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Cytarabine Analogues by Ion-Pairing LC-MS/MS

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Cytarabine Analogues by Ion-Pairing LC-MS/MS © Advanced Chromatography Technologies Ltd. 1 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Cytarabine Analogues by Ion-Pairing LC-MS/MS Cytarabine and cytidine are isobaric. Robust method with good separation achieved. LLOQ = 1 ng/ml human plasma ACE 3 C18 3μm, 50 x 2.1 mm Gradient analysis A = 0.1% perfluoropentanoic acid + 0.1% formic acid in water B = 0.1% perfluoropentanoic acid + 0.1% formic acid in acetonitrile T (mins) %B 0 0 0.5 0 3.0 13 4.0 90 5.0 0 Flow rate: 0.7 ml/min API 4000 MS TurboIonSpray, positive mode Source temperature: 550°C Reproduced with permission of Agilux Laboratories, USA © Advanced Chromatography Technologies Ltd. 2 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Tricyclic Antidepressants Key: ACE Excel SuperC18 1 Doxepin 2μm, 100 x 3.0mm 1a Doxepin isomer Gradient analysis 1 Imipramine 3 A = 20 mM ammonium formate pH 3.0 2 Desipramine 3 Amitriptyline B = 20 mM ammonium formate pH 3.0 in MeOH:water 9:1 v/v 4 Nortriptyline 1 5 Clomipramine 2 Time (mins) %B 6 0 50 6 70 7 70 7.5 4 5 Flow rate: 1.2ml/min Column temperature: 40°C Injection volume: 2μl Detection: UV, 260nm 1a 1 2 3 4 5 6 7 min © Advanced Chromatography Technologies Ltd. 3 ® ACE HPLC / UHPLC Columns www.ace-hplc.com 15-Hydroxy Lubiprostone in Human Plasma Lubiprostone, a fatty acid derived from prostaglandin E1, is rapidly metabolised to 15-hydroxy lubiprostone. Quantitation is based on 15-hydroxy lubiprostone, with the d4 analogue as internal standard Lowest calibration standard sample containing 2.0pg/ml in human 15-Hydroxy lubiprostone EDTA K3 plasma MW 392.5 ACE Excel 2 C18 2μm, 50 x 3.0mm Isocratic analysis 15-Hydroxy I.S. A = 0.1% formic acid in water lubiprostone B = Acetonitrile Flow rate: 0.65ml/min Column temperature: 35°C Injection volume: 15μl MDS Sciex API 5000 TurboIonSpray negative mode IonSpray voltage: -4500V Source temperature: 450°C Transitions monitored: 15-Hydroxy lubiprostone 391.2 → 373.2 I.S. (15-Hydroxy lubiprostone-d4) 395.2 → 377.2 Reproduced with permission of inVentiv Health Clinical, Quebec, Canada © Advanced Chromatography Technologies Ltd. 4 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Tricyclic Antidepressants ACE Excel C18-PFP 2μm, 100 x 3.0mm 1 Isocratic analysis B = 20 mM ammonium formate pH 3.0 in MeOH:water 54:46 v/v Flow rate: 1.2ml/min 2 Column temperature: 40°C Injection volume: 2μl Detection: UV, 260nm Key: 1 Doxepin 3 5 4 2 Imipramine 3 Protriptyline 6 4 Nortriptyline 5 Trimipramine 6 Clomipramine 0 1 2 3 4 5 min © Advanced Chromatography Technologies Ltd. 5 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Paracetamol and Related Compounds ACE Excel C18-PFP 4 2μm, 100 x 3.0mm Gradient analysis A = 20 mM ammonium acetate pH 6.0 1 B = 20 mM ammonium acetate pH 6.0 in MeOH:water 9:1 v/v 8 2 Time (mins) %B 3 0 9 5 5.5 63 6.5 63 7 7 9 10 Flow rate: 1.2ml/min Column