Wa 64081

ACQUITY UPLC BEH AMIDE COLUMNS
Application Notebook
List of Compounds Analyzed Using ACQUITY UPLC BEH Amide Columns
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
T hiourea............................................................................................................................................................................................... 37
Uric Acids............................................................................................................................................................................................. 38
Water Soluble Vitamins........................................................................................................................................................................ 39
1
Analysis of 5-Fluorouracil using ACQUITY UPLC BEH Amide Columns
C lic k on t he underlined blue t e x t for details on t he p roduc t s used in t his a p p licat ion
T e st condit ions St ruc t u r e
Columns: ACQUITY UPLC® BEH Amide,

H
2.1 x 50 mm, 1.7 μm N O
Part Number: 186004800

Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/ H2O with
NH
5 mM CH3COONH4 and 0.02% NH4OH,
F
pH 9.0
Flow Rate: 0.2 mL/min
O
Injection Volume: 5.0 µL (PLNO)
5 - F luorouracil
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
0.40 k prime = 2.2
0.30
AU
0.20
t0= 1.3 min

0.10
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
WA60121
© 2009 Waters Corporation. Waters, ACQUITY UPLC and T he Science of W hat’s Possible are registered trademarks of Waters Corporation.
2
Analysis of Acr ylamide, Methacr ylic Acid and Methacr ylamide
using ACQUITY UPLC BEH Amide Columns
T e st condit ions St ruc t u r e s
Columns: ACQUITY UPLC® BEH Amide, O O

2.1 x 150 mm, 1.7 µm
H 2C H2C
NH2 NH2
Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/H2O with 5 mM
O O
CH3COONH4 and 0.02% NH4OH,
CH3
pH 9.0 H 2C O H 2C
Flow Rate: 0.5 mL/min M e t h a c r y l a m i dNH
e2 A c r y l a m i d eNH2
Injection Volume: 5.0 µL (PLNO) H 2C
Sample Concentration: 30 μg/mL each CH3 OH
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH O
Column Temperature: 25 °C CH3

H 2C
OH
CH3
Met hac rylic acid
com p ounds
1
0.7 1. Methacrylamide
2. Acrylamide
2
3. Methacrylic acid
0.6
0.5
0.4
AU
0.3
0.2
t0=0.9 min 3
0.1
0 1 2 3 4 5 6 min
WA60108
3
Analysis of Allantoin using ACQUITY UPLC BEH Amide Columns
Column: ACQUITY UPLC® BEH Amide, H

H 2N HN N
2.1 x 150 mm, 1.7 µm
O
Isocratic Mobile Phase B: 90/10 MeCN/H2O O N
O H
Injection Volume: 5.0 µL (PLNO) Al l a n t o in
Sample Diluent: 90/10 MeCN/H2O
0.6
k prime = 3.2
0.5
0.4
AU
0.3
0.2
0.1
t0 = 1.9 min
0 2.0 4.0 6.0 8.0 10 12 14 16 min
WA60107
4
Cellulosic Hydrolysates Using ACQUITY UPLC BEH Amide Columns
T e st condit ions STRUC TURE S

O OH O OH
O O OH HO HO
OH
HO HO OH
C hromatograp hic Conditions HO OH HO OH
HO OH HO OH
OH OH
Column: ACQUITY UPLC® BEH Amide Xylose Fructose Glucose Mannose
2.1 x 100 mm, 1.7 µm
O OH HO O HO OH
Part Number: 186004801 HO
OH
O O HO
O O OH HO
Mobile Phase A: 80/20 MeCN/H2O with 0.2% HO HO OH O OH
OH O
HO OH
triethylamine [TEA] HO OH HO O
OH
OH O OH
Mobile Phase B: 30/70 MeCN/H2O with 0.2% HO O
Cellobiose Sucrose
triethylamine [TEA] HO OH
HO OH
Flow Rate: 0.12 mL/min HO OH
HO Raffinose
Gradient: 10 minute gradient, 80%-50% HO O
MeCN (w/0.2% TEA) with 30 minute O
O
HO HO
HO HO
re-equilibration HO O OH O
O
O
HO O OH
Time Profile HO
O O
(min) %A %B HO OH
HO OH HO
OH HO
OH
n
0.00 100.00 0.00 OH
n = 1 to 5
10.00 60.00 40.00 Melezitose Maltooligosaccharides

10.01 100.00 0.00
40.00 100.00 0.00
Com p ounds
Sample Concentration: 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O 1. Xylose 6. Cellobiose 11. Maltopentaose
Column Temperature: 35 °C 2. Fructose 7. Melezitose 12. Maltohexaose
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) 3. Mannose 8. Raffinose 13. Maltoheptaose
Weak Needle Wash: 75/25 MeCN/H2O (500 µL) 4. Glucose 9. Maltotriose
Seal Wash: 50/50 MeCN/H2O 5. Sucrose 10. Maltotetraose
Instrument: Waters ACQUITY UPLC with ELSD
5 7 8,9
2 3 4 6
1 11 12
EL SD Conditions 10 13
Gain: 200 11 12
9 10 13
Pressure: 40 psi
3
Drift Tube Temperature: 40 °C
Nebulizer: Cooling 7
Data Rate: 10 pps
8
Filter Time Constant: Normal
6
1
5
2 4
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 min
WA60127
5
Chemical Stability STUDY OF ACQUITY UPLC BEH AMIDE COLUMNS
T e st condit ions
Columns: ACQUITY UPLC® BEH Amide, Injection Volume: 2.0 µL (full loop injection mode)
2.1 x 50 mm, 1.7 µm Sample Concentration: 25 μg/mL each
Part Number: 186004800 Sample Diluent: 75/25 MeCN/MeOH
Mobile Phase A: 50/50 MeCN/H2O with 10 mM Column Temperature: 30 °C
CH3COONH4, pH 5.5 Weak Needle Wash: 95/5 MeCN/H2O
Mobile Phase B: 95/5 MeCN/H2O with 10 mM Detection: UV @ 254 nm
CH3COONH4, pH 5.5 Sampling Rate: 40 pts/sec
Flow Rate: 0.5 mL/min Filter Time Constant: 0.1
Gradient: Time Profile Instrument: Waters ACQUITY UPLC with
(min) %A %B ACQUITY UPLC PDA Detector
Initial 1 99
2.00 99 1
2.10 1 99 St ruc t u r e s
2.50 1 99
H H H
N O N O N
Com p ounds NH N
F
1. Uracil
2. 5-fluorocytosine
O NH2 NH
3. Cytosine
Uracil 5 - F l u o r o c y t o s in e
1 Injection 1
0.6 3 H H H
2 N O N O N O
0.4
AU
0.2 NH N N
F
0
Injection 1000
0.6 O NH2 NH2
0.4 C y t o s in e
AU
0.2
0.6 Injection 2000
0.4
AU
0.2
0
0 1 2 min
WA60106
6
Analysis of Food Sugars in Bran with Raisins Cereal
Using ACQUITY UPLC BEH Amide Columns
T e st condit ions
C hromatograp hic Conditions EL SD Conditions
Column: ACQUITY UPLC® BEH Amide Gain: 200

2.1 x 150 mm, 1.7 µm Pressure: 40 psi
Part Number: 186004802 Drift Tube Temperature: 40 °C
Mobile Phase A: 80/20 MeCN/H2O with 0.2% Nebulizer: Cooling
triethylamine [TEA] Data Rate: 10 pps
Mobile Phase B: 30/70 MeCN/H2O with 0.2% Filter Time Constant: Normal
triethylamine [TEA]
Flow Profile: 90% A/10% B (75% MeCN with
STRUC TURE S
0.2% TEA)
Fructose
Injection Volume: 2.0 µL (PLNO) Sucrose
Sample Concentration: Standards at 1 mg/mL each, cereal
extracted at 8 mg/mL
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) Sucrose Fructose
p-Toluamide
Weak Needle Wash: 75/25 MeCN/H2O (500 µL) (unretained compound)
O
Seal Wash: 50/50 MeCN/H2O
NH 2
Com p ounds H3C

