XBridge Amide HPLC CoLumns · mide W a 64098 LSU 0 500 1000 1500 LSU 0 500 1000 1500 LSU 0 500 1000 1500 0 2 4 6 8 10 12 14 16 min Saccharide Standard 1 mg/mL Robitussin ® Brand

XBridge Amide HPLC CoLumns A p p l i c a t i o n N o t e b o o k

SiO

O

O

NH 2Linker

Description Page Number Part Number

ELSD Applications

Cellulosic Hydrosylates 4 WA64102

Food Sugars and Polysaccharides

Beer 5 WA64101

Bran with Raisin Cereal 6 WA64089

Cough Syrup 7 WA64098

Honey 8 WA64099

Ketchup 9 WA64090

Maple Syrup 10 WA64100

Milk 11 WA64091

Molasses 12 WA64092

Potato Chips 13 WA64096

Prepared Foods 14 WA64093

Sports Drink 15 WA64094

Sugar Standard 16 WA64088

Sugar/Saccharide Standard 17 WA64097

Wine 18 WA64095

Stevia Related Compounds 19 WA64103

MS Applications

Food Sugars and Polysaccharides

Beer 20 WA64105

Organophosphonic Acids 21 WA64107

Sugar/Saccharide Standard 22 WA64104

Histidine Dipeptides 24 WA64109

Morphine 25 WA64111

Organic Acids 26 WA64110

Organophosphonic Acids 27 WA64108

Stevia and Related Compounds 28 WA64106

[ List of Compounds AnALyzed using XBridge Amide CoLumns ]

Description Page Number Part Number

UV Applications

5-Fluorouracil 29 WA64113

Acrylamide, Methacrylic Acid and Methacrylamide 30 WA64115

Allantoin 31 WA64112

Ascorbic and Isoascorbic Acid - Gradient 32 WA64118

Ascorbic and Isoascorbic Acid - Isocratic 33 WA64119

Ginsenoside Rb1 in Ginseng Root Powder Extract 34 WA64086

Ginsenoside Rb1 in Herbal Medicine Extract 35 WA64087

Nucleobases 36 WA64116

Nucleotide Phosphates 37 WA64121

Thiourea 38 WA64114

Uric Acids 39 WA64117

Water-Soluble Vitamins 40 WA64120

[ 4 ]

ELSD AnALySiS of C ELLuLoSiC HyDroLySAt ES uSing An X BriDgE AmiDE CoLumn

ELSD ConDit ionS

Gain: 100

Pressure: 30 psi

Drift Tube Temperature: 50 ˚C

Nebulizer: cooling

Data Rate: 10 pps

Filter Time Constant: 0.2 sec

SampLE Ex t raCt ion p roC EDurE

1. Dilute with 50:50 acetonitrile/water.

2. Filter using 0.45 µm PVDF syringe filter.

HpLC ConDit ionS

Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm

Mobile Phase A: 80/20 acetonitrile/water with 0.2 % triethylamine

Mobile Phase B: 30/70 acetonitrile/water with 0.2 % triethylamine

Gradient: Time Profile (min) %A %B 0.00 100 0 21.00 40 60 21.01 100 0 33.00 100 0

Flow Rate: 1.0 mL/min

Injection Volume: 15.0 µL

Sample Concentration: 0.8 mg/mL each

Column Temperature: 35 °C

Needle Wash: 75:25 acetonitrile:water

Seal Wash: 50:50 acetonitrile:water

Instrument: Alliance® System with 2424 ELSD

xBr

idge

am

ide

Wa

64

10

2

©2010 Waters Corporation. Waters and Alliance are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. VW-IH-PDF

LSU

0

200

400

600

800

1000

1200

1400

1600

1800

0 2 4 6 8 10 12 14 16 18 20 22 min

Compounds

1. Xylose2. Fructose 3. Mannose

4. Glucose 5. Sucrose 6. Cellobiose

7. Melezitose8. Raffinose9. Maltotriose

10.Maltotetraose11.Maltopentaose12.Maltohexaose

13.Maltoheptaose

1 2

3

4

5

6

7

8,9

10

11

12

13

Cellulosic Hydrolysates 0.8 mg/mL

St ruCturES

OHO

OH

HOOH

OH

Fructose

OHOHO

OHOH

OH

Mannose

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Maltooligosacchrides

OHOHO

HO

OH

OH

Glucose

OHOHO

HO

OH

OOHO

HO

OH

OH

Cellobiose

Xylose

OHOHO

OH OH

Sucrose

OHOHO HO

HO

OHOOH

HO

OHO

Raffinose

OOH

OOH

OH

O

OHOH

OH

OH

OHO

HO

HO

OHO

Melezitose

OHOHO

OH

OH

O

OH

OHOH

O

OHOHO

OH

OH

O

on-LinE orDEring information

Description Part Number

XBridge Amide Column, 3.5 µm, 4.6 x 250 mm 186004870

Acrodisc® LC 13 mm, 0.45 µm, 100/pkg WAT200512

12 x 32 LCMS Certified Glass Screw Neck Vial 600000751CV

http://www.waters.com/waters/partDetail.htm?cid=511505&id=38673http://www.waters.com/waters/partDetail.htm?cid=511544&id=33952http://www.waters.com/waters/partDetail.htm?cid=10000711&id=28885

