ACQUITY UPLC BEH AMIDE COLUMNS Application Notebook
ACQUITY UPLC BEH AMIDE COLUMNS A p p l i c a t i o n N o t e b o o k
5-Fluorouracil........................................................................................................................................................................................ 2
Acrylamide, Methacrylic Acid and Methacrylamide............................................................................................................................. 3
Allantoin ............................................................................................................................................................................................... 4
Cellulosic Hydrolysates ......................................................................................................................................................................... 5
Chemical Stability Study of ACQUITY UPLC BEH Amide Columns ...................................................................................................... 6
Food Sugars in Bran with Raisins Cereal .............................................................................................................................................. 7
Food Sugars in Ketchup ......................................................................................................................................................................... 8
Food Sugars in Milk .............................................................................................................................................................................. 9
Food Sugars in Molasses .....................................................................................................................................................................10
Food Sugars In Prepared Foods ...........................................................................................................................................................11
Food Sugars in Sports Drink ................................................................................................................................................................12
Food Sugars in Wine ...........................................................................................................................................................................13
Food Sugars .........................................................................................................................................................................................14
UPLC/MS Analysis of Food Sugars with Acetone as Organic Modifier ............................................................................................15
Food Sugars/Saccharides in Beer .......................................................................................................................................................16
UPLC/MS Analysis of Food Sugars/Saccharides in Beer ....................................................................................................................17
Food Sugars/Saccharides in Cough Syrup ..........................................................................................................................................18
Food Sugars/Saccharides in Honey ....................................................................................................................................................19
Food Sugars/Saccharides in Maple Syrup ..........................................................................................................................................20
Food Sugars/Saccharides in Potato Chips ...........................................................................................................................................21
HILIC Gradient Separation of Ascorbic Acid and Isoascorbic Acids ...................................................................................................22
HILIC Gradient Separation of Organophosphonic Acids .....................................................................................................................23
HILIC Isocratic Separation of Isoascorbic Acid and Ascorbic Acid .....................................................................................................24
HILIC Isocratic Separation of Organophosphonic Acids ......................................................................................................................25
Histidine Dipeptides ..........................................................................................................................................................................26
Mono-, Di- and Oligosaccharides ........................................................................................................................................................27
UPLC/MS Analysis of Mono-, Di- and Oligosaccharides ....................................................................................................................28
Mono-, Di- and Oligosaccharides with Acetone as Organic Modifiers .............................................................................................29
Morphine .............................................................................................................................................................................................30
Nucleobases Using 30 mm ACQUITY UPLC BEH Amide Columns ....................................................................................................31
Nucleobases ........................................................................................................................................................................................32
Nucleotide Phosphates ........................................................................................................................................................................33
Organic Acids ......................................................................................................................................................................................34
Stevia Related Compounds .................................................................................................................................................................35
UPLC/MS Analysis of Stevia Related Compounds ..............................................................................................................................36
