REVERSE PHASE CLUMN GUIDE
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Luna 5 μm Phenyl-Hexyl, 150x4.6 mm Luna 5 μm C8(2), 150x4.6 mm Luna 5 μm C18(2), 150x4.6 mm : 50:15:35 Methanol:Acetonitrile: 20mM Ammonium Formate ph 3.5 : 1.0 ml/min : 1. Rhodamine 123 2. Rhodamine B Base 3. Rhodamine 6G : DS150 4.65 μ Luna Phenyl-Hexyl150 4.65 μ : 50:50 Acetonitrile: 20 mm Potassium phosphate ph 2.5 : 1.0 ml/min : 1. Indoprofen 2. Ethyl paraben 3. Naproxen 4. Diflunisal 5. Indomethacin 6. Ibuprofen 3
S S S S S H H H H S H S H S H H S H 4 3 1 2 1 2 3 4 Dimensions: Eluent A: Eluent B: Gradient Profile: Flow Rate: Col. Temp.: Detection: 150 4.6 mm ID 50 mm Sodium Phosphate Acetonitrile Step No.Time (min) Pct A Pct B 1 ml/min ambient UV-Vis Abs.-Variable Wave.(UV) @ 210 nm (ambient) 0 2 4 6 Time (min) 1Benzoic Acid 2Diphenhydramine 3Dextromethorophan 4Phenylephrine 0 2 4 6 8 10 Time (min) 4
H H2 H2 H2 H2 H2 H2 H H H H2 H2 H2 H H2 H H H2 - H2 H H H2 H2 H2 H2 H2 H2 H H H2 H2 H2 to to 1 2 1 5 2 3 4 5 3 6 Baseline resolution of all peaks 0 2 4 6 8 min 10 6Thiamine-no elution 4 0 2 4 6 8 min 10 Dimension: 150 x 4.6 mm Mobile Phase: Acetonitrile/100 mm Ammonium Formate, ph 3.2 (90:10) Flow Rate: 1.0 ml/min Detection: UV @ 260 nm Temperature: Ambient Sample: 1. PABA 2. Nicotinamide 3. Riboflavin 4. Nicotinic Acid 5. Pyridoxine 6. Thiamine 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 800 600 400 200 Cotinine Peak Area 97,400 Elutes in ion suppression zone! Nornicotine Peak Area 4,281 Nicotine Peak Area 4,849 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 min 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 800 600 400 200 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 min Column: Gemini 5 µm C18 Dimension: 100 x 2.0 mm Part No.: 00D-4435-B0 Mobile Phase: A: Water with 0.1 % v/v Formic Acid B: Acetonitrile with 0.1 % v/v Formic Acid Gradient: A/B (97:3) hold 1 min, to (70:30) in 2 min, to (97:3) in 1 min, hold for 2.5 min Flow Rate: 0.4 ml/min Detection: Mass Spectrometer (MS) Temperature: Ambient Sample: 1. Nornicotine (MRM: 149.1/80.1) 2. Nicotine (MRM: 163.3/105.9) 3. Cotinine (MRM: 177.3/80.1) Column: Dimension: Part No.: Mobile Phase: Flow Rate: Luna 5 µm HILIC 100 x 2.0 mm 00D-4450-B0 Acetonitrile /100 mm Ammonium Formate, ph 3.2 (90:10) 0.4 ml/min Detection: Temperature: Sample: Mass Spectrometer (MS) Ambient 1. Cotinine (MRM: 177/80) 2. Nicotine (MRM: 163/80) 3. Nornicotine (MRM: 149/80) 5
100 1A : 90 %MeCN(2.5 min) MeCN 90 80 70 60 1B : Gradient (90 %50 % for 7.5 min) 1C : 50 %MeCN(2.5 min) 50 40 0 2 4 6 8 10 12 min Luna HILIC ph 3.2 2A : 100 mm Ammonium Formate, ph 3.2 2B : 100 mm Ammonium Acetate, ph 5.8 ph Luna HILIC ph 5.8 1. PABA 2. Nicotinamide 3. Riboflavin 4. Nicotinic Acid 5. Pyridoxine 6. Thiamine 7. Ascorbic Acid 8. Cyanocobalamin 9. Folic Acid Email : psc@glc.shimadzu.co.jp TEL : 03-5835-0126 6
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1. Metaproterenol 2.Pindolol 3.Metoprolol Dimensions Mobile Phase Flow Rate Detection Injection Temperature Sample : 150 x 4.6 mm : 20 mm Potassium phosphate ph 3 /Methanol (50:50) : 1.0 ml/min : UV @ 230 nm : 2 µl : 22 C : 1. Metaproterenol (0.4 µg), 2. Pindolol (0.6 µg), 3. Metoprolol (0.15 µg), 4. Alprenolol (0.3 µg), 5. Propranolol (4 µg), 6. Ethylparaben (0.4 µg), Dimensions Mobile Phase Flow Rate Detection Temperature Sample : 150 x 4.6 mm : 20 mm Potassium phosphate ph2.5/ Methanol (97:3) : 1.0 ml/min : UV @ 220 nm : 25 C : 1. Formic aci () 2. Acetic acid 8
