acquity uplc beh amide columns application notebook · click on the underlined blue text for...

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

http://www.waters.com/waters/partDetail.htm?cid=511505&id=38549


O

H2C

CH3

NH2

O

H2C

CH3

OH

O

H2CNH2


Analysis of Acrylamide, Methacrylic Acid and Methacrylamide

using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS


2.1 x 150 mm, 1.7 µm


Isocratic Mobile Phase: 95/2.5/2.5 MeCN/IPA/H2O with 5 mM

CH3COONH4 and 0.02% NH4OH,

pH 9.0



Sample Concentration: 30 μg/mL each









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




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


Isocratic Mobile Phase B: 90/10 MeCN/H2O




Sample Diluent: 90/10 MeCN/H2O








St ruCture

all anto in

NH

NHHN

O

O

O

H2N

Wa60107

4




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


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

triethylamine [TEA]

Mobile Phase B: 30/70 MeCN/H2O with 0.2%

triethylamine [TEA]


Gradient: 10 minute gradient, 80%-50%

MeCN (w/0.2% TEA) with 30 minute

re-equilibration

Time Profile

(min) %A %B


Sample Concentration: 1 mg/mL each



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



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


Chemical Stability STUDY OF ACQUITY UPLC BEH AMIDE COLUMNS

t eSt COndit iOnS


2.1 x 50 mm, 1.7 µm


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


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 Diluent: 75/25 MeCN/MeOH




Sampling Rate: 40 pts/sec




C yto s ine

N

NH

O

NH2

F

NH

NH

O

O

N

NH

O

NH2

Wa60106

6





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


Nebulizer: Cooling

Data Rate: 10 pps


St ruCtureS

Glucose

Glucose

CH3

NH2

O

p-Toluamide(unretained compound)


Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

t eSt COndit iOnS



2.1 x 150 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]


Flow Profile: 90% A/10% B (75% MeCN with

0.2% TEA)


Sample Concentration: Standards at 1 mg/mL each, cereal

extracted at 8 mg/mL







COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

Wa60120

7



Food Sugars

1

2 4

5 6

0.0 1.0 2.0 3.0 4.0 min

3

3

2 5

Tomato Ketchup


Analysis of Food Sugars in Ketchup Using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm


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 Profile: 95% A/5% B (77.5% acetone with

0.05% TEA)


Sample Concentration: Standards at 1 mg/mL each







elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

St ruCtureS

Wa60117

Glucose

Glucose

CH3

NH2

O



Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

8



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


Analysis of Food Sugars in MILK Using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]



0.2% TEA)




Column Temperature: 35°C





elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

Wa60118

St ruCtureS

Glucose

Glucose

CH3

NH2

O



Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

9



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


Analysis of Food Sugars in Molasses Using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS



2.1 x 150 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]



0.2% TEA)


Sample Concentration: Standards at 1 mg/mL each, molasses

at 5 mg/mL







elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

Wa60119

St ruCtureS

Glucose

Glucose

CH3

NH2

O



Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

10



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


Analysis of Food Sugars In Prepared FOods


t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]


Flow Profile: 95% A/5% B (77.5% acetone

with 0.05% TEA)









elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

Wa60115

St ruCtureS

Glucose

Glucose

CH3

NH2

O



Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

11



Food Sugars

1

2 4

5 6

Gatorade BrandSports Drink

0.0 1.0 2.0 3.0 4.0 min

3

3

2


Analysis of Food Sugars in Sports Drink


t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]


Flow Profile: 95% A/5% B (77.5% acetone with

0.05% TEA)









elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


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



Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

12



0 1 2 3 4 5 6 7 8 9 10 min

CabernetSauvignon

Chardonnay

1 2


Analysis of Food Sugars in Wine Using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS



2.1 x 150 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]



0.2% TEA)


Sample Concentration: 50% wine in diluent


Column Temperature: 35°C





COMpOundS

1. Fructose

2. Glucose

Glucose

GlucoseFructose

Fructose

St ruCtureS

elSd ConditionsGain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


Wa60114

13



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


Analysis of Food Sugars Using ACQUITY UPLC BEH Amide Columns

elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]



0.2 % TEA)









COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

Wa60109

St ruCtureS

Glucose

Glucose

CH3

NH2

O



Fructose

Fructose

Maltose

Maltose

Sucrose

Sucrose

Lactose

14



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


UPLC/MS Analysis of Food Sugars Using ACQUITY UPLC

BEH Amide Columns with Acetone as Organic Modifier

t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm



ammonium hydroxide [NH4OH]