Thiourea ..............................................................................................................................................................................................37
Uric Acids ............................................................................................................................................................................................38
Water Soluble Vitamins .......................................................................................................................................................................39
List of Compounds Analyzed Using ACQUITY UPLC BEH Amide Columns
1
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Analysis of 5-Fluorouracil using ACQUITY UPLC BEH Amide Columns
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
AU
0
0.10
0.20
0.30
0.40
t0= 1.3 min
k prime = 2.2
1.0 2.0 0 3.0 4.0 5.0 6.0 7.0 min
t eSt COndit iOnS
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 μm
Part Number: 186004800
Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/ H2O with
5 mM CH3COONH4 and 0.02% NH4OH,
pH 9.0
Flow Rate: 0.2 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 50 μg/mL
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 265 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
St ruCture
5 - f l uo ro u ra c i l
F
NH
NH
O
O
Wa60121
2
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
O
H2C
CH3
NH2
O
H2C
CH3
OH
O
H2CNH2
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Acrylamide, Methacrylic Acid and Methacrylamide
using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 150 mm, 1.7 µm
Part Number: 186004802
Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/H2O with 5 mM
CH3COONH4 and 0.02% NH4OH,
pH 9.0
Flow Rate: 0.5 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 30 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 210 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
St ruCtureS
M et ha c ryl amid e a c ryl amid e
M et h a c r yl i c a c id
AU
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 1 2 3 4 5 6 min
1
2
3t0=0.9 min
COMpOundS
1. Methacrylamide
2. Acrylamide
3. Methacrylic acid
O
H2C
CH3
NH2
O
H2C
CH3
OH
O
H2CNH2
Wa60108
3
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Allantoin using ACQUITY UPLC BEH Amide Columns
k prime = 3.2
t0 = 1.9 min
AU
0
0.1
0.2
0.3
0.4
0.5
0.6
0 2.0 4.0 6.0 8.0 10 12 14 16 min
t eSt COndit iOnS
Column: ACQUITY UPLC® BEH Amide,
2.1 x 150 mm, 1.7 µm
Part Number: 186004802
Isocratic Mobile Phase B: 90/10 MeCN/H2O
Flow Rate: 0.2 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 100 μg/mL
Sample Diluent: 90/10 MeCN/H2O
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 210 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
St ruCture
all anto in
NH
NHHN
O
O
O
H2N
Wa60107
4
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Cellulosic Hydrolysates Using ACQUITY UPLC BEH Amide Columns
Wa60127
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 min
1
2
2
4
5
6
7
8
3
9 10 11 1213
13
4
56
7 8,9
10 11 12 13
COMpOundS
1. Xylose
2. Fructose
3. Mannose
4. Glucose
5. Sucrose
6. Cellobiose
7. Melezitose
8. Raffinose
9. Maltotriose
10. Maltotetraose
11. Maltopentaose
12. Maltohexaose
13. Maltoheptaose
St ruCtureS
Xylose
O OH
OHHO
HO
O
OHHO
HO OH
OH
Fructose
O OH
OH
OH
HO
HO
Glucose
O OH
OH
OH
HO
HO
Mannose
O
OHOH
HO
HO
O OH
OH
OHO
HO
Cellobiose
O
HO OH
OHHO
O O
OH
OH
HO
HO
Sucrose
OOH
OHHO
OO
OHHO
HO
HO
O
OHHO
HO
OOH
Raffinose
OOH
OHHO
HOO
O
OHHO
HOO
O
OHHO
HO
HO
n
n = 1 to 5
Maltooligosaccharides
OHO
HO
OH
OH
O
HOOH
O OH
O
O
HOHO
HO
HO
Melezitose
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.12 mL/min
Gradient: 10 minute gradient, 80%-50%
MeCN (w/0.2% TEA) with 30 minute
re-equilibration
Time Profile
(min) %A %B
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
0.00
10.00
10.01
40.00
100.00
60.00
100.00
100.00
0.00
40.00
0.00
0.00
5
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
AU
0
0.2
0.4
0.61
23
Injection 1
AU
0
0.2
0.4
0.6Injection 1000
AU
0
0.2
0.4
0.6
0 1 2 min
Injection 2000
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Chemical Stability STUDY OF ACQUITY UPLC BEH AMIDE COLUMNS
t eSt COndit iOnS
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
CH3COONH4, pH 5.5
Mobile Phase B: 95/5 MeCN/H2O with 10 mM
CH3COONH4, pH 5.5
Flow Rate: 0.5 mL/min
Gradient: Time Profile
(min) %A %B
Initial 1 99
2.00 99 1
2.10 1 99
2.50 1 99
COMpOundS
1. Uracil
2. 5-fluorocytosine
3. Cytosine
N
NH
O
NH2
F
NH
NH
O
O
N
NH
O
NH2
5 - f l uo ro c yto s ineu ra c i l
St ruCtureS
Injection Volume: 2.0 µL (full loop injection mode)
Sample Concentration: 25 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 30 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 254 nm
Sampling Rate: 40 pts/sec
Filter Time Constant: 0.1
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
C yto s ine
N
NH
O
NH2
F
NH
NH
O
O
N
NH
O
NH2
Wa60106
6
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars in Bran with Raisins Cereal
Using ACQUITY UPLC BEH Amide Columns
3 6
1
2 4
5Food Sugar
Standard
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 min
4
Raisin Bran Brand Cereal8mg/mL2 3
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
St ruCtureS
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 150 mm, 1.7 µm
Part Number: 186004802
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.29 mL/min
Flow Profile: 90% A/10% B (75% MeCN with
0.2% TEA)
Injection Volume: 2.0 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each, cereal
extracted at 8 mg/mL
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Wa60120
7
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Food Sugars
1
2 4
5 6
0.0 1.0 2.0 3.0 4.0 min
3
3
2 5
Tomato Ketchup
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars in Ketchup Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 acetone/H2O with 0.05%
triethylamine [TEA]
Mobile Phase B: 30/70 acetone/H2O with 0.05%
triethylamine [TEA]
Flow Rate: 0.15 mL/min
Flow Profile: 95% A/5% B (77.5% acetone with
0.05% TEA)
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 85 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
St ruCtureS
Wa60117
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
8
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 min
Milk – 1% Fat5% in 50% ACN
Food SugarStandard
1
2
3
4
5
6
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars in MILK Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.13 mL/min
