P-W-221
UPLC ANALYSIS OF CARBOHYDRATES;
APPLICATIONS FOR SACCHARIDE ANALYSIS IN FOOD & BEVERAGE PRODUCTS AND PHARMACEUTICAL EXCIPIENTS
Christopher Hudalla, Jim Cook, Mike Dion, Pamela Iraneta, Kevin Jenkins, Paul Smith, Dan Walsh, and Kevin Wyndham
Waters Corporation Chemistry Organization R & D, Milford, MA 01757
• Complex
sample
matrices
result
in
chromatographic interferences requiring additional
sample preparation steps to clean up the sample
prior to analysis
• Silica based columns lack the robustness
necessary for high pH applications
• Amino or polyamine based columns are plagued
by high levels of column bleed and shortened
column lifetime
• Formation of Schiff bases and enamines can lead
to the loss of the reducing sugars at higher
temperatures or lower flow rates, resulting in
inaccurate quantitation and degraded columns
• Mutarotation of the reducing sugars may result
in poor peak shape and the undesired separation
of the α and β ring forms (anomers)
Here we present our work with the new
ACQUITY UPLC® BEH Amide stationary phase
which allows fast and efficient separations of
simple and complex carbohydrates. The robust
nature of the BEH particles enables high pH
saccharide separations, which are uniquely
suited for detection of these compounds in
their native form by negative ion ESI-MS. This
eliminates the need for pre- or post-column
derivatization for ESI-MS, as well as postcolumn adduct formation prior to APCI-MS.
4β 5 α
2α
5β
75% ACN
with no modifier
at 35°C
4α
1
3
2
75% ACN
with 0.2% TEA
at 35°C
5
4
1
3
2
4
80% Acetone
with 0.05% TEA
at 90°C
5
(>8 Days of testing)
100
90
85
80
1.0
2.0
3.0
4.0
Minutes
5.0
6.0
7.0
*
Honey Sample
70
8.0
s
pH ~ 10.35
w
and 90°C
*
75
Molasses Sample
65
0.0
Glucose Retention
Loss: 2.1%
Std.Dev.: 0.01
RSD: 0.46%
95
Food Sugar Standard
0
500
1000
Injection Number
2.1 x 150mm Column @ 0.29mL/min
Salt Interferences
1
ACQUITY UPLC® BEH Amide
3
2
4 5
*
*
1500
2000
METHODS
Chromatographic Conditions:
All chromatograms were collected on an ACQUITY
UPLC® system utilizing the 1.7µm BEH Amide
columns in various dimensions. Mobile phases were
either acetonitrile/water or acetone/water modified
with triethylamine (TEA) or ammonium hydroxide
(NH4OH). Flow rates and temperatures were varied
according to the experiment.
1
2
2.0
78%
Loss
6.0
Minutes
8.0
10.0
4
**
0.0
2.0
4.0
6.0
8.0
10.0
Minutes
37%
Loss
2
12.0
16.0
18.0
2.5
3.0
3.5
Minutes
4.0
4.5
5.0
5.5
4
5
4
1
1
3
2
45
0.0
5.0
10.0
Minutes
15.0
0.0
2.0
4.0
2) Glucose,
5
Food Sugars
3.00
2.00
4.00
3) Sucrose, 4) Maltose,
5) Lactose (1mg/mL each)
MS Analysis of Carbohydrates in Beer
Trisaccharides
Disaccharides
Stevia
(5mg/mL)
Stevia Standards
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Minutes
4.5
MS (77.5% ACN with 0.1% NH4OH)
5.0
5.5
6.0
SIR of 5 Channels ESTIC
1.15e6
100
m/z = 803.8
(Stevioside)
m/z = 950.1
Netherland Lager
Belgian Beer
Wheat Beer
(Rebaudioside A)
m/z = 318.5
1.0
1.5
0.0
Stevia
(Isosteviol)
0.5
(50µg/mL)
2.0
2.5
3.0
3.5
4.0
4.5
3
5.0
5.5
6.0
1
8.0
3
2
4
1.0
2.0
3.0
4.0
Samples 1% (v/v) in 50:50 ACN/H2O
Min
1.0
2.0
3.0
4.0
2
3
4
5
5.0
6.0
7.0
8.0
9.0
10.0
Minutes
Standards: 1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Maltotriose
