csh technology: acquity csh columns applications notebook€¦ · list of compounds analyzed using...
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CSH TeCHnology: ACQUITY CSH Columns Applications Notebook
List of compounds anaLyzed using csh coLumns
Lit Code Title Page
WA64123 Acetaminophen Impurities 2
WA64122 Acids, Bases, and Neutrals 3
WA64133 Aromatic Acids 4
WA64124 Basic Drugs 5
WA64125 Beta Blockers 6
WA64134 Buproprion Hydrochloride (Wellbutrin) in Extended Release Tablets 7
WA64135 Clozapine Impurities 8
WA64136 Food Additives 9
WA64126 Formic and Acetic Acid 10
WA64138 Mirtazapine Impurities 11
WA64127 Nonsteroidal Anti-Inflammatory Drugs (NSAIDS) 12
WA64139 Organic Acids 13
WA64128 Paroxetine Impurities 14
WA64129 Paroxetine Isomers 15
WA64130 Pharmaceutical Test Mix 16
WA64137 Statin Drugs 17
WA64131 Sulfa Drugs 18
WA64132 Tricyclic Antidepressants 19
WA64140 Veterinary Drugs 20
2
ACQU
ITY
CSH
WA6
4123
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ Fluoro-Phenyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005351
Mobile Phase A: 0.1% acetic acid in water
Mobile Phase B: acetonitrile
Gradien:
Injection Volume: 2 μL
Sample Diluent: water
Column Temperature: 40 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 270 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.6 98 2 Initial2.00 0.6 50 50 62.50 0.6 50 50 62.51 0.6 98 2 63.50 0.6 98 2 6
STRUCTURES
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min
1
2
3 4
Compounds
1. 4-Aminophenol (0.002 mg/mL)
2. Acetaminophen (0.2 mg/mL)
3. 4-Nitrophenol (0.002 mg/mL)
4. 4-Chloroacetanilide (0.002 mg/mL)
HO
NH
O
OH
NO2Cl
HN
O
4-Aminophenol Acetaminophen
4-Nitrophenol 4-Chloroacetanilide
OH
NH2
anaLysis of acetaminophen impurities using acQuit y csh coLumns
3
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4122
LC CONDITIONS
Columns: ACQUITY CSH™ C18, 2.1 x 50 mm, 1.7 μm
Part Number: 186005296
ACQUITY CSH Phenyl-Hexyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005406
ACQUITY CSH Fluoro-Phenyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005351
Mobile Phase A: 0.1% formic acid in water (pH ~ 2.7)
Mobile Phase B: acetonitrile
Gradient Table:
Injection Volume: 2 μL (PLNO)
Sample Diluent: 25/75 methanol/water
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 254 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with column manager
and ACQUITY UPLC PDA detector
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.5 95 5 Initial5.00 0.5 5 95 65.01 0.5 95 5 65.50 0.5 95 5 6
STRUCTURES
ACQUITY CSH C18
AU
0.00
0.15
0.30
0.45
AU
0.00
0.15
0.30
0.45 ACQUITY CSH Phenyl-Hexyl
1
5
6 7
2
3
1
2
3
4
4
5
6 7
AU
0.00
0.15
0.30
0.45
0.00 1.20 2.40 3.60 4.80 min
ACQUITY CSH Fluoro-Phenyl
1
2
3
4
5
6 7
Compounds
1. Imipramine (100 µg/mL)
2. Amitriptyline (100 µg/mL)
3. 1-Pyrenesulfonic acid (50 µg/mL)
4. Flavone (30 µg/mL)
5. Fenoprofen (100 µg/mL)
6. Diclofenac (100 µg/mL)
7. Octanophenone (100 µg/mL)
N
N N
SO3H
O
O
OCOOH
COOHHN
Cl
Cl
O
Imipramine Amitriptyline
1-Pyrenesulfonic acid
Flavone
Fenoprofen
DiclofenacOctanophenone
anaLysis of acids, Bases, and neutraLs using acQuit y csh coLumns
4
ACQU
ITY
CSH
WA6
4133
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ C18, 2.1 x 50 mm, 1.7 μm
Part Number: 186005296
Mobile Phase A: 0.1% acetic acid in water
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 4 μL
Sample Concentration: 10 µg/mL each
Sample Diluent: water
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 280 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.6 98 2 Initial5.00 0.6 40 60 65.50 0.6 40 60 65.51 0.6 98 2 66.00 0.6 98 2 6