Lactose
1. p-Toluamide 5. Maltose p-Toluamide
3. Glucose
(unretained compound)
2. Fructose 4. Sucrose 6. Lactose
Maltose Glucose
4
Maltose Glucose
Raisin Bran Brand Cereal

2 3 8mg/mL
2 4
3
6
Food Sugar
5 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
WA60120
7
Analysis of Food Sugars in Ketchup Using ACQUITY UPLC BEH Amide Columns

Fructose
Sucrose
C hromatograp hic Conditions
Column: ACQUITY UPLC® BEH Amide

2.1 x 50 mm, 1.7 µm
Sucrose Fructose
Mobile Phase A: 80/20 acetone/H2O with 0.05%
p-Toluamide
triethylamine [TEA] (unretained compound)
O
Mobile Phase B: 30/70 acetone/H2O with 0.05%
triethylamine [TEA] NH 2
Flow Profile: 95% A/5% B (77.5% acetone with H3C
Lactose
0.05% TEA) p-Toluamide
Injection Volume: 0.7 µL (PLNO) (unretained compound)
Sample Concentration: Standards at 1 mg/mL each
Maltose Glucose
Strong Needle Wash: 20/80 MeCN/H2O (800 µL)
Weak Needle Wash: 75/25 MeCN/H2O (500 µL)
Seal Wash: 50/50 MeCN/H2O Maltose Glucose
EL SD Conditions Com p ounds

Gain: 200 1. p-Toluamide 4. Sucrose
Pressure: 40 psi 2. Fructose 5. Maltose
Drift Tube Temperature: 40 °C 3 3. Glucose 6. Lactose
Nebulizer: Cooling
Data Rate: 10 pps
1 2
5
Tomato Ketchup
2
4
3
5 6
Food Sugars
0.0 1.0 2.0 3.0 4.0 min
WA60117
8
Analysis of Food Sugars in MILK Using ACQUITY UPLC BEH Amide Columns

Fructose
Sucrose

2.1 x 100 mm, 1.7 µm
Part Number: 186004801 Sucrose Fructose
Mobile Phase A: 80/20 MeCN/H2O with 0.2% p-Toluamide
triethylamine [TEA] O
Mobile Phase B: 30/70 MeCN/H2O with 0.2%

H3C
Flow Profile: 90% A/10% B (75% MeCN with Lactose
Sample Concentration: Standards at 1 mg/mL each Maltose Glucose
Column Temperature: 35°C
EL SD Conditions Com p ounds
Gain: 200 1. p-Toluamide 4. Sucrose

Pressure: 40 psi 2. Fructose 5. Maltose
Drift Tube Temperature: 40 °C 3. Glucose 6. Lactose
Nebulizer: Cooling 1
Data Rate: 10 pps
Milk – 1% Fat
5% in 50% ACN
2
4
3
6
5
Food Sugar
Standard
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
WA60118
9
Analysis of Food Sugars in Molasses Using ACQUITY UPLC BEH Amide Columns
T e st condit ions

triethylamine [TEA]
STRUC TURE S
0.2% TEA)
Fructose
Sucrose
Sample Concentration: Standards at 1 mg/mL each, molasses
at 5 mg/mL
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) Sucrose Fructose
p-Toluamide
Weak Needle Wash: 75/25 MeCN/H2O (500 µL) (unretained compound)
Seal Wash: 50/50 MeCN/H2O O

NH 2
H3C
Com p ounds Lactose
p-Toluamide
1. p-Toluamide 3. Glucose 5. Maltose (unretained compound)
2. Fructose 4. Sucrose 6. Lactose
Maltose Glucose
Maltose Glucose
2
3
Molasses
5 mg/mL
2 4
3
6
5 Food 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
WA60119
10
Analysis of Food Sugars In Prepared FOods

Fructose
Sucrose

2.1 x 50 mm, 1.7 µm
Sucrose Fructose
triethylamine [TEA] O

H3C
Flow Profile: 95% A/5% B (77.5% acetone Lactose
with 0.05% TEA) p-Toluamide
EL SD Conditions
Gain: 200
Pressure: 40 psi
Nebulizer: Cooling
Data Rate: 10 pps Strawberry
Filter Time Constant: Normal Smoothie
Hot Cross Buns

Com p ounds
1. p-Toluamide White Bread

2. Fructose
3. Glucose
4. Sucrose Lamb Curry Meal
5. Maltose 1 2
4
3
6. Lactose
5 6
Food Sugars
0.0 1.0 2.0 3.0 4.0 min
WA60115
11
Analysis of Food Sugars in Spor ts Drink

Fructose
Sucrose

2.1 x 50 mm, 1.7 µm
Sucrose Fructose
Mobile Phase A: 80/20 acetone/H2O with 0.05% p-Toluamide
triethylamine [TEA]
O
Flow Profile: 95% A/5% B (77.5% acetone with H3C
Lactose
EL SD Conditions
Gain: 200
Pressure: 40 psi
2
3
Nebulizer: Cooling
Data Rate: 10 pps
Com p ounds 1
Gatorade Brand
1. p-Toluamide 4. Sucrose Sports Drink
2. Fructose 5. Maltose 2
3. Glucose 6. Lactose 4
3
5 6
Food Sugars
0.0 1.0 2.0 3.0 4.0 min
WA60116
Gatorade is a registered trademark of Stokely-Van Camp, Inc.
12
Analysis of Food Sugars in Wine Using ACQUITY UPLC BEH Amide Columns
T e st condit ions

Gain: 200
Pressure: 40 psi
2.1 x 150 mm, 1.7 µm
Nebulizer: Cooling
Mobile Phase A: 80/20 MeCN/H2O with 0.2%
Data Rate: 10 pps
triethylamine [TEA]
triethylamine [TEA]
Flow Profile: 90% A/10% B (75% MeCN with STRUC TURE S
0.2% TEA) Fructose
Glucose
Sample Concentration: 50% wine in diluent
Column Temperature: 35°C
Fructose Glucose
Com p ounds
1. Fructose
2. Glucose
1 2
Cabernet
Sauvignon
Chardonnay
0 1 2 3 4 5 6 7 8 9 10 min
WA60114
13
Analysis of Food Sugars Using ACQUITY UPLC BEH Amide Columns
T e st condit ions

triethylamine [TEA]
0.2 % TEA) STRUC TURE S
Injection Volume: 1.3 µL (PLNO) Fructose
Sample Concentration: 1 mg/mL each Sucrose

Sucrose Fructose
Seal Wash: 50/50 MeCN/H2O p-Toluamide
O
NH 2
Com p ounds
H3C
1. p-Toluamide Lactose
p-Toluamide
2. Fructose (unretained compound)
3. Glucose
1 Maltose Glucose
4. Sucrose
5. Maltose
6. Lactose
Maltose Glucose
2
4
3
6
5
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
WA60109
14
UPLC /MS Analysis of Food Sugars Using ACQUITY UPLC
BEH Amide Columns with Acetone as Organic Modifier
T e st condit ions
C hromatograp hic Conditions Mass Spectrometer Conditions

Ionization Mode: ES -
Capillary: 2.8 kV
2.1 x 50 mm, 1.7 µm
Cone Voltage: 25 V
Source Temperature: 120 °C
Mobile Phase A: 80/20 acetone/H2O with 0.05%
Desolvation Temperature: 350 °C
ammonium hydroxide [NH4OH]
Desolvation Gas Flow: 500 L/Hr
Cone: 50 L/Hr
SIR (m/z): 179.2 (Fructose, Glucose);
341.3 (Sucrose, Maltose,
Flow Profile: 94% A/6% B (77% acetone with
Lactose)
0.05% NH4OH)
Dwell Time: 0.08 s
Sample Concentration: 10 µg/mL each
Column Temperature: 85 °C STRUC TURE S Fructose
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) Sucrose