[ 5 ]

ELSD AnALySiS of fooD SugA rS/SACCHA riDES in BEEr uSing An X BriDgE AmiDE CoLumn

ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps





HpLC ConDit ionS


Mobile Phase A: 80/20 acetonitrile/water with 0.2% triethylamine

Mobile Phase B: 30/70 acetonitrile/water with 0.2% triethylamine




Sample Concentration: 1 mg/mL each

Column Temperature: 35 ˚C

Needle Wash: 75:25 acetonitrile/water

Seal Wash: 50:50 acetonitrile/water


xBr

idge

am

ide

Wa

64

10

1

LSU

0500

10001500

LSU

0500

10001500

LSU

0500

10001500

LSU

0500

10001500

LSU

0500

10001500

0 2 4 6 8 10 12 14 16 18 20 min

Saccharide Standard 1 mg/mL

Wheat Beer 20% (v/v)

Belgian Beer 20% (v/v)

Chocolate Stout 50% (v/v)

Dark Ale 20% (v/v)

1

2 3 4

5 6 7 8 9 10

Compounds

1. p-Toluamide 2. Fructose 3. Glucose

4. Sucrose 5. Maltose 6. Maltotriose

7. Maltotetraose 8. Maltopentaose 9. Maltohexaose

10. Maltoheptaose

St ruCturES

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Fructose

Sucrose


p-Toluamide Glucose

Maltose

NH2O

©2010 Waters Corporation. Waters and Alliance are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. SC-IH-PDF







[ 6 ]

ELSD AnALySiS of fooD SugA rS in BrAn w it H rAiSin C ErEAL uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS




Flow Profile: 90% A/10% B (75% acetonitrile

with 0.2% triethylamine)








ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps



1. Weigh out sample (~ 3g) into 50 mL centrifuge tube.

2. Add 25 mL of 50:50 acetonitrile/water and homogenize.

3. Centrifuge at 3200 rpm for 30 minutes.

4. Collect supernatant and filter using 0.45 µm PVDF syringe filter.

xBr

idge

am

ide

Wa

64

08

9

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

2. Fructose

4. Sucrose

1. p-Toluamide 3. Glucose

5. Maltose

NH2O

LSU

0200400600800

10001200

LSU

0200400600800

10001200

0 2 4 6 8 10 12 14 16 min

Raisin Bran Brand Cereal 4 mg/mL

Food Sugar Standard 1 mg/mL

1

2 3 4

5 6

2 3

4







OHO

HO HO

OHO

OHO HO

HO

OH

6. Lactose

St ruCturES


[ 7 ]

ELSD AnALySiS of fooD SugA rS/SACCHA riDES in CougH Syru p uSing An X BriDgE AmiDE CoLumn

ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps





HpLC ConDitionS












xBr

idge

am

ide

Wa

64

09

8

LSU

0

500

1000

1500

LSU

0

500

1000

1500

LSU

0

500

1000

1500

0 2 4 6 8 10 12 14 16 min


Robitussin® Brand Cough Syrup 0.5% (v/v)

Generic Tussin Cough Syrup 0.2% (v/v)

1

2 3 4

5 6 7 8 10

2 3

2

3

5 6 7 8 9 10

Compounds:




10. Maltoheptaose

St ruCturES

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Fructose

Sucrose


p-Toluamide Glucose

Maltose

NH2O








[ 8 ]

ELSD AnALySiS of fooD SugA rS/SACCHA riDES in HonEy uSing An X BriDgE AmiDE CoLumn

ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps





HpLC ConDit ionS












xBr

idge

am

ide

Wa

64

09

9

LSU

0

500

1000

1500

LSU

0 500

1000

1500

LSU

0 500

1000

1500

LSU

0 500

1000

1500

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 min


1 2 3

4 5 6 7 8 9 10

Corn Syrup 5 mg/mL

Fake Honey 8 mg/mL

Pure Honey 2 mg/mL

2

3

5 6 7 8 9 10

2

3

5 6 7 8 9 10

2 3

Compounds: 1. p-Toluamide 2. Fructose 3.Glucose

4.Sucrose 5.Maltose 6.Maltotriose

7.Maltotetraose 8.Maltopentaose 9.Maltohexaose

10.Maltoheptaose

St ruCturES

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Fructose

Sucrose


p-Toluamide Glucose

Maltose

NH2O








[ 9 ]

ELSD AnALySiS of fooD SugA rS in K Et CHu p uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS




Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)








ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps







xBr

idge

am

ide

Wa

64

09

0

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

2. Fructose

4. Sucrose


5. Maltose

NH2O

LSU

0

500

1000

1500

LSU

0

500

1000

1500

0 2 4 6 8 10 12 14 16 min

Tomato Ketchup 2 mg/mL


1 2

3 4

5 6

2

3

5







OHO

HO HO

OHO

OHO HO

HO

OH

6. Lactose

St ruCturES


[ 10 ]