STRUCTURES
0.00
0.04
0.08
0.12
0.16
0.20
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
AU
1
2
3
4
5
67
COmPOUNDS
1. Protocatechuic acid
2. 3,4-Dihydroxyphenylacetic acid (DOPAC)
3. 4-Hydroxybenzoic acid
4. Vanillic acid
5. Syringic acid
6. Salicylic acid
7. 2,4-Dichlorophenoxyacetic acid (Weed B Gon)
Protocatechuic acid
COOH
OHOCH3
OCH3
HO
H3CO COOH COOHOH
4-Hydroxybenzoic acid
Vanillic acid
Syringic acid
Salicylic acid
2,4-Dichlorophenoxyacetic acid (Weed B Gon)
3,4-Dihydroxyphenylacetic acid (DOPAC)
Cl Cl
O COOH
COOH
OH
COOH
OHOH
HO
COOH
OH
anaLysis of aromatic acids using acQuit y csh coLumns
5
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4124
LC CONDITIONS
Column: ACQUITY CSH™ Phenyl-Hexyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005406
Mobile Phase A: 10 mM ammonium formate, pH 3
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 4 μL
Sample Diluent: water
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 260 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with ACQUITY
UPLC PDA
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.4 95 5 Initial3.00 0.4 45 55 63.50 0.4 45 55 63.51 0.4 95 5 64.50 0.4 95 5 6
STRUCTURES
0.00
0.04
0.08
0.12
0.16
0.20
0.0 0.5 1.0 1.5 2.0 2.5 3.0 min
AU
1
2
3
4
5
6
7
8
9
Compounds
1. 2-aminopyrazine (25 μg/mL)
2. Pindolol (10 μg/mL)
3. Quinine (10 μg/mL)
4. Quinine impurity
5. Labetalol (60 μg/mL)
6. Diltiazem impurity
7. Diltiazem (25 μg/mL)
8. Verapamil (50 μg/mL)
9. Amitriptyline (25 μg/mL)
N
S
NO
O
O
OCH3
HO
HN
H2N
O OH
OHN
OH
HN
N
HO
H3CO
N
H
H
H3CO
H3CO
CNN OCH3
OCH3
N
Pindolol2-aminopyrazine
Quinine Diltiazem
Labetalol Amitriptyline
Verapamil
N
N NH2
anaLysis of Basic drugs using acQuit y csh coLumns
6
ACQU
ITY
CSH
WA6
4125
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ Phenyl-Hexyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005406
Mobile Phase A: 10 mM ammonium formate, pH 3
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 4 μL
Sample Diluent: water
Sample Concentration: 10 µg/mL each
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 280 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.4 95 5 Initial3.00 0.4 15 85 63.50 0.4 15 85 63.51 0.4 95 5 64.50 0.4 95 5 6
STRUCTURES
0.00
0.02
0.04
0.06
0.08
0.10
0.0 0.5 1.0 1.5 2.0 2.5 3.0 min
AU
1
2
3
4
5
6 Compounds
1. Atenolol
2. Pindolol
3. Metoprolol
4. Oxprenolol
5. Labetalol
6. Propranolol
Atenolol
NH2
OON
H OH
HO
HN
H2N
O OH
OHN
OH
H3CO
OHN
O
OH
OHN
OH
HN
OOH
NH
Labetalol
Metoprolol
Oxprenolol
Pindolol
Propranolol
anaLysis of Beta BLockers using acQuit y csh coLumns
7
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4134
LC CONDITIONS
Column: ACQUITY CSH™ C18, 2.1 x 50 mm, 1.7 μm
Part Number: 186005296
Mobile Phase A: water
Mobile Phase B: acetonitrile
Mobile Phase C: 2% formic acid in water
Gradient:
Injection Volume: 5 μL
Sample Diluent: H2O
Sample Concentration: 20 µg/mL
Column Temperature: 30 °C
Wash Solvent: 50/50 water/acetonitrile
Purge Solvent: 50/50 water/acetonitrile
System: ACQUITY UPLC® H-Class with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.9 90 5 5 Initial3.00 0.9 0 95 5 63.50 0.9 0 95 5 63.51 0.9 90 5 5 65.00 0.9 90 5 5 6
SamPLE PREPaRaTION PROCEDURE
Ten extended release tablets (each containing 100 mg bupropion
hydrochloride) were homogenized in 50 mL of 50/50 methanol/
water to give 20 mg/mL of bupropion hydrochloride. The sample was
then centrifuged at 4000 rpm for 10 minutes. The supernatant was
then diluted to 0.02 mg/mL in water and filtered through a 0.45 µm
PVDF syringe filter prior to injection.