Sucrose
ACQUITY TQD Fructose
Lactose
Com p ounds
1. Fructose 3. Sucrose Maltose Lactose Glucose

2. Glucose 4. Maltose
5. Lactose
100
SIR of 2 Channels ES- 3
TIC Maltose Glucose
2.77e5
%
1 4
5
2
0
0. 0 0. 5 1. 0 1. 5 2. 0 2. 5 3. 0 3. 5 min
WA60113
15
Analysis of Food Sugars/Saccharides in Beer Using
ACQUITY UPLC BEH Amide Columns
Glucose
Maltose
Column: ACQUITY UPLC® BEH Amide Glucose

2.1 x 100 mm, 1.7 µm
Maltose Sucrose
triethylamine [TEA]
Sucrose
triethylamine [TEA] Fructose
Gradient: 10 minute gradient, 75%-45%
MeCN (w/0.2% TEA) with 25 minute
re-equilibration Lactose
Time Profile Fructose
Maltooligosaccharides
(min) %A %B p-Toluamide
0.00 90.00 10.00
O
10.00 30.00 70.00
10.01 90.00 10.00 NH 2
n
35.00 90.00 10.00 n= 1 to 5
Injection Volume: 1.3 µL (PLNO) H3C

Sample Concentration: Standards at 1 mg/mL, beer at 100% p-Toluamide
(No dilution) (unretained compound)
Column Temperature: 35 °C Com p ounds
Strong Needle Wash: Analysis
20/80 MeCN/H2O (800 µL) of Food Sugars/Saccharides in
Beer Using 1. p-Toluamide
ACQUITY UPLC5. BEH
MaltoseAmide 9. Maltopentaose
Columns 2. Fructose 6. Lactose 10. Maltohexahose
3. Glucose 7. Maltotriose 11. Maltoheptaose
4. Sucrose 8. Maltotetraose
EL SD Conditions Wheat Beer
Gain: 200
Belgian Beer
Pressure: 40 psi
Dutch Beer
Nebulizer: Cooling
Data Rate: 10 pps Chocolate Stout

Dark Ale
1 3 4 5 6 9 10
2 7 8 11
Standards
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
WA60125
16
UPLC /MS Analysis of Food Sugars/Saccharides in Beer
T e st condit ions
Column: ACQUITY UPLC® BEH Amide Ionization Mode: ES -

2.1 x 100 mm, 1.7 µm Capillary: 2.8 kV
Part Number: 186004801 Cone Voltage: 25 V (fructose, glucose, maltotriose);
Mobile Phase A: 80/20 MeCN/H2O with 0.1% 40V (sucrose and maltose)
ammonium hydroxide [NH4OH] Source Temp: 120 °C
Mobile Phase B: 30/70 MeCN/H2O with 0.1% Desolvation Temp: 350 °C
ammonium hydroxide [NH4OH] Desolvation Gas Flow: 500 L/Hr
Flow Rate: 0.13 mL/min Cone: 50 L/Hr
Gradient: 10 minute gradient, 75%-45% MeCN SIR (m/z): 179.0 (fructose, glucose);
(w/0.1% NH4OH) with 25 minute 341.1 (sucrose, maltose);
re-equilibration 503.2 (maltotriose)
Time Profile Dwell Time: 0.04 s
(min) %A %B
0.00 90.00 10.00
10.00 30.00 70.00
10.01 90.00 10.00 STRUC TURE S Maltooligosaccharides
35.00 90.00 10.00

Maltose
Sample Concentration: Standards at 10 µg/mL, Beer at
50% dilution n= 1 to 5 n
Sample Diluent: 50/50 MeCN/H2O Maltooligosaccharides

Maltose
Column Temperature: 35 °C Fructose
Sucrose
Weak Needle Wash: 75/2MeCN/H2O 5 (500 µL)
Instrument: Waters ACQUITY UPLC with SQ
Fructose Sucrose
Glucose
Trisaccharides
Disaccharides (m/z = 503.2)
(m/z = 341.1)
SIR of 3 Channels ES-
TIC
4.91e6
Lactose Glucose
%
1 Dutch Beer
0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 min

3 Com p ounds
TIC
5.18e5
1. Fructose 4. Maltose
2. Glucose 5. Maltotriose
%
1 2
4 5 Food Sugar
Standard 3. Sucrose
0
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 min
WA60126
17
Analysis of Food Sugars/Saccharides in Cough Syrup
Glucose
Maltose

2.1 x 100 mm, 1.7 µm
Maltose Sucrose
triethylamine [TEA]
Sucrose
triethylamine [TEA] Fructose
Gradient: 6 minute gradient, 80%-50% MeCN
(w/ 0.2% TEA) with 12 minute
(min) %A %B p-Toluamide
0.00 100.00 0.00 O
6.00 40.00 60.00
6.01 100.00 0.00 NH 2
n= 1 to 5 n
18.00 100.00 0.00
Injection Volume: 1.3 µL (PLNO) H3C
Sample Concentration: Standards at 1 mg/mL each, cough p-Toluamide
syrups at 1% (v/v) (unretained compound)
Strong Needle Wash: MeCN/H2O 20/80 (800 µL) Com p ounds
Weak Needle Wash: MeCN/H2O 75/25 (500 µL) 1. p-Toluamide
2 6. Maltotriose
Seal Wash: MeCN/H2O 50/50 2. Fructose 7. Maltotetraose
Instrument: Waters ACQUITY UPLC with ELSD 3. Glucose 8. Maltopentaose
4. Sucrose 9. Maltohexaose
EL SD Conditions 5. Maltose 10. Maltoheptaose
3
Gain: 200
1
Pressure: 40 psi
Generic Tussin
Drift Tube Temperature: 40 °C Cough Syrup
Nebulizer: Cooling
Data Rate: 10 pps Robitussin Brand
Filter Time Constant: Normal Cough Syrup
4
2
7 8
3 6 9
5 10
Saccharide
Standard
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
WA60121
© 2009 Waters Corporation. Waters, ACQUITY UPLC, and T he Science of W hat’s Possible are registered trademarks of Waters Corporation.
Robitussin is a registered trademark of Wyeth Corporation.
18
Analysis of Food Sugars/Saccharides in Honey
Glucose
Maltose

2.1 x 50 mm, 1.7 µm
Part Number: 186004800 Maltose Sucrose

triethylamine [TEA]
triethylamine [TEA] Sucrose
Gradient: 5 minute gradient, 80%-50% Fructose
Time Profile Maltooligosaccharides

(min) %A %B
0.00 100.00 0.00 Fructose
5.00 40.00 60.00
5.01 100.00 0.00
n
15.00 100.00 0.00 n= 1 to 5
Injection Volume: 0.7 µL (PLNO) Maltooligosaccharides
Sample Concentration: Honey and corn syrup at 5-10 mg/mL

each
Com p ounds
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) 1. Fructose 3. Sucrose 5. Maltotriose
Weak Needle Wash: 75/25 MeCN/H2O (500 µL) 2. Glucose 4. Maltose 6. Maltotetraose
Seal Wash: 50/50 MeCN/H2O 7. Maltopentaose
EL SD Conditions
3 4
Gain: 200 5
6 Corn Syrup
7 (10 mg/mL)
1
Pressure: 40 psi
Nebulizer: Cooling
Data Rate: 10 pps Fake Honey
(8 mg/ml)
Pure Honey
(5 mg/mL)
0.0 1.0 2.0 3.0 4.0 5. 0 6.0 7.0 min
WA60124
19
Analysis of Food Sugars/Saccharides in Maple Syrup
Glucose
Maltose

