Quantitative Liquid Chromatography
Analysis of Melamine in Dairy Products
Using Agilent's 1120 Compact LC and1200 Rapid Resolution LC
Application Note
Food SafetyAuthors

Abstract

Hua Wu, Rong An, Yao Xiao, Zhixu

In this application, three different LC methods are developed for the determination ofZhang, and Ping Li

melamine in dairy products. The first is a modification of the U.S. FDA method [1]. An

Agilent Technologies Co., Ltd.

Agilent LC system (1120 or 1200) is used with a ZORBAX SB-C8 LC column to run in3 Wang Jing Bei Lu,

reversed-phase ion-pair mode for routine quantitation of melamine. The second

Beijing 100102

method is targeted for high throughput using an Agilent Rapid Resolution LC (RRLC)

Chinasystem (1200SL) to speed melamine analysis by more than three times with a Rapid

EditorResolution High Throughput (RRHT) column. The third is an alternative ion-exchange
LC method where a ZORBAX 300SCX column is employed to successfully retain

Wei Luanmelamine using a simple mobile phase of buffered water/acetonitrile without theAgilent Technologies Co., Ltd.

presence of ion-pair reagent. Due to the complexity of dairy product matrices, a412 Ying Lun Road

cleanup step using solid phase extraction (SPE) is required for the above methods.

Shanghai 200131

The Agilent SampliQ SCX, a mixed-mode polymer SPE cartridge with combinedChinareversed-phase and strong cation exchange properties, is used to successfully remove
matrix interferences.

Introduction

Experimental

In March 2007, imported pet food ingredients contaminatedwith melamine caused renal failure in dogs and cats acrossthe United States. Once again, this compound is in the news
as an illicit adulterant in milk and milk products. The same
contaminant is now being detected in other food productsthat contain milk imported from China and as global concern
rises, widespread testing for melamine is proceeding.

Standard Preparation

The published analytical approaches include LC, LC/MS, and
GC/MS. The LC method is being used for quantitative analyses of melamine. Liquid chromatographic separation of thissmall polar compound can be achieved by reversed-phaseion-pair liquid chromatography. The U.S. FDA developed thismethodology for melamine in pet food in 2007. With a slight
modification of the proportion of mobile phase, the method
can be successfully applied to separate melamine from avariety of dairy product matrices.

The sample preparation process is a modification of the China
national standard [2].

A stock solution of melamine at 1,000 µg/mL is prepared inmethanol by sonication. Dilutions in mobile phase are madeup at 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 µg/mL
concentrations.
Sample Preparation

Sample Extraction Procedure
For liquid milk, milk powder, yogurt, ice cream, and creamy
candy samples:

The disadvantage of conventional HPLC is time and solvent
consumption. The Agilent 1200 Series Rapid Resolution LCsystem is designed for highest throughput without loss ofresolution or with better resolution in combination with theAgilent ZORBAX RRHT columns. In this application note, theconventional HPLC method is transferred from a 4.6 mm ×250 mm, 5 µm ZORBAX SB-C8 column to a 4.6 mm × 50 mm,
1.8 µm RRHT ZORBAX SB-C8 column with equivalent results,
and the LC run time is shortened from almost 20 minutes to6 minutes.
An alternative approach for liquid chromatographic separationof this small polar compound is ion exchange chromatography. Agilent ZORBAX 300SCX is an ionic bonded-phase column packing used for cation exchange high-performance liquid chromatography. This packing consists of an aromatic sulfonic acid moiety covalently bonded to ZORBAX porous silica.
This column is successfully applied to retain melamine using
a simple mobile phase of buffered water/acetonitrile without
the presence of ion-pair reagent.

•Weigh 2 ± 0.01 g of sample and add to a 50-mL centrifuge
tube, add 15 mL of 5% trichloroacetic acid in water and
5 mL of acetonitrile, then cap.

•Sonicate for 10 min and then place samples on verticalshaker for 10 min. Centrifuge for 10 min at 4000 rpm.

•Wet filter paper with 5% trichloroacetic acid in water,then filter the supernatant into a 25.0-mL volumetricflask, and bring to volume with 5% trichloroacetic acid inwater.

•Transfer a 5.0-mL aliquot of the extract into a glass tube,
and then add 5.0 mL purified water. Vortex to mix
thoroughly.

For cheese, cream, and chocolate samples:

For complex dairy product matrices, it is necessary to remove
interferences such as protein, sugar, and fat before LC injection. Solid-phase extraction (SPE) is a simple way to clean up
the complex matrix extract. SampliQ is a new family of SPE
cartridges from Agilent with a wide range of sorbent
chemistries. Among this family, the mixed-mode SampliQ
Strong Cation Exchange (SCX) cartridge is a sulfonic acidmodified divinyl benzene polymer with both ion exchange andreversed-phase retention properties. This makes the SampliQ
SCX very effective for cleanup after solvent extraction.

