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General Description
Agilent Technologies has developed a large number of keyHPLC applications for the pharmaceutical market. These appli-cations include analysis equipment, separation media and chro-matographic conditions. All proposed solutions are accompa-nied by examples, configuration overviews and method valida-tion data.
This guide gives a condensed overview of the analysis of pharmaceutical drugs using HPLC. It has been written to findstarting conditions for application development and to makeordering of the entire HPLC systems easy, correct and complete. The guide contains seven sections:
• Applications – include detailed chromatographic condi-tions, performance of the HPLC method, and reference to aconfiguration 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 highsample throughput system.
• Instrumentation – describes the analysis equipment usedfor the applications in this guide.
• Configuration examples – give an overview of recom-mended HPLC equipment with ordering information for thedescribed applications.
• Quick reference guide – lists the analysis conditions ofmore than 100 pharmaceutical drugs.
• Literature – provides an overview of publications, in whichthe results of the proposed solutions were published.
Pharmaceutical Applications with HPLC
Agilent TechnologiesInnovating the HP Way
Solutions Guide
April 2000
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!
3
HPLC Applications High performance liquid chromatography (HPLC) is an important toolfor the analysis of pharmaceutical drugs, for drug monitoring and forquality assurance. The method enables complex mixtures, for example,herbal medicine plant extracts, to be separated into individual com-pounds, which can be identified and quantified by suitable detectors anddata handling systems. Separation and detection occurs at ambient tem-perature or slightly above. Therefore, the method is ideally suited forcompounds of limited thermal stability.
State-of-the-art HPLC equipment can automate HPLC separations, usingautomatic samplers, injectors, microprocessor-controlled analytical con-ditions and ChemStations for data evaluation. Important requirementsfor 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 pharmaceuticallaboratories and companies, but also the precision of the results byeliminating human errors.
An overview of the pharmaceutical drugs used in this guide is presentedon pages 5-6.
Introduction
4
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 chromatographicresults, as well as safeguard data. It supports important end-user taskssuch as reviewing and summarizing results for statistical evaluation,archiving and restoring data and creating control charts and cross-sample reports. These services also aid users in validating their methods and doing on-going system suitability testing. Two examplesusing the database for the Agilent ChemStation are described on pages56-66.
The new Agilent 1100 Series combinatorial chemistry analysis system isa fully automated analysis solution designed to speed up the process ofdrug discovery. The combination Agilent 1100 Series HPLC, Agilent1100 Series LC/MSD detector and the Agilent 220 micro plate sampleroffers the full potential of well plate technology and is the ideal tool foranalyzing complex libraries. Details can be found on page 67.
Agilent ChemStation Database
Agilent 1100 SeriesCombinatorial ChemistryAnalysis System
5
Application Overview
Group Examples PageAnalgesic drugs Antipyrine, Hydroxyantipyrine, Acetaminophen 7
Androgen drugs Testosterone Acetate, Testosterone 8
Antianginal drugs Verapamil 9
Antiarrythmic drugs Quinidine, Disopyramide, Procainamide, N-Acetylprocainamide 10
Antiasthmatic drugs Caffeine, Theophylline, Enprofylline, Theobromine 11Antibacterial drugsPenicillin-like Ampicillin, Amoxicillin, Penicillin G, Penicillin V 12 Tetracyclines Minocycline, Tetracycline, Doxycycline 13 Miscellaneous Hydroxybenzotriazole, Chloramphenicol, Trimethoprim
Sulfamethoxazole, Furazolidone, Nalidixic Acid 14
Anticoagulant drugs Warfarin 17
Antidepressant drugs Bupropion, Trazodone, Maprotiline 18Antiepileptic drugs Caffeine, Phenytoin, Methylphenylsuccinimide, Phenylethylmalon-
amide, Carbamazepinepoxide, Ethosuximide, Phenobarbital, Carbamazepine, Primidone 20
Antiestrogen drugs Tamoxifen 26Antihistaminic drugs Tetracaine, Promethazine, Chlorpheniramine, Tripelenamine 27
Antihypertensive drugs Enalapril, Captopril 28
Antiinflammatory drugs Naproxen 29
Antiprotozoal drugs Metronidazole 30
Antitumor drugs Paclitaxel (Taxol) 31
Antitussive drugs Dextromethorphan 33Catecholamines Norepinephrine, Epinephrine, Dihydroxybenzylamine,
Dopamine 34Glucocorticoid drugs Beclomethasone Dipropionate, Prednisolone,
Prednisolone Acetate, Betamethasone, Betamethasone Valerate,Hydrocortisone, Hydrocortisone Acetate 35
H2-Antagonists Ranitidine, Cimetidine 37Hypnotic drugs Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital,
Amobarbital, Mephobarbital, Flunitrazepam 38Keratolytic drugs Salicylic Acid, Phtalic Acid, Benzoic Acid 41Muscle-relaxing drugs Papaverine 42Sedative drugs Diazepam, Oxazepam, Clonazepam, Flunitrazepam 43Sulfa drugs Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine,
Sulfamethazine 44
Therapeutic peptides Angiotensin II, Angiotensin I, Insulin, Oxytocin 45Tricyclic anti- Protriptyline, Nortriptyline, Doxepin, Imipramine,depressant drugs Amitriptyline, Trimipramine 46VitaminsFat soluble Vitamins A1, D3, E 47 Water soluble Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide,
Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine, Riboflavine, Thiamine, Thiotic Acid 48
6
Medical Herb Extracts Active Compounds PageAtropa belladonna Atropine 49
Cortex cinchonae Quinine, Quinidine 50
Dan Shen Protocatechuic Acid, Protocatechuic Aldehyde, Tanshinone I,
Tanshinone IIA, Cryptotanshinone 51
Ephedra sinica stapf Ephedrine, Norephedrine 53
Ginko bilobae Quercetin, Kaempferol 54
Rheum palmatum Rhein, Emodin 55
7
Analysis of analgesic drugs
Analgesic Drugs
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A= 0.05 M KH2P 04 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 10 min 40 % B Column wash at 12 min 10 % BUV detector variable wavelength detector,
204 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.07 %
Precision of area10 runs, 100 mg/l < 0.7 % Instrumentation:
see configuration example 2 on page 77
�
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�
���
�� �
�
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Antipyrine
Hydroxyantipyrine
Acetaminophen
1 Acetaminophen2 Antipyrine3 Hydroxyantipyrine
1
23
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
100
120
8
Column 4.6 x 125 mm Hypersil ODS, 5 µmMobile phase A= water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 50 % B
at 10 min 90 % B Column wash at 13 min 90 % B
at 15 min 50 % BUV detector variable wavelength detector
at 0 min 254 nmat 6 min 234 nm, standard cell
Column compartment temperature 40 °CStop time 15 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
Androgen Drugs
Analysis of androgen drugs
HPLC method performance
Limit of detection 0.25 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 50 mg/l < 0.08 %
Precision of area10 runs, 50 mg/l < 0.8 %
�
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Testosterone
R = H
Testosterone acetate
R = OAc
Testosteroneacetate
0 2 4 6 8 10 12 14
Absorbance[mAU]
0
10
20
30
40
50
60
70254 nm 234 nm
Time [min]
Testosterone
9
Antianginal Drugs
HPLC method performance
Limit of detection 0.01 mg/l (5-µl injection), (VWD) S/N=2
Precision of RT 0.03%10 runs, 100 mg/l
Precision of area10 runs, 100 mg/l 0.12%
Linearity(correlation factor) 0.99992
Instrumentation:see configuration example 3 on page 78
Column compartmenttemperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Column 4.6 x 75 mm Zorbax SB-C183.5 µm
Mobile phase A = 0.025M KH2P 04 in water (pH = 3), B = acetonitrile
Flow rate 1.0 ml/minGradient at 0 min 20 % B
at 10 min 80 % BColumn wash at 12 min 20 % BUV detector variable wavelength detector
204 nm, standard cellfluorescence detector228/312 nm, standard cell
Analysis of antianginal drugs
Verapamil
Time [min]
1 2 3 4 5 6 7 8
Emission[LU]
0.5
1
1.5
2
2.5
1
268/312 nm
Excitation Emission
Wavelength [nm]200 220 240 260 280 300 320 340 360 380
0
20
40
60
80
100312 nm228 nm
268 nm
120
Analysis of verapamil using the Agilent 1100 Series fluorescence detector (column: 2.1 x 50 mm Zorbax SB-C18, 5 µm)
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Verapamil
Verapamil
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
100
200
300
400
500
10
Column 4 x 125 mm Purospher RP-18, 5 µmMobile phase A = 0.05 M KH2P 04 in water (pH = 2.5 ), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 0 % B
at 5 min 10 % Bat 10 min 40 % B
Column wash at 11 min 40 % Bat 12 min 0 % B
UV detector variable wavelength detectorat 0 min 220 nmat 4 min 254 nmat 7.5 min 204 nmat 10 min 220 nm, standard cell
Column compartment temperature 60 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
Analysis of antiarrythmic drugs
Antiarrythmic Drugs
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=21 mg/l (procainamide)
Precision of RT10 runs, 100 mg/l < 0.06%
Precision of area10 runs, 100 mg/l < 0.5 %
�
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Quinidine
�
������
Disopyramide
���
�
�
�
�
Procainamide
R=H
N-Acetylprocainamide
R = OAc
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
25
50
75
100
125
150
175
200
1
3
4
254 nm
204 nm
220 nm
220nm2
51 Procainamide2 N-Acetyl- procainamide3 Quinidine4 Hydroquini- dine *5 Disopyramide
* Quinidine contains up to 10 % Hydroquinidine
11
Time [min]
0 1 2 3 4 5 6
Absorbance[mAU]
0
50
100
150
200
250
300
350
1 Theobromine2 Theophylline3 Enprofylline4 Caffeine
1
2
34
Antiasthmatic Drugs
Analysis of antiasthmatic drugs
at 7 min 8 % BUV detector variable wavelength detector
270 nm, standard cellColumn compartmenttemperature 50 °CStop time 7 min
Column 4 x 125 mm Hypersil ODS, 5µ mMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minIsocratic at 0 min 8 % B