2.1 x 100 mm, 1.7 µm
Part Number: 186004801 Maltose Sucrose

triethylamine [TEA]
triethylamine [TEA] Sucrose
Gradient: 10 minute gradient, 80%-50% Fructose
Time Profile Maltooligosaccharides

(min) %A %B
0.00 100.00 0.00 Fructose
10.00 40.00 60.00
10.01 100.00 0.00
n= 1 to 5 n
35.00 100.00 0.00
Injection Volume: 1.3 µL (PLNO) Maltooligosaccharides
Sample Concentration: Maple syrups at 5-10 mg/mL each
Com p ounds
Weak Needle Wash: 75/25 MeCN/H2O (500 µL) 1. Fructose 3. Sucrose 5. Maltotriose
Seal Wash: 50/50 MeCN/H2O 2. Glucose 4. Maltose 6. Maltotetraose
Instrument: Waters ACQUITY UPLC with ELSD 7. Maltopentaose
EL SD Conditions
Gain: 200 1
4
Pressure: 40 psi Log Cabin Brand 5
6 7
Maple Syrup
Drift Tube Temperature: 40 °C 3
Nebulizer: Cooling
Data Rate: 10 pps “Pure”
Filter Time Constant: Normal Maple Syrup
“Light”
Pancake Syrup
Vermont
Maple Syrup
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
WA60123
20
Analysis of Food Sugars/Saccharides in Potato Chips
T e st condit ions STRUC TURE S Glucose

Glucose
2.1 x 100 mm, 1.7 µm Lactose
triethylamine [TEA] O Sucrose
NH 2
triethylamine [TEA]
Flow Rate: 0.13 mL/min H3C
Gradient: 10 minute gradient, 80%-50% p-Toluamide Sucrose

MeCN (w/0.2% TEA) with 25 minute (unretained compound)
re-equilibration
Maltose
(min) %A %B
0.00 100.00 0.00
10.00 40.00 60.00
10.01 100.00 0.00
35.00 100.00 0.00 Maltose Fructose
Sample Concentration: Standards at 1 mg/mL each, potato
chips extracted at 120 mg/mL
Com p ounds
Column Temperature: 35 °C 1. p-Toluamide 3. Glucose 5. Maltose
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) 2. Fructose 4. Sucrose 6. Lactose
4
1
EL SD Conditions
Gain: 200
Pressure: 40 psi
Drift Tube Temperature: 40 °C BBQ Flavored
Nebulizer: Cooling Potato Chips
3 (extracted in 50:50 MeCN/H 2O)
2
Data Rate: 10 pps
Filter Time Constant: Normal 4
6
2
3 5
Food Sugar
Standard
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min
WA60122
21
HILIC Gradient Separation of Ascorbic Acid And Isoascorbic
Acids using ACQUITY UPLC BEH Amide Columns
HO
Columns: ACQUITY UPLC® BEH Amide,
O
2.1 x 100 mm, 1.7 μm O
HO HO
Mobile Phase A: 50/50 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH,
HO OH
pH 5.0
Mobile Phase B: 90/10 MeCN/H2O with 10 mM
Ascorbic Acid
pH 5.0
HO HO
Gradient: Time Profile O O
O O
(min) %A
HO %B HO
Initial 0.1 99.9
10.00 99.9 0.1
10.01 0.1 99.9 HO OH HO OH
15.00 0.1 99.9

Injection Volume: 5.0 μL (PLNO) Isoascorbic Acid
Sample Concentration: 30 μg/mL each
Weak Needle Wash: 95/5 MeCN/H2O com p ounds
Detection: UV @ 260nm
Sampling Rate: 20 points/sec 1. Isoascorbic acid
Filter Time Constant: 0.2 2. Ascorbic acid
2
0.35
1
0.30
0.25
0.20
AU
0.15
0.10
0.05
0 2 4 6 8 10 min
WA60105
22
HILIC Gradient Separation of Organophosphonic Acids
T e st condit ions
Columns: ACQUITY UPLC® BEH Amide, Ionization Mode: ES -

2.1 x 100 mm, 1.7 µm Capillary: 2.5 KV
Part Number: 186004801 Cone: 30 V (EMPA, IMPA, PMPA);
Mobile Phase A: 50/50 MeCN/H2O with 10 mM 40 V (CMPA); 35 V (MMPA)
CH3COONH4 and 0.04% NH4OH, Source Temperature: 120 °C
pH 9.0 Desolvation Temperature: 400 °C
Mobile phase B: 95/5 MeCN/H2O with 10 mM Desolvation Gas Flow: 800 L/Hr
CH3COONH4 and 0.04% NH4OH, Cone: 5 L/Hr
pH 9.0 SIR m/z: 122.9 (EMPA); 136.95 (IMPA);
Flow Rate: 0.5 mL/min 179.0 (PMPA); 177.0 (CMPA);
Gradient: Time Profile 150.95 (MMPA)
(min) %A %B Dwell Time: 0.1 s
Initial 0.1 99.9
10.00 99.9 90.0
10.01 0.1 99.9 St ruc t u r e s
15.00 0.1 99.9 O
O O
H 3C
Injection Volume: 5.0 µL (PLNO) H 3C P OH H3C
H 3C P O
HO P O
Sample Concentration: 2 μg/mL each O CH3
CH3
OH
Sample Diluent: 75/25 MeCN/MeOH CH3
H 3C
H 3C
Column Temperature: 65 °C O O
2- (m e Ot h y l) p r o p y l O O
Weak Needle Wash: 95/5 MeCN/H2O m e t h y l p h o s p h Ho Cni c3
C y cHl3Co h eP x yHOH
lC P HC P P in a c ol y l H C
3
a c iCH
O HC 3
dP ( MOM PA) 3 O
HO P O
3
Instrument: Waters ACQUITY UPLC with m et hyl p h o sO p h oni c 3 CH3

OH
m e t h yl
CH p h o s p h o ni c
3
OH CH3
ACQUITY SQD a c i d (C M PA)
H 3C OH
a c i d CH( P M PA) 3
H 3C
H 3C
O O
H3C P O H 3C P O
CH3
CH3
100 2 OH
OH
H 3C
4.63e6
Isopropyl Et hyl
m e t h y l p h o s p h o ni c m e t h y l p h o s p h o ni c
1
a c i d ( I M PA) a c i d ( E M PA)
com p ounds
Intensity %
1. Pinacolyl methylphosphonic acid (PMPA)

5 2. 2-(methyl)propyl methylphosphonic acid (MMPA)
3. Cyclohexyl methylphosphonic acid (CMPA)
4. Isopropyl methylphosphonic acid (IMPA)
5. Ethyl methylphosphonic acid (EMPA)
0
0 2 4 6 8 10 12 14 min
WA60104
23
HILIC Isocratic Separation of Isoascorbic acid and ascorbic
acid using ACQUITY UPLC BEH Amide Columns
H O
C hromatograp hic Conditions O

O
Column: ACQUITY UPLC® BEH Amide, H O
Isocratic Mobile Phase: 80/20 MeCN/H2O with 10 mM

KH2PO4, pH 4.6
Flow Rate: 0.2 mL/min H O O H
Injection Volume: 5.0 µL (PLNO) Ascorbic acid

H O
O
Weak Needle Wash: 95/5 MeCN/H2O O
H O
Detection: UV @ 260nm
H O O H
ACQUITY UPLC PDA Detector Isoascorbic acid
Com p ounds
1 2
0.5 1. Isoascorbic acid
2. Ascorbic acid
0.4
0.3
AU
0.2
ACQUITY UPLC BEH Amide

