Pharmaceutical Applications With HPLC

Pharmaceutical Applications with HPLC Solutions Guide
April 2000
General Description
Agilent Technologies has developed a large number of key HPLC applications for the pharmaceutical market. These applications include analysis equipment, separation media and chromatographic conditions. All proposed solutions are accompanied by examples, configuration overviews and method validation data. This guide gives a condensed overview of the analysis of pharmaceutical drugs using HPLC. It has been written to find starting conditions for application development and to make ordering of the entire HPLC systems easy, correct and complete. The guide contains seven sections: Applications include detailed chromatographic conditions, performance of the HPLC method, and reference to a configuration example at the end of the guide. Agilent ChemStation database gives a short description of the data organization system accompanied by two application examples. Agilent 1100 Series combinatorial chemistry analysis system describes features and key benefits of this high sample throughput system. Instrumentation describes the analysis equipment used for the applications in this guide. Configuration examples give an overview of recommended HPLC equipment with ordering information for the described applications. Quick reference guide lists the analysis conditions of more than 100 pharmaceutical drugs. Literature provides an overview of publications, in which the results of the proposed solutions were published.
Agilent Technologies
Innovating the HP Way
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Agilent ChemStation Database . . . . . . . . . . . . . . . . . . . . . . . . . . .56 Agilent 1100 Series Combinatorial Chemistry Analysis System .67 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76 Quick Reference Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81 Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84
Please note that the described instruments in this document are recommendations only!
Introduction
HPLC Applications
High performance liquid chromatography (HPLC) is an important tool for the analysis of pharmaceutical drugs, for drug monitoring and for quality assurance. The method enables complex mixtures, for example, herbal medicine plant extracts, to be separated into individual compounds, which can be identified and quantified by suitable detectors and data handling systems. Separation and detection occurs at ambient temperature or slightly above. Therefore, the method is ideally suited for compounds of limited thermal stability. State-of-the-art HPLC equipment can automate HPLC separations, using automatic samplers, injectors, microprocessor-controlled analytical conditions and ChemStations for data evaluation. Important requirements for automation are: excellent precision of the liquid chromatography system, data evaluation with report printouts, the possibility to store chromatograms and results, the possibility to detect leaks and other errors for safety reasons, and implemented OQ/PV tools in the HPLC system.
Automation not only increases the sample throughput in pharmaceutical laboratories and companies, but also the precision of the results by eliminating human errors. An overview of the pharmaceutical drugs used in this guide is presented on pages 5-6.
Agilent ChemStation Database
The client/server database for the Agilent ChemStation, for example, the Agilent ChemStore C/S is a data organization system which provides a solution to organize, manange and report chromatographic results, as well as safeguard data. It supports important end-user tasks such as reviewing and summarizing results for statistical evaluation, archiving and restoring data and creating control charts and crosssample reports. These services also aid users in validating their methods and doing on-going system suitability testing. Two examples using the database for the Agilent ChemStation are described on pages 56-66. The new Agilent 1100 Series combinatorial chemistry analysis system is a fully automated analysis solution designed to speed up the process of drug discovery. The combination Agilent 1100 Series HPLC, Agilent 1100 Series LC/MSD detector and the Agilent 220 micro plate sampler offers the full potential of well plate technology and is the ideal tool for analyzing complex libraries. Details can be found on page 67.
Agilent 1100 Series Combinatorial Chemistry Analysis System
Application Overview
Group
Analgesic drugs Androgen drugs Antianginal drugs Antiarrythmic drugs Antiasthmatic drugs Antibacterial drugs Penicillin-like Tetracyclines Miscellaneous Anticoagulant drugs Antidepressant drugs Antiepileptic drugs
Examples
Antipyrine, Hydroxyantipyrine, Acetaminophen Testosterone Acetate, Testosterone Verapamil
Page
7 8 9
Quinidine, Disopyramide, Procainamide, N-Acetylprocainamide 10 Caffeine, Theophylline, Enprofylline, Theobromine Ampicillin, Amoxicillin, Penicillin G, Penicillin V Minocycline, Tetracycline, Doxycycline Hydroxybenzotriazole, Chloramphenicol, Trimethoprim Sulfamethoxazole, Furazolidone, Nalidixic Acid Warfarin 11 12 13 14 17
Antiestrogen drugs Antihistaminic drugs Antihypertensive drugs Antiinflammatory drugs Antiprotozoal drugs Antitumor drugs Antitussive drugs Catecholamines Glucocorticoid drugs
Bupropion, Trazodone, Maprotiline 18 Caffeine, Phenytoin, Methylphenylsuccinimide, Phenylethylmalonamide, Carbamazepinepoxide, Ethosuximide, Phenobarbital, Carbamazepine, Primidone 20 Tamoxifen 26 Tetracaine, Promethazine, Chlorpheniramine, Tripelenamine 27 Enalapril, Captopril Naproxen Metronidazole Paclitaxel (Taxol) 28 29 30 31
Dextromethorphan 33 Norepinephrine, Epinephrine, Dihydroxybenzylamine, Dopamine 34 Beclomethasone Dipropionate, Prednisolone, Prednisolone Acetate, Betamethasone, Betamethasone Valerate, Hydrocortisone, Hydrocortisone Acetate 35 Ranitidine, Cimetidine Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital, Amobarbital, Mephobarbital, Flunitrazepam Salicylic Acid, Phtalic Acid, Benzoic Acid Papaverine Diazepam, Oxazepam, Clonazepam, Flunitrazepam Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine, Sulfamethazine Angiotensin II, Angiotensin I, Insulin, Oxytocin Protriptyline, Nortriptyline, Doxepin, Imipramine, Amitriptyline, Trimipramine Vitamins A1, D3, E Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide, Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine, Riboflavine, Thiamine, Thiotic Acid 37 38 41 42 43 44 45 46 47
H2-Antagonists Hypnotic drugs Keratolytic drugs Muscle-relaxing drugs Sedative drugs Sulfa drugs Therapeutic peptides Tricyclic antidepressant drugs Vitamins Fat soluble Water soluble
48
Medical Herb Extracts Active Compounds

