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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

APPLICATIONS

Salt Interferences

ELSD using 75% ACN at 1.4mL/min, 35°C 4.6 x 150mm Columns, 10µL injection 5 Food Sugars

with *Salts on:

Aminopropyl Column

Polyamine Column

Polymeric Amino Column

1 2 3

4 5

1

2 3 4,5

1 2 3

4 5

Minutes 0.0 2.0 4.0 6.0 8.0 10.0 12.0

*

*

* *

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (each 1mg/mL), * Salt Interferences

5 Food Sugars with and without salts

1

2 3

0.0 Minutes

2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

4 5

XBridge™ Amide Column With 0.1% TEA Mobile Phase Modifier

Low Salt Retention = No Salt Interference

* *

Food Sugars on 1.7µm BEH Amide

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

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (1mg/mL each)

Strawberry Smoothie

Hot Cross Buns

White Bread

Tomato Ketchup

Gatorade

Food Sugars

1

2

3 4 5

0.00 1.00 2.00 3.00 4.00 Minutes

Lamb Curry Meal

Raisin Bran Cereal

BEH Amide Column Chemistry 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).

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:

ACQUITY UPLC® BEH Amide

Loss of Reducing Sugars at Elevated Temperatures

● Substantial loss of the aldohexose reducing sugars is observed at elevated temperatures on amino and polyamine based columns ● No loss is observed on the ACQUITY UPLC® BEH Amide

Minutes 0.0 5.0 10.0 15.0

1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose (each 1mg/mL)

55°C

45°C

35°C

1

2

3

4,5

1

2

3

4,5

1

2 3

4 5

Polyamine

85% Loss

37% Loss 48%

Loss

78% Loss

ELSD: 1.4mL/min with 75% ACN

ELSD: 0.29mL/min with 0.2% TEA in 75% ACN

Minutes

0.0 2.0 4.0 6.0 8.0

2 3

4

1

2 3

4

1

2 3

4 5

5

5

ACQUITY BEH Amide

1

P-W-221

• 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 post-column adduct formation prior to APCI-MS.

INTRODUCTION 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. Typical methods of chromatographic analysis frequently utilize ligand/ion exchange or amino based column chemistries. Unique issues encountered with these methods include:

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.

Waters ACQUITY ELS Detection:

Gain: 200 Nebulizer: Cooling Pressure: 40 psi Data Rate: 10pps Drift Tube: 40°C Time Constant: Normal Data Processing: Savitsky-Golay Smoothing (Level 17) Mass Spec Detection (Waters TQD):

Ionization Mode: ES- Desol Temp: 350°C Capillary: 2.8kV Desol Gas Flow: 500L/Hr Cone Voltage: 25V Cone Gas Flow: 50L/Hr

High pH Stability of the 1.7µm ACQUITY UPLC® BEH Amide

● 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 ( pH~10.35), and a flow rate of 0.15mL/min

s w

*Glucose Retention 2000 injections under isocratic conditions

(>8 Days of testing)

Perc

en

t o

f O

rig

inal

Glu

cose

Rete

nti

on

(%

)

Food Sugar Standard

Glucose Retention Loss: 2.1%

Std.Dev.: 0.01 RSD: 0.46%

60

65

70

75

80

85

90

95

100

105

110

0 500 1000 1500 2000 Injection Number

Molasses Sample

*

Fru

cto

se

Su

cro

se

Glu

cose

Cereal Sample * F

ruct

ose

Su

cro

se

Glu

cose

Honey Sample

Fru

cto

se

Glu

cose

* pH ~ 10.35 and 90°C

s w

2.1 x 150mm Column @ 0.29mL/min

1

2

3

4

5

1

2

3

4 5

75% ACN with 0.2% TEA

at 35°C

80% Acetone with 0.05% TEA

at 90°C

1

3

2 α 4α

2β 4 β

5β

Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

α 5

75% ACN with no modifier

at 35°C

α-D-glucopyranose

β-D-glucopyranose

O

H O H

H H

H

H

O H O

H

O H

O H O

O

H O

H

H H

H

H

H O

H

O

H

O

H

Demonstration of Anomer Collapse on 1.7µm BEH Amide

1. Fructose 2. Glucose 3. Sucrose 4. Maltose 5. Lactose (each 1mg/mL)

Detection of Saccharides by Mass Spectrometry

● 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.

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)

SIR of 2 Channels ES-

TIC

6.09e5

0.1% Formic Acid

Unmodified

0.1% NH4OH

Minutes 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Good Ionization, Good Peak Shape

%

0

100

1 2

3

4 5

Poor Ionization, Poor Peak Shape

Good Ionization, Poor Peak Shape

Mobile Phase Modifier

CONCLUSIONS

● 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

Cough Syrup Analysis by ELSD 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

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

Min0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Generic Cough Syrup

Brand Name Cough Syrup

Standards

1

2 3

4 5 6 7

8

9

Samples 1% (v/v) in 50:50 ACN/H2O

Food Sugar Region

SIR of 3 Channels ES- TIC

5.62e6

Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

Standards

Dark Ale

Wheat Beer

Double Chocolate Stout

Standards: 1) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Maltotriose Each 10ppm in 50:50 ACN/Water

5

MS Analysis of Carbohydrates in Beer 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

Netherland Lager

Belgian Beer

Disaccharides Trisaccharides

1 4 3 2

Iso

stevio

l

Stevia (5mg/mL)

Stevia Standards

Ste

vio

sid

e

Reb

au

dio

sid

e A

Reb

au

dio

sid

e C

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 0

102030405060

708090

1000

LS

U

ELSD (77.5% ACN with 0.2% TEA)

MS and ELS Detection 2.1 x 100mm BEH Amide, Isocratic: 0.2mL/min at 35°C

MS (77.5% ACN with 0.1% NH4OH) SIR of 5 Channels ES- TIC

1.15e6

Minutes 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 0.0

0

%

100

m/z = 966.1 (Rebaudioside A)

m/z = 950.1 (Rebaudioside C)

m/z = 803.8 (Stevioside)

Stevia (50µg/mL)

m/z = 318.5 (Isosteviol)