Post on 22-Jan-2020
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
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 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Flow Rate: 1.0 mL/min
Injection Volume: 10.0 µL
Sample Concentration: 1 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
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
Maltooligosacchrides
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
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
[ 6 ]
ELSD AnALySiS of fooD SugA rS in BrAn w it H rAiSin C ErEAL uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile
with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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. 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
©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
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
OHO
HO HO
OHO
OHO HO
HO
OH
6. Lactose
St ruCturES
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
[ 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
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 ConDitionS
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 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Flow Rate: 1.0 mL/min
Injection Volume: 10.0 µL
Sample Concentration: 1 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
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
Saccharide Standard 1 mg/mL
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:
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
Maltooligosacchrides
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
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
[ 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
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
Flow Rate: 1.0 mL/min
Gradient: Time Profile (min) %A %B 0.00 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Injection Volume: 10.0 µL
Sample Concentration: 1 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
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
Saccharide Standard 1 mg/mL
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
Maltooligosacchrides
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
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
[ 9 ]
ELSD AnALySiS of fooD SugA rS in K Et CHu p uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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. 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
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
1. p-Toluamide 3. Glucose
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
Food Sugar Standard 1 mg/mL
1 2
3 4
5 6
2
3
5
©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
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
OHO
HO HO
OHO
OHO HO
HO
OH
6. Lactose
St ruCturES
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
[ 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
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 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Flow Rate: 1.0 mL/min
Injection Volume: 10.0 µL
Sample Concentration: 1 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
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
Saccharide Standard 1 mg/mL
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
4. Sucrose 5. Maltose 6. Maltotriose
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
Maltooligosacchrides
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
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
[ 11 ]
ELSD AnALySiS of fooD SugA rS in miLK uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
ELSD ConDit ionS
Gain: 100
Pressure: 30 psi
Drift Tube Temperature: 50 ˚C
Nebulizer: cooling
Data Rate: 10 pps
Filter Time Constant: 0.2 seconds
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
1. p-Toluamide 3. Glucose
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
Food Sugar Standard 1 mg/mL
1
2 3 4
5 6
6
©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
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
OHO
HO HO
OHO
OHO HO
HO
OH
6. Lactose
St ruCturES
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
[ 12 ]
ELSD AnALySiS of fooD SugA rS in moLASSES uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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.
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
Food Sugar Standard 1 mg/mL
1 2
3 4
5 6
2 3
4
©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
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
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
OHO
HO HO
OHO
OHO HO
HO
OH
6. Lactose
St ruCturES
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
[ 13 ]
ELSD AnALySiS of fooD SugA rS in potAto CHipS uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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. 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
09
6
©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
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
Food Sugar Standard 1 mg/mL
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
1. p-Toluamide 3. Glucose
5. Maltose
NH2O
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
St ruCturES
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
[ 14 ]
ELSD AnALySiS of fooD SugA rS in p rEpA rED fooDS uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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. 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
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
1. p-Toluamide 3. Glucose
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
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
St ruCturES
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
[ 15 ]
ELSD AnALySiS of fooD SugA rS in A Sport S DrinK uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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.
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
Food Sugar Standard 1 mg/mL
1 2 3 4
5 6
2 3
©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
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
1. p-Toluamide 3. Glucose
5. Maltose
NH2O
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
St ruCturES
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
[ 16 ]
ELSD AnALySiS of fooD SugA rS StAnDA rD uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Concentration: 1 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
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. 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
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
1. p-Toluamide 3. Glucose
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
Food Sugar Standard 1 mg/mL
©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
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
St ruCturES
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
[ 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
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. 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.