Columns Dimensions Mobile Phase Flow Rate Detection Sample : Synergi 4 µ Fusion-RP : 150 x 4.6 mm : 20 mm Potassium Phosphate, ph2.5 / Methanol (70:30) : 1.0 ml/min : UV @ 210 nm : 1. Phenylephrine 2. Phenylpropanolamine 3. Pseudoephedrine 9
Columns Dimensions Mobile Phase Flow Rate Detection Sample : Synergi 4 µ Fusion-RP : 150 x 4.6 mm : 20 mm Potassium Phosphate, ph2.5 / Acetonitrile (75:25) : 1.0 ml/min : UV @ 210 nm : 1. Maleic acid 2. Chlorpheniramine 3. Triprolidine 4. Diphenhydramine Columns Dimensions Mobile Phase Gradient Rate Flow Rate Detection : Synergi 4 µ Fusion-RP : 150 x 4.6 mm : A: 0.1% CH 3CH in Water B: 0.1% CH 3CH in Methanol : 95:5 (A/B) linear to 5:95 over 8 min hold for 5 min. : 0.5 ml/min : Ion Source: ESI Scan Rate: 13000 m/z/s Scan Range: 50-1000 10
4.Propranolol Dimensions Mobile Phase Gradient Flow Rate Detection Injection Temperature Sample : 50 x 4.6 mm : A= Water with 0.1% Formic acid B= Acetonitrile with 0.1% Formic acid : A/B (95:5) to 100% B in 5 min : 1.5 ml/min : UV @ 254 nm : 5 μl : 30 C : 1. Thiourea (0.25 μg), 2. Codeine (1 μg), 3. Chlorpheniramine (2 μg), 4. Propranolol (6 μg), 5. Desipramine (0.5 μg), 6. Ibuprofen (6 μg), 11
0.1% Formic acid 0.1% Acetic acid 0.1% TFA Column Dimensions rder No. Mobile Phase Gradient Flow Rate Detection Injection Temperature Sample : Synergi 4 μ Max-RP : 50 x 4.6 mm : 00B-4337-E0 : A= Water with 0.1 % TFA, Formic acid, or Acetic acid B = Acetonitrile with 0.1 % TFA, Formic acid, or Acetic acid : A/B (95:5) to 100% B in 5 min : 1.5 ml/min : UV @ 254 nm : 5 μl : 30 C : 1. Thiourea (0.25 μg), 2. Codeine (1 μg), 3. Chlorpheniramine (2 μg), 4. Propranolol (6 μg) 5. Desipramine (0.5 μg), 6. Ibuprofen (6 μg) CH2H CH2H H H H H F F Dexamethasone C22H29F5 Exact Mass: 392.20 Mol. Wt.: 392.46 Betamethasone C22H29F5 Exact Mass: 392.20 Mol. Wt.: 392.46 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 ( 1,000,000) 402.15 (1.00) 361.10 (1.00) 343.05 (1.00) 325.00 (1.00) 434.10 (1.00) 393.10 (1.00) 373.10 (1.00) 355.05 (1.00) 476.10 (1.00) 435.10 (1.00) 415.10 (1.00) 494.10 (1.00) 453.10 (1.00) 413.10 (1.00) 446.15 (1.00) 405.10 (1.00) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 2 : Prednisolone : Dexamethasone : Betamethasone : Triamcinolone acetonide : Fluocinolone acetonide : Hydrocortisone acetate Column Mobile phase Flow rate Column oven temperature Injection volume Probe voltage Nebulizer gas flow Drying gas pressure Probe temperature CDL temperature Block heater temperature CDL & Q-array voltage Interval SIM : Synergi MAX-RP (2.0 mm I.D. 150 mml.) : 0.1% formic acid-water / acetonitrile (70:30) : 0.3 ml/min : 40 C : 2 μl :+4.5 kv (APCI-Positive mode) : 2.5 L/min : 4 MPa : 350 C : 200 C : 200 C : using default values : 0.8 sec / 16 chs : m/z 402.1,361.1,343.1,325.1 for prednisolone m/z434.1, 393.1, 373.1, 355.1 for betamethasone & dexamethasone m/z 476.1, 435.1, 415.1 for triamcinolone acetonide m/z 494.1, 453.1, 413.1 for fluocinolone acetonide m/z 446.1, 405.1 for hydrocortisone acetate 12