STRUCTURES
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.0 0.5 1.0 1.5 2.0 2.5 3.0 min
Peak width (4.4%) = 1.3 secondsUSP tailing factor = 1.27
1 COmPOUND
1. Bupropion (pKa = 7.9)
ClHN
O
Bupropion
anaLysis of Bupropion hydrochLoride (WeLLButrin) in extended reLease taBLetsusing acQuit y csh coLumns
8
ACQU
ITY
CSH
W
A641
35
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ Phenyl-Hexyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005406
Mobile Phase A: water
Mobile Phase B: acetonitrile
Mobile Phase C: 2% formic acid in water
Gradient:
Injection Volume: 4 μL
Sample Diluent: 80/20 methanol/water
Sample Concentration: 400 µg/mL (impurities present at ~0.2%)
Column Temperature: 30 °C
Wash Solvent: 50/50 water/acetonitrile
Purge Solvent: 50/50 water/acetonitrile
System: ACQUITY UPLC® H-Class with
ACQUITY UPLC PDA and SQD
mS CONDITIONS
Capillary: 3.0 kV (ES+)
Cone: 20 V
Extractor: 3 V
Desolvation Temperature: 350 °C
Source Temperature: 150 °C
Desolvation Gas: 800 L/hr
Scan Range: 200 to 650 m/z
Scan Rate: 1000 Da/sec
STRUCTURES
AU
-0.005
0.000
0.005
0.010
0.015
0 1 2 3 4 5 min
UV 254 nm
1
2
3 4 5
Inte
nsity
Inte
nsity
Inte
nsity
Inte
nsity
0 1 2 3 4 5 min
Extracted Ion Chromatogramat m/z 313.1
Extracted Ion Chromatogramat m/z 345.1
Extracted IonChromatogramat m/z 539.2
Extracted IonChromatogramat m/z 245.0
0.0
5.0 x 105
1.0 x 106
0
1 x 106
0
2.0 x 105
4.0 x 105
0
2.0 x 105
4.0 x 105
5.0 x 105
COmPOUNDS
1. 8-Chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]-diazepine
2. Clozapine
3. 11-[2-[(2-Amino-4-chlorophenyl)amino]benzoyl]- 4-methylpiperazine
4. 11,11'-(Piperazine-1,4-diyl)bis(8-chloro-5H-dibenzo[b,e] [1,4]diazepine)
5. 8-Chloro-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.6 90 5 5 Initial5.0 0.6 45 50 5 66.0 0.6 0 95 5 66.1 0.6 90 5 5 68.0 0.6 90 5 5 6
N
NH
N
NH
Cl
8-Chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]-diazepine
N
NH
N
N
Cl
Clozapine
HN
Cl NH2
NON
1-[2-[(2-Amino-4-chlorophenyl)amino]benzoyl]-4-methylpiperazine
N
N
N
NH
Cl
N
HN
Cl
11,11'-(Piperazine-1,4-diyl)bis(8-chloro-5H-dibenzo
[b,e][1,4]diazepine)
HN
NH
O
Cl
8-Chloro-5,10-dihydro-11H-dibenzo[b,e][1,4]
diazepin-11-one
anaL aysis of cLozapine impurities using acQuit y csh coLumns
9
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4136
LC CONDITIONS
Column: ACQUITY CSH Fluoro-Phenyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005351
Mobile Phase A: 0.1% phosphoric acid in water
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 4 μL
Sample Diluent: water
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 240 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.6 98 2 65.00 0.6 2 98 65.50 0.6 2 98 65.51 0.6 98 2 66.00 0.6 98 2 6
STRUCTURES
0.00
0.10
0.20
0.30
0.40
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
1
2
3 4
5
COmPOUNDS
1. p-Hydroxybenzoic acid (10 µg/mL)
2. Sorbic acid (10 µg/mL)
3. Dehydroacetic acid (100 µg/mL)
4. Methyl paraben (25 µg/mL)
5. Saccharin (100 µg/mL)
HO
OH
O
COOH
HO
O
O
SNH
O
OO
p-Hydroxybenzoic acid
Sorbic acid
Methyl paraben
Saccharin
O
O O
ODehydroacetic acid
anaLysis of food additives using acQuit y csh coLumns
10
ACQU
ITY
CSH
WA6
4126
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ C18, 2.1 x 100 mm, 1.7 μm
Part Number: 186005297
Isocratic Mobile Phase: 0.1% phosphoric acid in water
Flow Rate: 0.6 mL/min
Injection Volume: 4 μL
Sample Concentration: 0.02%
Sample Diluent: water
