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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. 2 ® 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. Atenolol 6 2. Methylphenylsulfoxide 12 3. Eserine 10 11 4. Prilocaine 1 5. Bupivacaine 6. Tetracaine 5 7. 1,2,3,4-Tetrahydro-1-naphthol 7 8. Carvedilol 9. Nitrobenzene 10. Methdilazine

DAD1 C, Sig=254,8 Ref=460,100 (F:\131024-MJ\131024 2013-10-24 14-40-48\HIGH0000004.D) 11. Amitriptyline min 12. Valerophenone 1 2 3 4 5 6 min © Advanced Chromatography Technologies Ltd. 3 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Paraben Preservatives

1 ACE 3 Phenyl, 150 x 2.1mm Isocratic

Methanol – 25mM NH4OAc 2 (50:50), pH 6.8 Flow rate: 0.2ml/min Column temperature: 40°C Detection: UV at 240nm 3 Sample volume: 2μl

5 4

1. Methylparaben 6 2. Ethylparaben 3. n-Propylparaben 4. i-Butylparaben 5. N-Butylparaben 6. Benzylparaben

Reproduced with permission of Trevor Gibson, The Chemical Analysis Facility, University of Reading © Advanced Chromatography Technologies Ltd. 4 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Pharmaceutically Relevant mixture Separation

 ACE UltraCore SuperPhenylHexyl: selectivity with pH:

50x2.1mm, 2.5mm 4 9,6 Gradient analysis 11,8 5 A1= 10mM HCOONH4, pH3 (aq) B1= 10mM HCOONH4, pH 3 in pH 3 MeOH/water 9:1 v/v

10 1 2 A2= 0.1% NH3, pH 10.7 (aq) B2= 0.1% NH , pH10.7 in in 3 3 MeOH/water 9:1 v/v

7 T %B 0 3 5 100 7 100

40 °C 4 9 5 0.60 mL/min 11 254 nm

1. Benzamide 3 10 2. Caffeine 1 2 pH 10.7 3. Procainamide 4. N-acetylprocainamide 5. Propiophenone 6. Toluene 7. Remacemide 6 7 8 8. Ethylbenzene 9. Carvdilol 10. Nortriptyline 11. Clomipramine 1 2 3 4 5 6 min 7 © Advanced Chromatography Technologies Ltd. 5 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Clonidine Hydrochloride

Clonidine hydrochloride oral solution containing preservatives

Concentration clonidine HCl = 10μg/ml Concentration Preservatives ~ 1.5g/ml 2

1. Clonidine hydrochloride 2. Methyl hydroxybenzoate 3. Propyl hydroxybenzoate

ACE UltraCore SuperC18 2.5μm, 50 x 4.6mm 3 Gradient analysis

A = 0.2% w/v phosphate buffer-CH3OH- CH3CN (80:10:10) B = CH3CN Time (mins) %B 0 0 1 0.8 0 2.1 70 3.4 70 3.5 0 Flow rate: 2ml/min Column temperature: 20°C Injection volume: 100μl Detection: UV, 220nm

Reproduced with permission of Mary Nguyen, Guys Hospital, London © Advanced Chromatography Technologies Ltd. 6 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Procaine and p-Aminobenzoic acid

Procaine – sterile manufactured product System suitability tests Procaine ACE C18-PFP 3μm, 100 x 4.6mm Isocratic analysis p-Aminobenzoic acid Methanol - 0.6% acetic acid (19:81) (potential degradant) (adjusted to pH 4.7 with 20% NaOH) Flow rate: 1ml/min Detection: UV, 179nm

Detection limits for S/N = 10 Procaine = 0.0002mg/ml p-Aminobenzoic acid = 0.00005mg/ml

Reproduced with permission of John Peace, Huddersfield Royal Infirmary, UK © Advanced Chromatography Technologies Ltd. 7 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Water Soluble Vitamins ACE 3 C18-AR Pantothenic acid (vitamin B5) Cyanocobalamin (vitamin B12) H OH NH OH HO H C 3 CH3 O O D-Biotin (vitamin B7 or vitamin H) O 150x4.6mm, 3mm mAU HN NH 3 p-Aminobenzoic acid Gradient analysis 200 H H O Pyridoxine (vitamin B6) HO O

