LC-MS/MS Analysis of Glyphosate and Other Polar Contaminants in Food with a Novel Ion Exchange/HILIC ColumnXiaoning Lu, Dan Li, Connor Flannery

Restek Corporation

Introduction

Glyphosate is a broad-spectrum herbicide widely used throughout theworld. The International Agency for Research on Cancer classifiedglyphosate as a probable human carcinogen. The safety residual level ofglyphosate in food is regulated in USA, Europe, as well as other regions.Direct analysis of underivatized glyphosate, however, can be challengingdue to its minimum retention on a reversed-phase or Hydrophilicinteraction chromatography (HILIC) column and severe adsorption onto thestainless flow path. On the other hand, glyphosate and other polar anioniccontaminants tend to strongly retain on an anion-exchange column andrequire high salt mobile phase to elute, which is not friendly to massspectrometric analysis.

These challenges motivated the development of a hybrid ionexchange/HILIC column that offers balanced retention of glyphosate as wellas other polar contaminants. With the column, an LC-MS/MS method hasbeen established for the detection of glyphosate, aminomethylphosphonicacid (AMPA), glufosinate, as well as other 14 polar contaminants in variousfood matrices in a single run. Additionally, the non-specific binding ofglyphosate and other chelating compounds onto stainless steel isminimized with a simple passivation solution.

Conclusion

Hybrid Ion Exchange/HILIC Column

The hybrid ion exchange/HILIC column (RaptorTM Polar X, Restek) is built ona single ligand that is comprised of multiple functionalities including ion-exchange and polar chemical moieties (patent-pending). The hybrid ligandcan be applied to separate a wide range of compounds through ionexchange, HILIC, or the combination (mixed mode) of ion exchange andHILIC. There are many features and benefits the hybrid column offers,some of which are listed below:

Figure 1:The Polar X Hybrid Column offers Ion Exchange and HILIC retention mechanisms

Fig. 4, LC-MS/MS separation of glyphosate, AMPA and glufosinate andother 14 polar food contaminants on a hybrid IEX/HILIC column (2.1x30mm, 2.7 µm).

Detection of Glyphosate, AMPA and Glufosinate in Food

• To overcome the many challenges in the analysis of glyphosate and other polar contaminants in food, a novel hybrid ion exchange/HILIC column as well as a simple HPLC system passivation solution has been developed. The newly developed column offers simplicity in LC-MS mobile phase conditions, high lot-to-lot and run-to-run consistency, and versatility in the separation of a wide range of polar analytes.

• An LC-MS/MS method has been established for the analysis of glyphosate, AMPA and glufosinate as well as other 14 difficult polar contaminants in a single run.

• The applicability of the column and LC-MS/MS method has been verified with various food matrices.

PATENTS & TRADEMARKSRestek patents and trademarks are the property of Restek Corporation. (See www.restek.com/Patents-Trademarks for full list.) Other trademarks inRestek literature or on its website are the property of their respective owners. Restek registered trademarks are registered in the U.S. and may also beregistered in other countries.

• Single ligand

• High reproducibility

• Balanced retentions

• Versatile separations

• Fast equilibration

• MS friendly conditions

• Sharp peak shape

• Core-shell particles

Figure 2: Separation of water soluble vitamins B1, B3 and B9 on sevenlots of hybrid IEX/HILIC columns

Lot-to-lot variability of the hybrid IEX/HILIC columnThe hybrid IEX/HILIC is built on a single and pure ligand that is comprised ofmultiple functionalities for chromatography. A reproducible process has beendeveloped to create and immobilize the hybrid ligand onto core-shell particles.Fig. 2 shows the separation of three water-soluble vitamin Bs through ionexchange mechanisms, on seven lots of hybrid columns made at different timesby multiple operators. The variability (RSD) of the retention across the sevenlots of column is <1.0% for both B1 and B3 (1st and 2nd peak), and 2.4% for B9(3rd peak), respectively. The relatively low variability of B9’s retention isobtained despite its high retention with a k’ about 30.

Minutes

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 8.50 9.00 9.50 10.00

Mobile Phase: A) Water; B)Acetonitrile, each with 0.1% Formic Acid

Composition: 50 %B IsocraticFlow Rate: 0.4 mL/minDetection: UV

RSD: 2.4%B9 (K’~30)

RSD: <1%B3RSD: <1%

B1

HPLC Flow Path Passivation

LC-MS/MS Analysis of Glyphosate, AMPA and Glufosinate

Method Conditions:

MPA: 0.5% formic

acid in water

MPB: 0.5% formic

acid in ACN

Flow: 0.5 mL/min

Gradi

ent:

Time %B

0.0 65

5.0 10

6.5 10

6.51 65

8.0 65

Oven 35 °C

Inj.: 5 μL

Fig. 3: LC-MS/MS separation of glyphosate, AMPA and glufosinate on ahybrid IEX/HILIC column (2.1x30 mm, 2.7 µm). Note the sharp peakshapes, fast equilibrium time (1.5 min) between runs, and short cycletime (8 min).

LC-MS/MS Analysis of 17 Polar Contaminants in One Run

Method Conditions:

MPA: 0.5% formic acid in water

MPB: 0.5% formic acid in

acetonitrile

Flow: 0.5 mL/min

Gradient:

Time %B

0.0 65

5.0 10

11.5 10

11.51 65

13 65

Oven

Temp:

35 °C

Injection

Vol:

1 μL

Glyphosate, AMPA and glufosinate and other 14polar food contaminants are chromatographicallyresolved on a short hybrid IEX/HILIC column(2.1x30 mm, 2.7 µm) within 11 min under simpleand MS friendly mobile phase conditions. Theseanalytes are retained and separatedpredominantly through ion exchange interactionswith the hybrid column.

Fig. 5: QuPPe sample preparation and LC-MS/MS detection ofglyphosate, AMPA and glufosinate in various food matrices.

Fig. 6: Analyte retention over 500 injection. The data demonstrate aguard column protects and extends the column lifetime beyond 500injections.

AM

PA

20

0 p

pb

0.8

05

Bia

lap

ho

s1

00

pp

b 0

.84

7

Pe

rch

lora

te 5

pp

b 2

.59

3

Glu

fosi

nat

e 2

00

pp

b 3

.37

6

DFA 200 ppb5.018

Eth

ep

ho

n2

00

pp

b 5

.56

4

HEP

A 1

00

pp

b 4

.96

9

MP

PA

10

0 p

pb

4.0

76

Ch

lora

te 1

00

pp

b 5

.54

2

Gly

ph

osa

te 2

00

pp

b 6

.11

3

Bro

mid

e 2

00

0 p

pb

6.4

23

N-A

cety

l AM

PA

20

0 p

pb

6.9

32

Fose

tyl(

Al)

80

pp

b 7

.77

5

Ph

osp

ho

nic

acid

50

0 p

pb

8.2

75

N-A

cety

l Glu

fosi

nat

e 2

00

pp

b 9

.98

0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 min

(optional)