Column Temperature: 50 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 210 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
STRUCTURES
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 min
AU
1
2
COmPOUNDS
1. Formic acid
2. Acetic acid
Formic acid
OH
O
Acetic acid
O
OHH
anaLysis of formic and acetic acids using acQuit y csh coLumns
11
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4138
AU
0.000
0.008
0.016
0.0 1.5 3.0 4.5 min
1
2
3
4
5
6
7
8
COmPOUNDS
1. Mirtazapine Impurity A (0.15%)
2. Mirtazapine Impurity B (0.05%)
3. Mirtazapine Impurity C or F (0.09%)
4. Mirtazapine Impurity D (0.55%)
5. Mirtazapine (98.81%)
6. Unknown impurity (0.08%)
7. Mirtazapine Impurity E (0.13%)
8. Mirtazapine Impurity C or F (0.15%)
LC CONDITIONS
Column: ACQUITY CSH™ C18, 2.1 x 50 mm, 1.7 μm
Part Number: 186005296
Mobile Phase A: 0.1% ammonium hydroxide in water
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 1 μL
Sample Concentration: 1 mg/mL of mirtazapine USP resolution standard
Sample Diluent: 50/50 acetonitrile/water
Column Temperature 45 °C
Weak Needle Wash: 50/50 water/acetonitrile
Strong Needle Wash: 50/50 acetonitrile/water
Detection: UV @ 290 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
STRUCTURES
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.7 95 5 Initial6.5 0.7 22 78 67.5 0.7 22 78 67.6 0.7 95 5 68.0 0.7 95 5 6
NN
N
OH
NN
N
NN
N O
Mirtazapine Impurity A
Mirtazapine Impurity B
NN
NH
Mirtazapine Impurity D Mirtazapine Impurity E
NN
NO
Mirtazapine Impurity C
NN
N
Mirtazapine
NN
N
O
Mirtazapine Impurity F
anaLysis of mirtazapine impurities using acQuit y csh coLumns
12
ACQU
ITY
CSH
WA6
4127
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ Phenyl-Hexyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005406
Mobile Phase A: water
Mobile Phase B: acetonitrile
Mobile Phase C: 2% formic acid in water
Gradient:
Injection Volume: 5 μL
Sample Diluent: water
Column Temperature: 30 °C
Wash Solvent: 50/50 water/acetonitrile
Purge Solvent: 50/50 water/acetonitrile
Detection: UV @ 270 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® H-Class System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.6 70 25 5 Initial5.0 0.6 15 80 5 66.0 0.6 0 95 5 66.1 0.6 70 25 5 610.0 0.6 70 25 5 6
STRUCTURESAU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0 1 2 3 4 5 min
1
2
3
4
5
6
COmPOUNDS
1. Tolmetin (10 µg/mL)
2. Naproxen (10 µg/mL)
3. Fenoprofen (10 µg/mL)
4. Ibuprofen (100 µg/mL)
5. Indomethacin (10 µg/mL)
6. Diclofenac (10 µg/mL)
NO
COOH
Tolmetin
OCOOH
OH
O
H3CO
COOHCOOH
HN
Cl
Cl
N
O Cl
COOHH3CO
Fenoprofen Ibuprofen
Naproxen Diclofenac
Indomethacin
anaLysis of nonsteroidaL anti-infL ammatory drugs (nsaids) using acQuit y csh coLumns
13
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4139
COmPOUNDS
1. Tolmetin (10 µg/mL)
2. Naproxen (10 µg/mL)
3. Fenoprofen (10 µg/mL)
4. Ibuprofen (100 µg/mL)
5. Indomethacin (10 µg/mL)
6. Diclofenac (10 µg/mL)
LC CONDITIONS
Column: ACQUITY CSH™ C18, 2.1 x 150 mm, 1.7 μm
Part Number: 186005298
Mobile Phase: 0.1% phosphoric acid in water
Isocratic Flow Rate: 0.2 mL/min
Injection Volume: 1 to 3 μL
Sample Concentration: 125 µg/mL (250 µg/mL for lactic acid)
(250 µg/mL for lactic acid)
Sample Diluent: water
Column Temperature: 24 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 210 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
Sample Preparation Procedure: Samples were filtered through a 0.45 μm
PTFE syringe filter and injected directly.