Atropa belladonna Cortex cinchonae Dan Shen Atropine Quinine, Quinidine
Page
49 50
Protocatechuic Acid, Protocatechuic Aldehyde, Tanshinone I, Tanshinone IIA, Cryptotanshinone 51 53 54 55
Ephedra sinica stapf Ginko bilobae Rheum palmatum
Ephedrine, Norephedrine Quercetin, Kaempferol Rhein, Emodin
Analgesic Drugs
O N N
Absorbance [mAU] 120 100
1 Acetaminophen 2 Antipyrine 3 Hydroxyantipyrine 2 3
Antipyrine
80 60 O 40 N N 20 0
HO
Hydroxyantipyrine
6 Time [min]
10
Analysis of analgesic drugs
H HO N O
Acetaminophen Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4.6 x 75 mm Zorbax SB-C18, 3.5 m A= 0.05 M KH2P 04 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 10 min 40 % B at 12 min 10 % B variable wavelength detector, 204 nm, standard cell 25 C 12 min 5 min 5 l
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.07 % Precision of area 10 runs, 100 mg/l < 0.7 % Instrumentation: see configuration example 2 on page 77
Androgen Drugs
OR H H O Testosterone R=H Testosterone acetate R = OAc H Absorbance [mAU] 254 nm 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 Testosterone 234 nm Testosterone acetate
Time [min] Analysis of androgen drugs
Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume
4.6 x 125 mm Hypersil ODS, 5 m A= water, B = acetonitrile 1.0 ml/min at 0 min 50 % B at 10 min 90 % B at 13 min 90 % B at 15 min 50 % B variable wavelength detector at 0 min 254 nm at 6 min 234 nm, standard cell 40 C 15 min 5 min 5 l
Antianginal Drugs
OCH3 OCH3 Absorbance [mAU] 500 400 N 300 200 CN 100 0 H3CO Verapamil Analysis of antianginal drugs HPLC method performance Limit of detection 0.01 mg/l (5-l injection), (VWD) S/N=2 Precision of RT 10 runs, 100 mg/l 0.03% OCH 3 0 2 4 6 Time [min] 8 10 Verapamil
Column Mobile phase Flow rate Gradient Column wash UV detector
Precision of area 10 runs, 100 mg/l 0.12% Linearity (correlation factor) 0.99992
4.6 x 75 mm Zorbax SB-C18 3.5 m A = 0.025M KH2P 04 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 20 % B at 10 min 80 % B at 12 min 20 % B variable wavelength detector 204 nm, standard cell fluorescence detector 228/312 nm, standard cell
Column compartment temperature 25 C Stop time 12 min Post time 5 min Injection volume 5 l
Instrumentation: see configuration example 3 on page 78
Emission [LU]
120
Excitation 228 nm 268 nm
Emission 312 nm
2.5 2 1.5 1 0.5
100 80 60 40 20 0 200 220 240 260 280 300 320 Wavelength [nm] 340 360 380
Verapamil
268/312 nm 1 2 3 4 5 6 7 8
Time [min] Analysis of verapamil using the Agilent 1100 Series fluorescence detector (column: 2.1 x 50 mm Zorbax SB-C18, 5 m) 9
Antiarrythmic Drugs
Absorbance [mAU] N HO H3CO N Quinidine H 200 175 150 125 100 75 50 25 N N CONH 2 0 0 2
220 nm
254 nm 3
204 nm 5
220nm
1 Procainamide 2 N-Acetylprocainamide 3 Quinidine 4 Hydroquinidine * 5 Disopyramide
4 4 6 8 10
* Quinidine contains up to 10 % Hydroquinidine
Time [min] Analysis of antiarrythmic drugs
Disopyramide Column Mobile phase Flow rate Gradient Column wash O N H Column compartment temperature Stop time Post time Injection volume UV detector 4 x 125 mm Purospher RP-18, 5 m A = 0.05 M KH2P 04 in water (pH = 2.5 ), B = acetonitrile 1.0 ml/min at 0 min 0 % B at 5 min 10 % B at 10 min 40 % B at 11 min 40 % B at 12 min 0 % B variable wavelength detector at 0 min 220 nm at 4 min 254 nm at 7.5 min 204 nm at 10 min 220 nm, standard cell 60 C 12 min 5 min 5 l
NHR
Procainamide R=H N-Acetylprocainamide R = OAc HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 1 mg/l (procainamide) Precision of RT 10 runs, 100 mg/l < 0.06% Precision of area 10 runs, 100 mg/l < 0.5 % Instrumentation: see configuration example 2 on page 77
10
Antiasthmatic Drugs
O R O N N N N
Theobromine R=H Caffeine R=CH3 Enprofylline R=C3H 7
Absorbance [mAU] 2 350 300 250 200 150 100 50 1 3 4 1 2 3 4 Theobromine Theophylline Enprofylline Caffeine
O N O N
H N N
Theophylline
0 0 1 2 3 4 5 6
Time [min] Analysis of antiasthmatic drugs
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.1 % Precision of area 10 runs, 100 mg/l < 0.3 % Instrumentation: see configuration example 2 on page 77 Column m Mobile phase Flow rate Isocratic 4 x 125 mm Hypersil ODS, 5 A = water, B = acetonitrile 1.0 ml/min at 0 min 8 % B at 7 min 8 % B variable wavelength detector 270 nm, standard cell Column compartment temperature 50 C Stop time 7 min UV detector
Absorbance [mAU] 40 30 20 10 1
2 3 4
1 2 3 4
Theobromine Theophylline Enprofylline Caffeine
Standard
Solostin antiasthmatic drops 0 0 1 2 3 4 5 6 7 8
Time [min] Analysis of theophylline in Solosin antiasthmatic drops
11
Antibacterial Drugs with Pencillin-like Structure
O R
N H H S
COOH
Absorbance [mAU] 1 2 3 4 Amoxicillin Ampicillin Penicillin G Penicillin V
N H
1000 800 600 400 1 4 2 3
R =
HO
200
NH2
Amoxicillin
0 0 2 4 6 Time [min] Analysis of antibacterial drugs with pencillin-like structure 8 10 12
R =
NH2
Ampicillin
R = Penicillin G
4.6 x 75 mm Zorbax SB-C18 , 3.5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 5 % B at 10 min 60 % B at 12 min 10 % B variable wavelength detector 204 nm, standard cell 40 C 12 min 5 min 5 l
R =
HPLC method performance Limit of detection 1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.08 % Precision of area 10 runs, 100 mg/l < 1.1 % Instrumentation: see configuration example 2 on page 77
Penicillin V
12
Antibacterial Drugs - Tetracyclines
OH
OH O OH
CONH2 OH
Absorbance [mAU] 2 120 100 80 60 40 1 3 1 Minocycline 2 Tetracycline 3 Doxycycline
N(CH3 )2 Minocycline
N(CH3)2
OH
OH O OH
CONH2 OH
20 0
HO Tetracycline
4 Time [min]
10
N(CH3)2 Analysis of tetracyclines
OH
OH O OH
CONH2 OH
H Doxycycline
OH
N(CH3)2
4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 5 % B at 10 min 60 % B at 12 min 5 % B variable wavelength detector 350 nm, standard cell 25 C 12 min 5 min 5 l
13
Miscellaneous Antibacterial Drugs
N N OH Hydroxybenzotriazole N
Absorbance [mAU] 400 350 300 O H N CHCl2 OH OH 250 200 150 100 50 0 0 2 4 2 1 204 nm
368 nm 204 nm 254 nm 204 nm 1 Hydroxybenzotriazole 2 Trimethoprim 3 Furazolidone 4 Sulfamethoxazole 5 Chloramphenicol 6 Nalidixic acid
4 5
O2 N
Chloramphenicol
6 Time [min]
10
12
14
N N NH 2 H3CO OCH3
Trimethoprim O H2 N S O N Sulfamethoxazole N H
NH2
Analysis of miscellaneous antibacterial drugs
OCH3
O Column compartment temperature Stop time Post time Injection volume
4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 10 min 30 % B at 15 min 60 % B at 16 min 10 % B variable wavelength detector at 0 min 204 nm at 4.5 min 368 nm at 6 min 204 nm at 10 min 254 nm at 15 min 204 nm, standard cell 40 C 16 min 5 min 5 l
O O2 N O N N O
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 (VWD) Furazolidone Precision of RT 10 runs, 100 mg/l N COOH O Nalidixic acid < 0.08 %
Precision of area 10 runs, 100 mg/l < 0.3 % Linearity 1.00000 (correlation factor) 0.99990 (nalidixic acid) Instrumentation: see configuration example 3 on page 78
14
Excitation 230 nm 275 nm 343 nm
Emission 503 nm
Emission [LU]
230/503 nm
20 15 10 5 Trimethoprim 200 250 300 450 400 350 Wavelength [nm] Sulfamethoxazole
Trimethoprim 20
0 500
15
Sulfamethoxazole
10 Wavelength-switching 5 275/343 nm 250/343 nm 0 250/503 nm 0 2 4 6 8 10 12 14 16 18 Multiwavelength detection
Time [min] Analysis of trimethoprim and sulfamethoxazole using the Agilent 1100 Series fluorescence detector (column 2.1 x 50 mm Zorbax SB-C18, 5 m)
1 2 4 6 5
1 2 3 4 5 6
Hydroxybenzotriazole Trimethoprim Furazolidone Sulfamethoxazole Chloramphenicol Nalidixic acid
300 3 200 100 0 0 2 4 6
Standard Cortim Diolan Forte cystitis tablet 8 Time [min] 10 12 14
Analysis of trimethoprim and sulfamethoxazole in Cortim Diolan Forte cystitis tablet
15
Absorbance [mAU] 70 60 50 40 30 20 10 0 0 2 4 6 8 Time [min] Analysis of chloramphenicol in Noriplon pet ointment 10 12 3 1 2 4 5 6
1 2 3 4 5 6
Hydroxybenzotriazole Trimethoprim Furazolidone Sulfamethoxazole Chloramphenicol Nalidixic acid
Standard Noriplon pet ointment
14
16
Anticoagulant Drugs
COCH 3
Absorbance [mAU] 250 200 150 100 50 0 0 2 4 Time [min] Analysis of wafarin using the variable wavelength detector 6
Warfarin
OH
Wafarin
10
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 (VWD) Precision of RT 10 runs, 100 mg/l 0.02 % Precision of area 10 runs, 100 mg/l 0.46 %
4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 20 % B at 10 min 80 % B at 12 min 20 % B variable wavelength detector, 204 nm, standard cell fluorescence detector, 272/355 nm, standard cell 25 C 12 min 5 min 5 l
Emission [LU]
2
Excitation 272 nm
Emission 355 nm
3.5 3 2.5 2 1.5 1 0.5 Instrumentation: see configuration example 3 on page 78
Warfarin
1.5 1 0.5 0 200 400 300 Wavelength [nm] 500
272/355 nm
4 5 Time [min]
Analysis of wafarin using the Agilent 1100 Series fluorescence detector (column: 2.1 mm Zorbax SB-C18, 5 m) 17
Antidepressant Drugs
N O
Absorbance [mAU] 400 350 300 Cl 250 200 150 100 50 1
2 3
1 Bupropion 2 Trazodone 3 Maprotiline
Bupropion
H N
0 0 2 4 Time [min] Analysis of antidepressant drugs 6 8 10
Maprotiline Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 10 min 60 % B at 12 min 10 % B variable wavelength detector 210 nm, standard cell 25 C 12 min 5 min 5 l
N N N
N N
HPLC method performance Cl Trazodone Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.06 % Precision of area 10 runs, 100 mg/l < 0.6 % Instrumentation: see configuration example 2 on page 77
18
Antidepressant Drugs
Absorbance [mAU] 300 250 200 150 100 50 0 0 2
1 2 3 4
5 Chloramphenicol Papaverine Doxycyline 6 Sulfamethoxazole Nalidixic acid 7 Penicillin V Promethazine
8 Penicillin G 9 Verapamil 10 Dextrometorphan
1 2 3 4 5 6 7 8 9 10
6 Time [min]
10
Antidepressant drugs
Selectivity test results of antidepressant drugs
Antidepressant Drug Retention Time [min] Bupropion 5.41
Set Drug Papaverine Doxycycline
Retention Time [min] 5.43 5.48 5.68 6.05 6.24 6.93 7.11 7.61 7.66 7.83
Tradozone
5.80
Sulfamethoxazole Chloramphenicol Dextromethorphan
Maprotiline
7.64
Penicillin G Promethazine Nalidixic acid Penicillin V Verapamil
19
Antiepileptic Drugs
H O N N O Phenobarbital H O N N O Primidone H O H Absorbance [mAU] 400 350 300 250 200 150 100 50 0 0 5 10 15 20 Time [min] 25 30 35 40 1 24 3 2 3 2 4 1 3 56 1 5 4 89 2 4 13 56 7 8 9 6 7 8 9 4x125 mm Purospher RP-18, 5 m 4x125 mm Hypersil ODS, 5 m 1 Caffeine 2 Phenylethylmalonamide 3 Ethosuximide 7 89 3x125 mm Hypersil BDS, 3 m 4 Primidone 5 Phenobarbital 6 Methylphenylsuccinimide 7 Carbamazepineepoxide 8 Phenytoin 9 Carbamazepine
6 7 5
4.6x100 mm TSK gel ODS, 2 m
CONH2 CONH2
Phenylethylmalonamide
Analysis of antiepileptic drugs
Selected column Mobile phase Flow rate Gradient N CONH2 Carbamazepine O Column wash UV detector Column compartment temperature Stop time Post time Injection volume
4.6 x 125 mm Hypersil ODS, 5 m A = water, B = acetonitrile 0.8 ml/min at 0 min 15 % B at 25 min 40 % B at 30 min 80 % B at 32 min 15 % B variable wavelength detector, 204 nm, standard cell 60 C 32 min 5 min 5 l
N CONH2 Carbamazepineepoxide
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.12 % Precision of area 10 runs, 100 mg/l < 1.4 % Linearity > 0.99999 (correlation factor) Instrumentation: see configuration example 2 on page 77
20
Antiepileptic Drugs Ruggedness Test Results
Retention time [min] 17 14 11 8 5 2 58 60 Temperature [C] Dependence of retention time on temperature 62 Phenylethylmalonamide Ethosuximide Primidone Phenobarbital Methylphenylsuccinimide Phenytoin Carbamazepine
Retention time [min] 17 14 11 8 5 2 0.75 0.80 Flow rate [ml/min] Dependence of retention time on flow rate 0.85 Phenylethylmalonamide Ethosuximide Primidone Phenobarbital Methylphenylsuccinimide Phenytoin Carbamazepine
21
Antiepileptic Drugs
Area [counts] 4500 3500 2500 1500 500 204 206 208 210 Wavelength [nm] Dependence of area on wavelength Phenyl ethylmalonamide Ethosuximide Primidone Phenobarbital Methylphenylsuccinimid Phenytoin Carbamazepine
Retention time [min] 17 14 11 8 5 2 88:12 85:15 % Acetonitrile Dependence of retention time on % acetonitrile at start of runs 82:18 Phenylethylmalonamide Ethosuximide Primidone Phenobarbital Methylphenylsuccinimide Phenytoin Carbamazepine
22
Antiepileptic Drugs
Absorbance [mAU] 350 300 250 200 150 100 50 0 0 5
1 2 3 4
Sulfathiazole Sulfamethazine Sulfamerazine Sulfanilamide
5 6 7 8
9 Butalbital Sulfadiazine Hydrocortisone 10 Mephobarbital 11 Amobarbital Prednisolone 12 Allobarbital Butabarbital 13 Barbital