2•Weigh 2 ± 0.01 g of sample, grind with 8~12 g of sea
sand in a mortar, and then transfer into a 50-mL centrifuge tube.

•Wash the used mortar with 5 mL of 5% trichloroacetic
acid in water three times, transfer washings into a 50-mL
centrifuge tube, and then add 5 mL of acetonitrile.

•

Proceed with the sonication and other steps as describedin the previous preocedure..

•If the sample is very fatty, defat the extract using liquidliquid extraction with hexane saturated with 5%
trichloroacetic acid in water before cleanup by SPE.

Sample Cleanup Procedure

High-Throughput Method Using 1200SL RRLC:

A SampliQ SCX SPE cartridge (p/n 5982-3236, 3 mL, 60 mg, or
p/n 5982-3267, 6 mL, 150 mg) can be used to clean up sample
extracts; the latter is used in this application note. All SPE
elution steps, including conditioning, sample load, washing,
and the final elution, are performed with a flow rate of lessthan 1 mL/min except for drying the cartridge by applyingvacuum.
•Condition the SPE cartridge with 5 mL of methanol followed by 6 mL of water.

•Load the above sample extract to the conditioned cartridge. Wash the cartridge with 5 mL of water followed by
5 mL of methanol.

••Agilent 1200SL Series binary pump, degasser, wellplatesampler, thermostatted column compartment and diodearray detector (DAD)•ChemStation software (Ver. B.04.01 or later)

Dry the cartridge by applying vacuum, and then elute with
5 mL of 5% ammonium hydroxide in methanol.

ColumnZORBAX SB-C8 RRHT, 4.6 mm × 50 mm,
1.8 µm (p/n 827975-906)

Buffer

10 mM citric acid, 10 mM sodium octane
sulfonate, adjusted to pH 3.0Mobile phase

92:8 buffer:acetonitrile

Flow rate

1.5 mL/min

Injection volume

8 µL

Column temperature

30 °C

Detection wavelength

240 nmRun time

6 min•

Evaporate the eluate to dryness under a stream of nitrogen at approximately 50 ºC.

Ion Exchange Chromatography Method with 1120 Compact LCor 1200 LC:•Reconstitute the dried extract in 1.0 mL of mobile phase,
vortex for 1 min, and filter through a 0.2-µm regenerated
cellulose membrane filter (p/n 5064-8222) into a glass
LC vial.

•Agilent 1200 Series binary pump, degasser, wellplatesampler, thermostatted column compartment and variablewavelength detector (VWD) or equivalent 1120 Series
components•

EZChrom Elite Compact software or ChemStation
software (Ver. B.04.01 or later)

Instrumentation and Conditions
Conventional HPLC method using 1120 Compact LC or
1200 LC:••Column

ZORBAX 300SCX, 4.6 mm × 150 mm, 5 µm
(p/n 883952-704)

Buffer

50 mM ammonium formate solution,
adjust to pH 3.0 with formic acid

Mobile phase

15:85 buffer:acetonitrile

Flow rate

1.0 mL/min

EZChrom Elite Compact software or ChemStation
software (Ver. B.04.01 or later)

Injection volume

10 µL

Column temperature

30 °C

ColumnZORBAX SB-C8 (also known as ZORBAX
Rx-C8), 4.6 mm × 250 mm, 5 µm
(p/n 880975-906)

Detection wavelength

240 nm

Run time

5.5 min

Buffer

10 mM citric acid, 10 mM sodium octane
sulfonate, adjusted to pH 3.0

Results and Discussion

Mobile phase

92:8 buffer:acetonitrile

Flow rate

1.5 mL/min

Separation of Melamine in Dairy Products by ReversedPhase Ion-Pair LC

Injection volume

20 µL

Column temperature

30 °C

Agilent 1120 Compact LC system with gradient pump(degasser inside), autosampler, column compartment,
and variable wavelength detector (VWD) or equivalent
1200 Series components

Detection wavelength

240 nm

Run time

20 minMelamine is not retained by reversed-phase LC and thus
elutes with the solvent and unretained matrix interferences.
However, using an ion-pairing reagent with reversed-phase
chromatography, melamine can be well retained and separated from interferences. Figure 1 (a) is the chromatogram ofmelamine standard by reversed-phase ion-pair LC. Figure 1 (b)is the chromatogram of a positive yogurt sample after cleanup with the Agilent SampliQ SCX SPE cartridge.

3mAU
17.5
1512.5107.5
52.50_ 2.5(a)Melamine

24mAU
2.521.5
10.5
00.5
1Figure 1.