STRUCTURES
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
0.08
0.10
-0.02
0.00
0.02
0.04
0.06
0.08
10 2 3 4 5 6 7 8 9 10 min
1
2 3 4
5
6
6
6
3
Standard mix
Lemon-lime sports drink
Vitamin-fortified water (grape flavor)
COmPOUNDS
1. DL-Tartaric acid
2. L-Malic acid
3. Lactic Acid
4. Acetic Acid
5. Succinic Acid
6. Citric Acid
HOOH
O
OH
OH
O
HOOH
O
O
DL-Tartaric acid
OH
HO
OOH
O
L-Malic acid
OH
O
Acetic Acid
Succinic Acid
OHOH
O
Lactic Acid
HO COOH
COOH
COOH
Citric Acid
anaLysis of organic acids using acQuit y csh coLumns
14
ACQU
ITY
CSH
WA6
4128
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ Fluoro-Phenyl, 2.1 x 100 mm, 1.7 μm
Part Number: 186005352
Mobile Phase A: water
Mobile Phase B: methanol
Mobile Phase C: 2% acetic acid in water
Gradient:
Pre-Injector volume: 200 µL
Injection Volume: 5 μL
Sample Diluent: water
Sample Concentration: paroxetine at 0.2 mg/mL; impurities at
0.002 mg/mL each
Column Temperature: 40 °C
Wash Solvent: 50/50 water/acetonitrile
Purge solvent: 50/50 water/acetonitrile
Detection: UV @ 295 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® H-Class System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.6 85 10 5 Initial5.0 0.6 75 20 5 66.0 0.6 75 20 5 66.1 0.6 85 10 5 612.0 0.6 85 10 5 6
STRUCTURES
AU
-0.003
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0 1 2 3 4 5 6 min
1
2
3
4
COmPOUNDS
1. trans-4-phenyl-3-[(3,4-methylenedioxy) phenoxymethyl]-piperidine hydrochloride
2. cis-Paroxetine
3. Paroxetine hydrochloride
4. (3S-trans)-1-Methyl-3-[(1,3-benzodioxol-5- yloxy)methyl]-4-(4-fluorophenyl)piperidine
HN
O
O
O
trans-4-Phenyl-3-[[3,4-(methylenedioxy)phenoxy]methyl]piperidine
HN
O
O
O
F cis-Paroxetine
HN
F
O
O
O
Paroxetine
N
O
O
O
F(3S-trans)-1-Methyl-3-[(1,3-benzodioxol-5-yloxy)
methyl]-4-(4-fluorophenyl)piperidine
anaLysis of paroxetine impurities using acQuit y csh coLumns
ACQU
ITY
CHS
Colu
mns
15
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4129
COmPOUNDS
1. trans-4-phenyl-3-[(3,4-methylenedioxy) phenoxymethyl]-piperidine hydrochloride
2. cis-Paroxetine
3. Paroxetine hydrochloride
4. (3S-trans)-1-Methyl-3-[(1,3-benzodioxol-5- yloxy)methyl]-4-(4-fluorophenyl)piperidine
LC CONDITIONS
Column: ACQUITY CSH™ Fluoro-Phenyl, 2.1 x 100 mm, 1.7 μm
Part Number: 186005352
Mobile Phase A: water
Mobile Phase B: methanol
Mobile Phase C: 2% acetic acid in water
Gradient:
Pre-Injector volume: 200 µL
Injection Volume: 5 μL
Sample Diluent: water
Sample Concentration: 2 µg/mL each
Column Temperature: 40 °C
Wash Solvent: 50/50 water/acetonitrile
Purge Solvent: 50/50 water/acetonitrile
System: ACQUITY UPLC® H-Class System with
SQD STRUCTURES
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.6 85 10 5 Initial5.0 0.6 75 20 5 66.0 0.6 75 20 5 66.1 0.6 85 10 5 612.0 0.6 85 10 5 6
mS CONDITIONS
Capillary: 3.0 kV (ES+)
Cone: 30 V
Extractor: 3 V
Desolvation Temperature: 350 °C
Source Temperature: 150 °C
Desolvation Gas: 800 L/hr
Scan Range: 100 to 600 m/z
Scan Rate: 1000 Da/sec