1 2 3 4 5 6 7 8 9 10 11 12 13
Antiepileptics 10 Time [min] 15 20
Selectivity test results of antiepileptic drugs
Antidepressant Drug Retention Time [min] Caffeine 2.33
Set Drug Sulfanilamide Sulfadiazine Sulfathiazole
Retention Time [min] 1.70 2.36 2.50 3.20 3.30 4.33 5.57
Phenylethylmalonamide Ethosuximide Primidone
2.95 3.44 4.26
Sulfamerazine Barbital Sulfamethazine Allobarbital
Phenobarbital Methylphenylsuccinimide Carbamzepinepoxide Phenytoin
7.20 8.18 9.42 13.55 Butalbarbital Butalbital Prednisolone Hydrocortisone Mephobarbital Amobarbital 8.66 9.96 12.23 12.54 13.21 13.47
Carbamazepine
14.26
23
Antiepileptic Drugs
Absorbance [mAU] 300 250 200 150 100 50 0 0 2500000 2000000 1500000 1000000 500000 0 0
1 2 3 4 1 3
Phenylethylmalonamide Ethosuximide Primidone 7 Phenobarbital 6 8 4 5
5 6 7 8
Methylphenylsuccinimide Carbamazepineepoxide Carbamazepine Phenytoin
MS, TIC MS, BPC 1 2 3 Time [min] 4 5
Separation of antiepileptic drugs using LC/MS Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 2.1 x 50 mm, Zorbax SB-C18, 5 m A = 0.5 % HOAc in water, B = 0.5 % HOAC in methanol 0.5 ml/min at 0 min 15 % B at 7 min 45 % B at 8 min 15 % B diode array detector 204/8 nm, reference = 360/100 nm, standard cell 25 C 8 min 5 min 5 l
100 80 60 40 20 0 100 80 60 40 20 0 100 150 100 150
233.0
Max: 95834
N N O
O H
234.0 200 237.1 250 300 350 Max: 1.53526e+006
Phenobarbital M = 232 amu
N CONH2
238.1 200 250 300 350 Mass/charge ratio [m/z]
Carbamazepine M = 236 amu
MS spectra of two antiepileptic drugs
24
Antiepileptic Drugs
2500000 2000000 1500000 1000000 500000 1 2500000 2000000 1500000 1000000 500000 0 2 4 6 Time [min] Extracted ion chromatograms 2 3 4
MS, TIC 5
237 amu 253 amu 190 amu 233 amu 219 amu 142 amu 207 amu
10
Separation of antiepileptic drugs using LC/MS MS conditions Ionization mode Nebulizer pressure Drying gas temperature Drying gas flow Vcap Fragmentor Scan range m/z API-ES positive 35 psig 340 C 12 L/min 4000 volt 70 volt 100 400
25
Antiestrogen Drugs
Absorbance [mAU] 300 250 200 Tamoxifen
Tamoxifen
150 100 50 0 0 2 4 6 8 Time [min] Analysis of antiestrogen drugs 10 12 14
4 x 125 mm Purospher RP-18, 5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 20 % B at 14 min 60 % B at 16 min 20 % B variable wavelength detector 204 nm, standard cell 60 C 16 min 5 min 5 l
HPLC method performance Limit of detection 0.2 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l 0.08 % Precision of area 10 runs, 100 mg/l 0.46 % Instrumentation: see configuration example 2 on page 77
26
Antihistaminic Drugs
N N N
Absorbance [mAU] 200 175 1 2 3 150 125 100 75
1 2 3 4
Tripelenamine Chlorphaniramine Tetracaine Promethazine
Tripelenamine
N N Cl Chlorpheniramine
50 25 0 0 2 4 6 Time [min] Analysis of antihistaminic drugs 8 10
S N Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 10 min 55 % B at 12 min 10 % B variable wavelength detector 204 nm, standard cell 25 C 12 min 5 min 5 l
N Promethazine
N H
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.05 %
O O
Precision of area 10 runs, 100 mg/l < 0.2 %
N Tetracaine
27
Antihypertensive Drugs
O H O N N O
Absorbance [mAU] 140 120 100 80
Captopril
Enalapril
COOH
60 40
Enalapril SH
20 0 0 1 2 3 4 5 6 7 8
Time [min] O N COOH Analysis of antihypertensive drugs
Captopril
4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P O4 in water (pH = 2), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 8 min 55 % B at 9 min 10 % B variable wavelength detector, 204 nm, standard cell 60 C 9 min 5 min 5 l
HPLC method performance Limit of detection 0.5 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l Precision of area 10 runs, 100 mg/l < 0.06 % < 0.4 % Absorbance [mAU] 300 250 200 70 C 150 60 C Instrumentation: see configuration example 2 on page 77 100 50 0 0 2 4 Time [min] 6 8 50 C 40 C 30 C 10 Captopril Enalapril
Linearity (correlation factor) 1.00000
Increasing temperature leads to improved peak shape
28
Antiinflammatory Drugs
COOH H3CO Naproxen
Absorbance [mAU] 1000 Naproxen 800 600 400 200 0 0 2 4 6 Time [min] Analysis of antiinflammatory drugs 8 10
HPLC method performance Limit of detection S/N=2 Precision of RT 10 runs, 100 mg/l Precision of area 10 runs, 100 mg/l 0.01 mg/l (5-l injection),
0.03 % 0.17 % Instrumentation: see configuration example 2 on page 77
29
Antiprotozoal Drugs
HO O 2N N N Metronidazole
Absorbance [mAU] 140 120 100 80 60 40 20 0 0 2 4 Time [min] Analysis of antiprotozoal drugs 6 8 10 Metronidazole
0.06 % 0.15 % Instrumentation: see configuration example 1 on page 76
30
Antitumor Drugs
O O O OH HO O O H O O O H O OH
O N H
Absorbance [mAU] 120 100 80 60 40 20 0 0 2 4 Time [min] Analysis of antitumor drugs 6 8 10 Paclitaxel (Taxol)
Paclitaxel (Taxol)
4 x 125 mm Hypersil ODS, 5 m A = water, B = acetonitrile 1.0 ml/min at 0 min 50 % B at 10 min 90 % B at 12 min 50 % B variable wavelength detector 204 nm, standard cell 25 C 12 min 5 min 5 l
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l 0.07 % Precision of area 10 runs, 100 mg/l 0.79 % Linearity (correlation factor) 1.00000 Instrumentation: see configuration example 2 on page 77
31
Antitumor Drugs
Absorbance [mAU] 300 250 200 150 100 50 0 -50 0 400000 300000 200000 100000 0 0 0.5 1 1.5 2 2.5 Time [min] 3 3.5 4 MS, TIC MS, BPC 4.5 1 Time [min] 2 100 854.4 Paclitaxel (Taxol) 80 M = 853.9 amu 60 855.4 40 20 Max: 191719 0 860 880 900 920 940 Mass/charge ratio [m/z]
Separation of antitumor drugs using LC/MS
2.1 x 50 mm Zorbax SB-C18, 5 m A = 0.5 % HOAc in water, B = 0.5 % HOAc in methanol 0.5 ml/min at 0 min 50 % B at 2 min 65 % B at 4 min 65 % B at 5 min 50 % B diode array detector 210/16 nm, reference = 360/100 nm, standard cell 25 C 5 min 5 min 5 l
MS conditions Ionization mode Nebulizer pressure Drying gas temperature Drying gas flow Vcap Fragmentor Scan range m/z API-ES positive 35 psig 340 C 12 L/min 4000 volt 70 volt 700 900
32
Antitussive Drugs
CH3 H N H
Absorbance [mAU] 350 300 Dextromethorphan
H3CO Dextromethorphan
250 200 150 100 50 0 1 2 3 4 5 6
Time [min] Analysis of antitussive drugs
0.02 % 0.13 %
4.6 x 75 mm Zorbax SB-C18 , 3.5 m A = 0.025 M KH2P O4 in water , (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 8 min 80 % B at 10 min 10 % B variable wavelength detector 204 nm, standard cell 60 C 10 min 5 min 5 l
Instrumentation: see configuration example 2 on page 77 Absorbance [mAU] 400 350 300 250 200 150 100 50 0 0 2 4 Time [min] Dextromethorphan in Formel 44 plus cough sirup 33 6 8 Standard 'Formel 44 plus' cough syrup Dextromethorphan
Catecholamines
HO HO
Norepinephrine
NH2 OH
Absorbance [mAU] 1 140 120 100
2 3
1 Norepinephrine 2 Epinephrine 3 Dihydroxybenzylamine (internal standard) 4 Dopamine 4
HO HO
Epinephrine
80
N OH H
60 40 20 0 0 1 2 3 4 Time [min] Analysis of catecholamines 5 6 7
HO HO
Dopamine
NH2
HO HO
Dihydroxybenzylamine
NH2
4.6 x 75 mm Zorbax SB-C18 , 3.5 m A = 0.025 M KH2P O4 + 0.3 mM heptanesulfonic acid in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 1 % B at 5 min 2 % B at 7 min 15 % B at 8 min 1 % B variable wavelength detector 204 nm, standard cell 30 C 8 min 5 min 5 l
HPLC method performance Limit of detection 0.2 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 1000 mg/l< 0.1 %, 0.24 % (dopamine) Precision of area 10 runs, 1000 mg/l< 0.2 % Instrumentation: see configuration example 2 on page 77
34
Glucocorticoid Drugs (I)
OH O OH
HO
1 Hydrocortisone 2 Beclomethasone dipropionate 3 Hydrocortisone acetate
O Hydrocortisone
80 60 40 20 OCOC 2H5 O OCOC2H5 H Cl Analysis of glucocorticoid drugs 0 0 2 4 6 Time [min] 8 10
HO
O Beclomethasone dipropionate Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4 x 125 mm Hypersil ODS, 5 m A = water, B = acetonitrile 1.0 ml/min at 0 min 20 % B at 10 min 80 % B at 12 min 20 % B variable wavelength detector 254 nm, standard cell 25 C 12 min 5 min 5 l
OCOCH3 O OH
HO
O Hydrocortisone acetate HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l 0.03 % Precision of area 10 runs, 100 mg/l < 0.7 % Linearity (correlation factor)> 0.99997 Instrumentation: see configuration example 2 on page 77
35
Glucocorticoid Drugs (II)
OR O OH Absorbance [mAU] 120 100 O Prednisolone R=H Prednisolone acetate R = OAc 80 60 40 20 0 0 2 4 6 Time [min] OH O OH Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4 x 125 mm Hypersil ODS, 5 m A = water, B = acetonitrile 1.0 ml/min at 0 min 20 % B at 10 min 80 % B at 12 min 20 % B variable wavelength detector 254 nm, standard cell 25 C 12 min 5 min 5 l Analysis of glucocorticoid drugs 8 10 4 1 2 3 4 2 3 Prednisolone Betamethasone Prednisolone acetate Betamethasone valerate
HO
HO
F O Betamethasone
OH O OCOC4H9
HO
HPLC method performance F O Betamethasone valerate Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.04 %
Precision of area 10 runs, 100 mg/l < 0.4 % Linearity (correlation factor) > 0.99997 Instrumentation: see configuration example 2 on page 77
36
H2-Antagonists
N N H
Absorbance [mAU] 120 Cimetidine
100 80 N 60 CN 40 20 0 0 1 2 3 4 5 Time [min] 6 7 8 9 Ranitidine
H H
N N
Cimetidine
N Analysis of H2-antagonists O Column Mobile phase Flow rate Isocratic UV detector NO2 Column compartment temperature Stop time Injection volume 4.6 x 75 mm Zorbax SB-C18 , 3.5 m A = 0.025 M KH2P O4 in water, (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 8 % B at 10 min 8 % B variable wavelength detector 225 nm, standard cell 25 C 10 min 5 l
H H
N N
Ranitidine
HPLC method performance Limit of detection 0.2 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l 0.08 % Precision of area 10 runs, 100 mg/l < 0.7 % Instrumentation: see configuration example 2 on page 77
37
Hypnotic Drugs
O H O N N R1 Barbital R 1 = H, R2 = C2H 5, R3 = C2H 5 Phenobarbital R 1 = H, R2 = C6H 6, R3 = C2H 5 Mephobarbital R 1 = CH3, R2 = C6H 6, R3 = C2H 5 O H O N N H O Analysis of hypnotic drugs Butalbital R3 R2 O Absorbance [mAU] 7 300 250 200 150 100 50 0 0 2 4 6 8 Time [min] 10 12 14 16 1 3 8 2 4 5 6 1 2 3 4 5 6 7 8 Barbital Allobarbital Phenobarbital Butabarbital Butalbital Amobarbital Mephobarbital Flunitrazepam
O H O N N H C 2H 5 Amobarbital O Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4 x 125 mm Hypersil ODS, 5 m A = water, B = acetonitrile 1.0 ml/min at 0 min 10 % B at 16 min 50 % B at 18 min 10 % B variable wavelength detector 204 nm, standard cell 25 C 18 min 5 min 5 l
O H O N N H C 2H 5 O Butabarbital
HPLC method performance O H O N N H CH3 N O2N N F O Allobarbital Limit of detection 0.2 mg/l (5-l injection), S/N=2 0.4 mg/l (butalbital, amobarbital) Precision of RT 10 runs, 100 mg/l < 0.06 % Precision of area 10 runs, 100 mg/l < 0.5 % O Flunitrazepam Instrumentation: see configuration example 1 on page 76
38
Hypnotic Drugs
Absorbance [mAU] 250 200 150 100 50 0 1 0 1400000 1000000 600000 200000 -200000 1
3 45 6
1 2 3 4
Barbital Allobarbital Phenobarbital Butabarbital
5 Butalbital 6 Amobarbital 7 Mephobarbital
MS, TIC MS, BPC 2 3 4 5 Time [min] 6 7 8 9
Separation of hypnotic drugs using LC/MS Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 2.1 x 50 mm Zorbax SB-C18, 5 m A = 0.5 % HOAc in water, B = 0.5 % HOAc in methanol 0.5 ml/min at 0 min 5 % B at 5 min 60 % B at 8 min 60 % B at 10 min 5 % B diode array detector 210/16 nm, reference = 360/100 nm, standard cell 25 C 10 min 5 min 5 l
O H N
100 80 60 40 20 0 100 100 80 60 40 20 Instrumentation: see configuration example 4 on page 79 0 100 141.0
209.0 Max: 314456

