効率的なHPLCメソッドの開発手法 - chem …

1 hplc LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 1 hplc z z 9 - 9 - pH 9 9 9 z z z LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 2 hplc z z OK / NG z hplc z z LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 3 hplc pH 9 9 9 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 4 pH 9 9 9 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 5pH ( pH pH pH pH LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 6 pH pH - naphthalene benzyl alcohol benzoic acidacenaphthenenitrobenzene benzanilidepH)

2 30 1 2 3 4 5 6 7 8 9 100 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 pH 70 1 2 3 4 5 6 7 8 9 100 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (min)Time (min)Time (min)LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 7 SiO_ Na++ R3NH+SiO_ +NHR3+ Na+ (SiO-) (RNH+) (SiO) (R3NH+) SiOH + RCOO_SiO_ H+ _OOCR LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 8 pH - pH LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 9 pH 2 ZORBAX Eclipse XDB-C8, x 150 mm, 5 m 25 mM Na2 HPO4/ACN = 85/15 mL/min, 35 C 1.

3 Phenylpropanolamine2. Ephedrine3. Amphetamine 4. Methamphetamine5. Phenteramine11pH TF (5%)4133pH TF (5%)423532154324. ( i ) ( i ) (min)Time (min) pH LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 10 pH 2 pH 4 4pH 3 0C(CH3)HCOOHpH (CH3)2 IbuprofenpKa= (min)0246810 Time (min)0246810 ZORBAX SB-C8 x 150 mm, 5 m 5 mM KH2PO4/ ACN = 40/60 .0 mL/minLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 11 pH pH 3 8 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 12 ZORBAX Eclipse XDB-C18, x 75 mm, mpH 33pH 71342124012345 Time (min)05101520 Time (min) 20 mM phosphate buffer/MeOH 80/20 mL/min UV 254 nm 1 Nizatidine2 Famotidine3 Cimetidine4 PirenzipineTime (min)Time (min) 1.

4 Nizatidine2. Famotidine3. Cimetidine4. PirenzipineLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 13 pH pH LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 14 pH ZORBAX Extend-C18, x 150 mm, 5 m 10mL/min UV 254 nmpH11 ODS mL/min UV, 254 nm 1. Maleate2. Scopolamine3. Pseudoephedrine4. Doxylamine5. Chlorpheniramine6. Triprolidine7. DiphenhydraminepH 7, 20 mM Na2 HPO4/MeOH=30/70pH 11, 20 mM TEA/MeOH=30/7023474712,35135tR= pH 0 5 Time (min)0 5 10 Time (min)

5 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 15 p pH BC B pKa pKapH234567 pH pKa LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 16 ZORBAX RRHT Eclipse Plus C18, x 50 mm, m A 25 mM NaH2PO4, pH B mL/min 30 C UV 240 nm 1 Pindolol 2 Disopyridamide3 Propranolol 4 Diltiazem 5 DipyridamolemAU10012014034 2. 5. Dipyridamole5120406080250% MeOH(2,3), (3,4), (4,5) 5 ACN 123-200mAU1401345608010012014030% ACN123(1,2) -200204060 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 171220 pKa pH pKa pH LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 18 pH 9 9 9 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 19 pH pH pH 9 9 C3, CN, Ph)

6 H pH 9 9 pH LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 20 p zC18 zC8C18 C18 C18 zPhenyl - zC3 C18, C8 zCN LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 21 ) C18 C8 C18 C8 AQ 100% 100% LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 22 12 ZORBAX Eclipse XDB-CN /ACN=65/35min0510152034567 /ACN 65/35 x 150 mm, 5 m 2 ml/min 2 L 25 C1234,567 ZORBAX Eclipse XDB-Phenyl /ACN = 52/48 25 C UV, 210 nm 051015205123467 ZORBAX Eclipse XDB-C8 /ACN = 46/541.

7 Estriol ( g/ l), 2. b-Estradiol ( g/ l), 3. Ethinyl Estradiol ( g/ l), 4. Dienestrol ( g/ l), 051015206741235 ZORBAX Eclipse XDB-C18 5. Diethylstilbestrol ( g/ l) 6. Ethynylestradiol 3-methyl ether ( g/ l) 7. Ethynodiol Diacetate ( g/ l)051015201267 /ACN = 40/60LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 23 hplc / ZORBAX ZORBAX ZORBAX RRHT m ZORBAX RRHT SB-C18, mm 50 mm, mZORBAX RRHT SBC18, mm 50 mm, m LC 5 m LC 5 m ZORBAX SB-C18, mm 150 mm, 5 m LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 24 C18 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 25 LC Method Dev/JAIMA Show 2008 Sep/3/2008

8 Page 26 ZORBAX SB-C8, x 150 mm, 5 m /ACN 82/18 ZORBAX SBC8, x 150 mm, 5 m /ACN82/18 30 L 1. Caffeine 2. Salicylamide 1A. 100%ACN B. 2120 10Ti( i )0 10Ti( i ) Time (min)Time (min)LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 27 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 28 Agilent 1200 LC Method Development SolutionLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 29 Agilent 1200 LC Method Development Solution 12 8 3 288 288 LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 30

9 Agilent 1200 Method Development Solution gp ChromSword AutoAutoChromLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 31 ChromSword Auto Auto (pH, , 5 25 6 10 OffLi 6 10 Off -Line Distributed byLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 32 Distributed byChromSword Auto Agilent 1200 HPLCHPLC A il t 1200 hplc ChromSword Auto AutoRobustAgilent 1200 HPLCD istributed byLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 33 Distributed byChromSword Auto Distributed by LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 34 Distributed by ChromSword Auto 9 1 9 9 9 Word 9)

10 9 9 Distributed byLC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 35 Distributed by Agilent LC Method Dev/JAIMA Show 2008 Sep/3/2008 Page 36