Flow Profile: 90% A/10% B (75% MeCN with
0.2% TEA)
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35°C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Wa60118
St ruCtureS
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
9
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Molasses5 mg/mL
2
3
4
3
6
1
2 4
5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 min
Food SugarStandard
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars in Molasses Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 150 mm, 1.7 µm
Part Number: 186004802
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.29 mL/min
Flow Profile: 90% A/10% B (75% MeCN with
0.2% TEA)
Injection Volume: 2.0 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each, molasses
at 5 mg/mL
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Wa60119
St ruCtureS
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
10
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Food Sugars
1 2 4
5 6
White Bread
Lamb Curry Meal
StrawberrySmoothie
Hot Cross Buns
0.0 1.0 2.0 3.0 4.0 min
3
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars In Prepared FOods
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.15 mL/min
Flow Profile: 95% A/5% B (77.5% acetone
with 0.05% TEA)
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 85 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Wa60115
St ruCtureS
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
11
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Food Sugars
1
2 4
5 6
Gatorade BrandSports Drink
0.0 1.0 2.0 3.0 4.0 min
3
3
2
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars in Sports Drink
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 acetone/H2O with 0.05%
triethylamine [TEA]
Mobile Phase B: 30/70 acetone/H2O with 0.05%
triethylamine [TEA]
Flow Rate: 0.15 mL/min
Flow Profile: 95% A/5% B (77.5% acetone with
0.05% TEA)
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 85 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Wa60116
Gatorade is a registered trademark of Stokely-Van Camp, Inc.
St ruCtureS
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
12
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
0 1 2 3 4 5 6 7 8 9 10 min
CabernetSauvignon
Chardonnay
1 2
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars in Wine Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 150 mm, 1.7 µm
Part Number: 186004802
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.15 mL/min
Flow Profile: 90% A/10% B (75% MeCN with
0.2% TEA)
Injection Volume: 2.0 µL (PLNO)
Sample Concentration: 50% wine in diluent
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35°C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
COMpOundS
1. Fructose
2. Glucose
Glucose
GlucoseFructose
Fructose
St ruCtureS
elSd ConditionsGain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
Wa60114
13
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
1
2
3 4
56
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars Using ACQUITY UPLC BEH Amide Columns
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.13 mL/min
Flow Profile: 90% A/10% B (75% MeCN with
0.2 % TEA)
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Wa60109
St ruCtureS
Glucose
Glucose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Maltose
Maltose
Sucrose
Sucrose
Lactose
14
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
SIR of 2 Channels ES- TIC2.77e5
0
%
100
0.30.20.10.0 5 min.35.25.15.0
1
2
3
4
5
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
UPLC/MS Analysis of Food Sugars Using ACQUITY UPLC
BEH Amide Columns with Acetone as Organic Modifier
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 acetone/H2O with 0.05%
ammonium hydroxide [NH4OH]
Mobile Phase B: 30/70 acetone/H2O with 0.05%
ammonium hydroxide [NH4OH]
Flow Rate: 0.13 mL/min
Flow Profile: 94% A/6% B (77% acetone with
0.05% NH4OH)
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: 10 µg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 85 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with
ACQUITY TQD
Mass Spectrometer ConditionsIonization Mode: ES-
Capillary: 2.8 kV
Cone Voltage: 25 V
Source Temperature: 120 °C
Desolvation Temperature: 350 °C
Desolvation Gas Flow: 500 L/Hr
Cone: 50 L/Hr
SIR (m/z): 179.2 (Fructose, Glucose);
341.3 (Sucrose, Maltose,
Lactose)
Dwell Time: 0.08 s
St ruCtureSFructose
Fructose
Maltose
Maltose
Lactose
Lactose
Glucose
Glucose
Sucrose
Sucrose
COMpOundS
1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Lactose
Wa60113
15
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Analysis of Food Sugars/Saccharides inBeer Using ACQUITY UPLC BEH AmideColumns
Chocolate Stout
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min
Belgian Beer
Wheat Beer
Dark Ale
Dutch Beer
Standards
1 2 3 4 5 6 7 8 9 10 11
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars/Saccharides in Beer Using
ACQUITY UPLC BEH Amide Columns
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
7. Maltotriose
8. Maltotetraose
9. Maltopentaose
10. Maltohexahose
11. Maltoheptaose
Wa60125
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.13 mL/min
Gradient: 10 minute gradient, 75%-45%
MeCN (w/0.2% TEA) with 25 minute
re-equilibration
Time Profile
(min) %A %B
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL, beer at 100%
(No dilution)
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
0.00
10.00
10.01
35.00
90.00
30.00
90.00
90.00
10.00
70.00
10.00
10.00
Lactose
Sucrose
Sucrose
Maltooligosaccharides
nn= 1 to 5
Maltooligosaccharides
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Glucose
Glucose
St ruCtureS
Maltose
Maltose
16
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Sucrose
Food SugarStandard
SIR of 3 Channels ES- TIC5.18e5
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 min
%
0
21
3
4 5
Dutch Beer
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 min
%
0
SIR of 3 Channels ES- TIC4.91e6
1
Disaccharides(m/z = 341.1)
Trisaccharides(m/z = 503.2)
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
UPLC/MS Analysis of Food Sugars/Saccharides in Beer
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.1%
ammonium hydroxide [NH4OH]
Mobile Phase B: 30/70 MeCN/H2O with 0.1%
ammonium hydroxide [NH4OH]
Flow Rate: 0.13 mL/min
Gradient: 10 minute gradient, 75%-45% MeCN
(w/0.1% NH4OH) with 25 minute
re-equilibration
Time Profile
(min) %A %B
0.00 90.00 10.00
10.00 30.00 70.00
10.01 90.00 10.00
35.00 90.00 10.00
Injection Volume: 2.0 µL (PLNO)
Sample Concentration: Standards at 10 µg/mL, Beer at