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 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Flow Rate: 1.0 mL/min
Injection Volume: 10.0 µL
Sample Concentration: 1 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
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
Maltooligosacchrides
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
Saccharide Standard 1 mg/mL
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
©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
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
[ 18 ]
ELSD AnALySiS of fooD SugA rS in w inE uSing An X BriDgE AmiDE CoLumn
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
Flow Profile: 90% A/10% B (75% acetonitrile with 0.2% triethylamine)
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Sample Diluent: 50:50 acetonitrile/water
Column Temperature: 35 ˚C
Needle Wash: 75:25 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
Instrument: Alliance® System with 2424 ELSD
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, 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
Food Sugar Standard 1 mg/mL
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
1. p-Toluamide 3. Glucose
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
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
St ruCturES
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
[ 19 ]
ELSD AnALySiS of St Ev iA rELAt ED Com pounDS uSing An X BriDgE AmiDE CoLumn
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
Flow Rate: 1.0 mL/min
Isocratic: 95% A/5% B (77.5% acetonitrile with 0.2% triethylamine)
Injection Volume: 10.0 µL
Sample Concentration: 5 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
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. 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
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
©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
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
[ 20 ]
mS AnALySiS of fooD SugA rS/SACCHA riDES in BEEr uSing An X BriDgE AmiDE CoLumn
LC ConDit ionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase A: 80/20 acetonitrile/water with 0.1% ammonium hydroxide
Mobile Phase B: 30/70 acetonitrile/water with 0.1% ammonium hydroxide
Gradient: Time Profile (min) %A %B 0.00 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Flow Rate: 1.0 mL/min
Injection Volume: 20.0 µL
Column Temperature: 35 ˚C
Strong Needle Wash: 20/80 acetonitrile/water
Weak Needle Wash: 75/25 acetonitrile/water
Seal Wash: 50:5 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)
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
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 Clear Glass Screw Cap Vial 600000668CV
St ruCturES
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=28879
[ 21 ]
mS AnALySiS of orgAno pHoSpHoniC ACiDS uSing An X BriDgE AmiDE CoLumn
mS ConDitionS
Ionization Mode: negative electrospray
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
Desolvation Gas: 900 L/hr
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
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
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
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Column Temperature: 65 °C
Strong Needle Wash: 95/5 acetonitrile/water
Weak Needle Wash: 50:50 acetonitrile/water
Instrument: ACQUITY UPLC® System with 30-cm column cooler/heater and TQD
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
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 Clear Glass Screw Cap Vial 600000668CV
St ruCturES
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=28879
[ 22 ]
mS AnALySiS of fooD SugA rS/SACCHA riDE StAnDA rDS uSing An X BriDgE AmiDE CoLumn
LC ConDit ionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 150 mm
Mobile Phase A: 80/20 acetonitrile/water with 0.1% ammonium hydroxide
Mobile Phase B: 30/70 acetonitrile/water with 0.1% ammonium hydroxide
Gradient: Time Profile (min) %A %B 0.00 100 0 30.60 40 60 30.61 100 0 52.00 100 0
Flow Rate: 0.4 mL/min
Injection Volume: 10.0 µL
Column Temperature: 35 ˚C
Strong Needle Wash: 20/80 acetonitrile/water
Weak Needle Wash: 75/25 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
Instrument: ACQUITY UPLC® and TQD
mS ConDit ionS
Ionization Mode: negative electrospray
Capillary: 2.8 kV
Cone Voltage: 25 V (fructose, glucose); 15 V (sucrose, maltose); 40 V (maltooligosaccharides)
Source Temperature: 120 ˚C
Desolvation Temperature: 350 ˚C
Desolvation Gas: 500 L/hr
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
Maltooligosacchrides
Glucose
Maltose
xBr
idge
am
ide
Wa
64
10
4
SampLE Ex t raCt ion p roC EDurE
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.
[ 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
on-LinE orDEring information
Description Part Number
XBridge Amide Column, 3.5 µm, 4.6 x 150 mm 186004869
Acrodisc® LC 13 mm, 0.45 µm, 100/pkg WAT200512
12 x 32 LCMS Certified Clear Glass Screw Cap Vial 600000668CV
©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
http://www.waters.com/waters/partDetail.htm?cid=511505&id=38672http://www.waters.com/waters/partDetail.htm?cid=511544&id=33952http://www.waters.com/waters/partDetail.htm?cid=10000711&id=28879
[ 24 ]
mS AnALySiS of HiSt iDinE Dip Ept iDES uSing An X BriDgE AmiDE CoLumn
mS ConDitionS
Gain: 100
Pressure: 30 psi
Drift Tube Temperature: 50 ˚C
Nebulizer: cooling
Data Rate: 10 pps
Filter Time Constant: 0.2 sec
SampLE ExtraCtion proCEDurE
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.