Absorbance[mAU]
40
30
20
10
0
Time [min]0 2 4 6 81 3 5 7
Standard
’Solostin’ antiasthmatic drops
1
2
3 4
TheobromineTheophyllineEnprofyllineCaffeine
1234
Analysis of theophylline in ‘Solosin’ antiasthmatic drops
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.1 %
Precision of area10 runs, 100 mg/l < 0.3 %
Instrumentation:see configuration example 2 on page 77
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TheobromineR=H
CaffeineR=CH3
EnprofyllineR=C3H7
Theophylline
12
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 5 % B
at 10 min 60 % BColumn wash at 12 min 10 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 40 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
Analysis of antibacterial drugs with pencillin-like structure
Antibacterial Drugs with Pencillin-like Structure
HPLC method performance
Limit of detection 1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.08 %
Precision of area10 runs, 100 mg/l < 1.1 %
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Amoxicillin
R =
R =
R =
�R =
Ampicillin
Penicillin G
Penicillin V
1 Amoxicillin2 Ampicillin3 Penicillin G4 Penicillin V
1
2 3
4
Time [min]
0 2 4 6 8 10 12
Absorbance[mAU]
0
200
400
600
800
1000
13
Antibacterial Drugs - Tetracyclines
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 5 % B
at 10 min 60 % BColumn wash at 12 min 5 % BUV detector variable wavelength detector
350 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.07 %
Precision of area10 runs, 100 mg/l < 0.3 %
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Minocycline
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��
Tetracycline
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��
�������
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Doxycycline
1 Minocycline2 Tetracycline3 Doxycycline
1
2
3
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
100
120
Analysis of tetracyclines
14
Miscellaneous Antibacterial Drugs
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 10 min 30 % Bat 15 min 60 % B
Column wash at 16 min 10 % BUV detector variable wavelength detector
at 0 min 204 nmat 4.5 min 368 nmat 6 min 204 nmat 10 min 254 nmat 15 min 204 nm, standard cell
Column compartment temperature 40 °CStop time 16 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 3 on page 78
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2(VWD)
Precision of RT10 runs, 100 mg/l < 0.08 %
Precision of area10 runs, 100 mg/l < 0.3 %
Linearity 1.00000(correlation factor) 0.99990 (nalidixic acid)
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Hydroxybenzotriazole
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�
Chloramphenicol
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��������
���
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Trimethoprim
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Sulfamethoxazole
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Furazolidone
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Nalidixic acid
1 Hydroxyben- zotriazole2 Trimethoprim3 Furazolidone4 Sulfamethoxa- zole5 Chloramphenicol6 Nalidixic acid
1
Time [min]
0 2 4 6 8 10 12 14
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
2
3
4
5
6
204 nm
368 nm 204 nm
254 nm
204 nm
Analysis of miscellaneous antibacterial drugs
15
Miscellaneous Antibacterial Drugs
Analysis of trimethoprim and sulfamethoxazole using the Agilent 1100 Series fluorescence detector (column 2.1 x 50 mm Zorbax SB-C18, 5 µm)
Time [min]0 2 4 6 8 10 12 14
Absorbance[mAU]
0
100
200
300
400
500
1
2
3
4
5
6
1 Hydroxybenzotriazole2 Trimethoprim3 Furazolidone4 Sulfamethoxazole5 Chloramphenicol6 Nalidixic acid
Standard
’Cortim Diolan Forte’cystitis tablet
Analysis of trimethoprim and sulfamethoxazole in ‘Cortim Diolan Forte’ cystitis tablet
Time [min]
0 2 6 8 10 12 14 16 18
0
Emission[LU]
5
10
15
20
Wavelength-switching
Multiwavelength detection250/503 nm
250/343 nm
275/343 nm
230/503 nm
Trimethoprim
Sulfamethoxazole
Wavelength [nm]200 250 300 350 400 450 500
0
5
10
15
20
Excitation Emission
Trime-thoprim
Sulfame-thoxazole
230 nm 275 nm 343 nm 503 nm
4
16
Miscellaneous Antibacterial Drugs
Analysis of chloramphenicol in ‘Noriplon’ pet ointment
12
3
45 6
1 Hydroxybenzotriazole2 Trimethoprim3 Furazolidone4 Sulfamethoxazole5 Chloramphenicol6 Nalidixic acid
Standard
’Noriplon’ petointment
Time [min]
0 2 4 6 8 10 12 14
Absorbance[mAU]
0
10
20
30
40
50
60
70
17
Anticoagulant Drugs
Analysis of wafarin using the variable wavelength detector
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 20 % B
at 10 min 80 % BColumn wash at 12 min 20 % BUV detector variable wavelength detector, 204 nm, standard cell
fluorescence detector, 272/355 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 3 on page 78 Time [min]
1 2 3 4 5 6 7 8 9
Emission[LU]
0.5
1
1.5
2
2.5
3
3.5
272/355 nm
200 300 400 5000
0.5
1
1.5
Excitation Emission272 nm 355 nm Warfarin2
Wavelength [nm]
Analysis of wafarin using the Agilent 1100 Series fluorescence detector (column: 2.1 mm Zorbax SB-C18, 5 µm)
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2(VWD)
Precision of RT10 runs, 100 mg/l 0.02 %
Precision of area10 runs, 100 mg/l 0.46 %
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Wafarin
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
50
100
150
200
250 Warfarin
18
Antidepressant Drugs
Analysis of antidepressant drugs
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 10 min 60 % BColumn wash at 12 min 10 % BUV detector variable wavelength detector
210 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.06 %
Precision of area10 runs, 100 mg/l < 0.6 %
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Bupropion
�
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Maprotiline
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Trazodone
1 Bupropion2 Trazodone3 Maprotiline
1
2
3
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
19
Antidepressant Drugs
Selectivity test results of antidepressant drugs
Antidepressant Drug Retention Time [min] Set Drug Retention Time [min]
Bupropion 5.41 Papaverine 5.43
Doxycycline 5.48
Tradozone 5.80 Sulfamethoxazole 5.68
Chloramphenicol 6.05
Dextromethorphan 6.24
Maprotiline 7.64 Penicillin G 6.93
Promethazine 7.11
Nalidixic acid 7.61
Penicillin V 7.66
Verapamil 7.83
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
50
100
150
200
250
300
Antidepressant drugs
10 Dextrometorphan9 Verapamil8 Penicillin G
7 Penicillin V6 Sulfamethoxazole5 Chloramphenicol
4 Promethazine3 Nalidixic acid2 Doxycyline1 Papaverine
1 2 3 4 56 7 8 9 10
20
Antiepileptic Drugs
Analysis of antiepileptic drugs
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.12 %
Precision of area10 runs, 100 mg/l < 1.4 %
Linearity > 0.99999(correlation factor)
Selected column 4.6 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 0.8 ml/minGradient at 0 min 15 % B
at 25 min 40 % BColumn wash at 30 min 80 % B
at 32 min 15 % BUV detector variable wavelength detector, 204 nm, standard cellColumn compartment temperature 60 °CStop time 32 minPost time 5 minInjection volume 5 µl
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Phenobarbital
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Primidone
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Phenylethylmalonamide
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Carbamazepine
�
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Carbamazepineepoxide
Time [min]0 5 10 15 20 25 30 35 40
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
3x125 mm Hypersil BDS, 3 µm
4x125 mm Hypersil ODS, 5 µm
4x125 mm Purospher RP-18, 5 µm
4.6x100 mm TSK gel ODS, 2 µm
1 Caffeine 4 Primidone 7 Carbamazepine- 2 Phenyl- 5 Phenobarbital epoxide ethylmalonamide 6 Methylphenyl- 8 Phenytoin3 Ethosuximide succinimide 9 Carbamazepine
1 34 567
8 92
1234 5 6
7
89
23
1
54 6
78 9
123
4 56 7 89
21
Phenylethyl-malonamideEthosuximidePrimidonePhenobarbitalMethylphenyl-succinimidePhenytoinCarbamazepine
2
5
8
11
14
17
58 60 62
Temperature [ºC]
Retentiontime [min]
Antiepileptic Drugs — Ruggedness Test Results
Dependence of retention time on temperature
Phenylethyl-malonamideEthosuximidePrimidonePhenobarbitalMethylphenyl-succinimidePhenytoinCarbamazepine
2
5
8
11
14
17
0.75 0.80 0.85
Retentiontime [min]
Flow rate [ml/min]
Dependence of retention time on flow rate
22
Antiepileptic Drugs
Dependence of area on wavelength
2
5
8
11
14
17
88:12 85:15 82:18
Phenylethyl-malonamideEthosuximidePrimidonePhenobarbitalMethylphenyl-succinimidePhenytoinCarbamazepine
Retentiontime [min]
% Acetonitrile
Dependence of retention time on % acetonitrile at start of runs
Wavelength [nm]
Phenyl ethyl-malonamideEthosuximidePrimidonePhenobarbitalMethylphenyl-succinimidPhenytoinCarbamazepine
500
1500
2500
3500
4500
204 206 208 210
Area[counts]
23
Antiepileptic Drugs
Selectivity test results of antiepileptic drugs
Antidepressant Drug Retention Time [min] Set Drug Retention Time [min]
Caffeine 2.33 Sulfanilamide 1.70
Sulfadiazine 2.36
Sulfathiazole 2.50
Phenylethylmalonamide 2.95 Sulfamerazine 3.20
Ethosuximide 3.44 Barbital 3.30
Primidone 4.26 Sulfamethazine 4.33
Allobarbital 5.57
Phenobarbital 7.20
Methylphenylsuccinimide 8.18 Butalbarbital 8.66
Carbamzepinepoxide 9.42 Butalbital 9.96
Phenytoin 13.55 Prednisolone 12.23
Hydrocortisone 12.54
Mephobarbital 13.21
Amobarbital 13.47
Carbamazepine 14.26
1 Sulfathiazole 5 Sulfadiazine 9 Butalbital2 Sulfamethazine 6 Hydrocortisone 10 Mephobarbital3 Sulfamerazine 7 Prednisolone 11 Amobarbital4 Sulfanilamide 8 Butabarbital 12 Allobarbital 13 Barbital
Antiepileptics
Time [min]
0 5 10 15 20
Absorbance[mAU]
0
50
100
150
200
250
300
350
123456789
10111213
24
Antiepileptic Drugs
Separation of antiepileptic drugs using LC/MS
Column 2.1 x 50 mm, Zorbax SB-C18, 5 µmMobile phase A = 0.5 % HOAc in water, B = 0.5 % HOAC in methanolFlow rate 0.5 ml/minGradient at 0 min 15 % B
at 7 min 45 % BColumn wash at 8 min 15 % BUV detector diode array detector
204/8 nm, reference = 360/100 nm, standard cellColumn compartment temperature 25 °CStop time 8 minPost time 5 minInjection volume 5 µl
Mass/charge ratio [m/z]