ELSD AnALySiS of fooD SugA rS/SACCHA riDES in mA p L E Syru p uSing X BriDgE AmiDE CoLumn

ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps





HpLC ConDit ionS












xBr

idge

am

ide

Wa

64

10

0

LSU

0500

10001500

LSU

0500

10001500

LSU

0500

10001500

LSU

0500

10001500

LSU

0500

10001500

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 min


1 2 3 4 5 6 7 8 9 10

Vermont Maple Syrup 1 mg/mL

“Light” Pancake Syrup 5 mg/mL

“Pure” Maple Syrup 5 mg/mL

Log Cabin® Brand Maple Syrup 5 mg/mL

4

2 3 4

2 3

2

3

5 6 7 8 9 10

4

4

Compounds

1. p-Toluamide2. Fructose 3. Glucose


7. Maltotetraose8. Maltopentaose9. Maltohexaose

10. Maltoheptaose

St ruCturES

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Fructose

Sucrose


p-Toluamide Glucose

Maltose

NH2O








[ 11 ]

ELSD AnALySiS of fooD SugA rS in miLK uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS












ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps

Filter Time Constant: 0.2 seconds




xBr

idge

am

ide

Wa

64

09

1

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

2. Fructose

4. Sucrose


5. Maltose

NH2O

LSU

0200400600800

1000

LSU

0 200 400 600 800

1000

0 2 4 6 8 10 12 14 16 min

Low Fat Milk 1% in 50:50 MeCN:H2O


1

2 3 4

5 6

6







OHO

HO HO

OHO

OHO HO

HO

OH

6. Lactose

St ruCturES


[ 12 ]

ELSD AnALySiS of fooD SugA rS in moLASSES uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS












ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps





xBr

idge

am

ide

Wa

64

09

2

LSU

0 200 400 600 800

1000 1200 1400

LSU

0 200 400 600 800

1000 1200 1400

0 2 4 6 8 10 12 14 16 min

Molasses5 mg/mL


1 2

3 4

5 6

2 3

4


OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

2. Fructose

4. Sucrose


5. Maltose

NH2O






OHO

HO HO

OHO

OHO HO

HO

OH

6. Lactose

St ruCturES


[ 13 ]

ELSD AnALySiS of fooD SugA rS in potAto CHipS uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS












ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps







xBr

idge

am

ide

Wa

64

09

6


LSU

0200 400 600 800

1000 1200 1400 1600

LSU

0 200 400 600 800

1000 1200 1400 1600

0 2 4 6 8 10 12 14 16 min


1

2 3 4

5 6

Classical Potato Chips 33 mg/mL

4

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

OHO

OHO HO

HO

OH

2. Fructose

4. Sucrose

6. Lactose


5. Maltose

NH2O






St ruCturES


[ 14 ]

ELSD AnALySiS of fooD SugA rS in p rEpA rED fooDS uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS












ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps







xBr

idge

am

ide

Wa

64

09

3

LSU

0

1000

LSU

0

1000

LSU

0

1000

LSU

0

1000

LSU

0

1000

0 2 4 6 8 10 12 14 16 min

Lamb Curry Meal

Food Sugar Standard 1 mg/mL 1 2 3 4

5 6

2 3 4

2 3

5

2 3

5

2 3 6

4

White Bread

Hot Cross Buns

Strawberry Smoothie

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

OHO

OHO HO

HO

OH

2. Fructose

4. Sucrose

6. Lactose


5. Maltose

NH2O

©2010 Waters Corporation. Waters and Alliance are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. SC--IH-PDF






St ruCturES


[ 15 ]

ELSD AnALySiS of fooD SugA rS in A Sport S DrinK uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS












ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps





xBr

idge

am

ide

Wa

64

09

4

LSU

0

500

1000

1500

2000

LSU

0

500

1000

1500

2000

0 2 4 6 8 10 12 14 16 min

Gatorade Brand Sports Drink 30 µL/mL


1 2 3 4

5 6

2 3


OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

OHO

OHO HO

HO

OH

2. Fructose

4. Sucrose

6. Lactose


5. Maltose

NH2O






St ruCturES


[ 16 ]

ELSD AnALySiS of fooD SugA rS StAnDA rD uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS












ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps







xBr

idge

am

ide

Wa

64

08

8

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

OHO

OHO HO

HO

OH

2. Fructose

4. Sucrose

6. Lactose


5. Maltose

NH2O

LSU

0

200

400

600

800

1000

0 2 4 6 8 10 12 14 16 min

1

2

3

4

5

6








St ruCturES


[ 17 ]

ELSD AnALySiS of fooD SugA rS/SACCHA riDE StAnDA rDS uSing An X BriDgE AmiDE CoLumn

ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps







HpLC ConDit ionS












xBr

idge

am

ide

Wa

64

09

7

St ruCturES

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Fructose

Sucrose


p-Toluamide Glucose

Maltose

NH2O

LSU

0

200

400

600

800

1000

0 2 4 6 8 10 12 14 16 min

1

2 3

4

5 6


7 8 9

10

Compounds:




10.Maltoheptaose








[ 18 ]

ELSD AnALySiS of fooD SugA rS in w inE uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS







Sample Diluent: 50:50 acetonitrile/water





ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps




2. Filter using 0.45 µm PVDF syringe filter, if necessary.

xBr

idge

am

ide

Wa

64

09

5

LSU

0

500

1000

1500

LSU

0

500

1000

1500

LSU

0

500

1000

1500

0 2 4 6 8 10 12 14 16 min

Cabernet Sauvignon 10% in 50:50

MeCN:H2O


1 2 3 4

5 6

Chardonnay10% in 50:50

MeCN:H2O

2 3

2 3

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

OHO

OHO HO

HO

OH

2. Fructose

4. Sucrose

6. Lactose


5. Maltose

NH2O







St ruCturES


[ 19 ]

ELSD AnALySiS of St Ev iA rELAt ED Com pounDS uSing An X BriDgE AmiDE CoLumn

HpLC ConDit ionS





Isocratic: 95% A/5% B (77.5% acetonitrile with 0.2% triethylamine)







ELSD ConDit ionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps






4. Collect supernatant and filter using 0.45 µm

PVDF syringe filter.

xBr

idge

am

ide

Wa

64

10

3

LSU

0

500

1000

1500

2000

LSU

0

500

1000

1500

2000

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

Erythritol

Truvia 5 mg/mL

Stevia 5 mg/mL

Stevioside

Reba

udio

side

A

Reba

udio

side

LSU

0

50

100

LSU

0

50

100

4 6 8 10 min

Eryt

hrito

lSt

evio

side

Reba

udio

side

A

Reba

udio

side

C

Truvia5 mg/mL

Stevia5 mg/mL

St ruCturES

Stevioside

Erythritol

CH2OHHC OHHC OHCH2OH

H

H

CH2

O

OHOHO

HO

OH

O

OHOHO

HO

OH

O

OHOHO

OH

O

Rebaudioside A

H

H

CH2

O

OHOHO

HO

OH

O

OHOHO

HO

OH

O

OHO

OH

O

OHOHO

HO

OH

O

Rebaudioside C

H

H

CH2

O

OHOHO

HO

OH

O

OHOHO

HO

OH

O

OHO

OH

O

OHOHO

O

OH








[ 20 ]

mS AnALySiS of fooD SugA rS/SACCHA riDES in BEEr uSing An X BriDgE AmiDE CoLumn

LC ConDit ionS


Mobile Phase A: 80/20 acetonitrile/water with 0.1% ammonium hydroxide

Mobile Phase B: 30/70 acetonitrile/water with 0.1% ammonium hydroxide





Strong Needle Wash: 20/80 acetonitrile/water

Weak Needle Wash: 75/25 acetonitrile/water


Instrument: ACQUITY UPLC® System with 30-cm column cooler/heater and TQD

mS ConDit ionS

Ionization Mode: negative electrospray

Capillary: 3.0 kV

Cone Voltage: 25 V

Source Temperature: 120 ˚C

Desolvation Temperature: 450 ˚C

Desolvation Gas: 900 L/hr

Cone Gas: 50 L/hr

Dwell Time: 80 ms

SIR (m/z): 179.2 (fructose, glucose); 341.3 (sucrose, maltose); 503.4 (maltotriose)




OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

1. Fructose

3. Sucrose

5. Maltooligosacchrides

2. Glucose

4. Maltose

xBr

idge

am

ide

Wa

64

10

5

20 4 6 8 10 12 14 min

%

-5

95

%

-5

95 SIR of 3 Channels ES-

SIR of 3 Channels ES-

21

3

45

Disaccharides (m/z 341.3 )

Trisaccharides (m/z 503.4 )

Belgian Beer 20% diluted

Food Sugar Standard 10 µg/mL

©2010 Waters Corporation. Waters and ACQUITY UPLC are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. SC-IH-PDF





12 x 32 LCMS Certified Clear Glass Screw Cap Vial 600000668CV

St ruCturES


[ 21 ]

mS AnALySiS of orgAno pHoSpHoniC ACiDS uSing An X BriDgE AmiDE CoLumn

mS ConDitionS


Capillary: 2.5 kV

Cone Voltage: 30 V (EMPA,IMPA,PMPA); 35 V (MMPA); 40 V (CMPA)

Source Temperature: 120 °C

Desolvation Temperature: 450 °C


Cone Gas: 50 L/hr

Dwell Time: 100 ms

SIR (m/z): 122.9 (EMPA); 136.95 (IMPA); 150.95 (MMPA); 177.0 (CMPA); 179.0 (PMPA)

SampLE ExtraCtion proCEDurE



OPO

HO

2. 2-(methyl)propyl Methylphosphonic Acid (MMPA)

OPO

HO

3. Cyclohexyl Methylphosphonic Acid (CMPA)

OPO

HO

5. Ethyl Methylphosphonic Acid (EMPA)

OPO

HO

4. Isopropyl Methylphosphonic Acid (IMPA)

1. Pinacolyl Methylphosphonic Acid (PMPA)

OH

OPO

HO

LC ConDitionS


Mobile Phase A: 50/50 acetonitrile/water with 10 mM ammonium acetate and 0.04% ammonium hydroxide, pH 9.0