50% dilution
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/2MeCN/H2O 5 (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with SQ
COMpOundS
1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Maltotriose
Maltooligosaccharides
nn= 1 to 5
Maltooligosaccharides
Glucose
GlucoseLactose
St ruCtureS
Fructose
Fructose
Sucrose
Sucrose
Maltose
Maltose
Mass Spectrometer Conditions
Ionization Mode: ES-
Capillary: 2.8 kV
Cone Voltage: 25 V (fructose, glucose, maltotriose);
40V (sucrose and maltose)
Source Temp: 120 °C
Desolvation Temp: 350 °C
Desolvation Gas Flow: 500 L/Hr
Cone: 50 L/Hr
SIR (m/z): 179.0 (fructose, glucose);
341.1 (sucrose, maltose);
503.2 (maltotriose)
Dwell Time: 0.04 s
Wa60126
17
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Robitussin BrandCough Syrup
3 6
1
24
5Saccharide
Standard
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
7 8 9 10
Generic TussinCough Syrup
3
2
© 2009 Waters Corporation. Waters, ACQUITY UPLC, and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars/Saccharides in Cough Syrup
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.26 mL/min
Gradient: 6 minute gradient, 80%-50% MeCN
(w/ 0.2% TEA) with 12 minute
re-equilibration
Time Profile
(min) %A %B
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each, cough
syrups at 1% (v/v)
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: MeCN/H2O 20/80 (800 µL)
Weak Needle Wash: MeCN/H2O 75/25 (500 µL)
Seal Wash: MeCN/H2O 50/50
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Maltotriose
7. Maltotetraose
8. Maltopentaose
9. Maltohexaose
10. Maltoheptaose
Wa60121
0.00
6.00
6.01
18.00
100.00
40.00
100.00
100.00
0.00
60.00
0.00
0.00
Robitussin is a registered trademark of Wyeth Corporation.
Lactose
Sucrose
Sucrose
Maltooligosaccharides
nn= 1 to 5
Maltooligosaccharides
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Fructose
Fructose
Glucose
Glucose
St ruCtureS
Maltose
Maltose
18
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Sucrose
Lactose
Sucrose
Sucrose
Glucose
Glucose
1
2
3 4 5 6 7
Fake Honey(8 mg/ml)
Corn Syrup(10 mg/mL)
Pure Honey(5 mg/mL)
0.0 1.0 2.0 3.0 4.0 5. 0 6.0 7.0 min
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars/Saccharides in Honey
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.15 mL/min
Gradient: 5 minute gradient, 80%-50%
MeCN (w/0.2% TEA) with 10 minute
re-equilibration
Time Profile
(min) %A %B
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: Honey and corn syrup at 5-10 mg/mL
each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 45 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
St ruCtureS
COMpOundS
1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Maltotriose
6. Maltotetraose
7. Maltopentaose
Maltose
Maltose
Maltooligosaccharides
nn= 1 to 5
Maltooligosaccharides
0.00
5.00
5.01
15.00
100.00
40.00
100.00
100.00
0.00
60.00
0.00
0.00
Wa60124
Fructose
Fructose
19
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Lactose
Sucrose
Sucrose
Glucose
Glucose
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min
VermontMaple Syrup
“Light”Pancake Syrup
“Pure”Maple Syrup
Log Cabin BrandMaple Syrup
1
2
3
4 5 6 7
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars/Saccharides in Maple Syrup
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.13 mL/min
Gradient: 10 minute gradient, 80%-50%
MeCN (w/0.2% TEA) with 25 minute
re-equilibration
Time Profile
(min) %A %B
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: Maple syrups at 5-10 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
St ruCtureS
COMpOundS
1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Maltotriose
6. Maltotetraose
7. Maltopentaose
Maltose
Maltose
Maltooligosaccharides
nn= 1 to 5
Maltooligosaccharides
Wa60123
0.00
10.00
10.01
35.00
100.00
40.00
100.00
100.00
0.00
60.00
0.00
0.00
Fructose
Fructose
20
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
1
2
3
4
5
6
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min
Food SugarStandard
BBQ Flavored Potato Chips
(extracted in 50:50 MeCN/H2O)2 3
4
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Food Sugars/Saccharides in Potato Chips
Using ACQUITY UPLC BEH Amide Columns
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Lactose
Lactose
CH3
NH2
O
p-Toluamide(unretained compound)
p-Toluamide(unretained compound)
Sucrose
Sucrose
Fructose
Fructose
Glucose
Glucose
St ruCtureS
Maltose
Maltose
Wa60122
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.13 mL/min
Gradient: 10 minute gradient, 80%-50%
MeCN (w/0.2% TEA) with 25 minute
re-equilibration
Time Profile
(min) %A %B
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: Standards at 1 mg/mL each, potato
chips extracted at 120 mg/mL
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
0.00
10.00
10.01
35.00
100.00
40.00
100.00
100.00
0.00
60.00
0.00
0.00
21
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
AU
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 2 4 6 8 10 min
1
2
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
HILIC Gradient Separation of Ascorbic Acid And Isoascorbic
Acids using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 100 mm, 1.7 μm
Part Number: 186004801
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH,
pH 5.0
Mobile Phase B: 90/10 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH,
pH 5.0
Flow Rate: 0.2 mL/min
Gradient: Time Profile
(min) %A %B
Initial 0.1 99.9
10.00 99.9 0.1
10.01 0.1 99.9
15.00 0.1 99.9
Injection Volume: 5.0 μL (PLNO)
Sample Concentration: 30 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 260nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
COMpOundS
1. Isoascorbic acid
2. Ascorbic acid
St ruCtureS
a s c or b i c a c id
i so a s c o r b i c ac id
O
HO OH
O
HO
HO
O
HO OH
O
HO
HO
O
HO OH
O
HO
HO
O
HO OH
O
HO
HO
Wa60105
22
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
HILIC Gradient Separation of Organophosphonic Acids
using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
CH3COONH4and 0.04% NH4OH,
pH 9.0
Mobile phase B: 95/5 MeCN/H2O with 10 mM
CH3COONH4 and 0.04% NH4OH,
pH 9.0
Flow Rate: 0.5 mL/min
Gradient: Time Profile
(min) %A %B
Initial 0.1 99.9
10.00 99.9 90.0
10.01 0.1 99.9
15.00 0.1 99.9
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 2 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 65 °C
Weak Needle Wash: 95/5 MeCN/H2O
Instrument: Waters ACQUITY UPLC with
ACQUITY SQD
0
2
1
3
4
5
0
100
2 4 6 8 10 12 14 min
4.63e6
Inte
nsity
% COMpOundS
1. Pinacolyl methylphosphonic acid (PMPA)
2. 2-(methyl)propyl methylphosphonic acid (MMPA)