100 150 200 250 300 3500
20
40
60
80
100Max: 95834
233.0
234.0
100 150 200 250 300 3500
20
40
60
80
100Max: 1.53526e+006237.1
238.1
PhenobarbitalM = 232 amu
CarbamazepineM = 236 amu
N
Phenobarbital
N
N
O
OO
H
H
CONH2
MS spectra of two antiepileptic drugs
1 Phenylethylmalonamide 5 Methylphenylsuccinimide2 Ethosuximide 6 Carbamazepineepoxide 3 Primidone 7 Carbamazepine4 Phenobarbital 8 Phenytoin
12
3 4 56
7
8
0 1 2 3 4 5
Absorbance[mAU]
050
100150200250300
MS, TICMS, BPC
Time [min]1 2 3 4 5
0500000
1000000150000020000002500000
0
25
Antiepileptic Drugs
Separation of antiepileptic drugs using LC/MS
1 2 3 4 5
5000001000000150000020000002500000
Time [min]2 4 6 8 10
0
500000
1000000
1500000
2000000
2500000
207 amu142 amu
219 amu233 amu
190 amu253 amu
MS, TIC
Extracted ion chromatograms
237 amu
Instrumentation:see configuration example 4 on page 79
MS conditions
Ionization mode API-ES positiveNebulizer pressure 35 psigDrying gas temperature 340 °CDrying gas flow 12 L/minVcap 4000 voltFragmentor 70 voltScan range m/z 100 – 400
26
Antiestrogen Drugs
Analysis of antiestrogen drugs
Column 4 x 125 mm Purospher RP-18, 5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 20 % B
at 14 min 60 % BColumn wash at 16 min 20 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 60 °CStop time 16 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.2 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l 0.08 %
Precision of area10 runs, 100 mg/l 0.46 %
��
Tamoxifen
Tamoxifen
Time [min]
0 2 4 6 8 10 12 14
Absorbance[mAU]
0
50
100
150
200
250
300
27
Antihistaminic Drugs
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 10 min 55 % BColumn wash at 12 min 10 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.05 %
Precision of area10 runs, 100 mg/l < 0.2 %
��
�
Tripelenamine
�
�
��
Chlorpheniramine
�
�
�
Promethazine
�
�
�
�
�
Tetracaine
1 Tripelenamine2 Chlorphaniramine3 Tetracaine4 Promethazine
12
3
4
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
25
50
75
100
125
150
175
200
Analysis of antihistaminic drugs
28
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
50
100
150
200
250
300Enalapril
40 ºC
60 ºC
50 ºC
70 ºC
30 ºC
Captopril
Antihypertensive Drugs
Analysis of antihypertensive drugs
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 2), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 8 min 55 % BColumn wash at 9 min 10 % B UV detector variable wavelength detector, 204 nm, standard cellColumn compartment temperature 60 °CStop time 9 minPost time 5 minInjection volume 5 µl
Increasing temperature leads to improved peak shape
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.5 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.06 %
Precision of area10 runs, 100 mg/l < 0.4 %
Linearity (correlation factor) 1.00000
�
�
�
�
�
�����
Enalapril
�����
�
��
Captopril
Enalapril
Time [min]
0 1 2 3 4 5 6 7 8
Absorbance[mAU]
0
20
40
60
80
100
120
140 Captopril
29
Antiinflammatory Drugs
Analysis of antiinflammatory drugs
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 20 % B
at 10 min 80 % BColumn wash at 12 min 20 % BUV detector variable wavelength detector
230 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.01 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l 0.03 %
Precision of area10 runs, 100 mg/l 0.17 %
Linearity(correlation factor) 0.99997
����
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Naproxen
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
200
400
600
800
Naproxen1000
30
Antiprotozoal Drugs
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 5 % B
at 10 min 30 % BColumn wash at 12 min 5 % BUV detector variable wavelength detector
320 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 1 on page 76
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l 0.06 %
Precision of area10 runs, 100 mg/l 0.15 %
Linearity(correlation factor) 0.99996
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��
Metronidazole
Metronidazole
Time [min]0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
100
120
140
Analysis of antiprotozoal drugs
31
Antitumor Drugs
Analysis of antitumor drugs
Instrumentation:see configuration example 2 on page 77
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 50 % B
at 10 min 90 % BColumn wash at 12 min 50 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l 0.07 %
Precision of area10 runs, 100 mg/l 0.79 %
Linearity(correlation factor) 1.00000
��
��
��
��
�
��
���
�
�
��
�
�
�
Paclitaxel (Taxol)
Paclitaxel (Taxol)
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
100
120
32
Antitumor Drugs
Separation of antitumor drugs using LC/MS
Instrumentation:see configuration example 4 on page 79
Column 2.1 x 50 mm Zorbax SB-C18, 5 µmMobile phase A = 0.5 % HOAc in water, B = 0.5 % HOAc in methanolFlow rate 0.5 ml/minGradient at 0 min 50 % B
at 2 min 65 % BColumn wash at 4 min 65 % B
at 5 min 50 % BUV detector diode array detector
210/16 nm, reference = 360/100 nm, standard cellColumn compartment temperature 25 °CStop time 5 minPost time 5 minInjection volume 5 µl
0 1 2
Absorbance[mAU]
-500
50100150200250300
Time [min]0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
0100000200000300000400000
Time [min] Mass/charge ratio [m/z]860 880 900 920 940
020406080
100
Max: 191719
854.4
855.4
MS, TICMS, BPC
Paclitaxel (Taxol)M = 853.9 amu
MS conditions
Ionization mode API-ES positiveNebulizer pressure 35 psigDrying gas temperature 340 °CDrying gas flow 12 L/minVcap 4000 voltFragmentor 70 voltScan range m/z 700 – 900
33
Antitussive Drugs
Analysis of antitussive drugs
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water , (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 8 min 80 % BColumn wash at 10 min 10 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 60 °CStop time 10 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l 0.02 %
Precision of area10 runs, 100 mg/l 0.13 %
Linearity(correlation factor) 0.99998
����
�
�
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Dextromethorphan
Dextromethorphan
1 2 3 4 5 6
Absorbance[mAU]
0
50
100
150
200
250
300
350
Time [min]
Dextromethorphan in ‘Formel 44 plus’ cough sirup
'Formel 44 plus' cough syrup
Standard
Dextromethorphan
Time [min]
0 2 4 6 8
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
34
Catecholamines
Analysis of catecholamines
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 + 0.3 mM heptanesulfonic acid in water
(pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 1 % B
at 5 min 2 % Bat 7 min 15 % B
Column wash at 8 min 1 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 30 °CStop time 8 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.2 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 1000 mg/l< 0.1 %, 0.24 % (dopamine)
Precision of area10 runs, 1000 mg/l< 0.2 %
���
����
��
Norepinephrine
�
��
��
�� �
Epinephrine
��
��
���
Dopamine
�����
��
Dihydroxybenzylamine
1 Norepinephrine2 Epinephrine3 Dihydroxybenzylamine (internal standard)4 Dopamine
1 2 3
Time [min]
0 1 2 3 4 5 6 7
Absorbance[mAU]
0
20
40
60
80
100
120
140
4
35
Glucocorticoid Drugs (I)
Analysis of glucocorticoid drugs
Instrumentation:see configuration example 2 on page 77
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 20 % B
at 10 min 80 % BColumn wash at 12 min 20 % BUV detector variable wavelength detector
254 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l 0.03 %
Precision of area10 runs, 100 mg/l < 0.7 %
Linearity(correlation factor)> 0.99997
�
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��
��
Hydrocortisone
�
���
��
�
�������
�������
Beclomethasone dipropionate
�
���
������
��
Hydrocortisone acetate
1 Hydrocortisone2 Beclomethasone dipropionate3 Hydrocortisone acetate
23
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
100
120
1
36
Glucocorticoid Drugs (II)
Analysis of glucocorticoid drugs
Instrumentation:see configuration example 2 on page 77
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 20 % B
at 10 min 80 % BColumn wash at 12 min 20 % BUV detector variable wavelength detector
254 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l < 0.04 %
Precision of area10 runs, 100 mg/l < 0.4 %
Linearity(correlation factor) > 0.99997
�
���
��
��
PrednisoloneR=H
Prednisolone acetateR = OAc
�
���
��
��
Betamethasone
�
���
��
�������
Betamethasone valerate
1 Prednisolone2 Betamethasone3 Prednisolone acetate4 Betamethasone valerate
4
32
1
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
100
120
37
H2-Antagonists
Analysis of H2-antagonists
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water, (pH = 3), B = acetonitrileFlow rate 1.0 ml/minIsocratic at 0 min 8 % B
at 10 min 8 % BUV detector variable wavelength detector
225 nm, standard cellColumn compartment temperature 25 °CStop time 10 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 0.2 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l 0.08 %
Precision of area10 runs, 100 mg/l < 0.7 %
�
�
�
�
�
�
�
�
�
��
Cimetidine
�
�
�
�
�
�
�
���
Ranitidine
Cimetidine
Ranitidine
Time [min]0 1 2 3 4 5 6 7 8 9
Absorbance[mAU]
0
20
40
60
80
100
120
38
Hypnotic Drugs
Analysis of hypnotic drugs
Instrumentation:see configuration example 1 on page 76
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 16 min 50 % BColumn wash at 18 min 10 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 25 °CStop time 18 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.2 mg/l (5-µl injection), S/N=20.4 mg/l (butalbital,amobarbital)
Precision of RT10 runs, 100 mg/l < 0.06 %
Precision of area10 runs, 100 mg/l < 0.5 %
�
�
�
� �
��
��
���
BarbitalR1 = H, R2 = C2H5, R3 = C2H5
PhenobarbitalR1 = H, R2 = C6H6, R3 = C2H5
MephobarbitalR1 = CH3, R2 = C6H6, R3 = C2H5
Butalbital�
�
�
� �
�
�
�
�
�
� �
�
�����Amobarbital
�
�
�
� �
�
����
� Butabarbital
�
�
�
� �
�
�Allobarbital
�
����
����
Flunitrazepam
Time [min]
0 2 4 6 8 10 12 14 16
Absorbance[mAU]
0
50
100
150
200
250
300
1 Barbital2 Allobarbital3 Phenobarbital4 Butabarbital5 Butalbital6 Amobarbital7 Mephobarbital8 Flunitrazepam
12
3
4 5
6
7
8
39
Hypnotic Drugs
Separation of hypnotic drugs using LC/MS
Instrumentation:see configuration example 4 on page 79
N
N
O
H
O
H
O