Mobile Phase B: 95/5 acetonitrile/water with 10 mM ammonium acetate and 0.04% ammonium hydroxide, pH 9.0

Gradient: Time Profile (min) %A %B 0.00 0.1 99.9 60.00 10 90 60.01 0.1 99.9 72.00 0.1 99.9





Weak Needle Wash: 50:50 acetonitrile/water


xBr

idge

am

ide

Wa

64

10

7

©2010 Waters Corporation. Waters and ACQUITY UPLC are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. VW-IH-PDF

50 10 15 20 25 30 35 40 45 50 55 min

%

0

100 SIR of 5 Channels ES- 1

2 3

4

5 Organophosphonic Acid Standard 10 µg/mL each






St ruCturES


[ 22 ]

mS AnALySiS of fooD SugA rS/SACCHA riDE StAnDA rDS uSing An X BriDgE AmiDE CoLumn

LC ConDit ionS


Mobile Phase A: 80/20 acetonitrile/water with 0.1% ammonium hydroxide

Mobile Phase B: 30/70 acetonitrile/water with 0.1% ammonium hydroxide








Instrument: ACQUITY UPLC® and TQD

mS ConDit ionS


Capillary: 2.8 kV

Cone Voltage: 25 V (fructose, glucose); 15 V (sucrose, maltose); 40 V (maltooligosaccharides)




Cone Gas: 50 L/hr

Dwell Time: 200 ms

SIR (m/z): 178.96 (fructose, glucose); 341.04 (sucrose, maltose); 503.08 (maltotriose); 665.10 (maltotetraose); 827.22 (maltopentaose); 989.21 (maltohexaose); 1151.29 (maltoheptaose)

St ruCturES

OHO

OH

HOOH

OH

OHOHO HO

HO

OOHO HO

HO

OH

OHOHO HO

HO

OHOOH

HO

OHO

OHOHO

HO

OH

OH

OHO

HO HO

HO

OO HO HO

HO

O

HO HO

HO

OHO

nn = 1 to 5

Fructose

Sucrose


Glucose

Maltose

xBr

idge

am

ide

Wa

64

10

4






[ 23 ]

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 min

%

2

100

SIR of 7 Channels ES- TIC

1.44e6

1

2

3

4

5

6

7

8

9

Compounds: 1. Fructose 2. Glucose 3. Sucrose 4. Maltose 5. Maltotriose 6. Maltotetraose 7. Maltopentaose 8. Maltohexahose 9. Maltoheptaose

Food Sugar/Saccharide Standard10 µg/mL

xBr

idge

am

ide

Wa

64

10

4








[ 24 ]

mS AnALySiS of HiSt iDinE Dip Ept iDES uSing An X BriDgE AmiDE CoLumn

mS ConDitionS

Gain: 100

Pressure: 30 psi


Nebulizer: cooling

Data Rate: 10 pps







LC ConDitionS












xBr

idge

am

ide

Wa

64

10

9

5 10 15 20 25 30 35 min

%

0

100 SIR of 4 Channels ES+

Compounds: 1. Creatinine (1 µg/mL) 2. Creatine (10 µg/mL)

3. Anserine (10 µg/mL) 4. Carnosine (10 µg/mL)

Histidine dipeptides

1

2

3

4

0


St ruCturES

Creatine

Anserine

Creatinine

Carnosine

N NH

NH2

HO

O

NH

N NH

O

H2N NH

O COOHN

N

HO O

HNHN

N

NH2

O







[ 25 ]

mS AnALySiS of mor pHinEuSing An X BriDgE AmiDE CoLumn

LC ConDitionSColumn: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm

Mobile Phase A: 50/50 acetonitrile/water with 10 mM ammonium formate, pH 3.0

Mobile Phase B: 90/10 acetonitrile/water with 10 mM ammonium formate, pH 3.0

Gradient: Time Profile (min) %A %B 0.0 0.1 99.9 15.0 0.1 99.9 62.5 99.9 0.1 62.6 0.1 99.9 75 0.1 99.9

Flow Rate: 1.0 mL/minInjection Volume: 15.0 μLColumn Temperature: 30 °CStrong Needle Wash: 95/5 acetonitrile/methanolWeak Needle Wash: 95/5 acetonitrile/methanolSeal Wash: 50:50 acetonitrile/methanolInstrument: ACQUITY UPLC® System with 30-cm

column cooler/heater and TQD

mS ConDitionSIonization Mode: negative electrosprayCapillary: 3.0 kVCone Voltage: 30 V (6-acetylmorphine and morphine); 40 V (M3G)Source Temperature: 120 ˚CDesolvation Temperature: 350 ˚CDesolvation Gas: 800 L/hrCone Gas: 0 L/hrDwell Time: 0.1 sSIR (m/z): 287.5 (morphine);

329.5 (6-acetylmorphine); 463.6 (M3G)




xBr

idge

am

ide

Wa

64

11

1

St ruCturES

Morphine

Morphine-3β-D-glucuronide (M3G)