STRUCTURES
Inte
nsity
0
1x106
2x106
3x106
4x106
5x106
6x106
7x106
8x106
1 2 3 4 5 6 7 8 min 0
1
2
Extracted ion chromatogram at m/z 330.2
COmPOUNDS
1. cis-Paroxetine
2. (+)-trans-Paroxetine
(+)-trans-Paroxetinecis-Paroxetine
HN
O
O
O
F
HN
F
O
O
O
anaLysis of paroxetine isomers using acQuit y csh coLumns
16
ACQU
ITY
CSH
WA6
4130
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Columns: ACQUITY CSH™ C18, 2.1 x 50 mm, 1.7 μm
Part Number: 186005296
ACQUITY CSH Phenyl-Hexyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005406
ACQUITY CSH Fluoro-Phenyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005351
Mobile Phase A: water
Mobile Phase B: acetonitrile
Mobile Phase C: 2% formic acid in water
Gradient:
Injection Volume: 5 μL
Sample Diluent: water
Column Temperature: 30 °C
Wash Solvent: 50/50 water/acetonitrile
Purge Solvent: 50/50 water/acetonitrile
Detection: UV @ 254 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® H-Class System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.6 90 5 5 Initial5.0 0.6 0 95 5 66.0 0.6 0 95 5 66.1 0.6 90 5 5 610.0 0.6 90 5 5 6
STRUCTURES
AU
0.00
0.05
0.10
0.15
AU
0.00
0.05
0.10
0.15
AU
0.00
0.05
0.10
0.15
0 1 2 3 4 5 6 min
18
ACQUITY CSH C18
ACQUITY CSH Phenyl-Hexyl
ACQUITY CSH Fluoro-Phenyl
1
2 3
4
5
6
7 8
9
10
1 2 3
4
5
6
7 8
9
10
1 2
3
4
5
6
7 8
9
10
COmPOUNDS
1. 8-Bromoguanosine (6.25 µg/mL)
2. Acetanilide (3.75 µg/mL)
3. Triamcinolone (6.25 µg/mL)
4. Hydrocortisone (2.5 µg/mL)
5. 2-Amino-7-chloro-5-oxo-5H-[1]benzopyrano[ 2,3-b]pyridine-3-carbonitrile (2.5 µg/mL)
6. 3-Aminofluoranthene (6.25 µg/mL)
7. 6α-Methyl-17α-hydroxyprogesterone (5 µg/mL)
8. 2-Bromofluorene (3 µg/mL)
9. Perylene (2.5 µg/mL)
10. Naphtho[2,3-a]pyrene (2.5 µg/mL)
8-Bromoguanosine
N
HN
N
N
O
H2N
O
HO
OHOH
Br
Acetanilide
NH
O
Triamcinolone
O
O
HOH
HF
OHOH
OH
Hydrocortisone
O
O
HOH
HH
OH
OH
2-Amino-7-chloro-5-oxo-5H-[1]benzopyrano[2,3-b]
pyridine-3-carbonitrile
NO
NO
Cl
NH2
3-Aminofluoranthene
NH2
6α-Methyl-17α-hydroxyprogesterone
OH
H
H
O
OH
2-Bromofluorene
Br
Perylene
anaLysis of pharmaceuticaL test mix on acQuit y csh coLumns
Naphtho[2,3-a]pyrene
ACQU
ITY
CHS
Colu
mns
17
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
ACQU
ITY
CSH
WA6
4137
LC CONDITIONS
Column: ACQUITY CSH™ Fluoro-Phenyl, 2.1 x 50 mm, 1.7 μm
Part Number: 186005351
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 5 μL
Sample Concentration: 10 µg/mL each
Sample Diluent: 80/20 methanol/water
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 248 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
STRUCTURES
0.00
0.04
0.08
0.12
0.16
0 1 2 3 4 5 min
AU
1
2
3
4
COmPOUNDS
1. Pravastatin sodium (Pravachol)
2. Atorvastatin (Lipitor)
3. Lovastatin (Mevacor)
4. Simvastatin (Zocor)
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.6 95 5 Initial5.00 0.6 30 70 65.50 0.6 5 95 65.51 0.6 95 5 66.00 0.6 95 5 6
HO
HO
O
–OOCHO
OH
H
Na+
NCOOH
OH OH
NH
O
F
HO
OH
H
O
OHO
Pravastatin sodium
Atorvastatin
