/v c 20 mmph 6.00/5 v/v0/4 v/v 4
Ultron ES-VM 20 mm ph 4.60/ v/v 5 40 3 3 k' α Rs k' α 19 ES-VM 3. a 5 C 11 C 19 C 28 C 40 C k 1 18.67 11.49 7.79 4.97 2.59 k 2 23.94 14.59 9.68 6.05 3.06 α 1.28 1.27 1.25 1.22 1.18 1 446 722 59 1224 1899 2 380 563 798 890 1282 R s 1.12 1.35 1.44 1.21 1.08 a 20 mmph 4.60/ v/v Ultron ES-VM 6 ph 4.6 20 mm /0/20 v/v18 25 α ph 4.6 20 mm / 0/ v/v 15 7 [8] 18 C 22 C 25 C t 1 = 9.65 t 1 = 8.28 t 1 = 7.29 t 2 = 19.93 t 2 = 16.35 t 2 = 14.13 Time (min) Time (min) Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 6.0 150 mm 150 mm : Mobile phase: 20 mm (ph 20 phosphate buffer 4.6)/ (0/20 (ph 4.6)/acetonitrile (0/20 v/v) v/v) : Pressure: 18,22,25ºC 1 kg/cm 2 : Temperature: UV-220 Varied nm 6. HPLCUltron ES-VM 4.6 150 mm Ultron ES-VM 7 0.5 µl 4.6 150 mm Ultron ES-VM 1.5 3 nmol/g 15 30 [5] 2 nmol 1 µg HPLC 1 50 µl 25 µl [4] HPLC k' k' 5
Sample loading amount 0.75 µg 1.5 µg 5 µg µg 15 µg 20 Time (min) : Ultron ES-VM : Column: 4.6 150 Ultron mm ES-VM : Column size: 20 mm 4.6 KH 150 mm 2P 4 (ph 5.5)/C 2H 5H (0/4 v/v) : Mobile phase: 1.0 ml/min 20 mm KH 2 P 4 (ph 5.5)/C 2 H 5 H (0/4 v/v) Flow rate: 1.0 ml/min : Temperature: Ambient : UV-220 nm : 5-uL Injection volume: 5-uL loop : Sample: Tolperison 7. HPLC 2 Ultron ES-VM 8 2 1.0 ml/min Plate height cm cm 0.16 0.14 0.12 0. 0.08 0.06 0.04 0.02 4.6 mm id 6.0 mm id 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Mobile phase flow rate 8. Ultron ES-VM 150 mm 6
Ultron ES-VM 9 25 20 mm [5] ph 3.0α ph 3.0 4.6pH ph 4.0 ph 4.6 ph 7.0 0.02 M (ph 3.0) () ( ) ph 4.0 1) ( 50%) 2) ( ) () 1) 2) ( ) ph 4.0 ( ) () 0.02 M (ph 4.6) () ( ) ph 4.6 7.0 1) ( 50%) 2) ( ) () 1) 2) ( ) ph 4.6 7.0 ( ) () : 2- (20 mm):kh 2P 4/K 2HP 4 HCH/HCH 4 CH/Ca C 3H 4H(CH) 3KH 2P 4 7
49 43 Ultron ES-VM 4 5 4. Ultron ES-VM Rs 20 mm (ph 4.6)- (0/7.5 v/v) 2.74 20 mm (ph 4.6)- (0/25 v/v) 6.06 Bay K 8644 20 mm (ph 4.6)- (0/25 v/v) 5.92 20 mm (ph 4.6)- (0/20 v/v) 3.27 20 mm (ph 4.6)- (0/ v/v) 8.41 20 mm (ph 4.6)- (0/ v/v) 3.17 20 mm (ph 6.0)- (0/3 v/v) 3.08 20 mm (ph 5.5)- (0/ v/v) 1.26 20 mm (ph 4.6)- (0/ v/v) 6.48 20 mm (ph 5.5) 2.23 20 mm (ph 5.0)- (0/5 v/v) 2.36 20 mm (ph 5.5)- (0/3 v/v) 2.34 20 mm (ph 4.6)- (0/15 v/v) 2.85 20 mm (ph 4.6)- (0/15 v/v) 4.33 1,2-20 mm (ph 5.5) 1.74 20 mm (ph 5.5)- (0/ v/v) 2.04 20 mm (ph 5.5)- (0/5 v/v) 1.15 20 mm (ph 4.6) 1.42 20 mm (ph 3.0)- (0/30 v/v) 1.26 20 mm (ph 4.6)- (0/ v/v) 1.36 20 mm (ph 4.6) 1.73 20 mm (ph 5.5)- (0/5 v/v) 1.70 20 mm (ph 4.6)- (0/ v/v) 3.04 20 mm (ph 4.6)- (0/15 v/v) 2.15 20 mm (ph 3.0)- (0/ v/v) 1.73 20 mm (ph 3.0)- (0/ v/v) 1.37 20 mm (ph 4.6)- (0/35 v/v) 3.71 20 mm (ph 4.6) 1.40 20 mm (ph 4.6)- (0/ v/v) 1.70 20 mm (ph 5.7) 1.13 20 mm (ph 5.5)- (0/ v/v) 1.38 20 mm (ph 5.5)- (0/3 v/v) 2.04 20 mm (ph 3.0)- (0/8 v/v) 1.01 20 mm (ph 5.0)- (0/15 v/v) 1.02 20 mm (ph 4.6)- (0/25 v/v) 3.31 20 mm (ph 4.6)- (0/20 v/v) 1.32 20 mm (ph 4.6)- (0/20 v/v) 0.98 20 mm (ph 6.8)- (0/30 v/v) 1.24 20 mm (ph 5.5)- (0/30 v/v) 0.98 20 mm (ph 5.5)- (0/ v/v) 1.50 20 mm (ph 4.6)- (0/ v/v) 5.16 20 mm (ph 4.6)- (0/30 v/v) 3.69 20 mm (ph 4.6)- (0/5 v/v) 1.49 8