6-Acetylmorphine

HO

O

O

NCH3

H

O

HO

O

HO

NCH3

H

O

O

HO

NCH3

H

O

HO HO

OHOHO

10 20 30 40 50 60 min

%

0

100 SIR of 3 Channels ES+

Compounds:

1. 6-acetylmorphine (1 µg/mL) 2. Morphine (5 µg/mL) 3. M3G (10 µg/mL)

1

2

3

0

©2010 Waters Corporation. Waters and ACQUITY UPLC are registered trademarks ofWaters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. VW-IH-PDF







[ 26 ]

mS AnALySiS of orgAniC ACiDS uSing An X BriDgE AmiDE CoLumn

LC ConDitionSColumn: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm



Gradient: Time Profile (min) %A %B 0.00 0.1 99.9 3.00 0.1 99.9 3.10 40 60 14.00 70 30 14.10 0.1 99.9 26.00 0.1 99.9

Flow Rate: 1.0 mL/minInjection Volume: 15.0 μLColumn Temperature: 50 °CStrong Needle Wash: 95/5 acetonitrile/methanolWeak Needle Wash: 95/5 acetonitrile/methanolSeal Wash: 50:50 acetonitrile/methanolInstrument: ACQUITY UPLC® System with 30-cm

column cooler/heater and TQD

mS ConDitionSIonization Mode: negative electrosprayCapillary: 3.5 kVCone Voltage: 25 VCollision Energy: 10 eVSource Temperature: 130 °CDesolvation Temperature: 350 °CDesolvation Gas: 800 L/hrCone Gas: 50 L/hrCollision Gas: 0.1 mL/minDwell Time: 0.1 s

MRM (m/z): pyruvic acid: 86.92> 42.9; lactic acid: 88.92 > 42.9; succinic acid: 116.93 > 72.9; maleic acid and fumaric acid: 114.88 > 70.9




xBr

idge

am

ide

Wa

64

11

0

St ruCturES

Pyruvic Acid

Succinic Acid

Maleic Acid Lactic Acid

Fumaric Acid

HOOH

O

O

OHOH

O

O

OH

O

OHOH

O

O

HOOH

O

O

2 4 6 8 10 12 14 min

%

-5

95

%

-5

95

%

-5

95

%

-5

95

Compounds:

1. Maleic acid (5 µg/mL) 2. Pyruvic acid (200 µg/mL)

3. Lactic acid (200 µg/mL) 4. Succinic acid (200 µg/mL)

5. Fumaric acid (200 µg/mL)

Organic Acids

1

4

5

3

2

0

©2010 Waters Corporation. Waters and ACQUITY UPLC are registered trademarks ofWaters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. VW-IH-PDF







[ 27 ]

iSoC rAt iC mS AnALySiS of orgAno pHoSpHoniC ACiDS uSing An X BriDgE AmiDE CoLumn

mS ConDitionS


Capillary: 2.5 kV

Cone Voltage: 30 V (EMPA,IMPA,PMPA); 35 V (MMPA); 40 V (CMPA)

Source Temperature: 120 °C

Desolvation Temperature: 450 °C


Cone Gas: 50 L/hr

Dwell Time: 100 ms

SIR (m/z): 122.9 (EMPA); 136.95 (IMPA); 150.95 (MMPA); 177.0 (CMPA); 179.0 (PMPA)




St ruCturESLC ConDitionS


Mobile Phase: 90/10 acetonitrile/water with 10 mM ammonium acetate and 0.04% ammonium hydroxide, pH 9.0

Isocratic Flow Rate: 1.0 mL/min







OPO

HO

2. 2-(methyl)propyl Methylphosphonic Acid (MMPA)

OPO

HO

3. Cyclohexyl Methylphosphonic Acid (CMPA)

OPO

HO

5. Ethyl Methylphosphonic Acid (EMPA)

OPO

HO

4. Isopropyl Methylphosphonic Acid (IMPA)

1. Pinacolyl Methylphosphonic Acid (PMPA)

OH

OPO

HO

xBr

idge

am

ide

Wa

64

10

8


2.50 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

%

0

100 SIR of 5 Channels ES- Organophosphonic Acid Standard 10 µm/mL each

1

2 3

4

5

on- LinE orDEring information






[ 28 ]

mS AnALySiS of St Ev iA rELAt ED Com pounDS uSing An X BriDgE AmiDE CoLumn

mS ConDit ionS


Capillary: 3.0 kV

Cone Voltage: 25 V




Cone Gas: 50 L/hr

Dwell Time: 80 ms

SIR (m/z): 121.1 (erythritol); 803.8 (stevioside); 950.1 (rebaudioside C); 966.1 (rebaudioside A)