N
N
O
H
O
H
O
AllobarbitalM = 208 amu
100 200 300 4000
20
40
60
80
100
Max: 314456
209.0
141.0 210.1
Mass/charge ratio [m/z]100 200 300 400
0
2040
60
80
100
Max: 322675
225.1
226.1 ButalbitalM = 224 amu
MS spectra of two hypnotic drugs
1 2 34 5 6
7
0 1 2 3 4 5 6 7 8 9
Absorbance[mAU]
050
100150200250
1 Barbital 5 Butalbital2 Allobarbital 6 Amobarbital3 Phenobarbital 7 Mephobarbital4 Butabarbital
MS, TIC
MS, BPC
Time [min]
1 2 3 4 5 6 7 8 9-200000
200000
600000
1000000
1400000
Column 2.1 x 50 mm Zorbax SB-C18, 5 µmMobile phase A = 0.5 % HOAc in water, B = 0.5 % HOAc in methanolFlow rate 0.5 ml/minGradient at 0 min 5 % B
at 5 min 60 % BColumn wash at 8 min 60 % B
at 10 min 5 % BUV detector diode array detector
210/16 nm, reference = 360/100 nm, standard cellColumn compartment temperature 25 °CStop time 10 minPost time 5 minInjection volume 5 µl
40
Instrumentation:see configuration example 4 on page 79
Hypnotic Drugs
Separation of hypnotic drugs using LC/MS
HPLC method performance comparison forphenobarbital
VWD, 210 nm* MS (SIM, 233 amu)
Limit of detectionS/N = 2 0.2 mg/L 0.01 mg/L
Precision of RT10 runs, 100 mg/l 0.05 % 0.02 %
Precision of area10 runs, 100 mg/l 0.36 % 2.83 %
* using the method described on page 39
1 2 3 4 5 6 7 8 9200000
1000000
1800000
Time [min]2 4 6 8 10
0200000
600000
1000000
1400000
MS, TIC
Extracted ion chromatograms
185 amu
227 amu225 amu
213 amu233 amu
209 amu
247 amu
MS conditions
Ionization mode API-ES positiveNebulizer pressure 35 psigDrying gas temperature 340 °CDrying gas flow 12 L/minVcap 4000 voltFragmentor 70 voltScan range m/z 100 – 500
41
Analysis of keratolytic drugs
Keratolytic Drugs
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.05 M NH4OAc in water (pH = 2.2), B = acetonitrileFlow rate 1.2 ml/minIsocratic at 0 min 20 % B
at 10 min 20 % BUV detector variable wavelength detector
at 0 min 204 nmat 2.2 min 230 nmat 3.2 min 204 nm, standard cell
Column compartment temperature 50 °CStop time 10 minInjection volume 5 µl
Instrumentation:see configuration example 1 on page 76
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Phthalic acid
����
Benzoic acid
����
��
Salicylic acid
1 Phthalic acid2 Benzoic acid3 Salicylic acid
1
2
3
Time [min]0 2 4 6
Absorbance[mAU]
0
200
400
600
800
204 nm 230 nm
204 nm
1000
HPLC method performance
Limit of detection 0.2 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l < 0.1 %
0.16 % (phthalic acid)
Precision of area10 runs, 1000 mg/l< 0.8 %
42
Instrumentation:see configuration example 2 on page 77
Muscle-relaxing Drugs
Analysis of muscle-relaxing drugs
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 8 min 80 % BColumn wash at 10 min 10 % BUV detector variable wavelength detector
254 nm, standard cellColumn compartment temperature 60 °CStop time 10 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 100 mg/l 0.04 %
Precision of area10 runs, 100 mg/l 0.1 %
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Time [min]1 2 3 4 5 6 7
Absorbance[mAU]
0
100
200
300
400
500
600 Papaverine
Paperverine
43
Sedative Drugs
Analysis of sedative drugs
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.025 M KH2P O4 in water (pH = 3), B = acetonitrileFlow rate 1.2 ml/minGradient at 0 min 30 % B
at 10 min 55 % BColumn wash at 11 min 30 % BUV detector variable wavelength detector
210 nm, standard cellColumn compartment temperature 25 °CStop time 11 minPost time 5 minInjection volume 5 µl
1 Oxazepam2 Clonazepam3 Flunitrazepam4 Diazepam1
2
3 4
Standard
'Diazepam 5 Stada' Tablet
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
0
20
40
60
80
Analysis of diazepam in ‘Diazepam 5 Stada’ tablet
HPLC method performance
Limit of detection 0.1 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 10 mg/l < 0.08 %
Precision of area10 runs, 10 mg/l < 0.1 %
�
�
�
��
���
Diazepam
�
�
�
��
�
��
Oxazepam
�
����
����
Flunitrazepam
�
�
�
���
�
��
Clonazepam
Time [min]0 2 4 6 8 10
Absorbance[mAU]
0
300
400
500
1 Oxazepam2 Clonazepam3 Flunitrazepam4 Diazepam1
2
34
200
100
44
1 Sulfanilamide 2 Sulfadiazine 3 Sulfathiazole4 Sulfamerazine5 Sulfamethazine
12 3 4 5
T = 10 ºC
T = 20 ºC
T = 30 ºC
T = 40 ºC
T = 50 ºC
T = 60 ºC
Time [min]0 2 4 6 8 10 12 14 16
Absorbance[mAU]
0
200
400
600
800
1000
1200
Sulfa Drugs
Analysis of sulfa drugs
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 10 min 25 % BColumn wash at 12 min 10 % BUV detector variable wavelength detector
254 nm, standard cellColumn compartment temperature 50 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
Separation of sulfathiazole (3) and sulfamerazine (4) using different column temperatures
HPLC method performance
Limit of detection 0.2 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l < 0.07 %
1.2 % (sulfanilamide)
Precision of area10 runs, 100 mg/l < 0.4 %
��� ������
R = H
�
�R = Sulfathiazole
�
�R =
�
�R =
�
�R =
Sulfamethazine
Sulfadiazine
Sulfamerazine
1 Sulfanilamide2 Sulfadiazine3 Sulfathiazole4 Sulfamerazine5 Sulfamethazine
1
2 3 4
Time [min]
2 4 6 8 10 12
Absorbance[mAU]
0
50
100
150
200
250
5
Sulfanilamide
45
Therapeutic Peptides
Analysis of therapeutic peptides
Column 4.6 x 75 mm Zorbax SB-C18 , 3.5 µmMobile phase A = 0.1 % TFA in water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 20 % B
at 10 min 50 % BColumn wash at 12 min 20 % BUV detector variable wavelength detector
210 nm, standard cellColumn compartment temperature 25 °CStop time 12 minPost time 5 minInjection volume 5 µl
Instrumentation:see configuration example 2 on page 77
HPLC method performance
Limit of detection 2.5 mg/l (5-µl injection), S/N=2
Precision of RT10 runs, 250 mg/l < 0.12 %
Precision of area10 runs, 250 mg/l < 0.4 %
1 Oxytocin2 Angiotensin I3 Angiotensin II4 Insulin chain B (bovine)
1
2
3
4
Time [min]
0 2 4 6 8 10
Absorbance[mAU]
-100
-50
0
50
100
150
200
250
46
Tricyclic Antidepressant Drugs
Analysis of tricyclic antidepressant drugs
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 µmMobile phase A = 0.025M KH2P 04 in water (pH = 7), B = methanolFlow rate 1.0 ml/minGradient at 0 min 67 % B
at 15 min 85 % BColumn wash at 17 min 67 % BUV detector variable wavelength detector
210 nm, standard cellColumn compartment temperature 40 °CStop time 17 minPost time 10 minInjection volume 5 µl
Time [min]
0 2 4 6 8 10 12 14 16
Absorbance[mAU]
0
50
100
150
200
250
300
350
1 Protriptyline2 Nortriptyline3 Doxepin4 Imipramine5 Amitriptyline6 Trimipramine
12 3
4
5
6
Standard
'Aponal 100' Tablet
Analysis of doxepine in ‘Aponal 100’ tablets
HPLC method performance
Limit of detection <1.0 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l < 0.32 %
Precision of area10 runs, 100 mg/l < 0.6 %
�
�
�
�
Protriptyline
NortriptylineR = H
AmitriptylineR = CH3
�
�ImipramineR = H
TrimipramineR = CH3
�
�
Doxepin
1 Protriptyline2 Nortriptyline3 Doxepin4 Imipramine5 Amitriptyline6 Trimipramine
1
23
Time [min]
2 4 6 8 10 12 14 16
Absorbance[mAU]
0
50
100
150
200
4
5
6
47
Fat-Soluble Vitamins
Analysis of fat-soluble vitamins
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 µmMobile phase A = water, B = methanolFlow rate 1.0 ml/minGradient at 0 min 90 % B
at 15 min 100 % Bat 20 min 100 % B
Column wash at 21 min 90 % BUV detector variable wavelength detector
210 nm, standard cellColumn compartment temperature 20 °CStop time 21 minPost time 5 minInjection volume 5 µl
HPLC method performance
Limit of detection <4.0 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 1000 mg/l< 0.10 %
Precision of area10 runs, 1000 mg/l< 0.2 %
��
Retinol (A1)
�
�� �� Tocopherol
��
�
Cholecalciferol (D3)
1 Retinol (A)2 Cholecalciferol (D3)3 �-Tocopherol (E)
1
2
3
Time [min]
0 2.5 5 7.5 10 12.5 15 17.5 20
Absorbance[mAU]
0
200
400
600
800
48
Time [min]0 2 4 6 8 10
LU
0
20
40
60
80
270/530 nm
270/400 nm
1 Pyridoxamine (B6)2 Pyridoxal (B6)3 Pyridoxine (B6)4 Riboflavine (B2)
4100
2 31
Wavelength [nm]250 300 350 400 450 500 550
400 nm280 nm
Vitamin B
200
0
10
20
30
200 250 300 350 400 450 500 5500
10
20
30 262 nm362 nm
450 nm
530 nmVitamin B
Wavelength [nm]
40Excitation Emission Excitation
6
2
Emission
Water-Soluble Vitamins
Analysis of water-soluble vitamins
Instrumentation:see configuration example 3 on page 78
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.05M KH2P 04 in water (pH = 2.5), B = acetonitrileFlow rate 1.0 ml/minGradient 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 % BColumn wash at 19 min 60 % B
at 20 min 0.6 % BUV detector variable wavelength detector
204 nm, standard cellColumn compartment temperature 15 °CStop time 20 minPost time 10 minInjection volume 5 µl
Analysis of vitamins B2 and B6 using the Agilent 1100 Series fluorescence detector (column: 21 x 50 mm Zorbax SB-C18, 5 µm)
HPLC method performance
Limit of detection <6.0 mg/l (5-µl injection),S/N=2
Precision of RT10 runs, 100 mg/l < 0.40 %
Precision of area10 runs, 100 mg/l < 1.5 %
�
�
��� ��
��
���� Thiamine (B1)
�
��
�
�
�
��
�
��
����
�
����
�
Riboflavine (B2)
��
�
�
��
� �
��
�
Biotin
Pyridoxine,Pyridoxamine, Pyridoxal (B6)R = OH, NH2 or = 0
�
���
�Niacinamide
1 Pyridoxamine 2 Thiamine 3 Niacinamide 4 Pyridoxal 5 Pyridoxine 6 Aminobenzoic acid 7 Pantothenic acid 8 Folic acid 9 Riboflavine10 Biotin11 Thiotic acid
1
23
4
Time [min]
0 2 4 6 8 10 12 14 16 18
Absorbance[mAU]
0
50
100
150
200
250
3005
6
7
8
9 10 11
49
Atropa Belladonna Extract
Analysis of Atropa Belladonna extract
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 µmMobile phase A = 0.05M KH2P 04 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 10 % B
at 20 min 60 % BColumn wash at 23 min 60 % B
at 25 min 10 % BUV detector diode array detector
210 nm/16 (ref. 360 nm/100), standard cellColumn compartment temperature 40 °CStop time 25 minPost time 5 minInjection volume 5 µl
Extraction
1 g of the dried and powderedplant (from Caesar & Loretz
GmbH, Germany) was refluxedfor 30 min in 0.5 M acetic acid.After cooling the pH was ad-justed to 9 and the solution wasextracted five times with 50 mlchloroform. After drying oversodium sulfate the solvent wasremoved i. vac. and the residuedissolved in 2.5 ml methanol.After filtration 5 µl of the extractwere applied to HPLC.