N CH3 S OH A = 20mM KH2PO4, pH 2.83 (aq) B = 20mM KH2PO4, pH 2.83 in 175 HO OH 1:1 v/v MeOH:water 40C H2N NH2 205nm 1.5mL/min 150 1 HO O O Folic acid (vitamin B9 or vitamin M) O T (min) %B O NH 0 20 125 H3C N N HO NH HN NH 15 70 4 O H3C N N O H2N N N 100 HO Riboflavin (vitamin B2)

6 HO OH OH 7 75 1. Pyridoxine (vitamin B6) 2 5 2. Pantothenic acid (vitamin B5) 3. p-Aminobenzoic acid 50 4. Folic acid (vitamin B9 or vitamin M) 5. D-Biotin (vitamin B7 or vitamin H) 6. Cyanocobalamin (vitamin B12) 7. Riboflavin (vitamin B2) 25

0 2 4 6 8 10 12 14 16 min © Advanced Chromatography Technologies Ltd. 8 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Water Soluble Vitamins / Polar Molecules

O ACE 3 C18-AR O Nicotinamide HO O H Isonicotinamide HO OH O 3um, 150 x 4.6 mm NH H2N O 2 L-Ascorbic acid Isocratic analysis or vitamin C NH N HO N O Flow rate: 1.0 mL/min H mAU O N Temperature: 22°C Orotic acid Detection: UV 260 nm NH2 2 + N N MP: 96.5:3.5 v/v 0.1% H3PO4 (aq) : MeOH S O 10

H3C N H C 7 N 3 NH HO O Thiamine or 3 vitamin B1 N N OH H Hypoxanthine

NH2 4-aminobenzoic acid 4 8 O

HO OH 5 H3C N Pyridoxal or vitamin B6 Pyridoxamine or 1 6 OH OH Vitamin B6 CH3

N CH3 9 HO N HO Pyridoxine or OH vitamin B6

H2N

0 2 4 6 8 min © Advanced Chromatography Technologies Ltd. 9 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Non-Steroidal Anti-Inflammatory Drugs

CH3 ACE 3 C18-AR OH

H3C O O Naproxen mAU Ketoprofen

O CH3 Indomethacin OH Cl O O 2 N 150x4.6mm, 3mm CH3 H3C Gradient analysis O HO CH A = 0.1% v/ formic acid (aq) 3 Diclofenac 7 O Meclofenamic acid B = 0.1% v/v formic acid in MeOH OH Cl O O OH 40C Cl HCl NH 1.0mL/min H3C NH 254nm 4 Cl OH H3C Cl O T (min) %B Sulindac 0 52 6 28 74 3 S 33 74 O CH3 Mefenamic acid

F 8 O OH H F 9 CH3 N 1 H C NH F CH 5 3 Ibuprofen 3

HN NH2 O S S CH O O 3 O O OH Bendromethazide H3C *

0 5 10 15 20 25 30 min © Advanced Chromatography Technologies Ltd. 10 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Complex Steroid Mixture Separation ACE 3 C18-AR © Advanced Chromatography Technologies Ltd. 11 ® ACE HPLC / UHPLC Columns www.ace-hplc.com ACE® C18-PFP™: Halogenated Positional Isomers

 Chloroacetophenone halogenated isomers separation © Advanced Chromatography Technologies Ltd. 12 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Polar Analytes –1 Nucleosides & Vitamins

NH2 3 5 HO + N N O ACE C18-PFP, 3um, 150 x 4.6 mm 100% Aqueous S O Nicotinamide HO H C N O H 3 H3C Isocratic analysis Thiamine or HO OH NH2 vitamin B1 L-Ascorbic acid or OH Flow rate: 1 ml/min N vitamin C Temperature: 22°C 4 Detection: UV 254 nm

Cytosine Uracil O Mobile phase: 20 mM H3PO4 (aq) NH2 2 NH N

N O N O H H

6 NH 2 Cytidine N HO O N O

HO 7 OH 8 Pyridoxamine or O 2-Deoxycytidine Vitamin B6 N NH2 NH N CH3 N N HO HO N OH H O N O Hypoxanthine 9 H2N 1 HO