4. Collect supernatant and filter using 0.45 µmPVDF syringe filter.

LC ConDit ionS


Mobile Phase A: 80/20 acetonitrile/water with 0.1 % ammonium hydroxide

Mobile Phase B: 30/70 acetonitrile/water with 0.1 % ammonium hydroxide

Flow Profile: 95% A/5% B








xBr

idge

am

ide

Wa

64

10

6

10 2 3 4 5 6 7 8 9 min

%

0

100

%

0

100 SIR of 5 Channels ES-

SIR of 5 Channels ES-

Truvia 50 µg/mL

Stevia 50 µg/mL

Rebaudioside C

Stevioside

Rebaudioside A

Erythritol

Rebaudioside A 2.05e4

10x zoom

St ruCturES







SteviosideErythritol

CH2OHHC OHHC OHCH2OH

H

H

CH2

O

OHOHO

HO

OH

O

OHOHO

HO

OH

O

OHOHO

OH

O

Rebaudioside A

H

H

CH2

O

OHOHO

HO

OH

O

OHOHO

HO

OH

O

OHO

OH

O

OHOHO

HO

OH

O

Rebaudioside C

H

H

CH2

O

OHOHO

HO

OH

O

OHOHO

HO

OH

O

OHO

OH

O

OHOHO

O

OH


[ 29 ]

uv AnALySiS of 5-fLuorou rACiL uSing An X BriDgE AmiDE CoLumn

St ruCturE

5-Fluorouracil

HpLC ConDit ionS


Mobile Phase: 95/2.5/2.5 acetonitrile/isopropyl alcohol/water with 5 mM ammonium acetate, pH 9.0



Sample Concentration: 100 µg/mL


Needle Wash: 95/5 acetonitrile/water


UV Wavelength: 265 nm

Sampling Rate: 20 Hz


Instrument: Alliance® System with 2998 PDA

NH

HN

O

O

F


AU

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 4 6 8 10 12 14 16 18 20 22 24 min

5-Fluorouracil




xBr

idge

am

ide

Wa

64

11

3







[ 30 ]




uv AnALySiS of AC ryLAmiDE, mEt HAC ryLiC ACiD AnD mEt HAC ryLAmiDE uSing An X BriDgE AmiDE CoLumn

St ruCturES

1. Methacrylamide 2. Acrylamide

3. Methacrylic acid

HpLC ConDit ionS













NH2

O NH2

O

OH

O


AU

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

20 4 6 8 10 12 14 16 min

1

2

3

xBr

idge

am

ide

Wa

64

11

5







[ 31 ]

uv AnALySiS of ALLAntoin uSing An X BriDgE AmiDE CoLumn




Allantoin

HpLC ConDitionS


Mobile Phase: 90/10 acetonitrile/water











NH

HN

O

OHNH2N

O


AU

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 min

Allantoin

xBr

idge

am

ide

Wa

64

11

2

St ruCturE







[ 32 ]




grADiEnt AnALySiS of ASCorBiC AnD iSoASCorBiC ACiD uSing An X BriDgE AmiDE CoLumn

2. Ascorbic acid1. Isoascorbic acid

HpLC ConDit ionS


Mobile Phase A: 50/50 acetonitrile/water with 10 mM ammonium acetate, pH 5.0

Mobile Phase B: 90/10 acetonitrile/water with 10 mM ammonium acetate, pH 5.0

Gradient: Time Profile (min) % A % B 0.0 0.1 99.9 48.0 99.9 0.1 48.1 0.1 99.9 72.0 0.1 99.9











O

OHHO

O

HO

HO

OH OH

O

CH2OH

HHO

O

AU

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 5 10 15 20 25 30 35 40 45 min

1 2


xBr

idge

am

ide

Wa

64

11

8






St ruCturES


[ 33 ]

iSoC rAt iC uv AnALySiS of ASCorBiC AnD iSoASCorBiC ACiD uSing An X BriDgE AmiDE CoLumn

2. Ascorbic acid1. Isoascorbic acid

HpLC ConDit ionS


Mobile Phase: 80/20 acetonitrile/water with 2 mM monopotassium phosphate











O

OHHO

O

HO

HO

OH OH

O

CH2OH

HHO

O


AU

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60

0 2 4 6 8 10 12 14 16 18 20 min

1 2




xBr

idge

am

ide

Wa

64

11

9






St ruCturES


[ 34 ]

uv AnALySiS of ginSEnoSiDE rb1 in ginSEng root pow DEr EX t rAC t uSing An X BriDgE AmiDE CoLumn

Ginsenoside Rb1

Ginseng root powder extractTailing factor = 0.95

USP plate count = 6170

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 6.50 7.00 7.50 8.00 min

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

OH

OHOHO

OH

O

OHOHO

HO

OHO

OHOHO

HOO

OHOHO

HO

OH

O

HpLC ConDit ionS

Column: XBridge™ Amide, 3.5 µm, 4.6 x 150 mm

Mobile Phase: 80:20 acetonitrile/water




Sample Temperature: 10 °C







SampLE Ex t raCt ion p roC EDurE1. Weigh 200 mg ginseng root powder into an extraction vessel.

2. Add 1 mL 80% methanol, sonicate for 5 min.

3. Centrifuge at 10,000 rpm for 5 min.

4. Collect the supernatant.

5. Repeat steps 2-4 two more times.

6. Combine the extracts, mix well.

7. Filter through 13 mm nylon 0.2 µm filter for injection.







xBr

idge

am

ide

Wa

64

08

6

St ruCturE

Ginsenoside Rb1


[ 35 ]

uv AnALySiS of ginSEnoSiDE rb1 in HErBAL mEDiC inE EX t rAC t uSing An X BriDgE AmiDE CoLumn

SoLiD pHaSE Ex t raCt ion p roC EDurE

1. 360 mg Sep-Pak® Plus C18 cartridge.