LC ConDitionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
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 90 10 16.00 30 70 16.01 90 10 30.00 90 10
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Column Temperature: 65 °C
Strong Needle Wash: 95/5 acetonitrile/water
Weak Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
Instrument: ACQUITY UPLC® System with 30-cm column cooler/heater and TQD
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
©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
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
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 Clear Glass Screw Cap Vial 600000668CV
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=28879
[ 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)
SampLE ExtraCtion proCEDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
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 Clear Glass Screw Cap Vial 600000668CV
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=28879
[ 26 ]
mS AnALySiS of orgAniC ACiDS uSing 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 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 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
SampLE ExtraCtion proCEDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
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 Clear Glass Screw Cap Vial 600000668CV
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=28879
[ 27 ]
iSoC rAt iC mS AnALySiS of orgAno pHoSpHoniC ACiDS uSing An X BriDgE AmiDE CoLumn
mS ConDitionS
Ionization Mode: negative electrospray
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
Desolvation Gas: 900 L/hr
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
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
St ruCturESLC ConDitionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
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
Injection Volume: 15.0 µL
Column Temperature: 65 °C
Strong Needle Wash: 95/5 acetonitrile/water
Weak Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
Instrument: ACQUITY UPLC® System with 30-cm column cooler/heater and TQD
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
©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
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
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 Clear Glass Screw Cap Vial 600000668CV
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=28879
[ 28 ]
mS AnALySiS of St Ev iA rELAt ED Com pounDS uSing An X BriDgE AmiDE CoLumn
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): 121.1 (erythritol); 803.8 (stevioside); 950.1 (rebaudioside C); 966.1 (rebaudioside A)
SampLE Ex t raCt ion p roC EDurE
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 µmPVDF syringe filter.
LC ConDit ionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
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
Flow Rate: 1.0 mL/min
Injection Volume: 15.0 µL
Column Temperature: 35 ˚C
Strong Needle Wash: 20/80 acetonitrile/water
Weak Needle Wash: 75/25 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
Instrument: ACQUITY UPLC® System with 30-cm column cooler/heater and TQD
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
©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
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 Clear Glass Screw Cap Vial 600000668CV
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
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=28879
[ 29 ]
uv AnALySiS of 5-fLuorou rACiL uSing An X BriDgE AmiDE CoLumn
St ruCturE
5-Fluorouracil
HpLC ConDit ionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase: 95/2.5/2.5 acetonitrile/isopropyl alcohol/water with 5 mM ammonium acetate, pH 9.0
Flow Rate: 0.5 mL/min
Injection Volume: 50.0 µL
Sample Concentration: 100 µg/mL
Column Temperature: 25 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 265 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
NH
HN
O
O
F
©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
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
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
xBr
idge
am
ide
Wa
64
11
3
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
[ 30 ]
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase: 95/2.5/2.5 acetonitrile/isopropyl alcohol/water with 5 mM ammonium acetate, pH 9.0
Flow Rate: 1.2 mL/min
Injection Volume: 40.0 µL
Sample Concentration: 30 µg/mL
Column Temperature: 25 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 210 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
NH2
O NH2
O
OH
O
©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
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
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
[ 31 ]
uv AnALySiS of ALLAntoin uSing An X BriDgE AmiDE CoLumn
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
Allantoin
HpLC ConDitionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase: 90/10 acetonitrile/water
Flow Rate: 0.5 mL/min
Injection Volume: 40.0 µL
Sample Concentration: 100 µg/mL
Column Temperature: 25 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 210 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
NH
HN
O
OHNH2N
O
©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
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
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
[ 32 ]
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
grADiEnt AnALySiS of ASCorBiC AnD iSoASCorBiC ACiD uSing An X BriDgE AmiDE CoLumn
2. Ascorbic acid1. Isoascorbic acid
HpLC ConDit ionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
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
Flow Rate: 0.5 mL/min
Injection Volume: 60.0 µL
Sample Concentration: 30 µg/mL
Column Temperature: 25 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 260 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
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
©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
xBr
idge
am
ide
Wa
64
11
8
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
St ruCturES
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
[ 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
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase: 80/20 acetonitrile/water with 2 mM monopotassium phosphate
Flow Rate: 0.5 mL/min
Injection Volume: 60.0 µL
Sample Concentration: 30 µg/mL
Column Temperature: 25 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 260 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
O
OHHO
O
HO
HO
OH OH
O
CH2OH
HHO
O
©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
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
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
xBr
idge
am
ide
Wa
64
11
9
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
St ruCturES
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
[ 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
Isocratic Flow Rate: 1.4 mL/min
Injection Volume: 11.5 µL
Column Temperature: 60 °C
Sample Temperature: 10 °C
Needle Wash: 95:5 acetonitrile:water
Seal Wash: 10:90 acetonitrile:water
UV Wavelength: 203 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.1 sec
Instrument: Alliance® System with 2998 PDA
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.