3. Cyclohexyl methylphosphonic acid (CMPA)
4. Isopropyl methylphosphonic acid (IMPA)
5. Ethyl methylphosphonic acid (EMPA)
Mass Spectrometer Conditions
Ionization Mode: ES-
Capillary: 2.5 KV
Cone: 30 V (EMPA, IMPA, PMPA);
40 V (CMPA); 35 V (MMPA)
Source Temperature: 120 °C
Desolvation Temperature: 400 °C
Desolvation Gas Flow: 800 L/Hr
Cone: 5 L/Hr
SIR m/z: 122.9 (EMPA); 136.95 (IMPA);
179.0 (PMPA); 177.0 (CMPA);
150.95 (MMPA)
Dwell Time: 0.1 s
OH
O
PCH3
OO
O
PHO
H3CH3C
H3C
CH3
CH3
O
O
PH3C
OH H3C
CH3
O
O
PH3C
OHCH3
O
O
PH3C
OH
H3C
CH3
St ruCtureS
p ina c olyl m et hyl p ho s p honic
a c id ( p M pa)
2- (m et hyl) p ro pyl m et hyl p ho s p honic
a c id (M M pa)C y cl o hexyl
met hyl p hos p honic a c id (C M pa)
i so p ro pyl m et hyl p ho s p honic
a c id ( iM pa)
et hyl m et hyl p ho s p honic
a c id ( eM pa)
OH
O
PCH3
OO
O
PHO
H3CH3C
H3C
CH3
CH3
O
O
PH3C
OH H3C
CH3
O
O
PH3C
OHCH3
O
O
PH3C
OH
H3C
CH3
Wa60104
23
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
AU
0
0.1
0.2
0.3
0.4
0.5
AU
0
0.1
0.2
0.3
0.4
0.5
0 1 2 3 4 5 6 7 8 min
ACQUITY UPLC BEH Amide2.1 x 50 mm, 1.7 µm
Part Number: 186004800
1 2
1
2
ACQUITY UPLC BEH Amide2.1 x 100 mm, 1.7 µm
Part Number: 186004801
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
HILIC Isocratic Separation of Isoascorbic acid and ascorbic
acid using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide,
Isocratic Mobile Phase: 80/20 MeCN/H2O with 10 mM
KH2PO4, pH 4.6
Flow Rate: 0.2 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 30 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 260nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
COMpOundS
1. Isoascorbic acid
2. Ascorbic acid
St ruCtureS
O
OH O H
O
OH
OH
a s c or b i c a c id
O
OH O H
O
OH
OH
i so a s c o r b i c a c id
Wa60102
24
CliCk on the underlined blue text for details on the produCts used in this appliCation
2
1
3
4
5
Inte
nsity
%
0
100
0 1 2 3 4 5 6 7 8 9 min
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
t est Condit ions
C hromatographic Conditions
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Isocratic Mobile Phase: 90/10 MeCN/H2O with 10 mM
CH3COONH4 and 0.04% NH4OH,
pH 9.0
Flow Rate: 0.5 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 2 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 65 °C
Weak Needle Wash: 95/5 MeCN/H2O
Instrument: Waters ACQUITY UPLC with
ACQUITY SQD
Compounds
1. Pinacolyl methylphosphonic acid (PMPA)
2. 2-(methyl)propyl methylphosphonic acid (MMPA)
3. Cyclohexyl methylphosphonic acid (CMPA)
4. Isopropyl methylphosphonic acid (IMPA)
5. Ethyl methylphosphonic acid (EMPA)
mass spectrometer settingsIonization Mode: ES-
Capillary: 2.5 KV
Cone: 30 V (EMPA, IMPA, PMPA);
40 V (CMPA); 35 V (MMPA)
Source Temperature: 120 °C
Desolvation Temperature: 400 °C
Desolvation Gas Flow: 800 L/Hr
Cone: 5 L/Hr
SIR m/z: 122.9 (EMPA); 136.95 (IMPA);
179.0 (PMPA); 177.0 (CMPA);
150.95 (MMPA)
Dwell Time: 0.1 s
HILIC Isocratic Separation of Organophosphonic Acids
using ACQUITY UPLC BEH Amide Columns
Wa60100
O
O
P
OH
H3C
CH3
H3C
CH3
H3C
OP
O
OH
H3C
CH3
H3C
OP
O
OH
HO OP
O
CH3CH3
H3C H3C
H3C
H3C OHP
O
O
O
O
P
OH
H3C
CH3
H3C
CH3
H3C
OP
O
OH
H3C
CH3
H3C
OP
O
OH
HO OP
O
CH3CH3
H3C H3C
H3C
H3C OHP
O
O
st ruCtures
p ina c olyl m et hyl p ho s p honic
a c id ( p m pa)
2- (m et hyl) p ro pyl m et hyl p ho s p honic
a c id (m m pa)
C y cl o hexyl met hylp hosp honic
a c id (C m pa)
i so p ro pyl m et hyl p ho s p honic
a c id ( im pa)
et hyl m et hyl p ho s p honic
a c id ( em pa)
25
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
Inte
nsity
%
0
1
2
3
4
100
10 2 3 4 5 min
3.87e7
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Histidine dipeptides using ACQUITY UPLC BEH Amide Columns
COMpOundS
1. Creatinine (1 µg/mL)
2. Creatine (5 µg/mL)
3. Anserine (5 µg/mL)
4. Canosine (5 µg/mL)
NNH
NH
O
O
HO
NH2
N
N
NH
OO
HO
NH2
H3C
N NHO
NH2
OH CH3
NH
N
NHO
CH3
St ruCtureS
C a r no s ine C re at ine
C re at inine an s er ine
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 μm
Part Number: 186004800
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
CH3COONHz and 0.04 % NH4OH,
pH 9.0
Mobile Phase B: 95/5 MeCN/H2O with 10 mM
CH3COONH4 and 0.04 % NH4OH,
pH 9.0
Flow Rate: 0.5 mL/min
Gradient: Time Profile
(min) %A %B
Initial 0.1 99.9
5.00 65.0 35.0
5.01 0.1 99.9
6.00 0.1 99.9
Injection Volume: 5 µL
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 65 °C
Weak Needle Wash: 95/5 MeCN/H2O
Instrument: Waters ACQUITY UPLC
with ACQUITY SQD
Mass Spectrometer Settings
Ionization Mode: ES+
Capillary: 2.5 KV
Cone: 20 V (Carnosine; Creatinine,
Anserine); 25 V (Creatine)
Source Temperature: 120 °C
Desolvation Temperature: 400 °C
Desolvation Gas Flow: 800 L/Hr
Cone gas Flow: 5 L/Hr
SIR m/z: 227.1 (Carnosine); 132.1 (Creatine);
114.05 (Creatinine); 241.1(Anserine)
Dwell Time: 0.1 s
Wa60103
26
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min
1
23
4
5
67
98
10
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Mono-, Di- and Oligosaccharides
Using ACQUITY UPLC BEH Amide Columns
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
Wa60110
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.2%
triethylamine [TEA]
Flow Rate: 0.17 mL/min
Gradient: 5 minute gradient, 80%-50% MeCN
with 10 minute re-equilibration
Time Profile
(min) %A %B
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
0.00
5.00
5.01
15.00
100.00
40.00
100.00
100.00
0.00
60.00
0.00
0.00
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose
5. Maltose
6. Maltotriose
7. Maltotetraose
8. Maltopentaose
9. Maltohexahose
10. Maltoheptaose
St ruCtureS
Maltose
Maltose
Glucose
Glucose
Sucrose
Sucrose
CH3
NH2
O
p-Toluamide(unretained compound)
Maltooligosaccharidesp-Toluamide
(unretained compound)
nn= 1 to 5
Fructose
Fructose
27
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
9
%
0
100
12
3
4 5
6
7
8
SIR of 7 Channels ES-
TIC5.68e6
0.50 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
UPLC/MS Analysis of Mono-, Di- and Oligosaccharides
Using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 MeCN/H2O with 0.10%
ammonium hydroxide [NH4OH]
Mobile Phase B: 30/70 MeCN/H2O with 0.10%
ammonium hydroxide [NH4OH]
Flow Rate: 0.17 mL/min
Gradient: 5 minute gradient, 80%-50% MeCN
with 10 minute re-equilibration
Time Profile
(min) %A %B
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: 10 µg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with
ACQUITY TQD
Mass Spectrometer Conditions
Ionization Mode: ESCapillary: 2.8 kV
Cone Voltage: 25 V
Source Temperature: 120 °C