2 3 4 5 min © Advanced Chromatography Technologies Ltd. 13 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Separation of Sulphonamides

ACE Excel C18-PFP 3μm, 150 x 4.6mm Gradient analysis 1. Sulfadiazine A = Water 2. Sulfapyridine B = Acetonitrile 3. Sulfamerazine C = 10% formic acid 4. Sulfamoxole 9 Time (mins) %A %B %C 5. Sulfamethazine 0 84 15 1 6. Sulfamonomethoxine 12 74 25 1 7. Sulfachloropyridazine 14 59 40 1 8. Sulfamethoxazole 16 84 15 1 9. Sulfadimethoxine 18 84 15 1 Flow rate: 1ml/min Detection: UV, 254nm

1 3 8 6 5 7 2

Reproduced with permission of Antonio Martin Esteban, National Research Institute of Agriculture and Food Technology INIA, Spain © Advanced Chromatography Technologies Ltd. 14 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Aspirin And Related Substances Alternative selectivity with MeOH containing eluents

NH CH3 ACE Excel CN-ES O NH2 5 O OH O O OH H3C O

OH O CH3 H3C NH O OH O NH 2 O 1 3 6 OH CH3 4 ACE CN-ES 50x2.1mm, 3mm Gradient analysis

0.5 1 1.5 2 2.5 3 3.5 4 4.5 min A = 0.1% formic acid ACE Excel C18 5 B = 0.1% FA in MeOH 40 °C 0.6 mL/min 2 240 nm 5 – 38% B in 3.75 mins 3 6 Hold at 38% B until 5 mins 1 4

1. 2-Acetamidophenol 2. Acetanilide 3. Salicylamide 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min 4. Aspirin ACE Excel CN 5. Phenacetin 3 5 6. Salicylic acid 4,6 1

0.5 1 1.5 2 2.5 3 3.5 4 4.5 min © Advanced Chromatography Technologies Ltd. 15 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Small Pharmaceutically Relevant Analytes

Dipole + hydrophobic Interactions 1 ACE Excel CN-ES 2.245 2 3 6 5 7 ACE CN-ES 1.698 3.473 4.209 4 3.972 4.386 100x3mm, 3mm 3.306 Gradient analysis

A = 20mM ammonium formate (aq) B = 20mM ammonium formate in MeOH 40C 0.6 mL/min Polar embedded + hydrophobic Interactions T (min) %B 1/2 0 3 ACE Excel C18-Amide 2.006 5 100 3 5 4 6 6 100 3.019 3.564 4.180 4.440 7 6.5 3 4.518

1. Hydrochlorothiazide 2. Methylphenylsulphoxide 3. 1,3,5 Trinitrobenzene 4. Myricetin Hydrophobic + π-π + dipole + shape / position Interactions 5. P-Cresol 6. Sulindac 1/2 7. Toluene ACE Excel C18-PFP 2.096 3 5 4 6

2.401 3.529 3.898 7 4.736 4.678

1 2 3 4 min © Advanced Chromatography Technologies Ltd. 16 ® ACE HPLC / UHPLC Columns www.ace-hplc.com Salicylic acid in cell extracts

Extract from genetically engineered bacteria cells – targeted analysis for salicylic acid

ACE UltraCore SuperC18, 5μm, 150 x 4.6mm Gradient analysis (shown above) A = 0.1% TFA in water B = 0.1% TFA in acetonitrile Flow rate: 1ml/min Detection: UV, 250nm

Reproduced with permission of Trong Khoa Pham, University of Sheffield, UK © Advanced Chromatography Technologies Ltd. 17 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Guaifenesin

Key: 1 Guaifenesin 2 Benzoic acid

ACE UltraCore SuperC18, 2.5 μm, 50 x 3.0 mm Isocratic analysis

H2O/MeOH/Glacial acetic acid (60:40:1.5 v/v) Flow rate: 0.85 mL/min Column temperature: Ambient (22°C) 2 Detection: UV at 276 nm 1.124 Injection volume: 1.5 μL mAU 1 0.630 10

0.25 0.5 0.75 1 1.25 1.5 1.75 min

Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 18 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Hydrocortisone