©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
on-LinE orDEring information
Description Part Number
XBridge Amide Column, 3.5 µm, 4.6 x 150 mm 186004869
Acrodisc® LC 13 mm, 0.45 µm, 100/pkg WAT200512
12 x 32 LCMS Certified Glass Screw Neck Vial 600000751CV
xBr
idge
am
ide
Wa
64
08
6
St ruCturE
Ginsenoside Rb1
http://www.waters.com/waters/partDetail.htm?cid=511505&id=38672http://www.waters.com/waters/partDetail.htm?cid=511544&id=33952http://www.waters.com/waters/partDetail.htm?cid=10000711&id=28885
[ 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
Isocratic Flow Rate: 0.8 mL/min
Injection Volume: 20 µL
Column Temperature: 60 °C
Sample Temperature: 10 °C
Needle Wash: 95:5 acetonitrile:water
Seal Wash: 10:90 methanol:water
UV Wavelength: 203 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.1 sec
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
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
xBr
idge
am
ide
Wa
64
08
7
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
[ 36 ]
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
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
Column: 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: 95/5 acetonitrile/water with 10 mM ammonium formate, pH 3.0
Gradient: Time Profile (min) %A %B 0.0 1 99 50.0 50 50 50.1 1 99 60.0 1 99
Flow Rate: 1.2 mL/min
Injection Volume: 60.0 µL
Sample Concentration: 20 µg/mL
Column Temperature: 30 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 260 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2
Instrument: Alliance® System with 2998 PDA
©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
xBr
idge
am
ide
Wa
64
11
6
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
[ 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
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase: 80/20 acetonitrile/water with 2 mM monopotassium phosphate
Flow Rate: 1.2 mL/min
Injection Volume: 40.0 µL
Sample Concentration: 10 µg/mL
Column Temperature: 25 °C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 254 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
©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
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
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
[ 38 ]
SampLE Ex t raCt ion p roC EDurE
1. Dilute with 50:50 acetonitrile/water.
2. Filter using 0.45 µm PVDF syringe filter.
uv AnALySiS of t Hiou rEA uSing An X BriDgE AmiDE CoLumn
St ruCturE
Thiourea
HpLC ConDit ionS
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
Mobile Phase: 95/2.5/2.5 acetonitrile/isopropyl alcohol/water with 10 mM ammonium acetate, pH 9.0
Flow Rate: 0.5 mL/min
Injection Volume: 40.0 µL
Sample Concentration: 10 µg/mL
Column Temperature: 25 ˚C
Needle Wash: 95/5 acetonitrile/water
Seal Wash: 50:50 acetonitrile/water
UV Wavelength: 245 nm
Sampling Rate: 20 Hz
Filter Time Constant: 0.2 sec
Instrument: Alliance® System with 2998 PDA
H2N NH2
S
©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
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
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
[ 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
Column: XBridge™ Amide, 3.5 μm, 4.6 x 250 mm
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