O
N H
210.1 200 225.1

H
300
400
Allobarbital M = 208 amu

O N
Max: 322675
N H
226.1 200 300 400 Mass/charge ratio [m/z]
Butalbital M = 224 amu
MS spectra of two hypnotic drugs 39
Hypnotic Drugs
1800000 1000000 200000 1 1400000 1000000 600000 200000 0 2 4 6 Time [min] 8

10
MS, TIC 2 3 4 5 6 7 8 9
Extracted ion chromatograms

247 amu 227 amu 225 amu 213 amu 233 amu 209 amu 185 amu
Separation of hypnotic drugs using LC/MS
HPLC method performance comparison for phenobarbital VWD, 210 nm* Limit of detection S/N = 2 Precision of RT 10 runs, 100 mg/l Precision of area 10 runs, 100 mg/l 0.2 mg/L 0.05 % 0.36 % MS (SIM, 233 amu) 0.01 mg/L 0.02 % 2.83 % Instrumentation: see configuration example 4 on page 79
* using the method described on page 39
MS conditions Ionization mode Nebulizer pressure Drying gas temperature Drying gas flow Vcap Fragmentor Scan range m/z API-ES positive 35 psig 340 C 12 L/min 4000 volt 70 volt 100 500
40
Keratolytic Drugs
COOH COOH Absorbance [mAU] 1000 1 Phthalic acid 800 600 COOH 400 200 0 Benzoic acid 0 2 4 Time [min] 6 2 3 204 nm 230 nm 204 nm 1 Phthalic acid 2 Benzoic acid 3 Salicylic acid
COOH OH
Analysis of keratolytic drugs
Salicylic acid
Column Mobile phase Flow rate Isocratic UV detector
Column compartment temperature Stop time Injection volume
4.6 x 75 mm Zorbax SB-C18 , 3.5 m A = 0.05 M NH4OAc in water (pH = 2.2), B = acetonitrile 1.2 ml/min at 0 min 20 % B at 10 min 20 % B variable wavelength detector at 0 min 204 nm at 2.2 min 230 nm at 3.2 min 204 nm, standard cell 50 C 10 min 5 l
HPLC method performance Limit of detection 0.2 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.1 % 0.16 % (phthalic acid) Precision of area 10 runs, 1000 mg/l< 0.8 % Instrumentation: see configuration example 1 on page 76
41
Muscle-relaxing Drugs
OCH3 OCH3
Absorbance [mAU] 600 Papaverine
H3CO H3CO Paperverine
500 400 300 200 100 0 1 2 3 4 Time [min] 5 6 7
Analysis of muscle-relaxing drugs
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l 0.04 % Precision of area 10 runs, 100 mg/l 0.1 % Instrumentation: see configuration example 2 on page 77
42
Sedative Drugs
H3 C N
O Absorbance [mAU] N Diazepam 500 1 400 300 3 4 2
Cl
1 2 3 4
Oxazepam Clonazepam Flunitrazepam Diazepam
H N
O OH N Oxazepam
200 100 0 0 2 4 Time [min] Analysis of sedative drugs 6 8 10
Cl
CH3 N O2N N
O Flunitrazepam F
H N
O Clonazepam N
O2N
Cl
1 2 3 4 3 4
Oxazepam Clonazepam Flunitrazepam Diazepam
HPLC method performance Limit of detection 0.1 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 10 mg/l < 0.08 % Precision of area 10 runs, 10 mg/l < 0.1 %
60 40 20
Standard
'Diazepam 5 Stada' Tablet 0 0 2 4 Time [min] Analysis of diazepam in Diazepam 5 Stada tablet 6 8 10
43
Sulfa Drugs
H2N
SO2NHR
Absorbance [mAU] 250 200 1 1 2 3 4 5 2 4 Sulfanilamide Sulfadiazine Sulfathiazole Sulfamerazine Sulfamethazine
R=H
Sulfanilamide
R=
S
Sulfathiazole 150 100 50 N 0 Sulfamethazine N 2 4 6 8 10 3 5
R=
12
Time [min] Analysis of sulfa drugs
R=
N
Sulfadiazine
N
R=
Sulfamerazine
Column compartment temperature Stop time Post time Injection volume
4 x 125 mm Hypersil ODS, 5 m A = water, B = acetonitrile 1.0 ml/min at 0 min 10 % B at 10 min 25 % B at 12 min 10 % B variable wavelength detector 254 nm, standard cell 50 C 12 min 5 min 5 l
HPLC method performance Limit of detection 0.2 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.07 % 1.2 % (sulfanilamide) Precision of area 10 runs, 100 mg/l < 0.4 % Instrumentation: see configuration example 2 on page 77
Absorbance [mAU] 1200 1000 800 600 400 200 0 0 2 4
1 2 3 4 5 T = 60 C T = 50 C T = 40 C T = 30 C T = 20 C T = 10 C 6 8 10 Time [min] 12 14
1 2 3 4 5
Sulfanilamide Sulfadiazine Sulfathiazole Sulfamerazine Sulfamethazine
16
Separation of sulfathiazole (3) and sulfamerazine (4) using different column temperatures 44
Therapeutic Peptides
Absorbance [mAU] 250 200 150 100 50 0 -50 -100 0 2 1
2 3 1 2 3 4 4 Oxytocin Angiotensin I Angiotensin II Insulin chain B (bovine)
4 Time [min]
10
Analysis of therapeutic peptides
4.6 x 75 mm Zorbax SB-C18 , 3.5 m A = 0.1 % TFA in water, B = acetonitrile 1.0 ml/min at 0 min 20 % B at 10 min 50 % B at 12 min 20 % B variable wavelength detector 210 nm, standard cell 25 C 12 min 5 min 5 l
45
Tricyclic Antidepressant Drugs
Absorbance [mAU] 3 2 N H Protriptyline 200 150 100 50 0 N R Nortriptyline R=H Amitriptyline R = CH3 Analysis of tricyclic antidepressant drugs 2 4 6 8 10 12 14 16 1 4 6 5 1 2 3 4 5 6 Protriptyline Nortriptyline Doxepin Imipramine Amitriptyline Trimipramine
Time [min]
Column Mobile phase Flow rate Gradient N R Imipramine R=H Trimipramine R = CH3 O Column wash UV detector Column compartment temperature Stop time Post time Injection volume
4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 m A = 0.025M KH2P 04 in water (pH = 7), B = methanol 1.0 ml/min at 0 min 67 % B at 15 min 85 % B at 17 min 67 % B variable wavelength detector 210 nm, standard cell 40 C 17 min 10 min 5 l
N Doxepin HPLC method performance Limit of detection <1.0 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.32 % Precision of area 10 runs, 100 mg/l < 0.6 % Instrumentation: see configuration example 2 on page 77
Absorbance [mAU] 2 350 300 250 200 150 100 50 0 0 2 4 1
5 4
1 2 3 4 5 6 6 Standard
Protriptyline Nortriptyline Doxepin Imipramine Amitriptyline Trimipramine
'Aponal 100' Tablet 6 8 Time [min] 10 12 14 16
Analysis of doxepine in Aponal 100 tablets 46
Fat-Soluble Vitamins
Retinol (A1) Absorbance [mAU] 2 3 1 Retinol (A) 2 Cholecalciferol (D3) 3 a-Tocopherol (E)
800 600 1 400 200 OH 0 HO a- Tocopherol 0 2.5 5 7.5 10
12.5
15
17.5
20
Time [min] Analysis of fat-soluble vitamins
4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 m A = water, B = methanol 1.0 ml/min at 0 min 90 % B at 15 min 100 % B at 20 min 100 % B at 21 min 90 % B variable wavelength detector 210 nm, standard cell 20 C 21 min 5 min 5 l
HPLC method performance Limit of detection <4.0 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 1000 mg/l< 0.10 % Precision of area 10 runs, 1000 mg/l< 0.2 % Instrumentation: see configuration example 2 on page 77
HO
Cholecalciferol (D3)
47
Water-Soluble Vitamins
H3N N N
S OH 2 Cl Thiamine (B1) Absorbance [mAU] 1 300 250 8 6 200 OH 150 100 50 0 0 2 4 6 8 10 Time [min] OH Analysis of water-soluble vitamins 12 14 16 18 2 7 9 10 3 1 2 3 4 5 6 7 8 9 10 11 11 Pyridoxamine Thiamine Niacinamide Pyridoxal Pyridoxine Aminobenzoic acid Pantothenic acid Folic acid Riboflavine Biotin Thiotic acid
HO
HO OH N O N O H Riboflavine (B2)
N N
N HO R
Pyridoxine, Pyridoxamine, Pyridoxal (B6) R = OH, NH2 or = 0 H H Biotin
O H H S O O Niacinamide NH 2 N N N
OH
Column compartment temperature Stop time Post time Injection volume
4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.05M KH2P 04 in water (pH = 2.5), B = acetonitrile 1.0 ml/min at 0 min 0.6 % B, at 0.5 min 0.6 % B, at 4 min 6 % B at 12 min 30 % B, at 17 min 60 % B at 19 min 60 % B at 20 min 0.6 % B variable wavelength detector 204 nm, standard cell 15 C 20 min 10 min 5 l
Excitation 280 nm 30 20
Emission 400 nm Vitamin B 6 40 30 20 10
Excitation Vitamin B 2 262 nm 362 nm
450 nm
Emission 530 nm
LU 100 HPLC method performance Limit of detection <6.0 mg/l (5-l injection), S/N=2 Precision of RT 10 runs, 100 mg/l < 0.40 % Precision of area 10 runs, 100 mg/l < 1.5 % Instrumentation: see configuration example 3 on page 78 80
1
10 0 200 250 300 350 400 450 500 550 Wavelength [nm]
0 200 250 300 350 400 450 500 550 Wavelength [nm]
23 1 2 3 4 270/400 nm 270/530 nm Pyridoxamine (B6) Pyridoxal (B6) Pyridoxine (B6) Riboflavine (B2)
60 40 20 0 0
6 Time [min]
10
Analysis of vitamins B2 and B6 using the Agilent 1100 Series fluorescence detector (column: 21 x 50 mm Zorbax SB-C18, 5 m) 48
Atropa Belladonna Extract
N O O Atropine
OH
Absorbance [mAU] 1000 800 600 400 200 0 0 5 10 Time [min] Analysis of Atropa Belladonna extract 15 20 Atropa Belladonna extract Atropine standard Atropine
Extraction 1 g of the dried and powdered plant (from Caesar & Loretz GmbH, Germany) was refluxed for 30 min in 0.5 M acetic acid. After cooling the pH was adjusted to 9 and the solution was extracted five times with 50 ml chloroform. After drying over sodium sulfate the solvent was removed i. vac. and the residue dissolved in 2.5 ml methanol. After filtration 5 l of the extract were applied to HPLC.
4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 m A = 0.05M KH2P 04 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 10 % B at 20 min 60 % B at 23 min 60 % B at 25 min 10 % B diode array detector 210 nm/16 (ref. 360 nm/100), standard cell 40 C 25 min 5 min 5 l
Absorbance [mAU] 1200 1000 800 600 400 200 0 0 2.5 5 7.5
Norm 1000 800 600 400 200 0 Sample Atropine standard
Match factor: 999
Offset
200 225 250 275 300 325 350 375 Wavelength [nm]
10
12.5 Time [min]
15
17.5
20
22.5
Comparison of sample and standard spectra of atropine 49
Cortex Cinchonae Extract
N HO H3CO N Quinidine H
Absorbance [mAU] 400 350 300 250 200 150 100 50 1 3 4 2
1 2 3 4
Quinidine Quinine Hydroquinidine Hydroquinine
Cinchona extract
Quinidine standard Quinine standard 0 5 10 15 Time [min] 20 25 30
N HO H3CO N Quinine H
4 x 125 mm Purospher RP-18, 5 m A = 0.05M KH2P 04 in water (pH = 3), B = acetonitrile 0.8 ml/min at 0 min 4 % B at 25 min 10 % B at 45 min 30 % B at 46 min 60 % B at 49 min 60 % B at 50 min 4 % B diode array detector 210 nm/16 (ref. 360 nm/100), standard cell 25 C 50 min 5 min 5 l
Extraction The extract (from Caesar & Loretz GmbH, Germany) was diluted (1:10) and 5 l were applied to HPLC.
Quinidine standard 20 15 Absorbance Match factor: 992 10 [mAU] Offset 5 Sample 350 0 200 250 300 350 400 300 Wavelength [nm] 250 200 150 100 50 0 0 5 10 15 Time [min]
160 120 80 40 0
Quinine standard Match factor: 997 Sample 200 250 300 350 Wavelength [nm] Offset 400
20
25
Comparison of sample and standard spectra of quinidine and quinine 50
Dan Shen Dropping Pills Extract
COOH
Protocatechuic acid Absorbance [mAU]
OH OH
500 400 300
Protocatechuic aldehyde Dan Shen extract
CHO
Protocatechuic alde-
200 100 Protocatechuic aldehyde standard 0 2 4 6 8 10 12 14 16 18
OH OH
Time [min] O O O Tanshinone I