Each 10ppm in 50:50 ACN/Water
11.0
CONCLUSIONS
Standards
1. Fructose
2. Glucose
3. Sucrose
4. Maltose
5. Maltotriose
6. Maltotetraose
7. Maltopentaose
8. Maltohexahose
9. Maltoheptaose
Brand Name
Cough Syrup
5
1
Standards
m/z = 966.1
0.0
Food Sugar Region
Double Chocolate Stout
Generic
Cough Syrup
6.0
SIR of 3 Channels ESTIC
5.62e6
Dark Ale
(Rebaudioside C)
Minutes
1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose,
5) Lactose (each 1mg/mL)
4
2.1 x 100mm, 1.7µm BEH Amide, 10 minute gradient 75-45% ACN
with 0.1% NH4OH at 35°C, 0.13mL/min, 2µL injection volume
Samples diluted 1:1 in 100% ACN
5
4
35°C
20.0
1.00
1) Fructose,
2.1 x 100mm, 1.7µm BEH Amide, using ELSD detection
6min gradient, 80-50% ACN with 0.2%TEA at 0.26mL/min,
at 35°C with 1.3µL injection volume
3
4,5
3
2
Minutes
Cough Syrup Analysis by ELSD
48%
Loss
1
0.00
Minutes
55°C
2
Gatorade
6.0
45°C
2
14.0
3
60
50
40
30
20
10
0
0.0
0
0.0
1
5
1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose,
5) Lactose (each 1mg/mL), * Salt Interferences
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85%
Loss
4,5
2.0
1000
90
80
70
ACQUITY
BEH Amide
3
2
1
3
2
3
12.0
5 Food Sugars
with and without salts
1
1
Polyamine
2
XBridge™ Amide Column
With 0.1% TEA Mobile Phase Modifier
Low Salt Retention = No Salt Interference
Mass Spec Detection (Waters TQD):
Desol Temp:
350°C
Desol Gas Flow: 500L/Hr
Cone Gas Flow: 50L/Hr
1
Polymeric
Amino Column
4 5
4.0
200
Nebulizer:
Cooling
40 psi
Data Rate:
10pps
40°C
Time Constant: Normal
Savitsky-Golay Smoothing (Level 17)
Ionization Mode: ESCapillary:
2.8kV
Cone Voltage:
25V
4,5
3
*
0.0
3
2
*
Waters ACQUITY ELS Detection:
Gain:
Pressure:
Drift Tube:
Data Processing:
Polyamine Column
1
ELSD: 0.29mL/min
with 0.2% TEA in 75% ACN
ELSD: 1.4mL/min
with 75% ACN
0.1% Formic Acid
ELSD (77.5% ACN with 0.2% TEA)
is observed at elevated temperatures on amino and
polyamine based columns
● No loss is observed on the ACQUITY UPLC® BEH
Amide
Aminopropyl Column
Poor Ionization, Poor Peak Shape
2.1 x 100mm BEH Amide, Isocratic: 0.2mL/min at 35°C
*
● Substantial loss of the aldohexose reducing sugars
5 Food Sugars
with *Salts on:
Unmodified
Raisin Bran Cereal
MS and ELS Detection
Loss of Reducing Sugars at Elevated
Temperatures
ELSD using 75% ACN at 1.4mL/min, 35°C
4.6 x 150mm Columns, 10µL injection
Good Ionization, Poor Peak Shape
APPLICATIONS
Cereal Sample
60
Lamb Curry Meal
0.1% NH4OH
1.5
Tomato Ketchup
5
4
2
Good Ionization, Good Peak Shape
0
1.0
LSU
BEH particles are prepared from two high purity
monomers: one that forms SiO2 inorganic-units
and another that forms O1.5Si-CH2CH2-SiO1.5
organosiloxane-units. Particles are bonded with
a trifunctional amide bonding:
2000 injections under isocratic conditions
Hot Cross Buns
White Bread
100
*Glucose Retention
105
Strawberry Smoothie
Mobile Phase
Modifier
1
110
1
ELSD on ACQUITY UPLC® System using 0.15mL/min at 85°C
Isocratic: 77% Acetone with 0.05% TEA
2.1 x 50mm Column, 0.7µL injection
6.09e5
3
Rebaudioside A
2β
Food Sugars on 1.7µm BEH Amide
SIR of 2 Channels ES
TIC
Rebaudioside C
3
MS Detection of Food Sugars on
1.7µm ACQUITY UPLC® BEH Amide
Using 75% ACN at 0.13mL/min