Simvastatin
HO
OH
H
O
OHO
Lovastatin
anaLysis of statin drugs using acQut y csh coLumns
18
ACQU
ITY
CSH
WA6
4131
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
LC CONDITIONS
Column: ACQUITY CSH™ Fluoro-Phenyl, 2.1 x 100 mm, 1.7 μm
Part Number: 186005352
Mobile Phase A: 0.1% acetic acid in water
Mobile Phase B: acetonitrile
Gradient:
Injection Volume: 4 μL
Sample Diluent: water
Sample Concentration: 10 µg/mL each
Column Temperature: 30 °C
Weak Needle Wash: 95/5 water/acetonitrile
Strong Needle Wash: 95/5 acetonitrile/water
Detection: UV @ 254 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® System with
ACQUITY UPLC PDA
Time (min)
Flow(mL/min)
%A %B Curve
Initial 0.5 95 5 Initial5.00 0.5 40 60 65.50 0.5 40 60 65.51 0.5 95 5 66.00 0.5 95 5 6
STRUCTURES
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
AU
1
2
3
4
5
6
7
8
COmPOUNDS
1. Sulfanilamide
2. Sulfathiazole
3. Sulfamerazine
4. Sulfamethazine
5. Sulfamethizole
6. Succinylsulfathiazole
7. Sulfamethoxazole
8. Sulfasoxazole
Sulfanilamide
H2N
SNH
O O
N
N
H2N
SNH
O O
N
N
H2N
SNH
O O
NN
S
NH
SNH
O O
N
S
HOOC
O
H2N
SNH
O O ON
Sulfamerazine Sulfamethazine
Sulfamethizole
Succinylsulfathiazole
Sulfamethoxazole
H2N
SNH2
O O
Sulfathiazole
H2N
SNH
O O
N
S
SNH
NOO O
H2NSulfasoxazole
anaLysis of suLfa drugs using acQuit y csh coLumns
ACQU
ITY
CHS
Colu
mns
19
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
COmPOUNDS
1. Sulfanilamide
2. Sulfathiazole
3. Sulfamerazine
4. Sulfamethazine
5. Sulfamethizole
6. Succinylsulfathiazole
7. Sulfamethoxazole
8. Sulfasoxazole
ACQU
ITY
CSH
WA6
4132
LC CONDITIONS
Column: ACQUITY CSH™ C18, 2.1 x 50 mm, 1.7 μm
Part Number: 186005296
Mobile Phase A: water
Mobile Phase B: acetonitrile
Mobile Phase C: 2% formic acid in water
Gradient:
Injection Volume: 5 μL
Sample Diluent: water
Sample Concentration: 1 µg/mL each
Column Temperature: 40 °C
Wash Solvent: 50/50 water/acetonitrile
Purge Solvent: 50/50 water/acetonitrile
Detection: UV @ 254 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.1 sec
System: ACQUITY UPLC® H-Class System with
ACQUITY UPLC PDA and SQD
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.6 70 25 5 Initial2.0 0.6 60 35 5 63.0 0.6 0 95 5 63.1 0.6 70 25 5 65.0 0.6 70 25 5 6
mS CONDITIONS
Capillary: 3.0 kV (ES+)
Cone: 30 V
Extractor: 3 V
Desolvation Temperature: 350 °C
Source Temperature: 120 °C
Desolvation Gas: 800 L/hr
Scan Range: 100 to 600 m/z
Scan Rate: 5000 Da/sec
STRUCTURES
Inte
nsity
0.5 1.0 1.5 2.0 min 0.0
AU
1.0x107
2.0x107
3.0x107
4.0x107
0.000
0.004
0.008
0.012
UV
MS
1
2
3 4
5 6
0.5 1.0 1.5 2.0 min 0.0
COmPOUNDS
1. Doxepin
2. Desipramine
3. Imipramine
4. Nortriptyline
5. Amitriptyline
6. Trimipramine
O
N
Doxepin
NH
N
N NH
N
N
N
Desipramine
Imipramine
Nortriptyline
Amitriptyline
Trimipramine
anaLysis of tricycLic antidepressants using acQuit y csh coLumns
20
©2010 Waters Corporation. Waters, The Science of What’s Possible, ACQUITY CSH and ACQUITY UPLC are trademarks of Waters Corporation.