Absorbance [mAU] 400 350 300 250 200 150 100 50 0 Protocatechuic acid standard 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Dan Shen extract
Protocatechuic acid
Absorbance [mAU] 160 140 120 100 80 60 40 20 0 11 12 13 14 15 Time [min] 1 2 3
1 Tanshinone I 2 Cryptotanshinone 3 Tanshinone IIA
Dan Shen extract
Tanshinone IIA standard Cryptotanshinone standard Tanshinone I standard 16
O O
Tanshinone IIA
Time [min]
Analysis of Dan Shen dropping pills extract
O O O
Cryptotanshinone
4.6 x 75 mm Zorbax SB-C18, 3.5 m Mobile phase A = 0.025M KH2P 04 in water (pH = 3), B = methanol Flow rate 1.0 ml/min Gradient at 0 min 10 % B at 7 min 40 % B at 7.01 min 65 % B at 15 min 80 % B Column wash at 16 min 100 % B at 19 min 100 % B at 20 min 10 % B UV detector diode array detector 225 nm/16 (ref. 360 nm/100) 254 nm/16 (ref. 360 nm/100), standardcell Column compartment temperature 50 C Stop time 20 min Post time 5 min Injection volume 2 l
Column
Extraction 20 Dan Shen pills were dissolved in 5 ml methanol/water (3:1) in a volumetric flask. The solution was filtered and 2 l were applied to HPLC.
51
Dan Shen Dropping Pills Extract
Norm 4 3.5 3 2.5 2 1.5 1 0.5 0 220 240 Protocatechuic acid standard Match factor: 982
Offset Sample 260 280 300 320 340 360 380
140 120 100 80 60 40 20 0 220 240 260
Protocatechuic aldehyde standard
Match factor: 999
Sample 280 300 320 340 360 380
Offset
2.5 2 1.5 1 0.5 0 220 240 260 Sample
Tanshinone I standard
Match factor: 995
Offset
280
300
320
340
360
380
4 3 2 1 0 220 240 260 280 Sample
Cryptotanshinone standard
Match factor: 994
Offset
300
320
340
360
380
4 3 2 1 Sample 0 220 240 260 280
Tanshinone IIA standard
Match factor: 999
Offset
300
320
340
360
380
Wavelength [nm]
Comparison of sample and standard spectra
52
Ephedra Sinica Stapf Extract
N H
OH
Absorbance [mAU] Ephedrine 500
Ephedrine
400 300
Norephedrine Ephedra extract Norephedrine standard Ephedrine standard
H 2N
OH
200 100 0
Norephedrine
2.5
7.5
10
12.5
15
17.5
Time [min] Analysis of Ephedra Sinica Stapf extract
Extraction 50 ml of 0.5 M H2SO4 was added to 1 g dried plant and the mixture was stirred overnight. After filtration the pH was adjusted to 11-13 by adding 6 M NaOH. 8 g NaCl were added and the mixture extracted with ether (4 x 25 ml). The organic layer was dried over MgSO4, filtered and the solvent was removed i. vac. 5 ml H2O and 3 drops of 4 M HCl were added to the residue.
4.6 x 75 mm Zorbax SB-C18, 3.5 m A = 0.025 M KH2P 04 in water (pH = 3), B = acetonitrile 1.0 ml/min at 0 min 2 % B at 10 min 10 % B at 15 min 80 % B at 18 min 80 % B at 20 min 2 % B diode array detector 204 nm/8 (ref. 360 nm/100), standard cell 25 C 20 min 5 min 5 l
Norm 250 200 150 100 50 0 200
Match factor: 997
Absorbance [mAU] 300 250 200 150 100 Norephedrine 50 0 0 2 4 6 8 Ephedrine
Norepehdrine standard Sample 250 300 350 Wavelength [nm] Match factor: 983 Ephedrine standard Sample Offset 200 250 300 350 Wavelength [nm] 400 Offset 400
Norm 1000 800 600 400 200 0
10 12 Time [min]
14
16
18
Comparison of sample and standard spectra of norephedrine and ephedrine 53
Ginko Biloba Extract and Tablets
OH HO O OH OH Quercetin OH HO O OH OH Kaempferol O O OH Absorbance [mAU] 300 250 200 150 100 50 0 0 2 4 6 8 10 Time [min] 12
1 2 3
1 Quercetin 2 Kaempferol 3 Isorhamnetin (no standard available)
Ginko extract Ginko tablet
Quercetin standard Kaempferol standard 14 16 18
Extraction 4 g of Ginko Biloba extract (from Caesar & Loretz GmbH, Germany) were refluxed for 30 min in 70 ml methanol and 10 ml 25 % HCl. After cooling to room temperature the mixture was filtered and the filter washed with approximately 100 ml methanol. The solvent was partly removed i. vac. and diluted with methanol to 100 ml in a volumetric flask. 5 ml of this solution were filtered through a C18 disposable cartridge. The cartridge was washed with 4 ml methanol and the filtrate diluted to 10 ml in a volumetric flask. The same procedure was used to extract 10 ginko tablets (Promod Ginko biloba L., 40 mg ginko biloba extract per tablet, Sine Laboratories', Shanghai, China) Method and extraction from: A. Hasler, O. Stichler, J. Chromatogr. 508 (1990), 236-240
Analysis of Ginko Bilboba extract Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4 x 125 mm Hypersil ODS, 5 m A = 0.5 % H3P 04 in water, B = methanol 2.0 ml/min at 0 min 38 % B at 12 min 48 % B at 17 min 100 % B at 20 min 38 % B diode array detector 370 nm/16 (ref. off), standard cell 25 C 20 min 5 min 10 l
Norm 800
Absorbance [mAU] 600 500 400 300 200 100 0
600 400 200 0
Quercetin standard Match factor: 973
Sample 200
Offset 300 350 250 Wavelength [nm] 400
Norm 600 400 200 0 200
Kaempferol standard
Match factor: 980
Sample 350 250 300 Wavelength [nm] 400
12 10 Time [min]
14
16
18
Comparison of sample and standard spectra of quercetin and kaempferol 54
Rheum Palmatum Extract
OH
OH Absorbance [mAU] COOH 400 350 300 250 200 Rheum extract Rhein standard Emodin standard 0 2 4 6 8 10 12 14 Rhein Emodin
O Rhein
OH
OH
150 100 50
H3C O Emodin
OH
Time [min] Analysis of Rheum Palmatum extract
Extraction 5 g dried rhubarb root were extracted ultrasonically twice with 50 ml methanol for 30 minutes. The extract was filtered and the solvent evaporated i. vac. The residue was dissolved in 5 ml methanol and applied to HPLC.
4 x 125 mm Hypersil ODS, 5 m A = 0.05 M NH40Ac in water (pH = 2.5), B = acetonitrile 1.0 ml/min at 0 min 30 % B at 10 min 80 % B at 14 min 80 % B at 15 min 30 % B diode array detector 440 nm/16 (ref. off), standard cell 25 C 15 min 5 min 1 l
Absorbance [mAU] Emodin 250 200 150 100 Rhein
Norm 600 400 200 0 Sample 200 Norm 600 400 200 300
Match factor: 999
Rhein standard Offset 400 500 Wavelength [nm] Match factor: 999 Emodin standard 600
50 0 0 2 4 6 8 Time [min]
Sample 200 300 500 400 Wavelength [nm]
Offset 600
10
12
14
Comparison of sample and standard spectra of rhein and emodin 55
Agilent ChemStation Database
The database for the Agilent ChemStation is a relational database which stores data in organizational units (studies), including additional, customizable sample and analysis data. Required data sets can be retrieved quickly and easily for review. The user can set up studies to organize the data and create a query to select the runs of interest. The data can be explored by looking at samples or compounds, and, while reviewing, runs can be marked for approval or rejection. Data can be examined in graphical, tabular or chart format. When reviewing data tables, the user can specify the contents of the tables by specifying the columns he wants to see. Charts can be designed that allow the user to monitor instrument performance or view trends. The user can also generate statistical analyses, either in summary form or as a full regression analysis. When the required results are measured, the user can customize a report layout and generate a report, or export the data to another Windows application, for example, Microsoft Excel. Data can also be reprocessed and assembled into a batch which can be loaded into the Agilent ChemStation batch review. The following pages show two examples where the database of the Agilent ChemStation is used. Literature and a CD-ROM with more details are listed on page 82.
56
Using the Agilent ChemStation Database for Monitoring Drug Production
N N NH 2 H3CO OCH3 Trimethoprim OCH3
NH2
Scenario
A pharmaceutical company produces cystitis tablets containing the antibacterial drugs trimethoprim and sulfamethoxazole. The active compounds are bought from two different suppliers: company AAA and company BBB. The pharmaceutical company has five reaction vessels in which the compounds are mixed and the tablets are produced. The crude products from company AAA are used in vessels #1 and #2, the crude products from company BBB in vessels #3, #4 and #5. The amount of active compounds in the tablets are monitored by HPLC. Therefore, a tablet is dissolved in a certain amount of solvent and the concentration of trimethoprim and sulfamethoxazole is measured. The quality requirements are: trimethoprim: 90 110 mg/l sulfamethoxazole: 150 165 mg/l The results of the HPLC runs are sent to the database of the Agilent ChemStation. One sample from each vessel is measured every day. Some points are overlaid there are five measurement points per day. The following figures show the collected data using the database of the Agilent ChemStation. Figure 1 shows that the amounts of trimethoprim are well within the quality requirements (90-110 mg/l).
O H2 N S O N
N Sulfamethoxazole
Amount [mg/l]
Date
Figure 1 Amount of trimethoprim in cystitis tablets
57
Figure 2 shows the amount of trimethoprim in reaction vessel # 2.