0.1
2.1 x 50 mm, 1.7 µm
0
0.5 1
2
0.4
0.3
AU
0.2
0.1 ACQUITY UPLC BEH Amide

2.1 x 100 mm, 1.7 µm
0
0 1 2 3 4 5 6 7 8 min
WA60102
24
HILIC Isocratic Separation of Organophosphonic Acids
T e st condit ions
C hromatograp hic Conditions Mass Spectrometer Settings

Columns: ACQUITY UPLC® BEH Amide, Capillary: 2.5 KV
2.1 x 100 mm, 1.7 µm Cone: 30 V (EMPA, IMPA, PMPA);
Part Number: 186004801 40 V (CMPA); 35 V (MMPA)
Isocratic Mobile Phase: 90/10 MeCN/H2O with 10 mM Source Temperature: 120 °C
CH3COONH4 and 0.04% NH4OH, Desolvation Temperature: 400 °C
pH 9.0 Desolvation Gas Flow: 800 L/Hr
Flow Rate: 0.5 mL/min Cone: 5 L/Hr
Injection Volume: 5.0 µL (PLNO) SIR m/z: 122.9 (EMPA); 136.95 (IMPA);
Sample Concentration: 2 μg/mL each 179.0 (PMPA); 177.0 (CMPA);
Sample Diluent: 75/25 MeCN/MeOH 150.95 (MMPA)
Column Temperature: 65 °C Dwell Time: 0.1 s
ACQUITY SQD
St ruc t u r e s
O
100 2
H 3C P OH O
O H3C
O O
H 3C HO P O
H 3C P OH O O CH3
H 3C P
CH3
CH3 H3C
O H 3C
O H 3C HO P O H3C
OH
O CH3
H3C P
Cyclo hexyl 2- (m e t h y l) p r o p yCHl PCHin aH cC ol y l
3
3
m et hyl p ho s p honic
O
m et h yl p h o s p h o ni c O m e t h y l p h o s p hH oC ni c
OH 3
1 a c i d (C MPA) a c i d ( MMPA) a c i d ( PMPA)

H 3C P O H 3C P O
CH3 CH3
O O
OH OH
H 3C
H 3C P O H 3C P O
CH3 CH3
OH OH
H 3C
Intensity %
3 Isopropyl Et hyl
m e t h y l p h o s p h o ni c m e t h y l p h o s p h o ni c
a c i d ( IMPA) a c i d ( EMPA)
4
com p ounds
1. Pinacolyl methylphosphonic acid (PMPA)

5 2. 2-(methyl)propyl methylphosphonic acid (MMPA)
3. Cyclohexyl methylphosphonic acid (CMPA)
4. Isopropyl methylphosphonic acid (IMPA)
5. Ethyl methylphosphonic acid (EMPA)
0
0 1 2 3 4 5 6 7 8 9 min
WA60100
25
Analysis of Histidine dipeptides using ACQUITY UPLC BEH Amide Columns
T e st condit ions
C hromatograp hic Conditions Mass Spectrometer Settings
Column: ACQUITY UPLC® BEH Amide, Ionization Mode: ES +

2.1 x 50 mm, 1.7 μm Capillary: 2.5 KV
Part Number: 186004800 Cone: 20 V (Carnosine; Creatinine,
Mobile Phase A: 50/50 MeCN/H2O with 10 mM Anserine); 25 V (Creatine)
CH3COONHz and 0.04 % NH4OH, Source Temperature: 120 °C
pH 9.0 Desolvation Temperature: 400 °C
Mobile Phase B: 95/5 MeCN/H2O with 10 mM Desolvation Gas Flow: 800 L/Hr
CH3COONH4 and 0.04 % NH4OH, Cone gas Flow: 5 L/Hr
pH 9.0 SIR m/z: 227.1 (Carnosine); 132.1 (Creatine);
Flow Rate: 0.5 mL/min 114.05 (Creatinine); 241.1(Anserine)
Gradient: Time Profile Dwell Time: 0.1 s
(min) %A %B
Initial 0.1 99.9
5.00 65.0 35.0
5.01 0.1 99.9 St ruc t u r e s
6.00 0.1 99.9 O
H
Injection Volume: 5 µL N
HO OH CH3
Sample Diluent: 75/25 MeCN/MeOH
Column Temperature: 65 °C O NH N N NH
O
NH2
Instrument: Waters ACQUITY UPLC NH2
with ACQUITY SQD

C a r n o s in e C r e a t in e
CH3 H 3C
100 1
N NH NH2
N
3.87e7
O NH
N O
H N HO O
C r e a t inin e A n s e r in e
Intensity %
com p ounds
1. Creatinine (1 µg/mL)
2. Creatine (5 µg/mL)
3. Anserine (5 µg/mL)
2 4. Canosine (5 µg/mL)
3
0
0 1 2 3 4 5 min
WA60103
26
Analysis of Mono-, Di- and Oligosaccharides
T e st condit ions

triethylamine [TEA]
Gradient: 5 minute gradient, 80%-50% MeCN STRUC TURE S Fructose
with 10 minute re-equilibration Sucrose
Time Profile
(min) %A %B
0.00 100.00 0.00
5.00 40.00 60.00
5.01 100.00 0.00 Sucrose Fructose
p-Toluamide
15.00 100.00 0.00
O
Sample Concentration: 1 mg/mL each NH 2

H3C
n= 1 to 5 n
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) p-Toluamide
Maltooligosaccharides (unretained compound)
Maltose Glucose
4 Com p ounds
1. p-Toluamide 4. Sucrose 7. Maltotetraose

2. Fructose 5. Maltose 8. Maltopentaose
8 3. Glucose 6. Maltotriose 9. Maltohexahose
7
2
6 9 10. Maltoheptaose
3
5
10
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
WA60110
27
UPLC /MS Analysis of Mono-, Di- and Oligosaccharides
T e st condit ions
Column: ACQUITY UPLC® BEH Amide Ionization Mode: ESCapillary: 2.8 kV

2.1 x 50 mm, 1.7 µm Cone Voltage: 25 V
Part Number: 186004800 Source Temperature: 120 °C
Mobile Phase A: 80/20 MeCN/H2O with 0.10% Desolvation Temperature: 350 °C
ammonium hydroxide [NH4OH] Desolvation Gas Flow: 500 L/Hr
Mobile Phase B: 30/70 MeCN/H2O with 0.10% Cone: 50 L/Hr
ammonium hydroxide [NH4OH] SIR (m/z): 179.2 (fructose, glucose);
Flow Rate: 0.17 mL/min 341.3 (sucrose, maltose);
Gradient: 5 minute gradient, 80%-50% MeCN 503.4, 665.5, 827.6,
with 10 minute re-equilibration 989.7, 1151.8
Time Profile (maltooligosaccharides
(min) %A %B [n=1 to 5])
0.00 100.00 0.00 Dwell Time: 0.08 s
5.00 40.00 60.00
5.01 100.00 0.00
15.00 100.00 0.00
STRUC TURE S
Fructose
Glucose
Sample Concentration: 10 µg/mL each Maltose

Weak Needle Wash: 75/25 MeCN/H2O (500 µL) Glucose Fructose Maltose

Instrument: Waters ACQUITY UPLC with Sucrose
ACQUITY TQD
n= 1 to 5 n
Sucrose Maltooligosaccharides
3
100 SIR of 7 Channels ES-
TIC
5.68e6
Com p ounds
1. Fructose 4. Maltose 7. Maltopentaose

%
3. Sucrose 6. Maltotetraose 9. Maltoheptaose

4 5
2
1 7
8
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
WA60112
28
Analysis of Mono-, Di- and Oligosaccharides Using ACQUITY UPLC
BEH Amide Columns with Acetone as Organic Modifiers
T e st condit ions