2. Condition with 2 mL methanol.

3. Equilibrate with 2 mL of 30% methanol/70% water just before loading.

4. Load 5 mL of the solution from step 11 in the pretreatment stage.

5. Wash with 2 mL of 30% methanol/70% water.

6. Wash with 1 mL of sodium carbonate test solution (1 mol/L).

7. Wash with 10 mL of 30% methanol/70% water.

8. Elute with 5 mL methanol.

9. Place into auto sampler and inject.

HpLC ConDit ionS

Column: XBridge™ Amide, 3.5 µm, 4.6 x 250 mm

Mobile Phase: 80:20 acetonitrile/water


Injection Volume: 20 µL


Sample Temperature: 10 °C


Seal Wash: 10:90 methanol:water




Instrument: Alliance® Sytem with 2998 PDA

p rEt rEatmEnt1. Weigh 2 g of herbal medicine powder into a centrifuge tube.

2. Add 30 mL of 60% methanol/40% water.

3. Shake for 15 min.

4. Centrifuge at 4,000 rpm for 10 min.

5. Obtain the supernatant.

6. Repeat steps 2-5 with the residue using 15 mL of 60% methanol/40% water.

7. Combine the supernatant, and make exactly 50 mL by adding 60% methanol/40% water.

8. Take 10 mL of this solution and add 3 mL of sodium hydroxide test solution (1 mol/L).

9. Let stand for 30 min.

10. Add 3 mL of HCl test solution (1 mol/L).

11. Add 60% methanol/40% water to make exactly 20 mL.

St ruCturE

Ginsenoside Rb1

OH

OHOHO

OH

O

OHOHO

HO

OHO

OHOHO

HOO

OHOHO

HO

OH

O

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0 2 4 6 8 10 12 14 16 18 min

Herbal medicine extractTailing factor = 0.98

USP plate count = 15673

Ginsenoside Rb1

©2010 Waters Corporation. Waters, Alliance and Sep-Pak are registered trademarks of Waters Corporation. XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. VW-IH-PDF






xBr

idge

am

ide

Wa

64

08

7


[ 36 ]




uv AnALySiS of nuC L EoBASES uSing An X BriDgE AmiDE CoLumn

AU

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30 35 40 45 50 min

1

2

3

4 5

St ruCturES

HN

NH

O

O

HN

NH

O

O

HN

N NH

N

O

H2N

N

NH

NH2

O

HN

N

NH2

N

N

2. Uracil

4. Cytosine

1. Thymine 3. Adenine

5. tGuanine

HpLC ConDit ionS


Mobile Phase A: 50/50 acetonitrile/water with 10 mM ammonium formate, pH 3.0

Mobile Phase B: 95/5 acetonitrile/water with 10 mM ammonium formate, pH 3.0










Filter Time Constant: 0.2



xBr

idge

am

ide

Wa

64

11

6







[ 37 ]

uv AnALySiS of nuC L Eot iDE pHoSpHAt ES uSing An X BriDgE AmiDE CoLumn

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 min

2

3

1

4 5

St ruCturES

2. dAMP

4. dGMP

1. TMP 3. dCMP

5. dADP

HpLC ConDit ionS


Mobile Phase: 80/20 acetonitrile/water with 2 mM monopotassium phosphate















O

OH

OP

OOH

OP

O OHOH

N

N

NH2

N

N

N

N

NH2

N

N

O

OH

OP

O OHOH

N

NO

NH2

O

OH

O

OP

HOHO

HN

N

N

O

H2N N

O

OH

OP

OOHOH

HN

NO

O

O

OH

OP

OHOHO

xBr

idge

am

ide

Wa

64

12

1







[ 38 ]




uv AnALySiS of t Hiou rEA uSing An X BriDgE AmiDE CoLumn

St ruCturE

Thiourea

HpLC ConDit ionS













H2N NH2

S


AU

0.000.050.100.15

0.200.250.30

0.350.400.450.50

0.550.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 min

Thiourea

xBr

idge

am

ide

Wa

64

11

4







[ 39 ]

uv AnALySiS of u riC ACiDS uSing An X BriDgE AmiDE CoLumn

St ruCturES

1. Uric acid

4. 3,7-Dimethyluric acid

2. 1-Methyluric acid

3. 1,3-Dimethyluric acid

HpLC ConDit ionS


Mobile Phase A: 50/50 acetonitrile/water with 10 mM ammonium acetate, pH 5.0

Mobile Phase B: 90/10 acetonitrile/water with 10 mM ammonium acetate, pH 5.0

Gradient: Time Profile (min) % A % B 0.0 0.1

Documents

XBridge Amide HPLC CoLumns · mide W a 64098 LSU 0 500 1000 1500 LSU 0 500 1000 1500 LSU 0 500 1000 1500 0 2 4 6 8 10 12 14 16 min Saccharide Standard 1 mg/mL Robitussin ® Brand