Time [min]
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
Absorbance[mAU]
0
200
400
600
800
1000
1200
Wavelength [nm]200 225 250 275 300 325 350 375
Norm
0200400600800
1000
Atropine standard
SampleOffset
Match factor: 999
Comparison of sample and standard spectra of atropine
�
�
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��
Atropine
Time [min]
0 5 10 15 20
Absorbance[mAU]
0
200
400
600
800
1000
Atropine standard
Atropa Belladonna extract
Atropine
50
Time [min]0 5 10 15 20 25
Absorbance[mAU]
0
50
100
150
200
250
300
350
Wavelength [nm]200 250 300 350 400
04080
120160 Quinine standard
Sample
Wavelength [nm]200 250 300 350
05
101520 Quinidine standard
Sample Offset Offset
400
Match factor: 992Match factor: 997
Cortex Cinchonae Extract
Instrumentation:see configuration example 2 on page 77
Column 4 x 125 mm Purospher RP-18, 5 µmMobile phase A = 0.05M KH2P 04 in water (pH = 3), B = acetonitrileFlow rate 0.8 ml/minGradient at 0 min 4 % B
at 25 min 10 % Bat 45 min 30 % B
Column wash at 46 min 60 % Bat 49 min 60 % Bat 50 min 4 % B
UV detector diode array detector210 nm/16 (ref. 360 nm/100), standard cell
Column compartment temperature 25 °CStop time 50 minPost time 5 minInjection volume 5 µl
Extraction
The extract (from Caesar &
Loretz GmbH, Germany) wasdiluted (1:10) and 5 µl wereapplied to HPLC.
Comparison of sample and standard spectra of quinidine and quinine
�
����
�
���
Quinidine
�
����
�
���
Quinine
Time [min]
0 5 10 15 20 25 30
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
Quinidine standard
Cinchona extract1
2
34
1 Quinidine2 Quinine3 Hydroquinidine4 Hydroquinine
Quinine standard
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µm
Mobile phase A = 0.025M KH2P 04 in water (pH = 3), B = methanol
Flow rate 1.0 ml/minGradient at 0 min 10 % B
at 7 min 40 % Bat 7.01 min 65 % Bat 15 min 80 % B
Column wash at 16 min 100 % Bat 19 min 100 % Bat 20 min 10 % B
UV detector diode array detector225 nm/16 (ref. 360 nm/100)254 nm/16 (ref. 360 nm/100),standardcell
Column compartmenttemperature 50 °CStop time 20 minPost time 5 minInjection volume 2 µl
51
Time [min]
0.5 1 1.5 2 2.5 3 3.5 4 4.5
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
Protocatechuicacid
Dan Shen extract
Protocatechuic acid standard
0Time [min]
11 12 13 14 15 16
Absorbance[mAU]
0
20
40
60
80
100
120
140
1601
1 Tanshinone I2 Cryptotanshinone3 Tanshinone IIA32
Dan Shen extract
Cryptotanshinone standard
Tanshinone IIA standard
Tanshinone I standard
500
Protocatechuic aldehyde standard
Protocatechuicaldehyde
Dan Shen extract
Time [min]
0 2 4 6 8 10 12 14 16 18
Absorbance[mAU]
0
100
200
300
400
Analysis of Dan Shen dropping pills extract
Dan Shen Dropping Pills Extract
Extraction
20 Dan Shen pills were dissolvedin 5 ml methanol/water (3:1) in avolumetric flask. The solutionwas filtered and 2 µl wereapplied to HPLC.
��
��
���� Protocatechuic acid
��
��
��� Protocatechuic alde-
�
�
�
Tanshinone I
�
�
�Tanshinone IIA
�
�
�
Cryptotanshinone
52
220 240 260 280 300 320 340 360 380
020406080
100120140
Match factor: 999Protocatechuicaldehyde standard
Sample Offset
220 240 260 280 300 320 340 360 380
Norm
00.5
11.5
22.5
33.5
4 Match factor: 982Protocatechuicacid standard
Sample
Offset
Match factor: 995Tanshinone Istandard
Sample Offset
220 240 260 280 300 320 340 360 380
0
0.5
1
1.5
2
2.5
Wavelength [nm]
220 240 260 280 300 320 340 360 380
0
1
2
3
4
Match factor: 999Tanshinone IIAstandard
Sample Offset
220 240 260 280 300 320 340 360 380
0
1
2
3
4
OffsetSample
Cryptotanshinonestandard
Match factor: 994
Dan Shen Dropping Pills Extract
Comparison of sample and standard spectra
Instrumentation:see configuration example 2 on page 77
53
Norm
050
100150200250
Time [min]0 2 4 6 8 10 12 14 16 18
Absorbance[mAU]
50
100
150
200
250
300
Norepehdrine standard
Sample Offset
Wavelength [nm]200 250 300 350
Wavelength [nm]200 250 300 350
Norm
0200400600800
1000
Offset
Norephedrine
Ephedrine
Match factor: 997
Sample
Match factor: 983
Ephedrine standard
0
400
400
Ephedra Sinica Stapf Extract
Analysis of Ephedra Sinica Stapf extract
Instrumentation:see configuration example 2 on page 77
Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µmMobile phase A = 0.025 M KH2P 04 in water (pH = 3), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 2 % B
at 10 min 10 % BColumn wash at 15 min 80 % B
at 18 min 80 % Bat 20 min 2 % B
UV detector diode array detector204 nm/8 (ref. 360 nm/100), standard cell
Column compartment temperature 25 °CStop time 20 minPost time 5 minInjection volume 5 µl
Extraction
50 ml of 0.5 M H2SO4 was addedto 1 g dried plant and the mixturewas stirred overnight. After filtra-tion the pH was adjusted to 11-13by adding 6 M NaOH. 8 g NaClwere added and the mixtureextracted with ether (4 x 25 ml).The organic layer was dried overMgSO4, filtered and the solventwas removed i. vac. 5 ml H2Oand 3 drops of 4 M HCl wereadded to the residue.
Comparison of sample and standard spectra of norephedrine and ephedrine
���
�
Ephedrine
�����
Norephedrine
Norephedrine
Ephedrine
Ephedra extract
Norephedrine standard
Time [min]
0 2.5 5 7.5 10 12.5 15 17.5
Absorbance[mAU]
0
100
200
300
400
500
Ephedrine standard
54
Analysis of Ginko Bilboba extract
Ginko Biloba Extract and Tablets
Instrumentation:see configuration example 2 on page 77
Extraction
4 g of Ginko Biloba extract (fromCaesar & Loretz GmbH,
Germany) were refluxed for 30 min in 70 ml methanol and 10 ml 25 % HCl. After cooling toroom temperature the mixturewas filtered and the filter washedwith approximately 100 mlmethanol. The solvent was partlyremoved i. vac. and diluted withmethanol to 100 ml in a volumet-ric flask. 5 ml of this solutionwere filtered through a C18 dis-posable cartridge. The cartridgewas washed with 4 ml methanoland the filtrate diluted to 10 ml ina volumetric flask. The same pro-cedure was used to extract 10ginko tablets (‘Promod Ginkobiloba L.’, 40 mg ginko bilobaextract per tablet, Sine Labora-
tories', Shanghai, China)
Method and extraction from:A. Hasler, O. Stichler, J. Chroma-
togr. 508 (1990), 236-240
Time [min]0 2 4 6 8 10 12 14 16 18
Absorbance[mAU]
0
100
200
300
400
500
600
Wavelength [nm]200 250 300 350
Norm
0
200
400
600800
200 250 300 350
Norm
0
200
400
600
Quercetin standard
Sample
Sample
Kaempferolstandard
Offset
400
400Wavelength [nm]
Match factor: 973
Match factor: 980
Comparison of sample and standard spectra of quercetin and kaempferol
�
��
��
�
��
��
��
Quercetin
�
��
��
�
��
��
Kaempferol
1
1 Quercetin2 Kaempferol3 Isorhamnetin (no standard available)
Time [min]
0 2 4 6 8 10 12 14 16 18
Absorbance[mAU]
0
50
100
150
200
250
300
3
2
Ginko extract
Ginko tablet
Quercetin standard
Kaempferol standard
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = 0.5 % H3P 04 in water, B = methanolFlow rate 2.0 ml/minGradient at 0 min 38 % B
at 12 min 48 % BColumn wash at 17 min 100 % B
at 20 min 38 % BUV detector diode array detector
370 nm/16 (ref. off), standard cellColumn compartment temperature 25 °CStop time 20 minPost time 5 minInjection volume 10 µl
55
Time [min]0 2 4 6 8 10 12 14
Absorbance[mAU]
0
50
100
150
200
250
Wavelength [nm]200 300 400 500 600
Norm
0
200
400
600
200 300 400 500 600
Norm
0
200
400
600
Rhein standard
Sample Offset
Offset
Emodin standard
Rhein
Emodin
Match factor: 999
Match factor: 999
Wavelength [nm]
Sample
Rheum Palmatum Extract
Analysis of Rheum Palmatum extract
Instrumentation:see configuration example 2 on page 77
Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = 0.05 M NH40Ac in water (pH = 2.5), B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 30 % B
at 10 min 80 % BColumn wash at 14 min 80 % B
at 15 min 30 % BUV detector diode array detector
440 nm/16 (ref. off), standard cellColumn compartment temperature 25 °CStop time 15 minPost time 5 minInjection volume 1 µl
Extraction
5 g dried rhubarb root wereextracted ultrasonically twicewith 50 ml methanol for 30 minutes. The extract was filteredand the solvent evaporated i. vac. The residue was dissolvedin 5 ml methanol and applied toHPLC.