Mobile Phase A: 80/20 acetone/H2O with 0.05% Nebulizer: Cooling
Mobile Phase B: 30/70 acetone/H2O with 0.05% Filter Time Constant: Normal
triethylamine [TEA]
Gradient: 5 minute gradient, 80%-60% MeCN STRUC TURE S
with 10 minute re-equilibration Fructose
Time Profile Sucrose
(min) %A %B
0.00 100.00 0.00
5.00 60.00 40.00
5.01 100.00 0.00 Sucrose Fructose
15.00 100.00 0.00 p-Toluamide
O
Sample Concentration: 1 mg/mL each
Sample Diluent: 50/50 MeCN/H2O NH 2
Strong Needle Wash: 20/80 MeCN/H2O (800 µL) H3C
n= 1 to 5 n
Weak Needle Wash: 75/25 MeCN/H2O (500 µL) p-Toluamide
Maltooligosaccharides (unretained compound)
Maltose Glucose
Maltose Glucose
Com p ounds
1. p-Toluamide 4. Sucrose 7. Maltotetraose

2 4 2. Fructose 5. Maltose 8. Maltopentaose
5 6 7 8
9 10. Maltoheptaose
10
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
WA60111
29
Analysis of Morphine using ACQUITY UPLC BEH Amide Columns
T e st condit ions
Column: ACQUITY UPLC® BEH Amide, Ionization Mode: ES +

2.1 x 50 mm, 1.7 μm Capillary: 3.0 KV
Part Number: 186004800 Cone: 30 V (6-Acetyl morphine and Morphine),
Mobile Phase A: 50/50 MeCN/H2O with 10 mM 40 V (Morphine-3β-D-glucuronide)
NH4 COOH and 0.125% HCOOH, pH 3 Source Temperature: 120 °C
Mobile Phase B: 90/10 MeCN/H2O with 10 mM Desolvation Temperature: 350 °C
NH4 COOH and 0.125% HCOOH, pH 3 Desolvation Gas Flow (L/Hr): 800
Flow Rate: 0.6 mL/min SIR m/z: 329.5 (6-Acetyl morphine);
Gradient: Time Profile 287.5 (Morphine)
(min) %A %B 463.6 (Morphine-3β-D-glucuronide)
Initial 0.1 99.9 Dwell Time: 0.1 s
1.05 0.1 99.9
4.35 99.9 0.1
4.50 0.1 99.9
6.00 0.1 99.9 st ruc t u r e s
Injection Volume: 5 µL (PLNO)
HO O
Sample Diluent: 75/25 MeCN/MeOH with HO
O
OH
0.2% HCOOH O
Column Temperature: 30 °C O H
HO HO O H
NCH3
Weak Needle Wash: 95/5 MeCN/H2O NCH3
Instrument: Waters ACQUITY UPLC with HO
HO
ACQUITY SQD
M o r p hin e M o r p hin e - 3 ß - D - gl u c u r o ni d e
HO
100
1
1.36e7 O H
NCH3
6 - a c e t y l m o r p hin e
2
Intensity %
com p ounds
1. 6-acetylmorphine (100 ng/mL)

2. Morphine (100 ng/mL)
3. Morphine-3 ß-D-glucuronide (5 g/mL)
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
WA60101
30
Analysis of Nucleobases Using 30 MM ACQUITY UPLC BEH Amide Columns
T e st condit ions
Column: ACQUITY UPLC® BEH Amide, Injection Volume: 3.0 µL (PLNO)

2.1 x 30 mm, 1.7 µm Sample Concentration: 20 μg/mL each
Part Number: 186004839 Sample Diluent: 75/25 MeCN/MeOH
Mobile Phase A : 50/50 MeCN/H2O with 10 mM Column Temperature: 30 °C
HCOONH4 and 0.125% HCOOH, Weak Needle Wash: 95/5 MeCN/H2O
pH 3.0 Detection: UV @ 260 nm
Mobile Phase B : 95/5 MeCN/H2O with 10 mM Sampling Rate: 20 points/sec
HCOONH4 and 0.125% HCOOH, Filter Time Constant: 0.2
pH 3.0 Instrument: Waters ACQUITY UPLC with
Flow Rate: 1.0 mL/min ACQUITY UPLC PDA Detector
Gradient: Time %A %B
Initial 1 99
1.50 50 50 com p ounds
1.51 1 99
1. Thymine
2.70 1 99
2. Uracil
3. Adenine
4. Cytosine
5. Guanine
0.6
1
0.5
0.4
0.3
AU
5
4
0.2
0.1
0
0 0.1 0.2 0.3 0.4 0.5 min
WA60099
31
Analysis of Nucleobases using ACQUITY UPLC BEH Amide Columns
NH2
Columns: ACQUITY UPLC® BEH Amide, HN O
H
N O
2.1 x 50 mm, 1.7 µm N
N
NH NH
Part Number: 186004800 H 3C
N
Mobile Phase A : 50/50 MeCN/H2O with 10 mM O
O
N H
HCOONH4 and 0.125% HCOOH,

T h y m in e Uracil A d e nin e
pH 3.0
H O
Mobile Phase B : 95/5 MeCN/H2O with 10 mM N O
HCOONH4 in H2O and 0.125% HN

N
N
HCOOH, pH 3.0
H 2N N
N
Flow Rate: 0.5 mL/min H 2N H
Gradient: Time %A %B C y t o s in e G u a nin e

Initial 1 99
5.00 50 50
5.01 1 99
9.00 1 99
Injection Volume: 5.0 µL (PLNO) com p ounds
1. Thymine
Sample Diluent: 75/25 MeCN/MeOH
2. Uracil
3. Adenine
4. Cytosine
4. Guanine
Instrument: Waters ACQUITY UPLC
with ACQUITY UPLC PDA Detector
0.5
2
1
3
0.4
5
0.3
4
AU
0.2
0.1
0
0 1 2 3 min
WA60098
32
Analysis of Nucleotide Phosphates using ACQUITY UPLC BEH Amide Columns
Column: ACQUITY UPLC® BEH Amide,

2.1 x 150 mm, 1.7 μm
Isocratic Mobile Phase: 80/20 MeCN/H2O with
2 mM KH2PO4
Flow Rate: 0.5 mL/min 2 ’- D e o x y g u a n o s in e - 5’- T h y m i d in e 5’- m o n o -
Injection Volume: 5.0 µL (PLNO) Tr i p h o s p h a t e Tr i s o d i u m p hosp hate (T M P)
S al t D i h y d r a t e (d G TP )
Sample Diluent: 80/20 MeCN/H2O with 10 mM
CH3COONH4 and 0.02% CH3COOH
ACQUITY UPLC PDA Detector 2 ’- D e o x y c y t i d in e 2 ’- D e o x y g u a n o s in e
5’- m o n o p h o s p h a t e 5’- m o n o p h o s p h a t e s o d i u m
s o d i u m s al t (d C M P ) s al t h y d r a t e (d G M P )
0.10 3
2 ’- D e o x y a d e n o s in e 2 ’- D e o x y a d e n o s in e
5’- m o n o p h o s p h a t e 5’- d i p h o s p h a t e s o d i u m
0.08 (d A M P ) s al t (d ADP )
com p ounds
0.06
1. 2’-Deoxyguanosine-5’-Triphosphate Trisodium Salt Dihydrate
(dGTP) 100 µg/mL
AU
1 2 2. Thymidine 5’-monophosphate (TMP) 10 µg/mL

4
0.04
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
6
0.02 (dGMP) 15 µg/mL
6. 2’-Deoxyadenosine 5’-diphosphate sodium salt (dADP)
15 µg/mL
0
0 2 4 6 8 10 12 min
WA60097
33
Analysis of Organic Acids using ACQUITY UPLC BEH Amide Columns
UPLC condit ions MS condit ions
Column: ACQUITY BEH Amide, Instrument: ACQUITY TQD