temperature: 27°C Injection volume: 2μl 6 Detection: UV, 220nm 9 Key: 1 4-aminophenol 2 Hydroquinone 3 2-aminophenol 4 Paracetamol 5 2-acetamidophenol 6 Phenol 7 4-nitrophenol 1 2 3 4 5 6 7 8 2-nitrophenol 9 4-chloroacetanilide 10 4-chlorophenol © Advanced Chromatography Technologies Ltd. 6 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Pravastatin and Isomers by LC-MS/MS 6-epi-Pravastatin All 3 compounds have MW 424 MS/MS conditions alone insufficient for selective quantitation Baseline separation important Pravastatin 3α-iso-Pravastatin ACE C18 3 μm, 50 x 3.0 mm Isocratic analysis Acetonitrile-Methanol-THF-Water- Acetic acid (15:20:5:60:0.1) Flow rate: 0.6 ml/min Column temperature: Ambient Injection volume: 2 μl Sample: 1 μg/ml each isomer API 3000 triple quad MS TurboIonSpray – negative mode Extracted ion chromatogram of MRM m/z 423.3 → 321.1 Reproduced with permission of Biotrial Bioanalytical Services, Laval, QC, Canada © Advanced Chromatography Technologies Ltd. 7 ® ACE HPLC / UHPLC Columns www.ace-hplc.com NSAIDs ACE 5 Super C18 5μm, 150 x 4.6 mm Gradient analysis A = 0.1% formic acid in water B = 0.1% formic acid in acetonitrile Time (mins) %B mAU 7 0 20 20 70 1000 25 70 26 20 Flow rate: 1ml/min Column temperature: 40°C 800 Injection volume: 10μl Detection: UV, 254nm Key: 600 1 Aspirin 2 4 2 Phenacetin 3 Sulindac 9 4 Tolmetin 400 3 5 Naproxen 6 6 Nimesulide 8 10 7 Flurbiprofen 8 Diclofenac 200 9 Phenylbutazone 5 10 meclofenamic acid 1 0 5 10 15 20 25 min © Advanced Chromatography Technologies Ltd. 8 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Pristinamycin components in plasma by LC-MS/MS Pristinamycin antibiotic is a mixture of 2 components – pristinamycin lA and llA Virginiamycin used as internal standard Low calibration standard containing Processed study sample containing Pristinamycin lA Pristinamycin llA pristinamycin lA and llA 2.5ng/ml each of pristinamycin lA and llA in human NaF/K2C2O4 plasma ACE 3 C18 3μm, 30 x 3.0mm Gradient analysis A = 1mM NH4CO2H + 0.1% HCO2H in 65:35 H2O:CH3CN B = CH3CN T (mins) %B T (mins) %B 0 0 1.61 100 0.3 0 2.6 100 0.31 10 2.61 0 1.6 10 4 0 Flow rate: 1ml/min Column temperature: 25°C Injection volume: 10μl MDS Sciex API 4000 TurboIonSpray positive mode Transitions monitored: Pristinamycin lA 867.5 → 134.2 Pristinamycin llA 526.3 → 355.1 I.S. (Virginiamycin) 824.6 → 134.0 Reproduced with permission of inVentiv Health Clinical, Quebec, Canada © Advanced Chromatography Technologies Ltd. 9 ® ACE HPLC / UHPLC Columns www.ace-hplc.com NSAIDS – Fast analysis ACE Excel Super C18 2μm, 50 x 3.0 mm Gradient analysis A = 0.1% formic acid in water mAU 7 B = 0.1% formic acid in acetonitrile Time (mins) %B 700 0 20 0.25 20 3.5 70 600 4 70 4.25 20 500 Flow rate: 0.86ml/min Column temperature: 40°C 400 4 Injection volume: 1.4μl Detection: UV, 254nm 2 9 300 3 Key: 6 8 10 1 Aspirin 200 2 Phenacetin 5 3 Sulindac 4 Tolmetin 100 1 5 Naproxen 6 Nimesulide 7 Flurbiprofen 0 8 Diclofenac 9 Phenylbutazone 10 meclofenamic acid 0 1 2 3 4 min © Advanced Chromatography Technologies