ph 2 7.5 3 7 Ultron ES-VM 50 20 40 50 20 Ultron ES-VM 6.0 150 mm 1. T. Miwa, T. Miyakawa, and M. Kayano, Chem. Pharm. Bull., 35 (1987) 682. 2. T. Miwa, T. Miyakawa, and M. Kayano, J. Chromatogr., 408 (1987) 316. 3. M. kamoto and H. akzawa, J. Chromatogr., 504 (1990) 445. 4. Y. Hu and D. Kupfer, Drug Metabolism and Disposition, 30 (2002) 1329. 5. K. M. Kirkland, K. L. eilson, D. A. McCombs, and J. J. DeStefano, LC-GC, (1992) 322. 6. K. Ishii, S. Wakamoto, H. akai, and T. Sata, Chromatographia, 43 (1996) 413. 7. K. M. Kirkland, K. L. eilson, and D. A. McCombs., J. Chromatogr., 545 (1991) 43. 8. Y. Hu and D. Kupfer, Drug Metabolism and Disposition, 30 (2002) 1329. 9. J. Haginaka, J. Wakai, K. Takahashi, H. Yasuda, and T. Takagi, Chromatographia, 29 (1990) 587.. P. Bonato, R. Bortocan, C. Gaitani, F. Paias, M. Ina, and R. Lima, J. Braz. Chem. Soc., 13 (2), 2002. www.agilent.com/chem/jp S CH2 CH 20 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 6.0 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2P KH 4 2 (ph P 4 = (ph 4.6)/C = 4.6)/C 2H 5H 2 H= 5 H 0/20 = 0/20 v/v v/v : Pressure: 25 ºC1 kg/cm 2 : Temperature: UV-220 25 nm C. 9
Antiinflammatory Fenoprofen calcium _ C CH Ca 3+ 2H 2 () Cholinergic blocking (parasympatholytic) drugs Mepenzolate bromide C C Br _ Antiinflammatory Pranoprofen CH CH H + H 3 C 20 30 40 Time (min) Column: Ultron ES-VM : Ultron ES-VM Column size: 6.0 150 mm : 6.0 150 mm Mobile phase: 20 mm KH 2 P 4 (ph 5.6)/ : 20 mm KH C 2 H 5 H 2P (0/ 4 (ph 5.6)/ v/v) C Flow rate: 2H 5H (0/ v/v) 1.0 ml/min : 1.0 ml/min Temperature: 27 C : Detection: 27 ºC UV-220 nm (0.04 AUFS) : UV-220 nm (0.04 AUFS) 50 20 30 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 mm KH 2P KH 42 P (ph 4 (ph = 4.6) = 4.6) : Temperature: 27 ºC27 C : Detection: UV-220 UV-220 nm (0.04 nm (0.04 AUFS) AUFS) 20 30 Time (min) Column: Ultron ES-VM : Column size: Ultron 4.6 ES-VM 150 mm : Mobile phase: 4.6 150 20 mm mm KH 2 P 4 (ph 6.0)/ : 20 mm C 2 H KH 5 H 2P (0/5 4 (ph 6.0)/ v/v) Flow rate: C 2H 5H 1.0 (0/5 ml/min v/v) : Temperature: 1.0 ml/min 27 C : Detection: 27 ºC UV-220 nm (0.04 AUFS) : UV-220 nm (0.04 AUFS) Antihistamines Promethazine hydrochloride Local Anesthetic Bupivacaine Antiparkinson drugs Trihexyphenidyl S CH 2 CH 2 C H HCI CH 2 CH HCI H C (CH 2 ) 3 20 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 mm KH 2P KH 42 (ph P 4 4.6)/ (ph 4.6)/ C 2H 5H C 2 H 5 (0/20 H (0/20 v/v) v/v) : Temperature: Ambient : Detection: UV-220 UV-220 nm (0.04 nm (0.04 AUFS) AUFS) 20 Time (min) Column: : Ultron Ultron ES-VM ES-VM Column : size: 4.6 150 mm 150 mm Mobile : phase: 20 mm 20 mm KH 2P KH 42 P (ph 4 (ph 5.5)/ 5.5)/ C C (0/ (0/ v/v) v/v) Flow : rate: 1.0 ml/min 1.0 ml/min Temperature: : 25 ºC25 C Detection: : UV-220 UV-220 nm (0.04 nm (0.04 AUFS) AUFS) 5 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2P KH 4 2 (ph P 4 4.6)/ (ph 4.6)/ C 2H 5H C 2 H(0/ 5 H (0/ v/v) v/v) : Temperature: Ambient : Detection: UV-220 UV-220 nm (0.04 nm (0.04 AUFS) AUFS)