Desolvation Temperature: 350 °C
Desolvation Gas Flow: 500 L/Hr
Cone: 50 L/Hr
SIR (m/z): 179.2 (fructose, glucose);
341.3 (sucrose, maltose);
503.4, 665.5, 827.6,
989.7, 1151.8
(maltooligosaccharides
[n=1 to 5])
Dwell Time: 0.08 s
COMpOundS
1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Maltotriose
6. Maltotetraose
7. Maltopentaose
8. Maltohexahose
9. Maltoheptaose
Wa60112
0.00
5.00
5.01
15.00
100.00
40.00
100.00
100.00
0.00
60.00
0.00
0.00
nn= 1 to 5
Maltooligosaccharides
Sucrose
Sucrose
St ruCtureS
Glucose
Glucose
Fructose
Fructose
Maltose
Maltose
28
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
1
2
3
4
5 6 7 9810
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Mono-, Di- and Oligosaccharides Using ACQUITY UPLC
BEH Amide Columns with Acetone as Organic Modifiers
St ruCtureS
CH3
NH2
O
p-Toluamide(unretained compound)
Fructose
Fructose
Maltooligosaccharides
Maltose
Maltose
Glucose
Glucose
Sucrose
Sucrose
p-Toluamide(unretained compound)
nn= 1 to 5
elSd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
Wa60111
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A: 80/20 acetone/H2O with 0.05%
triethylamine [TEA]
Mobile Phase B: 30/70 acetone/H2O with 0.05%
triethylamine [TEA]
Flow Rate: 0.17 mL/min
Gradient: 5 minute gradient, 80%-60% MeCN
with 10 minute re-equilibration
Time Profile
(min) %A %B
Injection Volume: 0.7 µL (PLNO)
Sample Concentration: 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 85 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
0.00
5.00
5.01
15.00
100.00
60.00
100.00
100.00
0.00
40.00
0.00
0.00
COMpOundS
1. p-Toluamide
2. Fructose
3. Glucose
4. Sucrose 5. Maltose
6. Maltotriose
7. Maltotetraose
8. Maltopentaose
9. Maltohexahose
10. Maltoheptaose
29
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
1
2
3
Inte
nsity
%
0
100
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min0
1.36e7
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Morphine using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 μm
Part Number: 186004800
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
NH4COOH and 0.125% HCOOH, pH 3
Mobile Phase B: 90/10 MeCN/H2O with 10 mM
NH4COOH and 0.125% HCOOH, pH 3
Flow Rate: 0.6 mL/min
Gradient: Time Profile
(min) %A %B
Initial 0.1 99.9
1.05 0.1 99.9
4.35 99.9 0.1
4.50 0.1 99.9
6.00 0.1 99.9
Injection Volume: 5 µL (PLNO)
Sample Diluent: 75/25 MeCN/MeOH with
0.2% HCOOH
Column Temperature: 30 °C
Weak Needle Wash: 95/5 MeCN/H2O
Instrument: Waters ACQUITY UPLC with
ACQUITY SQD
Mass Spectrometer Conditions
Ionization Mode: ES+
Capillary: 3.0 KV
Cone: 30 V (6-Acetyl morphine and Morphine),
40 V (Morphine-3β-D-glucuronide)
Source Temperature: 120 °C
Desolvation Temperature: 350 °C
Desolvation Gas Flow (L/Hr): 800
SIR m/z: 329.5 (6-Acetyl morphine);
287.5 (Morphine)
463.6 (Morphine-3β-D-glucuronide)
Dwell Time: 0.1 s
COMpOundS
1. 6-acetylmorphine (100 ng/mL)
2. Morphine (100 ng/mL)
3. Morphine-3 ß-D-glucuronide (5 g/mL)
HO
O
O
NCH3
H
O
HO
O
HO
HNCH3
O
O
HO
H
O
HO HO
OHOHO
NCH3
6 - a c etylmor p hine
M or p hine M or p hine -3ß - d - gl u c u ronid e
St ruCtureS
Wa60101
30
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
AU
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.1 0.2 0.3 0.4 0.5 min
1
2
3
4
5
Analysis of Nucleobases Using 30 MM ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
Column: ACQUITY UPLC® BEH Amide,
2.1 x 30 mm, 1.7 µm
Part Number: 186004839
Mobile Phase A : 50/50 MeCN/H2O with 10 mM
HCOONH4 and 0.125% HCOOH,
pH 3.0
Mobile Phase B : 95/5 MeCN/H2O with 10 mM
HCOONH4 and 0.125% HCOOH,
pH 3.0
Flow Rate: 1.0 mL/min
Gradient: Time %A %B
Initial 1 99
1.50 50 50
1.51 1 99
2.70 1 99
COMpOundS
1. Thymine
2. Uracil
3. Adenine
4. Cytosine
5. Guanine
Injection Volume: 3.0 µL (PLNO)
Sample Concentration: 20 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 30 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 260 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
Wa60099
31
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
AU
0
0.1
0.2
0.3
0.4
0.5
0 1 2 3 min
12
3
4
5
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Nucleobases using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
Columns: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A : 50/50 MeCN/H2O with 10 mM
HCOONH4 and 0.125% HCOOH,
pH 3.0
Mobile Phase B : 95/5 MeCN/H2O with 10 mM
HCOONH4 in H2O and 0.125%
HCOOH, pH 3.0
Flow Rate: 0.5 mL/min
Gradient: Time %A %B
Initial 1 99
5.00 50 50
5.01 1 99
9.00 1 99
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 20 μg/mL each
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 30 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 260 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC
with ACQUITY UPLC PDA Detector
COMpOundS
1. Thymine
2. Uracil
3. Adenine
4. Cytosine
4. Guanine
St ruCtureS
N
NHN
N
NH2
NH
HN
O
O
H3CNH
HO
O
N
N
NHN
NH
O
H2NH2N
N
ONH
t hymine u ra c i l ad enine
C yto s ine G u anine
Wa60098
32
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
ad enine
AU
0
0.02
0.04
0.06
0.08
0.10
0 2 4 6 8 10 12 min
1 2
3
4
5
6
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Nucleotide Phosphates using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
Column: ACQUITY UPLC® BEH Amide,
2.1 x 150 mm, 1.7 μm
Part Number: 186004802
Isocratic Mobile Phase: 80/20 MeCN/H2O with
2 mM KH2PO4
Flow Rate: 0.5 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Diluent: 80/20 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 254 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
COMpOundS
1. 2’-Deoxyguanosine-5’-Triphosphate Trisodium Salt Dihydrate
(dGTP) 100 µg/mL
2. Thymidine 5’-monophosphate (TMP) 10 µg/mL
3. 2’-Deoxyadenosine 5’-monophosphate (dAMP) 10 µg/mL
4. 2’-Deoxycytidine 5’-monophosphate sodium salt (dCMP)
25 µg/mL
5. 2’-Deoxyguanosine 5’-monophosphate sodium salt hydrate
(dGMP) 15 µg/mL
6. 2’-Deoxyadenosine 5’-diphosphate sodium salt (dADP)
15 µg/mL
St ruCtureS
2’- d eoxy gu ano s ine -5’-tr i p ho s p hate tr i sodium S al t d i hyd rate (d G t p)
2’- d eoxy ad eno s ine 5’-mono p ho s p hate
(d aM p)
t hymidine 5’-mono -p ho s p h ate ( t M p)
2’- d eoxy c yt id ine 5’-mono p ho s p hate
so dium s al t (d C M p)
2’- d eoxy ad eno s ine 5’- d i p ho s p hate sodium
s al t (d a d p)
2’- d eoxy gu ano s ine 5’-mono p ho s p hate sodium
s al t hyd rate (d GM p)
Wa60097
33
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
8.82e5
1.65e6
9.53e4
5.43e4
1
2
3
4
5
Inte
nsity
%
0
100
0.5 1.0 1.5 2.0 2.5 min0
Inte
nsity
%
0
100
Inte
nsity
%
0
100
Inte
nsity
%
0
100