Flow Profile: 94% A/6% B (77% acetone with

0.05% NH4OH)


Sample Concentration: 10 µg/mL each







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



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


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



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]




re-equilibration

Time Profile

(min) %A %B


Sample Concentration: Standards at 1 mg/mL, beer at 100%

(No dilution)







elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


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


nn= 1 to 5


CH3

NH2

O



Fructose

Fructose

Glucose

Glucose

St ruCtureS

Maltose

Maltose

16



Sucrose

Food SugarStandard


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


1

Disaccharides(m/z = 341.1)

Trisaccharides(m/z = 503.2)


UPLC/MS Analysis of Food Sugars/Saccharides in Beer


t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm







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


Sample Concentration: Standards at 10 µg/mL, Beer at

50% dilution




Weak Needle Wash: 75/2MeCN/H2O 5 (500 µL)


Instrument: Waters ACQUITY UPLC with SQ

COMpOundS

1. Fructose

2. Glucose

3. Sucrose

4. Maltose

5. Maltotriose


nn= 1 to 5


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


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



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


t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]



(w/ 0.2% TEA) with 12 minute

re-equilibration

Time Profile

(min) %A %B


Sample Concentration: Standards at 1 mg/mL each, cough

syrups at 1% (v/v)



Strong Needle Wash: MeCN/H2O 20/80 (800 µL)

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

Seal Wash: MeCN/H2O 50/50


elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


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


nn= 1 to 5


CH3

NH2

O



Fructose

Fructose

Glucose

Glucose

St ruCtureS

Maltose

Maltose

18



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


Analysis of Food Sugars/Saccharides in Honey


t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]




re-equilibration

Time Profile

(min) %A %B


Sample Concentration: Honey and corn syrup at 5-10 mg/mL

each







elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


St ruCtureS

COMpOundS

1. Fructose

2. Glucose

3. Sucrose

4. Maltose

5. Maltotriose

6. Maltotetraose

7. Maltopentaose

Maltose

Maltose


nn= 1 to 5


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



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


Analysis of Food Sugars/Saccharides in Maple Syrup


t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]




re-equilibration

Time Profile

(min) %A %B


Sample Concentration: Maple syrups at 5-10 mg/mL each







elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


St ruCtureS

COMpOundS

1. Fructose

2. Glucose

3. Sucrose

4. Maltose

5. Maltotriose

6. Maltotetraose

7. Maltopentaose

Maltose

Maltose


nn= 1 to 5


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



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


Analysis of Food Sugars/Saccharides in Potato Chips


COMpOundS

1. p-Toluamide

2. Fructose

3. Glucose

4. Sucrose

5. Maltose

6. Lactose

Lactose

CH3

NH2

O



Sucrose

Sucrose

Fructose

Fructose

Glucose

Glucose

St ruCtureS

Maltose

Maltose

Wa60122

t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]




re-equilibration

Time Profile

(min) %A %B


Sample Concentration: Standards at 1 mg/mL each, potato

chips extracted at 120 mg/mL







elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


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



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


HILIC Gradient Separation of Ascorbic Acid And Isoascorbic

Acids using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS


2.1 x 100 mm, 1.7 μm



CH3COONH4 and 0.02% CH3COOH,

pH 5.0



pH 5.0



(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)





Detection: UV @ 260nm





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




HILIC Gradient Separation of Organophosphonic Acids


t eSt COndit iOnS



2.1 x 100 mm, 1.7 µm



CH3COONH4and 0.04% NH4OH,

pH 9.0

Mobile phase B: 95/5 MeCN/H2O with 10 mM


pH 9.0



(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







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)



Capillary: 2.5 KV

Cone: 30 V (EMPA, IMPA, PMPA);

40 V (CMPA); 35 V (MMPA)




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



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


1 2

1

2

ACQUITY UPLC BEH Amide2.1 x 100 mm, 1.7 µm



HILIC Isocratic Separation of Isoascorbic acid and ascorbic

acid using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS



Isocratic Mobile Phase: 80/20 MeCN/H2O with 10 mM

KH2PO4, pH 4.6












COMpOundS

1. Isoascorbic acid

2. Ascorbic acid

St ruCtureS

O

OH O H

O

OH

OH


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


t est Condit ions



2.1 x 100 mm, 1.7 µm


Isocratic Mobile Phase: 90/10 MeCN/H2O with 10 mM


pH 9.0








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)