ACE Ultracore SuperC18, 2.5 μm, 100 x 4.6 mm Isocratic analysis

Key: MeCN/MeOH/H2O (25:25:50 v/v) 1 Hydrocortisone Flow rate: 1 mL/min 2 Propylparaben Column temperature: Ambient (22°C) Detection: UV at 253 nm Injection volume: 5.8 μL

2 mAU 4.601

120 1 100 2.537

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 19 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Estradiol

Key: 1 Ethylparaben ACE Ultracore SuperC18, 2.5 μm, 100 x 4.6 mm 2 Estrone Isocratic analysis 3 Estradiol H2O/MeCN (45:55 v/v) Flow rate: 1.39 mL/min Column temperature: Ambient (22°C) Detection: UV at 205 nm Injection volume: 10.1 μL

1 2 mAU 1.096 1.511

200

150 3 1.804

100

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 20 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP 17a-Ethinylestradiol

ACE Ultracore SuperC18, 2.5 μm, 50 x 3.0 mm Key: 1 Ethylparaben Isocratic analysis 2 17α-Ethinylestradiol MeCN/H2O (50:50 v/v) Flow rate: 0.43 mL/min Column temperature: Ambient (22°C) Detection: UV at 280 nm Injection volume: 3.1 μL

1 mAU 0.862 70 2 1.348 60

1 2 3 4 min Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 21 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Naproxen

ACE Ultracore SuperC18, 2.5 μm, 50 x 3.0 mm Isocratic analysis

Key: H2O with glacial acetic acid (49:1)/MeCN (50:50 v/v) 1 Naproxen Flow rate: 0.51 mL/min 2 Butyrophenone Column temperature: Ambient (22°C) Detection: UV at 254 nm Injection volume: 2.8 μL

2 mAU 1.855 25

1 20 1.054

1 2 3 4 min Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 22 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Paracetamol / Aspirin / Caffeine

Key: ACE Ultracore SuperC18, 2.5 μm, 50 x 4.6 mm 1 Paracetamol Isocratic analysis 2 Caffeine H2O with glacial acetic acid (69:3)/MeOH (72:28 v/v) 3 Aspirin Flow rate: 2 mL/min 4 Benzoic acid Column temperature: 45°C 5 Salicylic acid Detection: UV at 275 nm

Injection volume: 2.5 μL

mAU 50 1 0.313 4 1.319 40

30 2 0.439 3

20 1.003

10 5 1.503

0 0.5 1 1.5 2 2.5 min

Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 23 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Hydroquinone

Key: ACE Excel C18, 2 μm, 50 x 3.0 mm 1 Hydroquinone Isocratic analysis

H2O/MeOH (45:55 v/v) Flow rate: 0.45 mL/min Column temperature: Ambient (22°C) Detection: UV at 280 nm Injection volume: 0.9 μL

mAU 0.606

80 HO

0.25 0.5 0.75 1 1.25 1.5 1.75 min

Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 24 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Doxepin

O Key: 1 Doxepin isomer ACE C8, 3 μm, 50 x 4.6 mm 2 Doxepin isomer Isocratic analysis 0.2 M Monobasic NaH2PO4 in water pH 2.5:MeOH (30:70 v/v) Flow rate: 0.56 mL/min 20.193 H3C N mAU Column temperature: 50°C Detection: UV at 254 nm CH3 17.5 Injection volume: 4.5 μL

12.5

7.5

5 22.715

2.5

5 10 15 20 25 min

Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis. © Advanced Chromatography Technologies Ltd. 25 ® ACE HPLC / UHPLC Columns www.ace-hplc.com USP Metronidazole

ACE C8, 3 μm, 75 x 4.6 mm Key: Isocratic analysis 1 Metronidazole H O/MeOH (4:1 v/v) OH 2 Flow rate 1.0 mL/min Column temperature: 30°C Detection: UV at 319 nm Injection volume: 15 μL N 2.301 mAU O2N CH3

700 N

600

500

400

300

200

100

0.5 1 1.5 2 2.5 3 3.5 min

Translated method according to USP <621> guidance. Always check the latest and official method information from the relevant pharmacopoeia prior to analysis.