OH HO
OOOH
O
O
OH
O
OH O
HOOH
O
O
HOOH
O
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Organic Acids using ACQUITY UPLC BEH Amide Columns
uplC COndit iOnS
Column: ACQUITY BEH Amide,
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile phase A: 50/50 MeCN/H2O with 10 mM
CH3COONH4, pH 9.0
Mobile phase B: 95/5 MeCN/H2O with 10 mM
CH3COONH4, pH 9.0
Gradient Flow Rate: 0.6 mL/min
Injection Volume: 5.0 μL
Column Temp: 50 °C
Sample Temp: 5 °C
Strong/Weak needle wash: 95/5 MeCN/H2O
Seal wash: 10/90 MeOH/H2O
Instrument: ACQUITY UPLC and TQD
Gradient: Time
(min) %A %B
Initial 0.1 99.9
0.4 0.1 99.9
0.5 40.0 60.0
2.0 70.0 30.0
2.01 0.1 99.9
5.0 0.1 99.9
St ruCtureS
M al ei c a c id
l a c t i c ac id S u c c ini c ac id
p y r uv ic a c id
MS COndit iOnS
Instrument: ACQUITY TQD
Ionization Mode: ES-
Superscript the -Capillary: 4.0 kV
Cone Voltage: -25 V
Collision Energy: 10 eV
Extractor: 3 V
RF Lens: 0.1 V
Source Temp: 130 °C
Desolvation Temp: 350 °C
Desolvation Gas: 650 L/hr
Cone Gas: 0 L/hr
Collision Gas: 0.1 mL/min
MRM condition: Pyruvic acid: 86.92 > 42.9
Lactic acid: 88.92 > 42.9
Succinic acid: 116.93 > 72.9
Maleic and Fumaric acid: 114.88 > 70.9
f um a r i c a c id
COMpOundS
1. Maleic acid (1 ppm)
2. Pyruvic acid (50 ppm)
3. Lactic acid (50 ppm)
4. Succinic acid (50 ppm)
5. Fumaric acid (50 ppm)
Wa60096
34
CliCk on the underlined blue text for details on the produCts used in this appliCation
Stevia(5 mg/mL)
(5 mg/mL) 0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
1400.0
1600.0
1800.0
2000.0
0.0 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
Stev
iosid
e
Reba
udio
side
A
Reba
udio
side
C
Eryt
hrito
l
Stev
iosid
e
Reba
udio
side
A
Reba
udio
side
C
Eryt
hrito
l
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
0.0
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 min
Stevia(5 mg/mL)
( 5mg/mL)
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Stevia Related Compounds Using ACQUITY UPLC BEH Amide Columns
t est Condit ions
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.20%
triethylamine [TEA]
Mobile Phase B: 30/70 MeCN/H2O with 0.20%
triethylamine [TEA]
Flow Rate: 0.20 mL/min
Flow Profile: 95% A/5% B (77.5% MeCN with
0.20% TEA)
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: 5 mg/mL
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with ELSD
elsd Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C
Nebulizer: Cooling
Data Rate: 10 pps
Filter Time Constant: Normal
st ruCtures
CH 2 OH HC OH HC OH
CH 2 OH
Erythritol
Stevioside
Rebaudioside A
Rebaudioside C
Wa60128
35
CliCk on the underlined blue text for details on the produCts used in this appliCation
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 min
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 min
%
0
100SIR of 5 Channels ES-
TIC7.83e5
Erythritol (m/z 121.1)
%
0
100 SIR of 5 Channels ES- TIC
1.15e6
Rebaudioside A(m/z 966.1)
Rebaudioside C(m/z 950.1)
Stevioside(m/z 803.8)
(10 g/mL)
Stevia(50 g/mL)
Rebaudioside A(m/z 966.1)
SIR of m/z803.8+950.1+966.1
4.71e3160Xzoom
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
UPLC/MS Analysis of Stevia Related Compounds Using
ACQUITY UPLC BEH Amide Columns
t est Condit ions
C hromatographic Conditions
Column: ACQUITY UPLC® BEH Amide
2.1 x 100 mm, 1.7 µm
Part Number: 186004801
Mobile Phase A: 80/20 MeCN/H2O with 0.10%
ammonium hydroxide [NH4OH]
Mobile Phase B: 30/70 MeCN/H2O with 0.10%
ammonium hydroxide [NH4OH]
Flow Rate: 0.20 mL/min
Flow Profile: 95% A/5% B (77.5% MeCN with
0.10% NH4OH)
Injection Volume: 1.3 µL (PLNO)
Sample Concentration: 50 µg/mL stevia, 10 µg/mL truvia
Sample Diluent: 50/50 MeCN/H2O
Column Temperature: 35 °C
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O
Instrument: Waters ACQUITY UPLC with
ACQUITY TQD
Mass spectrometer Conditions
Ionization Mode: ES-
Capillary: 2.8 kV
Cone Voltage: 25 V
Source Temperature: 120 °C
Desolvation Temperature: 350 °C
Desolvation Gas Flow: 500 L/Hr
Cone: 50 L/Hr
SIR (m/z): 317.5 (steviol);
803.8 (stevioside);
950.1 (rebaudioside C);
966.1 (rebaudioside A)
Dwell Time: 0.08 s
st ruCtures
Wa60129
CH 2 OH HC OH HC OH
CH 2 OH
Erythritol
Stevioside
Rebaudioside A
Rebaudioside C
36
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Thiourea using ACQUITY UPLC BEH Amide Columns
AU
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 4 5 6 7 8 min
k prime = 1.4
t0 = 1.8 min
t eSt COndit iOnS
Column: ACQUITY UPLC® BEH Amide,
2.1 x 150 mm, 1.7 µm
Part Number: 186004802
Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/H2O with 10 mM
CH3COONH4 and 0.01% NH4OH, pH 9.0
Flow Rate: 0.2 mL/min
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 10 μg/mL
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 245 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
St ruCture
t hio u re a
S
NH2H2N
Wa60095
37
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
AU
0
0.02
0.04
0.06
0.08
0 1 2 3 4 5 min
1
2
3 4
Analysis of Uric Acids using ACQUITY UPLC BEH Amide Columns
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
t eSt COndit iOnS
Column: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 µm
Part Number: 186004800
Mobile Phase A : 50/50 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH,
pH 5
Mobile Phase B : 90/10 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH,
pH 5
Flow Rate: 0.5 mL/min
Gradient: Time Profile
(min) %A %B
Initial 0.1 99.9
5.00 50.0 50.0
5.01 0.1 99.9
9.00 0.1 99.9
Injection Volume: 5.0 µL (PLNO)
Sample Concentration: 5 μg/mL each
Sample Diluent: 100% MeCN
Column Temperature: 25 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 285 nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
COMpOundS
1. Uric Acid
2. 1-Methyluric acid
3. 1,3-Dimethyluric acid
4. 3,7-Dimethyluric acid
NH
NH
HNNH
O
O
O
H3C
NH
NH
NNH
O
O
O
CH3
H3C
N NH
N NH
O
O
O
CH3
N NH
HN N
O
O
O
CH3
NH
NH
HNNH
O
O
O
H3C
NH
NH
NNH
O
O
O
CH3
H3C
N NH
N NH
O
O
O
CH3
N NH
HN N
O
O
O
CH3
NH
NH
HNNH
O
O
O
H3C
NH
NH
NNH
O
O
O
CH3
H3C
N NH
N NH
O
O
O
CH3
N NH
HN N
O
O
O
CH3
NH
NH
HNNH
O
O
O
H3C
NH
NH
NNH
O
O
O
CH3
H3C
N NH
N NH
O
O
O
CH3
N NH
HN N
O
O
O
CH3
St ruCtureS
u r ic ac id
1,3 - d im et hyl u r i c a c id 3,7- d im et hyl u r i c a c id
1- M et hyl u r i c a c id
Wa60094
38
CliCk On the underlined blue text fOr detailS On the prOduCtS uSed in thiS appliCatiOn
3,7- d im et hyl u r i c a c id
AU
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 min
1
2
3
4
5
6
7
8
© 2009 Waters Corporation. Waters, ACQUITY UPLC and The Science of What’s Possible are registered trademarks of Waters Corporation.