Comparison of sample and standard spectra of rhein and emodin
�
�
��
����
��
Rhein
�
�
����
�����
Emodin
RheinEmodin
Rheum extract
Rhein standard
Emodin standard
Time [min]
2 4 6 8 10 12 14
Absorbance[mAU]
0
50
100
150
200
250
300
350
400
0
56
The database for the Agilent ChemStation is a relational databasewhich stores data in organizational units (studies), including additional,customizable sample and analysis data. Required data sets can beretrieved quickly and easily for review. The user can set up studies toorganize the data and create a query to select the runs of interest. Thedata can be explored by looking at samples or compounds, and, whilereviewing, runs can be marked for approval or rejection. Data can beexamined in graphical, tabular or chart format. When reviewing datatables, the user can specify the contents of the tables by specifying thecolumns he wants to see. Charts can be designed that allow the user tomonitor instrument performance or view trends. The user can also gen-erate statistical analyses, either in summary form or as a full regressionanalysis. When the required results are measured, the user can cus-tomize a report layout and generate a report, or export the data toanother Windows application, for example, Microsoft® Excel. Data canalso be reprocessed and assembled into a batch which can be loadedinto the Agilent ChemStation batch review. The following pages showtwo examples where the database of the Agilent ChemStation is used.Literature and a CD-ROM with more details are listed on page 82.
Agilent ChemStation Database
Using the Agilent ChemStation Database for Monitoring DrugProduction
57
Scenario
� pharmaceutical company produces cystitis tablets containing theantibacterial drugs trimethoprim and sulfamethoxazole. The activecompounds are bought from two different suppliers: company AAA andcompany BBB. The pharmaceutical company has five reaction vesselsin which the compounds are mixed and the tablets are produced. Thecrude products from company AAA are used in vessels #1 and #2, thecrude products from company BBB in vessels #3, #4 and #5. Theamount of active compounds in the tablets are monitored by HPLC.Therefore, a tablet is dissolved in a certain amount of solvent and theconcentration of trimethoprim and sulfamethoxazole is measured. Thequality 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 AgilentChemStation. 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 theAgilent ChemStation.
Figure 1 shows that the amounts of trimethoprim are well within thequality requirements (90-110 mg/l).
Figure 1Amount of trimethoprim in cystitis tablets
�
�
����
��������
���
���
��� � �
�
�
�
��
Trimethoprim
Sulfamethoxazole
Amount [mg/l]
Date
58
Figure 2 shows the amount of trimethoprim in reaction vessel # 2.
Figure 2Amount of trimethorpim in reaction vessel #2
Figure 3Amount of sulfamethoxazole in cystitis tablet
Figure 3 illustrates the results for sulfamethoxazole, which show thaton days 4 and 5 some measurement points are below the qualityrequirements of 150 - 165mg/l.
Amount [mg/l]
Date
Amount [mg/l]
Date
59
Figures 4 and 5 show that for vessels #1 and #2 the amounts of sul-famethoxazole are too low on days 4 and 5.
Figure 4Amount of sulfamethoxazole in reaction vessel # 1
Figure 5Amount of sulfamethoxazole in reaction vessel # 2
Amount [mg/l]
Date
Amount [mg/l]
Date
60
Figure 6 displays the amount of sulfamethoxazole by supplier, whichshows that the tablets containing sulfamethoxazole from company AAAare below the quality requirements.
Figure 6Amount of sulfamethoxazole in cystitis tablet
Conclusion
The measurements on days 4 and 5 which are below the qualityrequirments are from reactions vessels #1 and # 2. Only runs using sulfamethoxazole from company AAA are below thequality requirements. Comparing the chromatograms of two runs showsthat there is an additional peak. This leads to the final conclusion thatthe sulfamethoxazole from company AAA was not pure.
Amount [mg/l]
Company AAA Company BBB
61
Using the Agilent ChemStation Database for Monitoring SerumConcentration
Scenario
A company produces antianginal pills containing verapamil.They have to monitor the concentration of the active compound in theserum of the patients. The serum concentration is monitored over atime span of 12 hours in six patients, which took one pill each.Patients’ data are:
Patient Sex Age (years) Smoker/non-smoker
1 male (1) 20 - 30 (1) non-smoker (1)
2 male (1) 30 - 40 (2) smoker (2)
3 female (2) 40 - 50 (3) non-smoker (1)
4 male (1) 40 - 50 (3) smoker (2)
5 female (2) 30 - 40 (2) smoker (2)
6 female (2) 20 - 30 (3) non smoker (1)
The results of the HPLC runs were sent to the database of the AgilentChemStation. 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 theyare higher than for others (grey circles).
Figure 7Serum concentration of verapamil for all patients
�
��
����
����
���� ����
Verapamil
Concentration [mg/l]
Time [hours]
62
Figure 8Serum concentration of verapamil for patients in the age group 20 - 30 years
Figure 9Serum concentration of verapamil for patients in the age group 30 - 40 years
All three age groups include patients with higher and lower serum con-centrations. This is shown in figures 8 - 10. The decomposition ratedoes, therefore, not depend on age.
Concentration
Time [hours]
Concentration
Time [hours]
63
Figure 10Serum concentration of verapamil for patients age group 40 - 50 years
Figures 11 and 12 show that the rate of decomposition does not dependon the factor smoker or non-smoker.
Figure 11Serum concentration of verapamil in non-smokers
Concentration
Time [hours]
Concentration
Time [hours]
64
Figure 12Serum concentration of verapamil in smokers
Figures 13 and 14 show that the rate of decomposition is higher formales than for females, which indicates that the decomposition rate isdependent on sex.
Figure 13Serum concentration of verapamil in male patients
Concentration
Time [hours]
Concentration
Time [hours]
65
Figure 14Serum concentration of verapamil in female patients
Figures 15 and 16 show example diagrams of a male and a femalepatient.
Figure 15Serum concentration of verapamil in patient 1 (male)
Time [hours]
Concentration [mg/l]
Time [hours]
Concentration [mg/l]
66
Figure 16Serum 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 thedecomposition rate is only dependent on the sex of the patient. Therate of verapamil decomposition is higher for male patients than forfemale patients.
Time [hours]
Concentration [mg/l]
67
Agilent 1100 Series Combinatorial Chemistry Analysis System
The Agilent 1100 Series combinatorial chemistry analysis systemincreases sample throughput, shortens response time and enables significant ease of control. The system can store and inject over 4600samples for unattended high throughput. Easy-to-use point-and-clicksoftware lets the user control the complete system, including samplehandling, data analysis and reporting.
The new liquid chromatograph/mass spectrometer (Agilent 1100 SeriesLC/MSD) hard - and software solution provides rapid molecular weightconfirmation of combinatorial libraries. Users can identify the productsof combinatorial syntheses and estimate their purities rapidly and easi-ly before screening. This gives them fast confirmation of combinatorialmethods 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 HPLCwith diode-array detector, the Agilent ChemStation, the Agilent 3DLC ChemStation software, the Agilent 1100 Series LC/MSD and thenew combinatorial chemistry software are supplied and supportedby 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: asampler mode for high speed sample analysis and a fractionationmode for sample isolation and purification.
68
• Easy to use - The system is built around an easy-to-use graphicaluser interface. A single screen shows the system status, layout of upto 12 96-well plates on the sampler and an overview of the sampleand fraction positions. Based on the target molecular weights sup-plied 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 matricesby 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 combinatorialchemistry analysis system turn to page 80. Literature with further infor-mation is listed on page 84.
69
Instrumentation
The Agilent 1100 Series HPLC system and Agilent ChemStations are ideally suited for the analysis of pharmaceutical samples. They meet themost demanding laboratory objectives, such as:
• helping to complying with regulatory and quality standards, usingbuilt-in, fast, features, that offer fast, labor-saving validation
• reducing operating cost – no need for helium due to all electricdesign, use of 2-mm columns reduces solvent costs,
• saving operator training time through intuitive user interface andbuilt-in online tutorials,
• maximizing productivity with fast system setup and intelligentautomation,
• maximizing instrument uptime through use of field-proven parts,multimedia instructions for effective maintenance, software foronsite 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 thewavelength range of 190 - 950 nm,
• thermostatted autosampler with a cooling temperature down to 4 ºCwhich avoids decomposition of thermally labile samples,
• temperature-controlled column compartment with a standard Peltierelement for heating and cooling, which eliminates analytical errorsdue 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.
Agilent 1100 Series modules and systems
70
Instrumentation
Agilent ChemStations are recommended for instrument control, dataevaluation and data traceability. Features for the pharmaceutical labo-ratory include:
• system suitability software for automated and customized methodvalidation and system suitability testing,
• report generator to print analysis reports according to regulatory andclient requirements,
• software for automated identification and quantification based ondiode-array and/or mass selective detection, spectral libraries for UV-visible and MS spectra (Agilent ChemStation for LC 3D and LC/MSD systems only),
• control and data acquisition of fluorescence and refractive indexdetector,
• 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 anetwork. A lab-wide raw data backup solution is also offered.