Ltd. 10 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Diuretics Key: 1 Hydrochlorothiazide ACE Excel C18-PFP 2 Amiloride 6 2μm, 50 x 3.0 mm 3 Furosemide Gradient analysis 4 Indapamide A = 10 mM ammonium formate pH 3.0 5 Bendroflumethazide 3 B = 10 mM ammonium formate pH 3.0 6 Spironolactone in MeOH:water 9:1 v/v 1 Time (mins) %B 0 5 0.5 5 5 70 5.5 70 6 5 Flow rate: 1.0ml/min Column temperature: 60°C 4 Injection volume: 2μl Detection: UV, 254nm 2 5 0 2 4 6 min Hydrochlorothiazide Amiloride Furosemide Indapamide Bendroflumethazide Spironolactone © Advanced Chromatography Technologies Ltd. 11 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Diuretics (Isocratic) Key: ACE Excel C18-PFP 1 Furosemide 2μm, 50 x 3.0 mm 2 Indapamide Isocratic analysis 3 Bendroflumethazide 10 mM ammonium formate pH 3.0 4 Spironolactone in methanol:water 45:55 v/v Flow rate: 1.0ml/min Column temperature: 60°C 1 Injection volume: 2μl Detection: UV, 254nm 2 Furosemide Indapamide 3 4 Bendroflumethazide Spironolactone 0 1 2 3 4 5 min © Advanced Chromatography Technologies Ltd. 12 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Antihistamines Key: ACE UltraCore SuperC18 1 Pseudoephedrine 2.5μm, 100 x 3.0mm 2 Scopolamine Gradient analysis 3 Doxylamine A = 20 mM ammonium formate pH 3.0 4 Chlorpheniramine 5 Triprolidine 7 B = 20 mM ammonium formate pH 3.0 6 Diphenhydramine in MeOH:water 9:1 v/v 7 Acrivastine 8 Promethazine Time (mins) %B 9 Fexofenadine 11 0 0 10 Cetirizine 7.5 100 11 Loratadine 8.5 100 9.0 Flow rate: 1.2ml/min 8 Column temperature: 25°C 5 Injection volume: 2μl 3 Detection: UV, 254nm 4 10 9 6 1 2 1 2 3 4 5 6 7 8 © Advanced Chromatography Technologies Ltd. 13 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Analgesics / Cough1 & Cold Medicine Ingredients HO O HO O O O NH2 NH CH3 CH3 NH CH O OH 3 HO O 7. salicylamide H3C N 9. phenol N OH mAU O O H C NH HO 3 OH 8. acetanilide 1. paracetamol O N N 5. 2-acetamidophenol 4. caffeine OH CH3 OH HO O O NH2 11 6. 3-hydroxybenzoic acid O OH 2. 4-aminobenzoic acid 3. 4-hydroxybenzoic acid 10. aspirin O CH3 H3C OH 8 12. sorbic acid 7 O 13 11. benzoic acid 14 O OH 3 NH CH3 1 10 12 OH O 5 6 H3C O 2 15 14. phenacetin 4 13. salicylic acid 9 O H ACE C18-AR OH 13,14 15. salicylaldehyde 11,12 8 3,4 7 150x4.6mm, 3mm 1,2 Gradient analysis 6 10 5 A = 0.1% v/v formic acid (aq) B = 0.1% v/v formic acid in MeCN 40C 15 240nm 9 1.0mL/min T (min) %B ACE C18 0 5 9 35 14 35 4 5 6 7 8 9 10 11 12 min 15 5 © Advanced Chromatography Technologies Ltd. 14 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Pharmaceutically Relevant Mixture ACE UltraCore SuperC18: selectivity with pH: 8,9 50x2.1mm, 2.5mm Gradient analysis pH 3 A1= 10mM HCOONH4, pH3 (aq) B1= 10mM HCOONH4, pH 3 in 3 MeCN/water 9:1 v/v 2 10 12 A2= 0.1% NH3, pH 10.7 (aq) 11 4 B2= 0.1% NH3, pH10.7 in in MeCN/water 9:1 v/v 6 1 5 7 T %B 0 3 5 100 6 100 DAD1 C, Sig=254,8 Ref=460,100 (F:\131024-MJ\131024_LOW_PH 2013-10-24 16-30-36\LOW0000005.D) 40C 8 0.60 mL/min 3 254 nm 4 2 9 pH 10.7 1.
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