Amount [mg/l]
Date
Figure 2 Amount of trimethorpim in reaction vessel #2
Figure 3 illustrates the results for sulfamethoxazole, which show that on days 4 and 5 some measurement points are below the quality requirements of 150 - 165mg/l.
Amount [mg/l]
Date
Figure 3 Amount of sulfamethoxazole in cystitis tablet
58
Figures 4 and 5 show that for vessels #1 and #2 the amounts of sulfamethoxazole are too low on days 4 and 5.
Amount [mg/l]
Date
Figure 4 Amount of sulfamethoxazole in reaction vessel # 1
Amount [mg/l]
Date
Figure 5 Amount of sulfamethoxazole in reaction vessel # 2
59
Figure 6 displays the amount of sulfamethoxazole by supplier, which shows that the tablets containing sulfamethoxazole from company AAA are below the quality requirements.
Amount [mg/l]
Company AAA
Company BBB
Figure 6 Amount of sulfamethoxazole in cystitis tablet
Conclusion
The measurements on days 4 and 5 which are below the quality requirments are from reactions vessels #1 and # 2. Only runs using sulfamethoxazole from company AAA are below the quality requirements. Comparing the chromatograms of two runs shows that there is an additional peak. This leads to the final conclusion that the sulfamethoxazole from company AAA was not pure.
60
Using the Agilent ChemStation Database for Monitoring Serum Concentration
OCH3 OCH3
Scenario
A company produces antianginal pills containing verapamil. They have to monitor the concentration of the active compound in the serum of the patients. The serum concentration is monitored over a time span of 12 hours in six patients, which took one pill each. Patients data are:
CN
Patient 1 2 3
Sex male (1) male (1) female (2) male (1) female (2) female (2)
Age (years) 20 - 30 (1) 30 - 40 (2) 40 - 50 (3) 40 - 50 (3) 30 - 40 (2) 20 - 30 (3)
Smoker/non-smoker non-smoker (1) smoker (2) non-smoker (1) smoker (2) smoker (2) non smoker (1)
H3CO Verapamil
OCH 3
4 5 6
The results of the HPLC runs were sent to the database of the Agilent ChemStation. Figure 7 shows the serum concentration for all patients. The highest concentration is reached after one hour, then it decreases. From 3 to 12 hours the concentrations differ for some patients they are higher than for others (grey circles).
Concentration [mg/l]
Time [hours]
Figure 7 Serum concentration of verapamil for all patients
61
All three age groups include patients with higher and lower serum concentrations. This is shown in figures 8 - 10. The decomposition rate does, therefore, not depend on age.
Concentration
Time [hours]
Figure 8 Serum concentration of verapamil for patients in the age group 20 - 30 years
Concentration
Time [hours]
Figure 9 Serum concentration of verapamil for patients in the age group 30 - 40 years
62
Concentration
Time [hours]
Figure 10 Serum concentration of verapamil for patients age group 40 - 50 years
Figures 11 and 12 show that the rate of decomposition does not depend on the factor smoker or non-smoker.
Concentration
Time [hours]
Figure 11 Serum concentration of verapamil in non-smokers
63
Concentration
Time [hours]
Figure 12 Serum concentration of verapamil in smokers
Figures 13 and 14 show that the rate of decomposition is higher for males than for females, which indicates that the decomposition rate is dependent on sex.
Concentration
Time [hours]
Figure 13 Serum concentration of verapamil in male patients
64
Time [hours]
Figure 14 Serum concentration of verapamil in female patients
Figures 15 and 16 show example diagrams of a male and a female patient.
Time [hours]
Figure 15 Serum concentration of verapamil in patient 1 (male)
65
Time [hours]
Figure 16 Serum concentration of verapamil in patient 3 (female)
Conclusion
The rate of decomposition of verapamil is not the same for all patients. The diagrams resulting from different queries in the database of the Agilent ChemStation (age, non-smoker/smoker, sex) show that the decomposition rate is only dependent on the sex of the patient. The rate of verapamil decomposition is higher for male patients than for female patients.
66
Agilent 1100 Series Combinatorial Chemistry Analysis System
The Agilent 1100 Series combinatorial chemistry analysis system increases sample throughput, shortens response time and enables significant ease of control. The system can store and inject over 4600 samples for unattended high throughput. Easy-to-use point-and-click software lets the user control the complete system, including sample handling, data analysis and reporting. The new liquid chromatograph/mass spectrometer (Agilent 1100 Series LC/MSD) hard - and software solution provides rapid molecular weight confirmation of combinatorial libraries. Users can identify the products of combinatorial syntheses and estimate their purities rapidly and easily before screening. This gives them fast confirmation of combinatorial methods and efficient verification of large combinatorial synthesis projects. Key benefits of the system include: Complete system integration All system components, i.e. the Agilent 220 microtiter plate sampler, the Agilent 1100 Series HPLC with diode-array detector, the Agilent ChemStation, the Agilent 3D LC ChemStation software, the Agilent 1100 Series LC/MSD and the new combinatorial chemistry software are supplied and supported by Agilent Technologies. Users get a single point of control from system setup to sample reporting. Modes of operation The system has two modes of operation: a sampler mode for high speed sample analysis and a fractionation mode for sample isolation and purification.
67
Easy to use - The system is built around an easy-to-use graphical user interface. A single screen shows the system status, layout of up to 12 96-well plates on the sampler and an overview of the sample and fraction positions. Based on the target molecular weights supplied by the user, the system confirms the presence of these targets. Highly reliable and easy to couple to HPLC maximizes productivity. The system can handle a broad range of sample types and matrices by typical flow rates without clogging, contamination or signal loss. As a result, current HPLC methods can be used without rework. For a typical configuration of an Agilent 1100 Series combinatorial chemistry analysis system turn to page 80. Literature with further information is listed on page 84.
68
Instrumentation
Agilent 1100 Series modules and systems
The Agilent 1100 Series HPLC system and Agilent ChemStations are ideally suited for the analysis of pharmaceutical samples. They meet the most demanding laboratory objectives, such as: helping to complying with regulatory and quality standards, using built-in, fast, features, that offer fast, labor-saving validation reducing operating cost no need for helium due to all electric design, use of 2-mm columns reduces solvent costs, saving operator training time through intuitive user interface and built-in online tutorials, maximizing productivity with fast system setup and intelligent automation, maximizing instrument uptime through use of field-proven parts, multimedia instructions for effective maintenance, software for onsite and remote troubleshooting, and achieving highest system performance, for example, in detector sensitivity for lowest detection limits. Further features for the pharmaceutical laboratory include: high sensitivity diode-array detector for qualitative analysis in the wavelength range of 190 - 950 nm, thermostatted autosampler with a cooling temperature down to 4 C which avoids decomposition of thermally labile samples, temperature-controlled column compartment with a standard Peltier element for heating and cooling, which eliminates analytical errors due to temperature fluctuations above, at and below ambient temperature. variable volume autosampler with integrated injector program, and online vacuum degasser, which removes trace levels of air for higher detector sensitivity and stability of retention times.
69
Instrumentation
Agilent ChemStation for HPLC
Agilent ChemStations are recommended for instrument control, data evaluation and data traceability. Features for the pharmaceutical laboratory include: system suitability software for automated and customized method validation and system suitability testing, report generator to print analysis reports according to regulatory and client requirements, software for automated identification and quantification based on diode-array and/or mass selective detection, spectral libraries for UVvisible and MS spectra (Agilent ChemStation for LC 3D and LC/MSD systems only), control and data acquisition of fluorescence and refractive index detector, enhanced OQ/PV procedures, Agilent 1100 Series control over LAN, HTML reporting capability, optional database as a central secure data organization, review, reporting and archiving tool for chemical analysis results, and optional software extension for networked Agilent ChemStations allowing users to monitor and control analyses in real time across a network. A lab-wide raw data backup solution is also offered.
70
Instrumentation
Detector Overview
Sensitive and selective detectors are very important for the analysis of pharmaceutical samples with HPLC, because of the need to analyze at low concentration levels in complex matrices. Most detectors applied in HPLC are also used for pharmaceutical analysis. The table below gives an overview of HPLC detectors with the most important characteristics and application examples. To provide highest sensitivity for the different compound classes several detection techniques are necessary.
Detector type Sensitivity Variable wavelength detector Diode-array detector high, low ng range
Selectivity low, one signal
Qualitative information low, only by RTs
Compounds antidepressant keratolytic muscle-relaxing
high, low ng range
high, several signals high, RTs and spectra Ginko billobae at different wavelengths Cortex cinchonae Atropa belladonna very high, light high, RTs and absorbance followed online spectra by emission of light must be possible,multisignal acquisition very high, selective search for specified masses antianginal anticoagulant water-soluble vitamins
Fluorescence very high detector low pg range
LC-MS very high, spectrometry pg range
very high, molecular antiepileptic weight and fragantitumor mentation information hypnotic
Variable wavelength detector
The variable-wavelength detector (VWD) is recommended for routine analysis of known mixtures. The detector can be operated and controlled from the Agilent control module and from the Agilent ChemStations. Other features for pharmaceutical laboratories include: high sensitivity for lowest detection limits, and wavelength time programming.
Diode array detector
The diode array detector (DAD) is recommended for the analysis of compounds in difficult matrices, whenever identification via retention time may not be sufficient. For additional confirmation spectra can be taken during the run and compared to those stored in spectral libraries. The evaluation of spectra can be done manually or automatically. The detector is controlled via the Agilent ChemStation for LC 3D systems. Other features include: high sensitivity for lowest detection limits, built-in validation tools, high resolution spectra, wavelength range up to 950 nm, and simultaneous multiwavelength detection allows use of signal ratios for purity control.
71
Instrumentation
Fluorescence detector
The Agilent 1100 Series fluorescence detector provides high-sensitivity multi-signal detection, online fluorescence spectra, straightforward operation and maintenance, and built-in features for GLP compliance. Other features include: simplified optical design for optimized baseline stability, long-life Xenon lamp for highest sensitivity, variable-mode monochromators for selective multisignal detection and online spectra acquisition, easy front access to flow cell for fast inspection or exchange, the timetable allows to setup signal parameters as well as excitation and emission scan ranges for maximum selectivity and sensitivity, the offline fluorescence scan gives complete characterization within a few minutes, for both excitation and emission of single compounds, and built-in diagnostic tools minimize downtime and increase operator safety.
Mass selective detector
The Agilent 1100 Series LC/MSD detector ideally complements a diodearray UV-visible detector. It gives laboratories the molecular weight, structural information, selectivity and sensitivity needed for quick compound identification, purity checks for chromatographic peaks and quantification of trace-level components in complex matrices. Other features include: standard positive and negative ionization modes and a choice of atmospheric pressure ionization electrospray (API-ES) and atmospheric pressure chemical ionization (APCI) modes, allow the analyses of wide variety of compound classes, mass spectral information increases confidence in results and shortens analysis time, and in-source fragmentation (collision-induced dissociation) provides powerful structural information.
Thermostatted column compartment
The Agilent 1100 Series thermostatted column compartment is designed for separations requiring stable temperature to achieve reproducible retention times or separations which improve at temperatures above or below ambient. Other features include: temperature range from 10 C below ambient to 80 C, large compartment for up to three 30 cm columns, easy access to fittings for quick column changeover, convenient Peltier cooling, and column identification module and tag with injection record for regulated laboratories.
72
Instrumentation
Autosampler
Agilent's unique injection principle has been used successfully for many years in thousands of instruments worldwide. This proven design has many outstanding advantages compared to conventional autosamplers, such as : Precise sample handling for dependable 24 hour/day chromatography. The autosamplers repeatability is typically less than 0.5 % for volumes as low as 1 l and less than 0.2 % for 10 l and above. Ease-of-use for fast familiarization, with handheld control module or Agilent ChemStation graphical user interface saves valuable operator time. It offers color-coded sample, calibration and control vials and current sample vials. Low internal volume of 300 l for minimum contribution to a system's total internal volume. Continuous flow-through design. All parts in contact with the sample are flushed during the run, minimizing carry-over to almost zero. Injector program for simple and reliable sample manipulation. 100-sample capacity for full capacity automation, with exchangeable trays for both large and small vials. Single valve design reduces downtime significantly. More help for GLP, which includes self-diagnostics, built-in logbooks and electronic SOPs on CD-ROM. Error-free transfer of methods with PC card makes compliance easier and less costly.
Thermostatted Autosampler
Analyzing thermally-labile compounds has always been a problem for pharmaceutical and biopharmaceutic analysts alike. Traditional cooling methods often leave condensation in the vials. Agilent's new thermostatted autosampler provides Peltier temperature control from 4 - 40 C of up to 100 samples (2-ml vials). By using dry air, condensation build-up is prevented. Other features include: Cooling temperature goes down to 4 C, which avoids decomposition of thermally labile samples. Decomposition caused by UV irradiation is minimized with the use of a light protection cover for the sampler tray. Cooling of highly volatile solvents in capped vials can minimize loss due to evaporation and consequently prevent any change in the sample concentration that may occur over time.
73
Instrumentation
Agilent 220 Micro Plate Sampler
The Agilent 220 micro plate sampler is an ideal tool to analyze large numbers of structurally distinct compounds. Integrated into the Agilent 1100 Series HPLC system it offers the full potential of well-plate technology. Other features include: High sample capacity, which increases sample throughput and shortens response times. This is especially useful for drug discovery, combinatorial chemistry, medicinal chemistry and pharmacokinetic studies. The choice of various plates, vials and tubes, including up to 12 micro plates The flexibility of sampler mode for high-speed sample analysis or fraction collector mode for sample isolation and purification. This gives the user a versatile tool to support screening, analysis and research. An autosampler injector program for sample preparations such as derivatization, dilution and mixing. The ability to store and inject over 4,600 samples (using 384-wellplates) for unattended high throughput. Easy-to-see color-coded sample and fraction tracking for a fast, clear overview of the analysis results.