Antipsychotics Thioridazine hydrochloride Diuretic drugs Ethiazide Skeletal muscle relaxants Eperisone hydrochloride S S CH 2 CH 2 H 2 Cl S H S H C2H 5 C H H 3 CH 2 C C CH CH 2 HCl HCI 20 30 40 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2PKH 4 (ph 2 P5.5)/ 4 (ph 5.5)/ C CH(0/30 3 C (0/30 v/v) v/v) : Temperature: 31 ºC 31 C : Detection: UV-254 UV-254 nm (0.04 nm AUFS) (0.04 AUFS) 20 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2PKH 4 (ph 2 P 4.6) 4 (ph 4.6) : Temperature: 25 ºC25 C : Detection: UV-220 UV-220 nm (0.04 nm (0.04 AUFS) AUFS) 20 Time (min) : Column: Ultron Ultron ES-VM ES-VM : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2P KH 4 (ph 2 P5.5)/ 4 (ph 5.5)/ C 2H 5H C 2 H(0/5 5 H (0/5 v/v) v/v) : Temperature: 25 ºC 25 C : Detection: UV-220 UV-220 nm (0.04 nm AUFS) (0.04 AUFS) 1,2-1,2-Diphenylethylamine Prenylamine lactate 2-2-Phenyl propionic acid CH (CH 2 ) 2 H CH CH 2 H 3 C CH CH CH 2 CH H 2 20 30 40 Time (min) 20 30 Time (min) Column: Ultron ES-VM Column : size: Ultron 4.6 ES-VM 150 mm Mobile : phase: 4.6 150 20 mm mm KH 2 P 4 (ph 5.5) Flow : rate: 20 mm 1.0 ml/min KH 2P 4 (ph 5.5) Temperature: : 1.0 ml/min 25 C Detection: : 25 ºC UV-220 nm (0.04 AUFS) : UV-220 nm (0.04 AUFS) Column: Ultron ES-VM Column size: 4.6 150 mm : Ultron ES-VM Mobile phase: 20 mm KH 2 P 4 (ph 5.0)/ : 4.6 150 mm C (0/15 v/v) : 20 mm KH 2P 4 (ph 5.0)/ Flow rate: 1.0 ml/min C (0/15 v/v) Temperature: 25 C : 1.0 ml/min (0.04 AUFS) : 25 ºC : UV-220 nm (0.04 AUFS) 20 Time (min) : Column: Ultron ES-VM Ultron ES-VM : Column size: 4.6 150 4.6 mm 150 mm : Mobile phase: 20 mm 20 mm KH 2 P 4 (ph 4.6) KH2P4 (ph 4.6) : Temperature: 25 ºC 25 C : Detection: UV-220 UV-220 nm (0.04 nm AUFS) (0.04 AUFS) 11
() rnidazol (Antibiotic) a 2 P a 20.208 23.128 () Bicalutamide (Antiandrogen) CF 3 H 3 C F S 2 CH 2 C C C H H 8.060 15.200 aftopidil (Alpha1a-adrenergic (α1a-) receptor antagonist) H CH 2 CHCH 2 H 3 C 7.404.193 0 20 30 Minute Column: Ultron ES-VM (5 µm) : Ultron ES-VM (5 µm) Column size: 4.6 150 mm : 4.6 150 mm Mobile phase: 20 mm KH 2 P 4 (ph 3.0)/ : 20 mm KH C 2P (88/12 4 (ph 3.0)/ v/v) CH Flow rate: 3C (88/12 v/v) 1.0 ml/min : 1.0 ml/min Temperature: 25 C : 25 ºC Sample: : UV-220 rnidazol nm disodium phosphate : 0.2 mg/ml Injection vol: 0.2 mg/ml 3 µl : 3 µl 0 5 15 20 25 Minute : Column: Ultron Ultron ES-VM ES-VM (5 µm) (5 µm) : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 mm KH 2P KH 42 (ph