Agilent ChemStation for HPLC
71
Instrumentation
Sensitive and selective detectors are very important for the analysis ofpharmaceutical samples with HPLC, because of the need to analyze atlow concentration levels in complex matrices. Most detectors appliedin HPLC are also used for pharmaceutical analysis. The table belowgives an overview of HPLC detectors with the most important charac-teristics and application examples. To provide highest sensitivity for thedifferent compound classes several detection techniques are necessary.
The variable-wavelength detector (VWD) is recommended for routineanalysis of known mixtures. The detector can be operated and controlledfrom 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.
The diode array detector (DAD) is recommended for the analysis ofcompounds in difficult matrices, whenever identification via retentiontime may not be sufficient. For additional confirmation spectra can betaken during the run and compared to those stored in spectral libraries.The evaluation of spectra can be done manually or automatically. Thedetector 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.
Detector type Sensitivity Selectivity Qualitative Compoundsinformation
Variable high, low, low, antidepressantwavelength low ng range one signal only by RTs keratolyticdetector muscle-relaxing
Diode-array high, high, several signals high, RTs and spectra Ginko billobaedetector low ng range at different wavelengths Cortex cinchonae
Atropa belladonna
Fluorescence very high very high, light high, RTs and antianginaldetector low pg range absorbance followed online spectra anticoagulant
by emission of light must water-solublebe possible,multisignal vitaminsacquisition
LC-MS very high, very high, selective very high, molecular antiepilepticspectrometry pg range search for specified weight and frag- antitumor
masses mentation information hypnotic
Detector Overview
Diode array detector
Variable wavelength detector
72
Instrumentation
The Agilent 1100 Series fluorescence detector provides high-sensitivity multi-signal detection, online fluorescence spectra, straightforwardoperation 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 com-pounds, and
• built-in diagnostic tools minimize downtime and increase operatorsafety.
The Agilent 1100 Series LC/MSD detector ideally complements a diode-array UV-visible detector. It gives laboratories the molecular weight,structural information, selectivity and sensitivity needed for quick com-pound identification, purity checks for chromatographic peaks andquantification 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 theanalyses of wide variety of compound classes,
• mass spectral information increases confidence in results and shortens analysis time, and
• in-source fragmentation (collision-induced dissociation) providespowerful structural information.
The Agilent 1100 Series thermostatted column compartment is designedfor separations requiring stable temperature to achieve reproducibleretention times or separations which improve at temperatures above orbelow ambient. Other features include:• temperature range from 10 ºC below ambient to 80 ºC,• 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 regu-
lated laboratories.
Fluorescence detector
Mass selective detector
Thermostatted column compartment
73
Instrumentation
Agilent's unique injection principle has been used successfully for manyyears in thousands of instruments worldwide. This proven design hasmany outstanding advantages compared to conventional autosamplers,such as :
• Precise sample handling for dependable 24 hour/day chromatogra-phy. The autosampler’s repeatability is typically less than 0.5 % for volumes as low as 1 µl and less than 0.2 % for 10 µl and above.
• Ease-of-use for fast familiarization, with handheld control module orAgilent ChemStation graphical user interface saves valuable operatortime. It offers color-coded sample, calibration and control vials andcurrent sample vials.
• Low internal volume of 300 µl for minimum contribution to a system's total internal volume.
• Continuous flow-through design. All parts in contact with the sampleare 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 withPC card makes compliance easier and less costly.
Analyzing thermally-labile compounds has always been a problem forpharmaceutical and biopharmaceutic analysts alike. Traditional coolingmethods often leave condensation in the vials. Agilent's new thermostattedautosampler provides Peltier temperature control from 4 - 40 ºC of up to100 samples (2-ml vials). By using dry air, condensation build-up is prevented.
Other features include:• Cooling temperature goes down to 4 ºC, 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.
Autosampler
Thermostatted Autosampler
74
Instrumentation
The Agilent 220 micro plate sampler is an ideal tool to analyze largenumbers of structurally distinct compounds. Integrated into the Agilent1100 Series HPLC system it offers the full potential of well-plate tech-nology.
Other features include:• High sample capacity, which increases sample throughput and short-
ens response times. This is especially useful for drug discovery, com-binatorial chemistry, medicinal chemistry and pharmacokinetic stud-ies.
• The choice of various plates, vials and tubes, including up to 12micro plates
• The flexibility of sampler mode for high-speed sample analysis orfraction collector mode for sample isolation and purification. Thisgives the user a versatile tool to support screening, analysis andresearch.
• An autosampler injector program for sample preparations such asderivatization, dilution and mixing.
• The ability to store and inject over 4,600 samples (using 384-well-plates) for unattended high throughput.
• Easy-to-see color-coded sample and fraction tracking for a fast, clearoverview of the analysis results.
Whether operating as a standalone module with the handheld controlmodule 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 offour solvents for utmost flexibility and increased convenience, forexample, 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 connec-tions.
• Both pistons of the two individual pumps are servo-controlled tomeet the highest chromatographic demands in gradient formation.
• Ideal for reproducible gradient at low flow rates due to an internalvolume of only 180 to 480 µl (without mixer), highest compositionstability and a flow range starting at typically 50 µl/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 solventdelivery for standard-bore columns without solvent degassing.
Agilent 220 Micro PlateSampler
Binary Pump
75
Instrumentation
An optimized design ensures virtually pulse-free and stable solvent flow,with dual floating pistons in series, precisely servo-controlled. Thisdesign with variable stroke volume allows pulse-free solvent delivery and efficient mixing. An inlet valve, electronically activated andsynchronized to piston stroke, eliminates vapor formation with volatilesolvents and requires no maintenance. High-speed proportioning valvesoffer convenient low-pressure mixing of up to four solvents and highestflexibility 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 systemand method development.
• A wide flow range up to 10 ml/min and a delay volume of 800 to 1100-µl support narrow-bore, standard and semipreparative applica-tions.
• Easy programming and control through the handheld control moduleor the Agilent ChemStation help save time.
• Robust materials withstand most demanding applications. Stainlesssteel, titanium, gold, ruby, sapphire, ceramics, PEEK and PTFEensure long lifetime. The seal wash option for high salt mobile phases keeps maintenance to a minimum which reduces operationcosts.
• More help for GLP – built-in leak and pressure test with graphicalpresentation of pressure profiles, and early maintenance feedback.
Dissolved gases are evacuated through special membrane tubes, provid-ing 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.
Quaternary Pump
Vacuum degasser
76
Configuration Example 1
G1354A
G1313A
G1316A
G1315A G1319A Printer
HP-IB10833D
CAN5181-1516
CAN5181-1516
CAN5181-1516
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 AgilentChemStation software, G2180AA spectral processingsoftware, HP Kayak computer, CD-ROM drive, HPLaserJet printer, Microsoft Windows 4.0 NT.
Optional database for Agilent ChemStation:
Agilent ChemStore C/S database client software.
Description Order
G1354A
G1313A
G1316AG1315AG1314A #018
G1319A
G2181BA
Minimum instrument configuration:
• quaternary pump with degasser
• autosampler
• thermostatted column compartment
• variable wavelength detector
• Agilent ChemStation
77
Configuration Example 2
Description Order
G1354A
G1327A
G1316A
G1315A G1319A Printer
HP-IB10833D
CAN5181-1516
CAN5181-1516
CAN5181-1516
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 AgilentChemStation software, G2180AA spectral processingsoftware, 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.
G1354A
G1327AG1316A
G1315AG1314A #018
G1319A
G2181BA
Minimum instrument configuration:
• quaternary pump with degasser
• autosampler
• thermostatted column compartment
• variable wavelength detector
• Agilent ChemStation
78
Configuration Example 3
Description Order
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 AgilentChemStation software, G2180AA spectral processingsoftware, 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.
G1354A
G1327AG1316AG1315AG1314A #018G1321A
G1319A
G2181BA
G1354A
G1327A
G1316A
G1315A
G1319A Printer
HP-IB10833D
CAN5181-1516
CAN5181-1516
CAN5181-1516
CAN5181-1516
G1321A
Minimum instrument configuration:
• quaternary pump with degasser
• autosampler
• thermostatted column compartment
• variable wavelength detector
• fluorescence detector
• Agilent ChemStation
79
Configuration Example 4
Description Order
G1354A
G1327A
G1316A
G1315A G1319A Printer
HP-IB10833D
CAN5181-1516
CAN5181-1516
CAN5181-1516
CAN5181-1516
G1946A
HP-IB10833D
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 AgilentChemStation software, G2180AA spectral processingsoftware, 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 SeriesLC/MSD. LC/MSD ChemStation add-on software.
Optional database for Agilent ChemStation:
Agilent ChemStore C/S database client software.
G1354A
G1327AG1316AG1315AG1314A #018
G1319A
G1946AG1948AG2715AA
G2181BA
80
Configuration Example for Agilent 1100 Series Combinatorial Chemistry Analysis System
Description Order
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 AgilentChemStation software, G2180AA spectral processingsoftware, 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.