Binary Pump
Whether operating as a standalone module with the handheld control module or as part of an Agilent ChemStation-controlled HPLC system, the binary pump is designed for top performance high-pressure mixing. It is the pump of choice for reproducible gradients at low flow rates. An optional programmable solvent selection valve combines two out of four solvents for utmost flexibility and increased convenience, for example, when flushing the column. Other features include: Two dual piston, in-series pumps in one housing provide a minimum of benchspace and lowest internal and external capillary connections. Both pistons of the two individual pumps are servo-controlled to meet the highest chromatographic demands in gradient formation. Ideal for reproducible gradient at low flow rates due to an internal volume of only 180 to 480 l (without mixer), highest composition stability and a flow range starting at typically 50 l/min. A flow range of up to 5.0 ml/min, which is ideal for standard-bore chromatography. Excellent high-pressure mixing capabilities and reliable solvent delivery for standard-bore columns without solvent degassing.
74
Instrumentation
Quaternary Pump
An optimized design ensures virtually pulse-free and stable solvent flow, with dual floating pistons in series, precisely servo-controlled. This design with variable stroke volume allows pulse-free solvent delivery and efficient mixing. An inlet valve, electronically activated and synchronized to piston stroke, eliminates vapor formation with volatile solvents and requires no maintenance. High-speed proportioning valves offer convenient low-pressure mixing of up to four solvents and highest flexibility in solvent choices. Other features include: Convenient access to four solvents for isocratic or gradient analysis speeds up preparation of mobile phases, flushing the HPLC system and method development. A wide flow range up to 10 ml/min and a delay volume of 800 to 1100-l support narrow-bore, standard and semipreparative applications. Easy programming and control through the handheld control module or the Agilent ChemStation help save time. Robust materials withstand most demanding applications. Stainless steel, titanium, gold, ruby, sapphire, ceramics, PEEK and PTFE ensure long lifetime. The seal wash option for high salt mobile phases keeps maintenance to a minimum which reduces operation costs. More help for GLP built-in leak and pressure test with graphical presentation of pressure profiles, and early maintenance feedback.
Vacuum degasser
Dissolved gases are evacuated through special membrane tubes, providing constant near-zero levels of air for improved baseline and drift. Other features include: Low internal volume for fast changeover of mobile phases. High degassing efficiency for trouble-free operation and highest performance, eliminating the need for helium. Four solvent selection for maximum versatility.
75
Configuration Example 1
Description Agilent 1100 Series quaternary pump. Includes Agilent 1100 Series vacuum degasser. Agilent 1100 Series autosampler. Agilent 1100 Series thermostatted column compartment. Agilent 1100 Series diode array detector. Standard flow cell, 10mm pathlength. Agilent ChemStation for LC 3D system. Includes G2170AA single instrument Agilent ChemStation software, G2180AA spectral processing software, HP Kayak computer, CD-ROM drive, HP LaserJet printer, Microsoft Windows 4.0 NT. Optional database for Agilent ChemStation: Agilent ChemStore C/S database client software. Order G1354A G1313A G1316A G1315A G1314A #018 G1319A
G2181BA
CAN 5181-1516
G1354A
CAN 5181-1516 Minimum instrument configuration: quaternary pump with degasser autosampler thermostatted column compartment variable wavelength detector Agilent ChemStation CAN 5181-1516
G1313A
G1316A HP-IB 10833D G1315A G1319A Printer
76
Description Agilent 1100 Series quaternary pump. Includes Agilent 1100 Series vacuum degasser. Agilent 1100 Series thermostatted autosampler. Agilent 1100 Series thermostatted column compartment. Agilent 1100 Series diode array detector. Standard flow cell, 10mm pathlength. Agilent ChemStation for LC 3D system. Includes G2170AA single instrument Agilent ChemStation software, G2180AA spectral processing software, HP Kayak computer, CD-ROM drive, HP LaserJet printer, Microsoft Windows 4.0 NT. Optional database for Agilent ChemStation: Agilent ChemStore C/S database client software. Order G1354A G1327A G1316A G1315A G1314A #018 G1319A
G2181BA
CAN 5181-1516
G1354A
CAN 5181-1516 Minimum instrument configuration: quaternary pump with degasser autosampler thermostatted column compartment variable wavelength detector Agilent ChemStation CAN 5181-1516
G1327A
77
Description Agilent 1100 Series quaternary pump. Includes Agilent 1100 Series vacuum degasser. Agilent 1100 Series thermostatted autosampler. Agilent 1100 Series thermostatted column compartment. Agilent 1100 Series diode array detector. Standard flow cell, 10mm pathlength. Agilent 1100 Series fluorescence detector. Agilent ChemStation for LC 3D system. Includes G2170AA single instrument Agilent ChemStation software, G2180AA spectral processing software, HP Kayak computer, CD-ROM drive, HP LaserJet printer, Microsoft Windows 4.0 NT. Optional database for Agilent ChemStation: Agilent ChemStore C/S database client software. Order G1354A G1327A G1316A G1315A G1314A #018 G1321A G1319A
G2181BA
CAN 5181-1516
G1354A
CAN 5181-1516
G1327A
CAN 5181-1516 Minimum instrument configuration: quaternary pump with degasser autosampler thermostatted column compartment variable wavelength detector fluorescence detector Agilent ChemStation CAN 5181-1516
G1316A
G1315A
HP-IB 10833D G1321A G1319A Printer
78
Description Agilent 1100 Series quaternary pump. Includes Agilent 1100 Series vacuum degasser. Agilent 1100 Series thermostatted autosampler. Agilent 1100 Series thermostatted column compartment. Agilent 1100 Series diode array detector. Standard flow cell, 10mm pathlength. Agilent ChemStation for LC 3D system. Includes G2170AA single instrument Agilent ChemStation software, G2180AA spectral processing software, HP Kayak computer, CD-ROM drive, HP LaserJet printer, Microsoft Windows 4.0 NT. Agilent 1100 Series LC/MSD spectrometer. API-Electrospray source for Agilent 1100 Series LC/MSD. LC/MSD ChemStation add-on software. Optional database for Agilent ChemStation: Agilent ChemStore C/S database client software. Order G1354A G1327A G1316A G1315A G1314A #018 G1319A
G1946A G1948A G2715AA
G2181BA
CAN 5181-1516
G1354A
CAN 5181-1516
G1327A
CAN 5181-1516
CAN 5181-1516
HP-IB 10833D
G1946A
79
Configuration Example for Agilent 1100 Series Combinatorial Chemistry Analysis System
Description Agilent 1100 Series quaternary pump. Includes Agilent 1100 Series vacuum degasser. Agilent 1100 Series thermostatted autosampler. Agilent 1100 Series thermostatted column compartment. Agilent 1100 Series diode array detector. Standard flow cell, 10mm pathlength. Agilent 1100 Series LC/MSD spectrometer. API-Electrospray source for Agilent 1100 Series LC/MSD. Agilent 220 micro plate sampler system. Agilent 220 microplate sampler fraction collection accessory. Agilent ChemStation for LC 3D system. Includes G2170AA single instrument Agilent ChemStation software, G2180AA spectral processing software, HP Kayak computer, CD-ROM drive, HP LaserJet printer, Microsoft Windows 4.0 NT. LC/MSD ChemStation add-on software. Data analysis software. Optional database for Agilent ChemStation: Agilent ChemStore C/S database client software. Order G1354A G1327A G1316A G1315A G1314A #018 G1946A G1948A G2250A G2251A
G1319A
G2715AA INB2202
G2181BA
CAN 5181-1516
G1354A
CAN 5181-1516
G1316A
HCP-CBLE-RS 5181-1527 HP-IB 10833D
G2250A
Remote 05989-60090
G1315A
HCP-CBLE-CS 5181-1520
G1946
HP-IB 10833D G1319A
Printer
80
Quick Reference Guide
The following table gives an overview of basic analysis parameters of a large number of pharmaceutical drugs. They are listed according to their substance class. The information includes: the stationary phase the mobile phase the column temperature the detector As shown in the table below, the variable wavelength detector is the detector most commonly used. The diode array and fluorescence detectors are also used for certain applications. Other recommended modules include a vacuum degasser, a quaternary pump, a thermostatted autosampler, a thermostatted column compartment and an Agilent ChemStation for LC systems.
Drug Stationary Phase Mobile Phase Temperature Detector
Analgesic drugs Antipyrine, Hydroxyantipyrine, Acetaminophen Androgen drugs Testosterone acetate, Testosterone Antianginal drugs Verapamil
Zorbax SB-C18
0.05M KH2P 04 25 C pH=3/acetonitrile
VWD, 204 nm
Hypersil ODS
water/acetonitrile 40 C
VWD (TT) 254 nm, 234 nm VWD, 204 nm FLD, 228/312 nm 60 C VWD (TT) 220 nm, 254 nm 204 nm VWD 270 nm VWD, 204 nm
Zorbax SB-C18
Antiarrythmic drugs Quinidine, Disopyramide Purosphere RP-18 0.05 KH2P 04 Procainamide pH=2.5/acetonitrile N-Acetylprocainamide Antiasthmatic drugs Caffeine, Theophylline, Hypersil ODS water/acetonitrile 50 C Enprofylline,Theobromine Antibacterial drugs Penicillin-like: Zorbax SB-C18 0.025 KH2P 04 40 C Ampicillin, Amoxicillin, pH=3/acetonitrile Penicillin G, Penicillin V Tetracyclines: Zorbax SB-C18 0.025 KH2P 04 25 C Minocycline, Tetracycline, pH=3/acetonitrile Doxycycline Miscellaneous: Zorbax SB-C18 0.025 KH2P 04 40 C Hydroxybenzotriazole , pH=3/acetonitrile Chloramphenicol, Trimethoprim Sulfamethoxazole, Furazolidone, Nalidixic Acid Anticoagulant drugs Warfarin Zorbax SB-C18 0.025 KH2P 04 25 C pH=3/acetonitrile Antidepressant drugs Bupropion, Trazodone, Zorbax SB-C18 0.025 KH2P 04 25 C Maprotiline pH=3/acetonitrile
VWD, 350 nm
VWD (TT) 204 nm 368 nm 254 nm
VWD, 204 nm FLD, 272/355 nm VWD, 210 nm
81
Drug
Stationary Phase
Mobile Phase
Temperature Detector
Antiepileptic drugs Caffeine, Phenytoin, Hypersil ODS water/acetonitrile 60 C Methylphenylsuccinimide, Phenylethylmalondiamide, Carbamazepinepoxid, Ethosuximide, Phenobarbital, Carbamazepine, Primidone Antiestrogen drugs Tamoxifen Purosphere RP-18 0.025M KH2P 04 60 C pH = 3/acetonitrile Antihistaminic drugs Tetracaine, Promethazine, Zorbax SB-C18 0.025M KH2P 04 25 C Chlorpheniramine, pH = 3/acetonitrile Tripelenamine Antihypertensive drugs Enalapril, Captopril Zorbax SB-C18 0.025M KH2P 04 60 C pH = 2/acetonitrile Antiinflammatory drugs Naproxen Zorbax SB-C18 0.025M KH2P 04 25 C pH = 3/acetonitrile Antiprotozoal drugs Metronidazole Zorbax SB-C18 0.025M KH2P 04 25 C pH = 3/acetonitrile Antitumor drugs Paclitaxel (Taxol) Hypersil ODS water/acetonitrile 25 C Antitussive drugs Dextromethorphan
VWD, 204 nm
VWD, 204 nm
VWD, 204 nm
VWD, 204 nm
VWD, 230 nm
VWD, 320 nm
VWD, 204 nm
Zorbax SB-C18
0.025M KH2P 04 60 C pH = 3/acetonitrile 0.025M KH2P 04 30 C + 0.3 mM heptanesulfonic acid pH = 3/acetonitrile water/acetonitrile 25 C
VWD, 204 nm
Catecholamines Norepinephrine Zorbax SB-C18 Epinephrine, Dihydroxybenzylamine, Dopamine Glucocorticoid drugs Beclomethasone-dipropionate,Hypersil ODS Prednisolone, Prednisolon-acetate, Betamethasone, Betamethason-valerat, Hydrocortisone, Hydrocortisone-acetate H2-Antagonists Ranitidine, Cimetidine Zorbax SB-C18 Hypnotic drugs Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital, Amobarbital, Methylphenobarbital, Flunitrazepam
VWD, 204 nm
VWD, 254 nm
0.025M KH2P 04 25 C pH = 3/acetonitrile water/acetonitrile 25 C
VWD, 225 nm
Hypersil ODS
VWD, 204 nm
82
Drug
Stationary Phase
Mobile Phase
Keratolytic drugs Salicylic Acid, Zorbax SB-C18 Phtalic Acid, Benzoic Acid Muscle-relaxing drugs Papaverine Zorbax SB-C18 Sedative drugs Diazepam, Oxazepam, Zorbax SB-C18 Clonazepam, Flunitrazepam Sulfa drugs Sulfanilamide, Sulfadiazine, Hypersil ODS Sulfathiazole, Sulfamerazine, Sulfamethazine, Therapeutic peptides Angiotensin II, Angiotensin I, Zorbax SB-C18 Insulin, Oxytocin Tricyclic antidepressant drugs Protriptyline, Nortriptyline, Zorbax Eclipse Doxepin, Imipramine, XDB C-18 Amitriptyline, Trimipramine Vitamins Fat soluble Zorbax Eclipse Vitamins A1, D3 E XDB C-18 Water soluble Zorbax SB-C18 Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide, Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine, Riboflavine, Thiamine, Thiotic Acid
0.05M NH40Ac 50 C pH=2.2/acetonitrile 0.025M KH2P 04 60 C pH=3/acetonitrile 0.025M KH2P 04 25 C pH=3/acetonitrile water/acetonitrile 50 C
VWD (TT), 204 nm, 230 nm VWD, 254 nm
VWD, 210 nm
VWD, 254 nm
0.1 % TFA/ acetonitrile 0.025M KH2P 04 pH=7/methanol
25 C
VWD, 210 nm
40 C
VWD, 210 nm
water/methanol 0.05M KH2P 04 pH=2.5/ acetonitrile
20 C 15 C
VWD, 210 nm VWD, 204 nm
Medical Herb/ Active Compound Atropa belladonna Atropine Cortex cinchonae Quinine, Quinidine
Stationary Phase
Mobile Phase
Zorbax Eclipse XDB-C18
DAD, 210 nm/16
Purosphere RP-18 0.05M KH2P 04 25 C pH=3/acetonitrile 0.025M KH2P 04 pH=3/methanol 50 C
DAD, 210 nm/16
Dan Shen Protocatechuic Acid, Zorbax SB-C18 Protocatechuic Aldehyde, Tanshinone I, Tanshinone IIA, Cryptotanshinone Ephedra sinica stapf Ephedrine, Norephedrine Zorbax SB-C18 Ginko bilobae Quercetin, Kaempferol Rheum palmatum Rhein, Emodin
DAD, 225 nm/16 254 nm/16
0.025M KH2P 04 25 C pH=3/acetonitrile 0.5 % H3P 04/ methanol 25 C
DAD, 204 nm/8
Hypersil ODS
DAD, 370 nm/16
Hypersil ODS
0.05M NH4O A c 25 C pH=2.5/acetonitrile
DAD, 440 nm/16
83
Literature
Literature for further reading is available on our worldwide web site at http://www.chem.agilent.com/cag/literature/apglit.html
Publication Title
Agilent publication number
Pharmaceutical Applications Analysis of Antibacterial Drugs by HPLC Analysis of Antiasthmatic Drugs by HPLC Analysis of Antiarrythmic Drugs by HPLC Analysis of Antianginal Drugs by HPLC Analysis of Anticoagulant Drugs by HPLC Analysis of Therapeutic Peptides by HPLC Analysis of Sedative Drugs by HPLC Analysis of Sulfa Drugs by HPLC Analysis of Muscle-relaxing Drugs by HPLC Analysis of Keratolytica by HPLC Analysis of H2-Antagonist Drugs by HPLC Analysis of Antitussive Drugs by HPLC Analysis of Antiprotozoal Drugs by HPLC Analysis of Antiinflammatory Drugs by HPLC Analysis of Antiestrogen Drugs by HPLC Analysis of Antiepileptic Drugs by HPLC Analysis of Protocatechuic Acid, Protocatechuic Aldehyde and Tanshinones in Dan Shen pills by HPLC Analysis of Ephedrine and Norephedrine in Ephedra Sinica Stapf by HPLC Analysis of Antitumor Drugs by HPLC Analysis of Antihypertensive Drugs by HPLC Analysis of Antihistaminic Drugs by HPLC Analysis of Antidepressant Drugs by HPLC Analysis of Androgen Drugs by HPLC Analysis of Analgesic Drugs by HPLC Analysis of Catecholamines by HPLC 5968-1078E 5968-1079E 5968-1080E 5968-1081E 5968-1082E 5968-1109E 5968-1110E 5968-1111E 5968-1112E 5968-1113E 5968-1114E 5968-1115E 5968-1116E 5968-1117E 5968-1118E 5968-1119E
5968-2882E 5968-2884E 5968-2886E 5968-2887E 5968-2889E 5968-2963E 5968-2964E 5968-2965E 5968-2966E
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Analysis of Glucocorticoids by HPLC Analysis of Hypnotic Drugs by HPLC Analysis of Tricyclic Antidepressant Drugs by HPLC Analysis of Fat-Soluble Vitamins by HPLC Analysis of Water-Soluble Vitamins by HPLC Analysis of Rhein and Emodin in Rhubarb Root Extract (Rheum palmatum) by HPLC
5968-2967E 5968-2968E 5968-2969E 5968-2970E 5968-2971E
5968-2972E
Analysis of Quercetin and Kaempferol in Ginko Extract and Tablets (Ginko biloba) by HPLC 5968-2973E Analysis of Quinine and Quinidine in Cinchona Bark Extract (Cortex cinchonae) by HPLC Analysis of Atropine in Belladonna Extract (Atropa Belladonna) Drugs by HPLC
5968-2974E 5968-2975E
Agilent ChemStation Database From Data to Decisions (CD-ROM) 5968-1713E 5966-
Increase Lab Productivity by Turning Chemical Analysis Data into Scientific Knowledge 1990E Drive Down Costs and Raise Productivity Quality Control of Pharmaceutical Drugs Turning Analysis and Performance Data into Comprehensible Charts
5968-4782E
5968-7570E
Agilent 1100 Series Combinatorial Chemistry Analysis System Agilent 220 Micro Plate Sampler with the Agilent 1100 Series System for Flexible High Throughput HPLC Analysis Isolation and Characterization of Natural Products Using the Agilent 220 Micro Plate Sampler with the Agilent 1100 Series HPLC System High Throughput Screening of a Peptide Library with the Agilent 1100 Series Combinatorial Chemistry Analysis System Agilent 220 Micro Plate Sampling System 5968-8661E 5968-9101E
5968-5322E
5968-6252E
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Pharmaceutical Applications With HPLC