2.1 x 100 mm, 1.7 µm Ionization Mode: ES -
Part Number: 186004801 Superscript the -Capillary: 4.0 kV
Mobile phase A: 50/50 MeCN/H2O with 10 mM Cone Voltage: -25 V
CH3COONH4, pH 9.0 Collision Energy: 10 eV
Mobile phase B: 95/5 MeCN/H2O with 10 mM Extractor: 3V
CH3COONH4, pH 9.0 RF Lens: 0.1 V
Gradient Flow Rate: 0.6 mL/min Source Temp: 130 °C
Injection Volume: 5.0 μL Desolvation Temp: 350 °C
Column Temp: 50 °C Desolvation Gas: 650 L/hr
Sample Temp: 5 °C Cone Gas: 0 L/hr
Strong/Weak needle wash: 95/5 MeCN/H2O Collision Gas: 0.1 mL/min
Seal wash: 10/90 MeOH/H2O MRM condition: Pyruvic acid: 86.92 > 42.9
Instrument: ACQUITY UPLC and TQD Lactic acid: 88.92 > 42.9
Gradient: Time Succinic acid: 116.93 > 72.9
(min) %A %B Maleic and Fumaric acid: 114.88 > 70.9
Initial 0.1 99.9
0.4 0.1 99.9
0.5 40.0 60.0 St ruc t u r e s
2.0 70.0 30.0 O
2.01 0.1 99.9
5.0 0.1 99.9
OH HO
OH
O O
com p ounds
O
1. Maleic acid (1 ppm) 4. Succinic acid (50 ppm)
M al e i c A c i d Pyruvic Acid
2. Pyruvic acid (50 ppm) 5. Fumaric acid (50 ppm)
3. Lactic acid (50 ppm) O O
100 4 8.82e5 HO
OH OH
Intensity %
0 O
100 1
1.65e6
OH
L actic Acid S u c c ini c A c i d
Intensity %
0
O
100
3
9.53e 4
HO
Intensity %
OH
0
100
2
5.43e4 O
Intensity %
Fumaric Acid
0
0 0.5 1.0 1.5 2.0 2.5 min
WA60096
34
Analysis of Stevia Related Compounds Using ACQUITY UPLC BEH Amide Columns
T e st condit ions STRUC TURES

CH2OH
Column: ACQUITY UPLC® BEH Amide HC OH
2.1 x 100 mm, 1.7 µm HC OH
CH2OH
Erythritol
triethylamine [TEA]
triethylamine [TEA] Stevioside

Flow Profile: 95% A/5% B (77.5% MeCN with
0.20% TEA)
Sample Concentration: 5 mg/mL
Instrument: Waters ACQUITY UPLC with ELSD Rebaudioside A
ELSD Conditions
Gain: 200
Rebaudioside C
Pressure: 40 psi
Nebulizer: Cooling
Data Rate: 10 pps 50.0
Filter Time Constant: Normal 2000.0

45.0
40.0
35.0
Stevioside
1800.0 30.0
Erythritol
Rebaudioside A
Rebaudioside C
25.0
1600.0 Stevia
Stevioside
20.0
Erythritol
(5 mg/mL)
15.0
1400.0
10.0
Rebaudioside A
5.0
1200.0 ( 5mg/mL)
0.0
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 min
1000.0
Rebaudioside C
800.0
600.0
Stevia
400.0 (5 mg/mL)
200.0
(5 mg/mL)
0.0
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
WA60128
35
UPLC /MS Analysis of Stevia Related Compounds Using
ACQUITY UPLC BEH Amide Columns
T e st condit ions STRUC TURES

CH2OH
Column: ACQUITY UPLC® BEH Amide HC OH
HC OH
2.1 x 100 mm, 1.7 µm CH2OH
Erythritol
Stevioside
Flow Profile: 95% A/5% B (77.5% MeCN with
0.10% NH4OH)
Sample Concentration: 50 µg/mL stevia, 10 µg/mL truvia
Instrument: Waters ACQUITY UPLC with Rebaudioside A
ACQUITY TQD
Mass Spectrometer Conditions

Rebaudioside C
Capillary: 2.8 kV
Cone Voltage: 25 V
Source Temperature: 120 °C
Desolvation Temperature: 350 °C
Desolvation Gas Flow: 500 L/Hr 100 Rebaudioside A TIC
(m/z 966.1)
7.83e5
Erythritol
Cone: 50 L/Hr (m/z 121.1)
SIR of m/z
SIR (m/z): 317.5 (steviol); 160X 803.8+950.1+966.1

4.71e3
zoom
%
803.8 (stevioside);
(10 g/mL)
950.1 (rebaudioside C);
0
966.1 (rebaudioside A) 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
Dwell Time: 0.08 s 100 SIR of 5 Channels ES-

TIC
Stevioside 1.15e6
(m/z 803.8)
Rebaudioside C
(m/z 950.1)
%
Stevia Rebaudioside A
(50 g/mL) (m/z 966.1)
0
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
WA60129
36
Analysis of Thiourea using ACQUITY UPLC BEH Amide Columns
Column: ACQUITY UPLC® BEH Amide, S

2.1 x 150 mm, 1.7 µm
Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/H2O with 10 mM
CH3COONH4 and 0.01% NH4OH, pH 9.0
H 2N NH2
Injection Volume: 5.0 µL (PLNO) T hi o u r e a
0.6
k prime = 1.4
0.5
0.4
0.3
AU
0.2
0.1
t0 = 1.8 min
0
0 1 2 3 4 5 6 7 8 min
WA60095
37
Analysis of Uric Acids using ACQUITY UPLC BEH Amide Columns
O O O O
H H 3C H 3C H
H H
N N N N
Column: ACQUITY UPLC® BEH Amide, HN O HN O N O N O
O O O
2.1 x 50 mm, 1.7 µm H H H 3C H 3C H H
N N
N N N
Part Number: 186004800 O HN N O HNH N O N N O NH N H
H H H
H H O O O
Mobile Phase A : 50/50 MeCN/H2O with 10 mM
N N N N
O N O H N H O N
H
O H N
H
H
H H
pH 5
Mobile Phase B : 90/10 MeCN/H2O with 10 mM Uric Acid 1- M e t h y l u r i c a c i d
pH 5 O O O O
CH3 CH
Flow Rate: 0.5 mL/min H 3C H 3C H H
N N N
Gradient: Time Profile N O N O HN O HN O N
CH3 CH
O O O
(min) %A %B H 3C H 3C H H
N N N
Initial 0.1 99.9 O N N O NH N H O HN N O HNH N N
H
5.00 50.0 50.0 O O O
CH3 N CH3 N CH3 N CH3 N
5.01 0.1 99.9 O N O H N H O N O H N H
9.00 0.1 99.9 1, 3 - D i m e t h y l u r i c a c i d 3,7- D i m e t h y l u r i c a c i d
CH3 CH3 CH3 CH3
Sample Diluent: 100% MeCN
Column Temperature: 25 °C com p ounds
Weak Needle Wash: 95/5 MeCN/H2O 1. Uric Acid
Detection: UV @ 285 nm 2. 1-Methyluric acid
Sampling Rate: 20 points/sec 3. 1,3-Dimethyluric acid
Filter Time Constant: 0.2 4. 3,7-Dimethyluric acid
0.08 1
2
0.06
3 4
0.04
AU
0.02
0 1 2 3 4 5 min
WA60094
38
HN N N
H
Analysis of Water Soluble Vitamins using ACQUITY UPLC BEH Amide
O
Columns
N CH CH
N HC 3
N CH
HO CH
NH2 O
OH
HC
O NH
C lic k on t he underlined blue t e x t for details on t he p roduc t s used
O in t his a p p licat
O ion OH
O NH
O
O CH
P N
OH
OH
T e st condit ions St ruc t u r e s O
N
O CH
HO HO
N CH3 OH
Column: ACQUITY UPLC BEH Amide,
N
®
N
N CH3
N N O N CH3
N HO O N
O
2.1 x 50 mm, 1.7 μm HO HO OH
HO
NH2 O O OH
NH2 OH HOH C O
Part Number: 186004800 3 N NH2 N OH
Mobile Phase A: 50/50 MeCN/H2O with 10 mM O O O O OH O OH