Analysis of Water Soluble Vitamins using ACQUITY UPLC BEH Amide Columns
t eSt COndit iOnS
Column: ACQUITY UPLC® BEH Amide,
2.1 x 50 mm, 1.7 μm
Part Number: 186004800
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
CH3COONH4 and 0.04 % NH4OH,
pH 9.0
Mobile Phase B: 90/10 MeCN/H2O with 10 mM
CH3COONH4 and 0.04 % NH4OH,
pH 9.0
Flow Rate: 0.5 mL/min
Gradient: Time Profile
(min) %A %B
Initial 0.1 99.9
3.50 70.0 30.0
3.51 0.1 99.9
7.50 0.1 99.9
Injection Volume: 5 µL (PLNO)
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH
Column Temperature: 30 °C
Weak Needle Wash: 95/5 MeCN/H2O
Detection: UV @ 265nm
Sampling Rate: 20 points/sec
Filter Time Constant: 0.2
Instrument: Waters ACQUITY UPLC with
ACQUITY UPLC PDA Detector
COMpOundS
1. Nicotinamide (25 µg/mL)
2. Pyridoxal (50 µg/mL)
3. Riboflavin (50 µg/mL)
4. Nicotinic acid (50 µg/mL)
5. Thiamine (50 µg/mL)
6. Ascorbic acid (25 µg/mL)
7. B12 (50 µg/mL)
8. Folic Acid (25 µg/mL)
St ruCtureS
O
N
NH2
N
O
OHHO
CH3
OH
OH
OH
HO
N N
N
O
NH
OH3C
H3C
O
N
OH
N
N NH2
N+
SH3C OH
CH3
OO
HO HO
HO
HO
nic ot in amid e
b12
N
NH
N
N
O
HN
NH
NH
O
O
O
HOHO
O
O
O
O
O
O
O
OO
O
O
O
HN
HN
HN
NH
NH
NH
CH
CH
CH
N N
N
NN
N
N
NH
CHCH
HC
HCHC
HC
HC
CH
H
OH
OHP
f ol i c ac id
N
NH
N
N
O
HN
NH
NH
O
O
O
HOHO
O
O
O
O
O
O
O
OO
O
O
O
HN
HN
HN
NH
NH
NH
CH
CH
CH
N N
N
NN
N
N
NH
CHCH
HC
HCHC
HC
HC
CH
H
OH
OHP
p yr idox al
O
N
NH2
N
O
OHHO
CH3
OH
OH
OH
HO
N N
N
O
NH
OH3C
H3C
O
N
OH
N
N NH2
N+
SH3C OH
CH3
OO
HO HO
HO
HO
O
N
NH2
N
O
OHHO
CH3
OH
OH
OH
HO
N N
N
O
NH
OH3C
H3C
O
N
OH
N
N NH2
N+
SH3C OH
CH3
OO
HO HO
HO
HO
nic ot ini c a c id
r ib o f l av in
O
N
NH2
N
O
OHHO
CH3
OH
OH
OH
HO
N N
N
O
NH
OH3C
H3C
O
N
OH
N
N NH2
N+
SH3C OH
CH3
OO
HO HO
HO
HO
a s c or b i c a c id
O
N
NH2
N
O
OHHO
CH3
OH
OH
OH
HO
N N
N
O
NH
OH3C
H3C
O
N
OH
N
N NH2
N+
SH3C OH
CH3
OO
HO HO
HO
HO
t hiamine
O
N
NH2
N
O
OHHO
CH3
OH
OH
OH
HO
N N
N
O
NH
OH3C
H3C
O
N
OH
N
N NH2
N+
SH3C OH
CH3
OO
HO HO
HO
HO
Wa60093
39
Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07
Australia 61 2 9933 1777
Belgium 32 2 726 1000
Brazil 55 11 5094-3788
Canada 1 800 252 4752 x2205
China 86 10 8586 8899
CIS/Russia +7 495 3367000
Czech Republic 420 2 617 1 1384
Denmark 45 46 59 8080
Finland 09 5659 6288
France 33 1 30 48 72 00
Germany 49 6196 400600
Hong Kong 852 29 64 1800
Hungary 36 1 350 5086
India and India Subcontinent 91 80 2837 1900
Ireland 353 1 448 1500
Italy 39 02 27 421 1
Japan 81 3 3471 7191
Korea 82 2 820 2700
Mexico 52 55 5200 1860
The Netherlands 31 76 508 7200
Norway 47 6 384 60 50
Poland 48 22 833 4400
Puerto Rico 1 787 747 8445
Singapore 65 6273 1221
Spain 34 93 600 9300
Sweden 46 8 555 11 500
Switzerland 41 56 676 70 00
Taiwan 886 2 2543 1898
United Kingdom 44 208 238 6100
All other countries: Waters Corporation U.S.A. 1 508 478 2000
1 800 252 4752
www.waters.com
Sales Offices
©2009 Waters Corporation. ACQUITY, ACQUITY UPLC, Alliance,
Atlantis, IS, Nova-Pak, Oasis, Platform LC, Quattro Ultima, Sentry, SunFire,
SymmetryShield, T he Science of What’s Possible, Waters, XTerra and ZQ.
All other trademarks are acknowledged.
WA64081 October 2009 KK-IH-PDF
The quality management system of Waters’ manufacturing facilitiesin Taunton, Massachusetts and Wexford, Ireland complies with the International Standard ISO 9001:2000 Quality Management and Quality Assurance Standards. Waters’ quality management system is periodically audited by the registering body to ensure compliance.