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Pharmaceutical Applications With HPLC

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Pharmaceutical Applications with HPLC Solutions Guide

Agilent ChemStation Database

Agilent 1100 Series Combinatorial Chemistry Analysis System

Medical Herb Extracts Active Compounds

Protocatechuic Acid, Protocatechuic Aldehyde, Tanshinone I, Tanshinone IIA, Cryptotanshinone 51 53 54 55

Ephedra sinica stapf Ginko bilobae Rheum palmatum

Ephedrine, Norephedrine Quercetin, Kaempferol Rhein, Emodin

Absorbance [mAU] 120 100

1 Acetaminophen 2 Antipyrine 3 Hydroxyantipyrine 2 3

Analysis of analgesic drugs

Time [min] Analysis of androgen drugs

Column Mobile phase Flow rate Gradient Column wash UV detector

Instrumentation: see configuration example 3 on page 78

Excitation 228 nm 268 nm

2.5 2 1.5 1 0.5

1 Procainamide 2 N-Acetylprocainamide 3 Quinidine 4 Hydroquinidine * 5 Disopyramide

Time [min] Analysis of antiarrythmic drugs

Theobromine R=H Caffeine R=CH3 Enprofylline R=C3H 7

Time [min] Analysis of antiasthmatic drugs

Theobromine Theophylline Enprofylline Caffeine

Solostin antiasthmatic drops 0 0 1 2 3 4 5 6 7 8

Time [min] Analysis of theophylline in Solosin antiasthmatic drops

Antibacterial Drugs with Pencillin-like Structure

Absorbance [mAU] 1 2 3 4 Amoxicillin Ampicillin Penicillin G Penicillin V

1000 800 600 400 1 4 2 3

0 0 2 4 6 Time [min] Analysis of antibacterial drugs with pencillin-like structure 8 10 12

Antibacterial Drugs - Tetracyclines

Absorbance [mAU] 2 120 100 80 60 40 1 3 1 Minocycline 2 Tetracycline 3 Doxycycline

N(CH3)2 Analysis of tetracyclines

Miscellaneous Antibacterial Drugs

Analysis of miscellaneous antibacterial drugs

Column Mobile phase Flow rate Gradient Column wash UV detector

O Column compartment temperature Stop time Post time Injection volume

Miscellaneous Antibacterial Drugs

Excitation 230 nm 275 nm 343 nm

10 Wavelength-switching 5 275/343 nm 250/343 nm 0 250/503 nm 0 2 4 6 8 10 12 14 16 18 Multiwavelength detection

Absorbance [mAU] 500 400

Hydroxybenzotriazole Trimethoprim Furazolidone Sulfamethoxazole Chloramphenicol Nalidixic acid

300 3 200 100 0 0 2 4 6

Standard Cortim Diolan Forte cystitis tablet 8 Time [min] 10 12 14

Analysis of trimethoprim and sulfamethoxazole in Cortim Diolan Forte cystitis tablet

Miscellaneous Antibacterial Drugs

Absorbance [mAU] 70 60 50 40 30 20 10 0 0 2 4 6 8 Time [min] Analysis of chloramphenicol in Noriplon pet ointment 10 12 3 1 2 4 5 6

Hydroxybenzotriazole Trimethoprim Furazolidone Sulfamethoxazole Chloramphenicol Nalidixic acid

Standard Noriplon pet ointment

3.5 3 2.5 2 1.5 1 0.5 Instrumentation: see configuration example 3 on page 78

1.5 1 0.5 0 200 400 300 Wavelength [nm] 500

Absorbance [mAU] 400 350 300 Cl 250 200 150 100 50 1

1 Bupropion 2 Trazodone 3 Maprotiline

0 0 2 4 Time [min] Analysis of antidepressant drugs 6 8 10

Absorbance [mAU] 300 250 200 150 100 50 0 0 2

5 Chloramphenicol Papaverine Doxycyline 6 Sulfamethoxazole Nalidixic acid 7 Penicillin V Promethazine

8 Penicillin G 9 Verapamil 10 Dextrometorphan

Selectivity test results of antidepressant drugs

Antidepressant Drug Retention Time [min] Bupropion 5.41

Set Drug Papaverine Doxycycline

Sulfamethoxazole Chloramphenicol Dextromethorphan

Penicillin G Promethazine Nalidixic acid Penicillin V Verapamil

4.6x100 mm TSK gel ODS, 2 m

Analysis of antiepileptic drugs

Antiepileptic Drugs Ruggedness Test Results

Absorbance [mAU] 350 300 250 200 150 100 50 0 0 5

Sulfathiazole Sulfamethazine Sulfamerazine Sulfanilamide