Aplicações Farmaceuticas Com HPLC

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

�

�

�

�

�

��

��

�

�

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




Precision of area10 runs, 50 mg/l < 0.8 %

�

�

��

� �

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


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


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)

�

��

��

��

��

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



Analysis of antiarrythmic drugs

Antiarrythmic Drugs


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%


�

��

�

��

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


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






�

�

�

�

�

�

�

�

�

�

�

��

�

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




Analysis of antibacterial drugs with pencillin-like structure

Antibacterial Drugs with Pencillin-like Structure


Limit of detection 1 mg/l (5-µl injection), S/N=2



�

�

��

��

�

��

�

�

��

��

��

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








��

��

��

��

��

��

Minocycline

��

��

��

��

��

��

Tetracycline

��

��

��

��

��

��

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


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




Limit of detection 0.1 mg/l (5-µl injection), S/N=2(VWD)



Linearity 1.00000(correlation factor) 0.99990 (nalidixic acid)

�

��

��

Hydroxybenzotriazole

��

��

��

��

�

Chloramphenicol

�

�

��

��

��

��

Trimethoprim

��

�

�

�

��

Sulfamethoxazole

��

�

Furazolidone

� �

��

�

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


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


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


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)


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 %

�

��

��

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









��

��

�

Bupropion

�

�

Maprotiline

�

�

��

�

�

�

�

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






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

�

�

�

� �

�

�

Phenobarbital

�

�

�

�

�

�

Primidone

��

��

Phenylethylmalonamide

�

��

Carbamazepine

�

��

�

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


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


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


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








��

Tamoxifen

Tamoxifen

Time [min]

0 2 4 6 8 10 12 14

Absorbance[mAU]

0

50

100

150

200

250

300

27

Antihistaminic Drugs









��

�

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


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






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










��

��

Naproxen

Time [min]

0 2 4 6 8 10

Absorbance[mAU]

0

200

400

600

800

Naproxen1000

30

Antiprotozoal Drugs






Limit of detection 0.1 mg/l (5-µl injection),S/N=2




�

��

��

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


Column 4 x 125 mm Hypersil ODS, 5 µmMobile phase A = water, B = acetonitrileFlow rate 1.0 ml/minGradient at 0 min 50 % B








��

��

��

��

�

��

��

�

�

��

�

�

�

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


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 5 min 50 % BUV detector diode array detector


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


33

Antitussive Drugs

Analysis of antitussive 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








��

�

�

��

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


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





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









Linearity(correlation factor)> 0.99997

�

��

��

��

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









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


225 nm, standard cellColumn compartment temperature 25 °CStop time 10 minInjection volume 5 µl






�

�

�

�

�

�

�

�

�

��

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






Limit of detection 0.2 mg/l (5-µl injection), S/N=20.4 mg/l (butalbital,amobarbital)



�

�

�

� �

��

��

��

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


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




40


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


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 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


��

��

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




0.16 % (phthalic acid)


42


Muscle-relaxing Drugs

Analysis of muscle-relaxing drugs








�

��

��

��

��

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





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





�

�

�

��

��

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





Separation of sulfathiazole (3) and sulfamerazine (4) using different column temperatures




1.2 % (sulfanilamide)


��

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








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


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



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


Limit of detection <1.0 mg/l (5-µl injection),S/N=2



�

�

�

�

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


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





Precision of RT10 runs, 1000 mg/l< 0.10 %


��

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


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


Analysis of vitamins B2 and B6 using the Agilent 1100 Series fluorescence detector (column: 21 x 50 mm Zorbax SB-C18, 5 µm)





�

�

��

��

�� 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


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



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

�

�

�

��

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


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


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


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



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


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 18 min 80 % Bat 20 min 2 % B

UV detector diode array detector204 nm/8 (ref. 360 nm/100), standard cell


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


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


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



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


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



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.


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]


Time [hours]


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]


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


Description Order

G1354A

G1327A

G1316A


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.



G1354A

G1327AG1316A

G1315AG1314A #018

G1319A

G2181BA



• autosampler




78


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.




G1354A

G1327AG1316AG1315AG1314A #018G1321A

G1319A

G2181BA

G1354A

G1327A

G1316A

G1315A

G1319A Printer

HP-IB10833D

CAN5181-1516

CAN5181-1516

CAN5181-1516

CAN5181-1516

G1321A



• autosampler



• fluorescence detector


79


Description Order

G1354A

G1327A

G1316A


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 1100 Series LC/MSD spectrometer.API-Electrospray source for Agilent 1100 SeriesLC/MSD. LC/MSD ChemStation add-on 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.



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


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


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


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

Microsoft and Windows NT are U.S. registered trademarks of Microsoft Corp.

For the latest information and services visit our world wide web site:http://www.agilent.com/chem

Agilent TechnologiesInnovating the HP Way

Copyright © 1998-2000 Agilent TechnologiesAll Rights Reserved. Reproduction, adaptationor translation without prior written permissionis prohibited, except as allowed under thecopyright laws.

Printed In Germany 04/2000

Publication Number 5968-2635E

Aplicações Farmaceuticas Com HPLC

Documents

Transcript of Aplicações Farmaceuticas Com HPLC