and 35°C (2.1 x 50mm Column)
Stevioside
H
Isosteviol
OH
H
(each 1mg/mL)
%
OH
OH
Sucrose
OH
H
HO
Glucose
H
H
Fructose
H
O
HO
Sucrose
H
O
H
OH
Fructose
HO
HO
H
● Saccharide molecules ionize readily under high pH
conditions, resulting in increased MS sensitivity
without the need for additional pre- or post-column
derivatization or adduct formation.
● Unlike silica, BEH particle technology has been
shown to have excellent high pH stability
● To confirm the stability of the amide bonding
on BEH, repetitive injections of real samples
were performed.
Intermittent Food Sugar
Standard injections were included to monitor
column performance during testing
● Testing was performed using a 2.1 x 150mm
column under isocratic conditions with 0.05%
TEA in 80% Acetone at 90°C (ws pH~10.35), and a
flow rate of 0.15mL/min
Fructose
Glucose
Sucrose
Maltose
Lactose
Glucose
H OH
1.
2.
3.
4.
5.
Glucose
The Bridged Ethyl Hybrid technology was
selected as the base particle due to its
increased thermal and pH stability relative to
silica. The amide bonding was selected due to
its higher stability relative to the amino phases
(reduced column bleed) and the longer lifetime
(no bonded phase reaction with the reducing
sugars; no formation of Schiff Bases).
β-D-glucopyranose
Detection of Saccharides by Mass Spectrometry
%
Typical methods of chromatographic analysis
frequently utilize ligand/ion exchange or
amino based column chemistries.
Unique
issues encountered with these methods
include:
α-D-glucopyranose
Fructose
Carbohydrates are the most abundant class of
organic compounds in nature.
‹
They are found naturally in many raw food
stuffs, from fruits and vegetables to grains and
dairy products.
‹
They are a common ingredient in most
prepared foods, both commercially and in the
home. Almost everything we consume has a
carbohydrate component.
● The pharmaceutical industry frequently uses
simple or complex mixtures of sugars or sugar
derivatives as pharmaceutical excipients. The
analysis
of
these
compounds
in
drug
formulations provides needed information for
regulatory compliance.
‹
Many consumer products contain alternative
sweeteners, both artificial and natural.
‹
It is this ubiquitous nature of carbohydrates
that makes their analyses critical to our
understanding of the nutritional consequences
of the foods we consume.
‹
BEH Amide Column Chemistry
High pH Stability of the
1.7µm ACQUITY UPLC® BEH Amide
Percent of Original
Glucose Retention (%)
INTRODUCTION
Demonstration of Anomer Collapse
on 1.7µm BEH Amide
5
5.0
6
7
6.0
8
9
Standards
7.0
8.0
● Anomer peaks arising from reducing sugars can be
collapsed into a single peak by using either high
temperature or high pH mobile phases
● The BEH particle technology provides a stable and
robust substrate for the trifunctional amide bonding,
providing extended column lifetime under a wide
variety of conditions
● Low salt retention eliminates salt interferences from
complex sample matrices
● Both ELS and MS detection allow gradient elution for
analysis of more complex polysaccharides
● Saccharides ionize readily under high pH conditions
enabling MS detection without pre– or post-column
modification
©2009 Waters Corporation