anaLysis of veterinary drugs using acQuit y csh coLumns
LC CONDITIONSColumns: ACQUITY CSH™ C18, 2.1 x 100 mm, 1.7 μm Part Number: 186005297Mobile Phase A: waterMobile Phase B: acetonitrile Mobile Phase C: 2.0% formic acid in water (pH ~ 2.7)Gradient:
Injection Volume: 5 μL Sample Diluent: 10/90 methanol/waterColumn Temperature: 30 °CWash: 1% formic acid in waterSystem: ACQUITY UPLC® H-Class with TQD (MassLynx™ v. 4.1)
TQD CONDITIONSCapillary: 3.00 kVExtractor: 3.00 VRF: 0.10 VSource Temperature: 150 °CDesolvation Temperature: 350 °CCone Gas Flow: 30 L/HrDesolvation Gas Flow: 800 L/Hr
Collision Gas Flow: 0.10 mL/min
1
7
8
3
6
4
5 9
2
100
%
00 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min
COmPOUNDS
1. Ciprofloxacin (4 µg/mL)
2. Enrofloxacin (2 µg/mL)
3. Erythromycin (2 µg/mL)
4. Oxacillin (4 µg/mL)
5. Oxytetracycline (4 µg/mL)
6. Penicillin G (2 µg/mL)
7. Sulfamerazine (2 µg/mL)
8. Sulfamethazine (0.5 µg/mL)
9. Tetracycline (4 µg/mL)
Time (min)
Flow(mL/min)
%A %B %C Curve
Initial 0.3 80 15 5 Initial2.5 0.3 55 40 5 63.9 0.3 0 95 5 64.9 0.3 0 95 5 65.0 0.4 80 15 5 66.0 0.4 80 15 5 66.1 0.3 80 15 5 67.0 0.3 80 15 5 6
Compound MRM transition (m/z) Cone (V) CID (eV)
Ciprofloxacin332.00>288.21332.00>314.32
20 20
2525
Enrofloxacin360.10>316.38360.10>342.39
15 15
2525
Erythromycin734.68>158.18734.68>576.49
30 30
20 15
Oxacillin402.40>160.25402.40>243.24
25 25
15 15
Oxytetracycline461.10>426.43461.10>443.67
25 25
35 30
Penicillin-G335.17>160.16335.17>176.03
30 30
30 25
Sulfamerazine265.18>92.10
265.18>156.2225 25
20 30
Sulfamethazine279.00>92.21
279.00>124.3130 30
30 25
Tetracycline445.20>410.26445.20>427.80
30 30
35 30
N
F
HNN
O
OH
O
Ciprofloxacin
N
F
NN
O
OH
O
Enrofloxacin
HH
OH O OH O
NH2
OOH
NHOOH
OH
Oxytetracycline
NH N
S
COOH
HH
O
O
Penicillin G
NO
N
SHN
O
OCOOH
HH
Oxacillin
H2N
SNH
O O
N
N
Sulfamerazine
H2N
SNH
O O
N
N
Sulfamethazine
HH
OH O OH O
NH2
OOH
NHOOH
Tetracycline
O
O
OHHO OH
O
O
O
O
NHO
OCH3
OH
Erythromycin
ACQU
ITY
CSH
WA6
4140
21
ACQU
ITY
CSH
NOTE
S
22
ACQU
ITY
CSH
NOTE
S
23
ACQU
ITY
CSH
NOTE
S
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June 2010 720003537EN KK-PDF