68101214(b)

16min

18

Melaminemin24681012141618
Separation of (a) 20 µg/mL melamine standard, and (b) positive yogurt sample after cleanup by SampliQ SCX SPE cartridge. Retention time of melamine is 14.2 minutes.

High-Throughput Analysis by Agilent 1200SL RRLC withRRHT Column

Figure 2 (b) is the chromatogram of the same yogurt samplein Figure 1 (b). In order to make sure that the column is clear
for the next injection, the total run time is extended to 6 minutes. The high throughput RRLC method is applied in the variety of dairy products matrices, including yogurt, liquid milk,and milk powder to demonstrate that the same resolution isachieved as with the conventional HPLC method. The calibration curve for the RRLC method is shown in Figure 3. The calibration includes 0.8, 2.0, 20.0, 40.0, and 80.0 µg/mL. The
instrumental LOD (limit of detection) of the RRLC method is0.03 µg/mL.

With the Agilent 1200 Series RRLC system, high throughput ispossible. In combination with the Agilent ZORBAX RRHT
columns, excellent chromatographic resolution can beachieved at much shorter run times than with a conventional
LC system. A RRLC method is developed to dramaticallyincrease the sample throughput for the determination ofmelamine in dairy products. Figure 2 (a) is the chromatogramof a melamine standard by the RRLC method with the retention time of melamine at 2.8 minutes.
mAU
3.5

2.873

(a)

32.52Melamine

1.5
10.5
00.5mAU
3.5

11.5

2

2.5

33.5minMelamine
2.877(b)32.5
23.151

1.5
10.5
00.5
Figure 2.

1

1.5

22.5

3

3.5

min

Separation of (a) 0.8 µg/mL melamine standard, and (b) positive yogurt sample after cleanup by SampliQ SCX SPE cartridge. Retention time of melamine is 2.8 minutes.

4y = 17.76x _3.07
R 2 = 0.99996

Area

51200
1000

4800
600

3400
200

12Measured point (0.833, 11.732)

00Figure 3.

50
Amount (wt %)Calibration curve of RRLC method.

time of the ion exchange chromatography is only 5.5 minuteswith an LOD of 0.05 µg/mL, as shown in Figure 5. The calibration curve for the IEC method is shown in Figure 6. The calibration points include 0.5, 1.0, 5.0, 10, 50, and 100 µg/mL.
Although the separation is shown to be interference free forraw milk and liquid milk without any additive, it is still recommended that the cleanup step be included to ensure robust
methodology for running many samples and samples of different matrices.

Ion Exchange Chromatographic Method
An alternative to ion-pair reversed-phase chromatography formelamine is ion exchange chromatography (IEC). The Agilent
ZORBAX 300SCX is used for cation exchange high-performance liquid chromatography (HPLC). This column is
employed to separate melamine in dairy product matrices
with sufficient retention to separate matrix interferences.
Figure 4 shows the separation of melamine from interferences without the SPE cleanup step. Generally, the total runmAU
80

Melamine
60

40

2000Figure 4.

1234Separation of melamine in milk powder sample by IEC without cleanup by SPE.

55min

mAU
0.4

Melamine, S/N = 3.9

0.3
0.2
0.1
0-0.1
-0.2
-0.3
-0.4
-0.5
01

Figure 5.

234

5Limit of detection (LOD) for melamine at the concentration of 0.05 µg/mL.

1600
1400

y = 15.054x
R 2 = 0.9998

1200

Area

1000
800
600
400
200
00Figure 6.

20

4060
Concentration (µg/mL)

80

100

Calibration curve of IEC method.

6120minConclusions
Three approaches are described in this application note; thefirst is a reversed-phase ion-pair LC method employingAgilent 1120 compact LC or 1200 HPLC with an SB-C8 column. The second is a high-throughput method, which reduces
the LC run time from 20 minutes to 6 minutes using theAgilent 1200 RRLC with the ZORBAX RRHT SB-C8 column.
The last is an IEC method using the Agilent ZORBAX 300SCX
column. Each successfully separates melamine from matrix
interferences and provides identification by retention timeand quantitative results. The results of this study, including
sample cleanup with SampliQ SCX SPE cartridges and thethree separation approaches, show that a complete solution
from Agilent for the determination of melamine in dairy products is provided. The reversed-phase ion-pair method isbased on the FDA and China national standards. However, theIEC method is simple, quick, sensitive, and robust. With thismethod, melamine can be successfully retained using a simple mobile phase without the presence of an ion-pair reagent.

References
1.

Updated FCC Developmental Melamine Quantitation(HPLC-UV), April 2, 2007

2.

GB/T 22388-2008 Determination of melamine in raw milk
and dairy products, October 7, 2008

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© Agilent Technologies, Inc., 2008
Published in the USA
October 23, 2008
5989-9949EN