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


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



Inte

nsity

%

0

1

2

3

4

100

10 2 3 4 5 min

3.87e7


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



2.1 x 50 mm, 1.7 μm



CH3COONHz and 0.04 % NH4OH,

pH 9.0


CH3COONH4 and 0.04 % NH4OH,

pH 9.0



(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




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)




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



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


Analysis of Mono-, Di- and Oligosaccharides


elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


Wa60110

t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]



with 10 minute re-equilibration

Time Profile

(min) %A %B









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


Maltooligosaccharidesp-Toluamide

(unretained compound)

nn= 1 to 5

Fructose

Fructose

27



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


UPLC/MS Analysis of Mono-, Di- and Oligosaccharides


t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm









Time Profile

(min) %A %B


Sample Concentration: 10 µg/mL each







ACQUITY TQD


Ionization Mode: ESCapillary: 2.8 kV

Cone Voltage: 25 V




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


Sucrose

Sucrose

St ruCtureS

Glucose

Glucose

Fructose

Fructose

Maltose

Maltose

28



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


Analysis of Mono-, Di- and Oligosaccharides Using ACQUITY UPLC

BEH Amide Columns with Acetone as Organic Modifiers

St ruCtureS

CH3

NH2

O


Fructose

Fructose


Maltose

Maltose

Glucose

Glucose

Sucrose

Sucrose


nn= 1 to 5

elSd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


Wa60111

t eSt COndit iOnS



2.1 x 50 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]




Time Profile

(min) %A %B









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



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


Analysis of Morphine using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS



2.1 x 50 mm, 1.7 μm



NH4COOH and 0.125% HCOOH, pH 3


NH4COOH and 0.125% HCOOH, pH 3



(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




ACQUITY SQD


Ionization Mode: ES+

Capillary: 3.0 KV

Cone: 30 V (6-Acetyl morphine and Morphine),

40 V (Morphine-3β-D-glucuronide)



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




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


2.1 x 30 mm, 1.7 µm


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


pH 3.0


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











Wa60099

31



AU

0

0.1

0.2

0.3

0.4

0.5

0 1 2 3 min

12

3

4

5


Analysis of Nucleobases using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS


2.1 x 50 mm, 1.7 µm




pH 3.0


HCOONH4 in H2O and 0.125%

HCOOH, pH 3.0


Gradient: Time %A %B

Initial 1 99

5.00 50 50

5.01 1 99

9.00 1 99









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



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


Analysis of Nucleotide Phosphates using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS


2.1 x 150 mm, 1.7 μm


Isocratic Mobile Phase: 80/20 MeCN/H2O with

2 mM KH2PO4



Sample Diluent: 80/20 MeCN/H2O with 10 mM

CH3COONH4 and 0.02% CH3COOH








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



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


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


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


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



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)


Analysis of Stevia Related Compounds Using ACQUITY UPLC BEH Amide Columns

t est Condit ions



2.1 x 100 mm, 1.7 µm



triethylamine [TEA]


triethylamine [TEA]


Flow Profile: 95% A/5% B (77.5% MeCN with

0.20% TEA)


Sample Concentration: 5 mg/mL







elsd Conditions

Gain: 200

Pressure: 40 psi


Nebulizer: Cooling

Data Rate: 10 pps


st ruCtures

CH 2 OH HC OH HC OH

CH 2 OH

Erythritol

Stevioside

Rebaudioside A

Rebaudioside C

Wa60128

35


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


UPLC/MS Analysis of Stevia Related Compounds Using

ACQUITY UPLC BEH Amide Columns

t est Condit ions



2.1 x 100 mm, 1.7 µm







Flow Profile: 95% A/5% B (77.5% MeCN with

0.10% NH4OH)


Sample Concentration: 50 µg/mL stevia, 10 µg/mL truvia







ACQUITY TQD

Mass spectrometer Conditions


Capillary: 2.8 kV

Cone Voltage: 25 V




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



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


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












St ruCture

t hio u re a

S

NH2H2N

Wa60095

37



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


t eSt COndit iOnS


2.1 x 50 mm, 1.7 µm




pH 5



pH 5



(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



Sample Diluent: 100% MeCN








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



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


Analysis of Water Soluble Vitamins using ACQUITY UPLC BEH Amide Columns

t eSt COndit iOnS


2.1 x 50 mm, 1.7 μm




pH 9.0



pH 9.0



(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)









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


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.

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