Column Dimensions rder N. Mobile Phase Flow Rate Temperature Detection Sample : Synergi 4 µ Hydro-RP : 250 x 4.6 mm : 00G-4375-E0 : 20 mm Potassium Phosphate, ph2.9 : 0.7 ml/min : 22 ºC : UV @ 220 nm : 1. xalic acid 2. Tartaric acid 3. Glycolic acid 4. Formic acid 5. Pyruvic acid 6. Malonic acid 7. Acetic acid 8. Maleic acid 9. Citric acid 13
Mobile phase Sample : 20 mm Potassium phosphate : Nucleotides Dimensions Mobile Phase Gradient Flow Rate Temperature Detection Injection Sample : 150 x 4.6mm : A=10 mm Triethylammonium formate ph 6.0 B=Acetonitrile with 10 mm Triethylammonium formate : A/B (85:15) to A/B (35:65) in 15 minutes : 1.5 ml/min : Ambient : UV @ 230 nm : 1 µl of beta-blockers mix each at 0.8 µg/µl : 1. Atenolol 2. Pindolol 3. Nadolol 4. Acebutolol 5. Metoprolol 6. Labetalol 14
1. Pyridine 56 µg/ml 3. Sulfathiazole 100 µg/ml 5. Benzyl alcohol 1.2 mg/ml 7. Nortriptyline 100 µg/ml 9. 5-Methyl-salicylaldehyde 110 µg/ml 10. Hexaphenone 100 µg/ml 2. Quinidine 100 µg/ml 4. Triprolidine 125 µg/ml 6. Phenol 0.5 mg/ml 8. 3-Methyl-4-nitrobenzoic acid 100 µg/ml Email : psc@glc.shimadzu.co.jp TEL : 03-5835-0126 1.10e6 1.00e6 3.0e5 1.0e5 0.52 284.2/135. 2 6.0e5 4.0e5 Chlord iazepoxid e 300.2/227. 0 0.68 1.10e6 1.00e6 2.7e5 0.29 284.2/135. 2 Chlord iazepoxid e 300.2/227. 0 0.43 0.57 9.00e5 8.00e5 6.0e5 xazepam 287.0/241. 2 0.92 4.0e5 Nordazepa m 271.2/140. 2 0.93 9.00e5 8.00e5 4.0e5 xazepam 287.0/241. 2 0.76 4.0e5 Nordazepa m 271.2/140. 2 0.75 7.00e5 4.0e5 7.00e5 6.00e5 6.00e5 5.00e5 4.0e5 Clonazepam 316.1/270. 1 0.95 1.0e6 Te mazepa m 301.2/255. 0 0.99 5.00e5 2.5e5 Clonazepam 316.1/270. 1 0.81 1.0e6 Te mazepa m 301.2/255. 0 0.82 4.00e5 5.0e5 4.00e5 5.0e5 3.00e5 3.00e5 2.00e5 5.5e5 Flunitrzepam 314.2/268. 3 0.99 4.0e5 Diazepam 285.2/193. 2 1.03 2.00e5 2.5e5 Flunitrzepam 314.2/268. 3 0.84 3.9e5 Diazepam 285.2/193. 2 0.85 1.00e5 0 1.0 2.0 min 4.0e5 1.00e5 0 1.0 2.0 min 15 Column: Luna 3 µm C18220 x 2.0 mm MercuryMS cartridge Column: Synergi 2.5 µm Fusion-RP 10 x 2.0 mm MercuryMS cartridge Gradient: 15:85 to 95:5% B in 1.0 min, re-equilibration for 1.0 min2.0 min cycle Gradient: 15:85 to 95:5% B in 1.0 min, re-equilibration for 1.0 min 2.0 min cycle Injection: 10 µl of 100 ng/ml Benzodiazepines from human plasma after SPE clean-up Injection: 10 µl of 100 ng/ml Benzodiazepines in urine after SPE clean-up Flow rate: 0.6 ml/min Flow rate: 0.6 ml/min
120% 100% 80% Percent Efficiency Gemini NX-C18 60% Column A Column B 40% Column C Column D 20% Column E Column F GeminiNX-C18 0% 0 100 200 300 400 500 600 Time of exposure (hour) 16
} } H3C µ 9 µ 9 1 2 3 4 6 5 7 8 10 App ID 16641 1 2 3 4,5 6 7 8 10 App ID 16642 0 10 20 min Conditions for all columns Dimensions Mobile Phase Gradient Flow Rate Temperature Detection : 150 x 4.6 mm : A: 10 mm Ammonium Bicarbonate ph 10.5 B: Acetonitrile : A/B (85:15) to (70:30) in 15 min to (50:50) in 5 min, Hold for 5 min : 1.5 ml/min : Ambient : UV @ 230 nm 0 10 20 min Sample : 1.Bisoprolol Contaminant 2.Sotalol 3.Atenolol 4.Labetolol (Diastereoisomeric Pair) 5.Nadolol (Diastereoisomeric Pair) 6.Pindolol 7.Metoprolol 8.Bisoprolol 9.Propranolol 10.Alprenalol H2N H H N N H H3C H 4.Labetalol H H H 5.Nadolol 17
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