NH O HO HO OH
H3C N
CH3COONH4 and 0.04 % NH4OH,
P y r i d o x al N i c o t ini c a cOi d Ascorbic acid
pH 9.0 OH OH
OH
Mobile Phase B: 90/10 MeCN/H2O with 10 mM HO HO HO NN CH3
CH
O NN
NN HO HO
3
OH HO
OH
CH3COONH4 and 0.04 % NH4OH, HOHO
HO O N OH CH3
HO O O
OH NH2
NH
OHN OH N OO HO O O
pH 9.0 OH 2 NOH N
OH
+
H3C O N O
H3C N NN ON
Flow Rate: 0.5 mL/min OO
H3C O
O
H3 C
H N
N NH
N OH
O
OH
NH
N 2 S OH
Gradient: Time Profile HO HO
NH HO HO HO NH HO
H3C N i c o t in a m i d e
N
NH NH O
(min) %A %B
H3C N HC
OH 3
N F ol i c A c i d
O OH O
O O
Initial 0.1 99.9 HOHO HO HO HO
HO
OH
3.50 70.0 30.0 OH
HO
HO OO OO
CH3OH
3.51 0.1 99.9 CH3 OH CH3
HH3C C NN+N
NN OO
7.50 0.1 99.9 N N 3
N+
N N +
Injection Volume: 5 µL (PLNO) H3 C H3 C N NH2 S NH

HO
HO HO
HO
N H CNH S NH
OH OH H3C NH2
NN N S
2
H3C OH
Sample Diluent: 75/25 MeCN/MeOH with 0.2% HCOOH 3
OO
Column Temperature: 30 °C T hi a m in e
Weak Needle Wash: 95/5 MeCN/H2O R i b o f l a v in
O
Detection: UV @ 265nm HN
CH3
CH 3
CH O
HC
Sampling Rate: 20 points/sec NN NN+
+
Filter Time Constant: 0.2 NH

HH3C
3
C
NN
HN NH2
NH SS OH O
OH
Instrument: Waters ACQUITY UPLC with N
2
N
O HC
ACQUITY UPLC PDA Detector HC N NH
HN N N
H
CH
O HC
CH
CH
HC
NH O
O NH
O
O CH
6 P N
0.6
OH
O
1 O N CH
0.5
O
5 OH
0.4
4 B12
AU
0.3
com p ounds
0.2
2
1. Nicotinamide (25 µg/mL) O 5. Thiamine (50 µg/mL)
7
N
0.1
3
2. Pyridoxal (50 µg/mL)
HN N
6. Ascorbic acid (25 µg/mL)
8
3. Riboflavin (50 µg/mL) N 7. B12 (50 µg/mL) O
H NH
N
0 4. Nicotinic acid (50 µg/mL) 8. Folic Acid (25 µg/mL)
0 1 2 3 min NH
O
O
HO
WA60093
HO
39
Sales Offices
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(Central South Eastern Europe, CIS
Norway 47 6 384 60 50
and Middle East) 43 1 877 18 07
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China 86 10 8586 8899
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CIS/Russia +7 495 3367000
Taiwan 886 2 2543 1898
Czech Republic 420 2 617 1 1384
United Kingdom 44 208 238 6100
Denmark 45 46 59 8080
All other countries:
Finland 09 5659 6288
Waters Corporation U.S.A.
France 33 1 30 48 72 00 1 508 478 2000
1 800 252 4752
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www.waters.com
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India and India Subcontinent

91 80 2837 1900
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Mexico 52 55 5200 1860
©2009 Waters Corporation. ACQUITY, ACQUITY UPLC, Alliance,

Atlantis, IS, Nova-Pak, Oasis, Platform LC, Quattro Ultima, Sentry, SunFire,
SymmetryShield, T he Science of W hat’s Possible, Waters, XTerra and ZQ.
T he quality management system of Waters’ manufacturing facilities All other trademarks are acknowledged.
in 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 WA64081 October 2009 KK-IH-PDF
audited by the registering body to ensure compliance.

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ACQUITY UPLC BEH AMIDE COLUMNS

T e st condit ions St ruc t u r e

Columns: ACQUITY UPLC® BEH Amide,

Part Number: 186004800

0.40 k prime = 2.2

t0= 1.3 min

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

T e st condit ions St ruc t u r e s

Columns: ACQUITY UPLC® BEH Amide, O O

Column Temperature: 25 °C CH3

T e st condit ions St ruc t u r e

Column: ACQUITY UPLC® BEH Amide, H

0 2.0 4.0 6.0 8.0 10 12 14 16 min

T e st condit ions STRUC TURE S

10.00 60.00 40.00 Melezitose Maltooligosaccharides

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 min

0.6 Injection 2000

C hromatograp hic Conditions EL SD Conditions

Column: ACQUITY UPLC® BEH Amide Gain: 200

Com p ounds H3C

Raisin Bran Brand Cereal

T e st condit ions STRUC TURE S

Column: ACQUITY UPLC® BEH Amide

EL SD Conditions Com p ounds

0.0 1.0 2.0 3.0 4.0 min

T e st condit ions STRUC TURE S

Column: ACQUITY UPLC® BEH Amide

Flow Rate: 0.13 mL/min

EL SD Conditions Com p ounds

Gain: 200 1. p-Toluamide 4. Sucrose

C hromatograp hic Conditions EL SD Conditions

Column: ACQUITY UPLC® BEH Amide Gain: 200

Instrument: Waters ACQUITY UPLC with ELSD

T e st condit ions STRUC TURE S

Column: ACQUITY UPLC® BEH Amide

Flow Rate: 0.15 mL/min

Hot Cross Buns

1. p-Toluamide White Bread

0.0 1.0 2.0 3.0 4.0 min

T e st condit ions STRUC TURE S

Column: ACQUITY UPLC® BEH Amide

0.0 1.0 2.0 3.0 4.0 min

C hromatograp hic Conditions EL SD Conditions

C hromatograp hic Conditions EL SD Conditions

Column: ACQUITY UPLC® BEH Amide Gain: 200

Sample Diluent: 50/50 MeCN/H2O

C hromatograp hic Conditions Mass Spectrometer Conditions

Weak Needle Wash: 75/25 MeCN/H2O (500 µL)

1. Fructose 3. Sucrose Maltose Lactose Glucose

Column: ACQUITY UPLC® BEH Amide Glucose

Injection Volume: 1.3 µL (PLNO) H3C

EL SD Conditions Wheat Beer

Filter Time Constant: Normal

C hromatograp hic Conditions Mass Spectrometer Conditions

Column: ACQUITY UPLC® BEH Amide Ionization Mode: ES -

35.00 90.00 10.00

Sample Diluent: 50/50 MeCN/H2O Maltooligosaccharides

SIR of 3 Channels ES-

Column: ACQUITY UPLC® BEH Amide Glucose

C hromatograp hic Conditions

Column: ACQUITY UPLC® BEH Amide Glucose

Mobile Phase A: 80/20 MeCN/H2O with 0.2%

Time Profile Maltooligosaccharides

Injection Volume: 0.7 µL (PLNO) Maltooligosaccharides