P 4 4.6)/ (ph 4.6)/ C (88/12 C (88/12 v/v) v/v) : Temperature: 25 ºC25 C : Detection: UV-220 UV-220 nm nm : Sample: CH Bicalutamide in 33C 0.1 mg/ml 0.1 mg/ml : Injection vol: 3 µl3 µl 0 5 15 Minute Column: : Ultron Ultron ES-VM ES-VM (5 µm) (5 µm) Column : size: 4.6 150 150 mm mm Mobile : phase: 20 mm 20 mm KH 2P KH 42 P (ph 4 (ph 7.0)/ 7.0)/ C (75/25 C (75/25 v/v) v/v) Flow : rate: 1.0 ml/min 1.0 ml/min Temperature: : 25 ºC 25 C Detection: : UV-220 UV-220 nm nm Sample: : 0.5 aftopidil mg/ml Injection : vol: 1 µl1 µl 20 Cetirizine hydrochloride ( ) (Antihistamine) Cl CH CH 2 CH 2 CH 2 CH 2HCI.700 12.595 Fulvastatin sodium (HMG-CoA ) (HMG-CoA reductase inhibitor) H Ca F 34.546 39.913 Amlodipine besilate (Calcium channel blocker) ( ) H CH 2 CH 2 CH 2 H 2 C CC 2 H 5 6.783 11.331 Cl. S 3 H 0 5 15 Minute Column: Ultron ES-VM (5 µm) : Column size: Ultron 4.6 ES-VM 150 mm (5 µm) : Mobile phase: 4.6 150 20 mm mmkh 2 P 4 (ph 4.6)/ : 20 mm CHKH 3 C 2P (90/ 4 (ph 4.6) v/v) Flow rate: /C 1.0 ml/min (90/ v/v) : Temperature: 1.0 ml/min 25 C : Detection: 25 ºCUV-235 nm : Sample: : UV-235 Cetirizine nm hydrochloride 0.2 mg/ml mg/ml : Injection vol: 2 µl 2 µl 20 0 20 30 40 50 60 Minute Column: : Ultron Ultron ES-VM ES-VM (5 µm) (5 µm) Column : size: 4.6 150 150 mm mm Mobile : phase: 20 mm 20 mm KH 2P KH 4 2 P (ph 4 (ph 3.0)/ 3.0)/ C C (88/12 (88/12 v/v) v/v) Flow : rate: 1.0 1.0 ml/min ml/min Temperature: : 25 ºC 25 C Detection: : UV-235 UV-235 nm nm Sample: : Fulvastatin sodium 0.5 mg/ml 0.5 (Solvent: mg/ml ( : ) methanol) Injection : vol: 1 µl1 µl 0 5 15 20 Minute Column: : Ultron Ultron ES-VM ES-VM (5 µm) (5 µm) Column : size: 4.6 150 150 mm mm Mobile : phase: mm mm a 2HP a 2 HP 4 (ph 4 (ph 7.0)/ 7.0)/ C CH (78/22 3 C (78/22 v/v) v/v) Flow : rate: 1.0 ml/min 1.0 ml/min Temperature: : 25 ºC 25 C Detection: : UV-237 UV-237 nm nm Sample: : 0.02 Amlodipine besilate mg/ml ( : ) 0.02 mg/ml (Solvent: Methanol) Injection : vol: 2 µl2 µl 12
Fexofenadine hydrochloride ( ) (Antihistaminic) Sodium rabeprazole ( ) (Proton pump inhibitor) H H C (CH 2 ) 3 CH C CH HCl H 2 CH 2 CH 2 CH 3 C CH 2 S a 7.313 8.718 22.587 27.812 0 4 8 12 16 : Column: Ultron Ultron ES-VM ES-VM (5 µm) (5 µm) : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2P KH 4 (ph 2 P4.6)/ 4 (ph 4.6)/ C CH(95/5 3 C (95/5 v/v) v/v) : Temperature: 25 ºC25 C : Detection: UV-220 UV-220 nm nm : Sample: Fexofenadine hydrochloride 0.3 mg/ml 0.3 mg/ml : Injection vol: 1 µl 1 µl 0 20 30 40 Minute : Column: Ultron Ultron ES-VM ES-VM (5 µm) (5 µm) : Column size: 4.6 150 mm 150 mm : Mobile phase: 20 mm 20 KH mm 2P KH 4 (ph 2 P6.0)/ 4 (ph 6.0)/ C CH(95/5 3 C (95/5 v/v) v/v) : Temperature: 25 ºC25 C : Detection: UV-225 UV-225 nm nm : Sample: Sodium rabeprazole 0.2 mg/ml 0.2 mg/ml : Injection vol: 3 µl 3 µl 13
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