G1354A
G1327AG1316AG1315AG1314A #018G1946AG1948AG2250AG2251A
G1319A
G2715AAINB2202
G2181BA
G1354A
G1316A
G1315A
G1319A PrinterHP-IB10833D
CAN5181-1516
CAN5181-1516
Remote05989-60090
G1946
HP-IB10833D
G2250AHCP-CBLE-RS5181-1527
HCP-CBLE-CS5181-1520
81
Quick Reference Guide
Drug Stationary Mobile Temperature DetectorPhase Phase
Analgesic drugsAntipyrine, Zorbax SB-C18 0.05M KH2P 04 25 °C VWD, 204 nmHydroxyantipyrine, pH=3/acetonitrileAcetaminophen Androgen drugsTestosterone acetate, Hypersil ODS water/acetonitrile 40 °C VWD (TT)Testosterone 254 nm, 234 nmAntianginal drugsVerapamil Zorbax SB-C18 0.025M KH2P 04 25 °C VWD, 204 nm
pH=3/acetonitrile FLD, 228/312 nmAntiarrythmic drugsQuinidine, Disopyramide Purosphere RP-18 0.05 KH2P 04 60 °C VWD (TT)Procainamide pH=2.5/acetonitrile 220 nm, 254 nmN-Acetylprocainamide 204 nmAntiasthmatic drugsCaffeine, Theophylline, Hypersil ODS water/acetonitrile 50 °C VWDEnprofylline,Theobromine 270 nmAntibacterial drugsPenicillin-like: Zorbax SB-C18 0.025 KH2P 04 40 °C VWD, 204 nmAmpicillin, Amoxicillin, pH=3/acetonitrilePenicillin G, Penicillin VTetracyclines: Zorbax SB-C18 0.025 KH2P 04 25 °C VWD, 350 nmMinocycline, Tetracycline, pH=3/acetonitrileDoxycyclineMiscellaneous: Zorbax SB-C18 0.025 KH2P 04 40 °C VWD (TT)Hydroxybenzotriazole , pH=3/acetonitrile 204 nmChloramphenicol, Trimethoprim 368 nmSulfamethoxazole, Furazolidone, 254 nmNalidixic AcidAnticoagulant drugsWarfarin Zorbax SB-C18 0.025 KH2P 04 25 °C VWD, 204 nm
pH=3/acetonitrile FLD, 272/355 nmAntidepressant drugsBupropion, Trazodone, Zorbax SB-C18 0.025 KH2P 04 25 °C VWD, 210 nmMaprotiline pH=3/acetonitrile
The following table gives an overview of basic analysis parameters of alarge number of pharmaceutical drugs. They are listed according totheir 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 thedetector most commonly used. The diode array and fluorescence detectors are also used for certain applications. Other recommendedmodules include a vacuum degasser, a quaternary pump, a thermo-statted autosampler, a thermostatted column compartment and an Agilent ChemStation for LC systems.
82
Drug Stationary Mobile Temperature DetectorPhase Phase
Antiepileptic drugsCaffeine, Phenytoin, Hypersil ODS water/acetonitrile 60 °C VWD, 204 nmMethylphenylsuccinimide,Phenylethylmalondiamide, Carbamazepinepoxid, Ethosuximide, Phenobarbital, Carbamazepine, PrimidoneAntiestrogen drugsTamoxifen Purosphere RP-18 0.025M KH2P 04 60 °C VWD, 204 nm
pH = 3/acetonitrileAntihistaminic drugsTetracaine, Promethazine, Zorbax SB-C18 0.025M KH2P 04 25 °C VWD, 204 nmChlorpheniramine, pH = 3/acetonitrileTripelenamineAntihypertensive drugsEnalapril, Captopril Zorbax SB-C18 0.025M KH2P 04 60 °C VWD, 204 nm
pH = 2/acetonitrileAntiinflammatory drugsNaproxen Zorbax SB-C18 0.025M KH2P 04 25 °C VWD, 230 nm
pH = 3/acetonitrileAntiprotozoal drugsMetronidazole Zorbax SB-C18 0.025M KH2P 04 25 °C VWD, 320 nm
pH = 3/acetonitrileAntitumor drugsPaclitaxel (Taxol) Hypersil ODS water/acetonitrile 25 °C VWD, 204 nm
Antitussive drugsDextromethorphan Zorbax SB-C18 0.025M KH2P 04 60 °C VWD, 204 nm
pH = 3/acetonitrileCatecholaminesNorepinephrine Zorbax SB-C18 0.025M KH2P 04 30 °C VWD, 204 nmEpinephrine, + 0.3 mM heptane-Dihydroxybenzylamine, sulfonic acidDopamine pH = 3/acetonitrileGlucocorticoid drugsBeclomethasone-dipropionate,Hypersil ODS water/acetonitrile 25 °C VWD, 254 nmPrednisolone, Prednisolon-acetate,Betamethasone, Betamethason-valerat, Hydrocortisone, Hydrocortisone-acetateH2-AntagonistsRanitidine, Cimetidine Zorbax SB-C18 0.025M KH2P 04 25 °C VWD, 225 nm
pH = 3/acetonitrileHypnotic drugsBarbital, Allobarbital, Hypersil ODS water/acetonitrile 25 °C VWD, 204 nmPhenobarbital, Butabarbital, Butalbital, Amobarbital, Methylphenobarbital, Flunitrazepam
83
Drug Stationary Mobile Temperature DetectorPhase Phase
Keratolytic drugsSalicylic Acid, Zorbax SB-C18 0.05M NH40Ac 50 °C VWD (TT),Phtalic Acid, Benzoic Acid pH=2.2/acetonitrile 204 nm, 230 nmMuscle-relaxing drugsPapaverine Zorbax SB-C18 0.025M KH2P 04 60 °C VWD, 254 nm
pH=3/acetonitrileSedative drugsDiazepam, Oxazepam, Zorbax SB-C18 0.025M KH2P 04 25 °C VWD, 210 nmClonazepam, Flunitrazepam pH=3/acetonitrileSulfa drugsSulfanilamide, Sulfadiazine, Hypersil ODS water/acetonitrile 50 °C VWD, 254 nmSulfathiazole, Sulfamerazine, Sulfamethazine, Therapeutic peptidesAngiotensin II, Angiotensin I, Zorbax SB-C18 0.1 % TFA/ 25 °C VWD, 210 nmInsulin, Oxytocin acetonitrileTricyclic antidepressant drugsProtriptyline, Nortriptyline, Zorbax Eclipse 0.025M KH2P 04 40 °C VWD, 210 nmDoxepin, Imipramine, XDB C-18 pH=7/methanolAmitriptyline, TrimipramineVitaminsFat soluble Zorbax Eclipse water/methanol 20 °C VWD, 210 nmVitamins A1, D3 E XDB C-18Water soluble Zorbax SB-C18 0.05M KH2P 04 15 °C VWD, 204 nmAminobenzoic Acid, Biotin, pH=2.5/Folic Acid, Niacinamide, acetonitrilePantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine, Riboflavine, Thiamine, Thiotic Acid
Medical Herb/ Stationary Mobile Temperature DetectorActive Compound Phase Phase
Atropa belladonnaAtropine Zorbax Eclipse 0.05M KH2P 04 40 °C DAD, 210 nm/16
XDB-C18 pH=3/acetonitrileCortex cinchonaeQuinine, Quinidine Purosphere RP-18 0.05M KH2P 04 25 °C DAD, 210 nm/16
pH=3/acetonitrileDan ShenProtocatechuic Acid, Zorbax SB-C18 0.025M KH2P 04 50 °C DAD, 225 nm/16Protocatechuic Aldehyde, pH=3/methanol 254 nm/16Tanshinone I, Tanshinone IIA, CryptotanshinoneEphedra sinica stapfEphedrine, Norephedrine Zorbax SB-C18 0.025M KH2P 04 25 °C DAD, 204 nm/8
pH=3/acetonitrileGinko bilobaeQuercetin, Kaempferol Hypersil ODS 0.5 % H3P 04/ 25 °C DAD, 370 nm/16
methanolRheum palmatumRhein, Emodin Hypersil ODS 0.05M NH4OAc 25 °C DAD, 440 nm/16
pH=2.5/acetonitrile
84
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 5968-1078E
Analysis of Antiasthmatic Drugs by HPLC 5968-1079E
Analysis of Antiarrythmic Drugs by HPLC 5968-1080E
Analysis of Antianginal Drugs by HPLC 5968-1081E
Analysis of Anticoagulant Drugs by HPLC 5968-1082E
Analysis of Therapeutic Peptides by HPLC 5968-1109E
Analysis of Sedative Drugs by HPLC 5968-1110E
Analysis of Sulfa Drugs by HPLC 5968-1111E
Analysis of Muscle-relaxing Drugs by HPLC 5968-1112E
Analysis of Keratolytica by HPLC 5968-1113E
Analysis of H2-Antagonist Drugs by HPLC 5968-1114E
Analysis of Antitussive Drugs by HPLC 5968-1115E
Analysis of Antiprotozoal Drugs by HPLC 5968-1116E
Analysis of Antiinflammatory Drugs by HPLC 5968-1117E
Analysis of Antiestrogen Drugs by HPLC 5968-1118E
Analysis of Antiepileptic Drugs by HPLC 5968-1119E
Analysis of Protocatechuic Acid, Protocatechuic Aldehyde and Tanshinones in Dan Shen pills by HPLC 5968-2882E
Analysis of Ephedrine and Norephedrine in Ephedra Sinica Stapf by HPLC 5968-2884E
Analysis of Antitumor Drugs by HPLC 5968-2886E
Analysis of Antihypertensive Drugs by HPLC 5968-2887E
Analysis of Antihistaminic Drugs by HPLC 5968-2889E
Analysis of Antidepressant Drugs by HPLC 5968-2963E
Analysis of Androgen Drugs by HPLC 5968-2964E
Analysis of Analgesic Drugs by HPLC 5968-2965E
Analysis of Catecholamines by HPLC 5968-2966E
85
Analysis of Glucocorticoids by HPLC 5968-2967E
Analysis of Hypnotic Drugs by HPLC 5968-2968E
Analysis of Tricyclic Antidepressant Drugs by HPLC 5968-2969E
Analysis of Fat-Soluble Vitamins by HPLC 5968-2970E
Analysis of Water-Soluble Vitamins by HPLC 5968-2971E
Analysis of Rhein and Emodin in Rhubarb Root Extract (Rheum palmatum)by HPLC 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 5968-2974E
Analysis of Atropine in Belladonna Extract (Atropa Belladonna) Drugs by HPLC 5968-2975E
Agilent ChemStation Database
From Data to Decisions (CD-ROM) 5968-1713E
Increase Lab Productivity by Turning Chemical Analysis Data into Scientific Knowledge 5966-1990E
Drive Down Costs and Raise Productivity 5968-4782E
Quality Control of Pharmaceutical Drugs–Turning Analysis and Performance Datainto Comprehensible Charts 5968-7570E
Agilent 1100 Series Combinatorial Chemistry Analysis System
Agilent 220 Micro Plate Sampler with the Agilent 1100 Series System for FlexibleHigh Throughput HPLC Analysis 5968-5322E
Isolation and Characterization of Natural Products Using the Agilent 220 MicroPlate Sampler with the Agilent 1100 Series HPLC System 5968-6252E
High Throughput Screening of a Peptide Library with the Agilent 1100 Series Combinatorial Chemistry Analysis System 5968-8661E
Agilent 220 Micro Plate Sampling System 5968-9101E
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