Analysis of HMF by HPLC

Vural Gökmen

OO OH

COST Action 927 Training SchoolBuilding Skills on the Analysis of Thermal Process Contaminants in Foods22-26 October 2007, Ankara

Background

COOH

R1

NH2

O

OR

2

COOH

R1

N

H

R2

OH

OH

COOH

R1

N

H

R2

O

R1

N

H

R2

O

R1

N

H

R2

O

H2O

CO2

O OHO

Amino compound

Carbonyl compound

N-Glycosylated

Schiff base

Decarboxylated Schiff base

Hydroxymethylfurfural

- 3H2O O OHO

-D-Glucofuranose Hydroxymethylfurfural

O

OH

OH

OH

OH

OH

Maillard reaction

Hexose dehydration

At lower temperature processes, Maillard reaction is mainly responsible for HMF formation.

At higher temperature processes, hexose dehydration is mainly responsible for HMF formation. High acidity accelerates the formation.

It forms during thermal process and storage!

Amino acids

&

Sugars

Thermal Process / Storage

HMF

Some facts on HMF

• No need to heat at elevated temperatures to form HMF as in the case of acrylamide.

• All the foods at which acrylamide is present contains certain levels of HMF.

• Even more foods (jams, jellies, fruit juices, honey, etc) contain HMF.

• Its level tends to increase during storage.

• Foods having high sugars are at high risk.

Molecule Properties

• Chemical structure

– Aldehyde

– Alcohol

– Furan ring

• Soluble in water, alcohols

• MW=126

– [M+H] m/z 127, [-OH+H] m/z 109

• Absorbs well

– λmax = 285 nm

OO OH

Proximate Composition

• Carbohydrates

– Sugars (up to 20 %)

– Polysaccharides

• Starch (up to 20 %)

• Proteins (up to 15 %)

• Lipids (up to 40 %)

• HMF (mg/g levels)WATER

Thermally Processed Food

PART I

AQUEOUS EXTRACTIONwithout

SOLID PHASE EXTRACTION

Case Study

• Aqueous Extraction

• Carrez Clarification

• Cold Centrifugation (~0oC)

• SPE Cleanup

– hydrophobic & cation exchange action

• LC/UV Detection

– 285 nm

• LC/MS Detection

– APCI+, m/z 127, m/z 109

Scheme for HMF Analysis

Sampling

Homogenization

Extraction

Extract Cleanup

Detection

Representative

Water / Organic Solvent

Carrez clarificaion SPE

LC/UVLC/MS

Equipments

• Solid sample grinder

• Analytical balance

• Wortex mixer

• Ultra Turrax homogenizer

• Centrifuge with cooling

• HPLC

• LC/MSAgilent 1100 HPLC

Diode Array Detector

Chemicals and Consumables

• Water • Methanol • Acetonitrile• Formic or acetic acid • Carrez I & Carrez II • Disposables

– tube, syringe, eppendorf, tip, vial, filter

– SPE cartridge • Waters Oasis HLB

Sample Homogenization

4oC

Extraction

Vortex for 3 min

1.0 g

9 ml 10 mM water

0.5 ml Carrez I & II

Centrifuge 5000 rpm x 10 min

(~ 0oC)

Fat layer

Solid

recentrifuge

Raw extract

SPE Cleanup Strategy for HMF

ConditionWater Wash

0.5 mlDiethylether

Elution

Co-extractives

HMF

Sample Load1.0 ml

Dry cartridge under N2

DiscardEvaporateunder N2

Redissolve in water

LC/UV AnalysisLC

LC/UV Chromatogram

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

10

20

30

40

50

UV detection at 285 nm

Crisp bread

1 µg/ml standard

Figure. LC-UV chromatogram of crisp bread containing 21.5 mg/kg of HMF.Chromatographic conditions; column Atlantis dC18 (4.6 x 250 mm, 5 µm),mobile phase mixture of 0.1% formic acid and acetonitrile (90:10, v/v) at a flowrate of 1.0 ml/min at 25oC, detection 285 nm

Low concentrations

Figure. Chromatogram of a snack with low HMF content (1.1 mg/kg). Chromatographicconditions; column Atlantis dC18 (4.6 x 250 mm, 5 µm), mobile phase mixtureof 0.1% formic acid and acetonitrile (90:10, v/v) at a flow rate of 1.0 ml/min at25oC, detection 285 nm

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

5

10

15

20

25

30

35

40

Some comments

• Presence of interfering co-extractives may cause over-estimation of HMF in foods.

• This is specifically important for the samples with lower HMF concentrations.

• In this case, better sample preparation approaches, which is able to eliminate interfering co-extractives, are required.

Case Study-1

• Analysis of HMF in crisp bread

Portions of ground sample are

ready for analysis.

PART II

AQUEOUS EXTRACTIONwith

SOLID PHASE EXTRACTION

Advanced analysis of HMF

EXTRACTION

SPE CLEANUP

LC/UV/MS DETECTION

LC/UV/MS Analysis

• UV detection at 285 nm

• MS detection in SIM mode APCI+

– m/z 127 and m/z 109

• Rapid resolution chromatography

– Atlantis dC18, 2.1 x 150 mm (3 µm)

– Mobile phase

• 10 mM Formic Acid : Acetonitrile (95:5, v/v)

• 0.2 ml/min at 30oC

Good linearity

min7.5 8 8.5 9 9.5

mAU

0

2.5

5

7.5

10

12.5

15

17.5

20

min6 7 8 9 10 11

0

50000

100000

150000

200000

250000

LC/UV @ 285 nm

LC/MS @ SIM m/z 127

100 – 500 ppb

100 – 500 ppb

Over-estimation of HMFmAU

0

5

10

15

20

25

30

35

min0 2.5 5 7.5 10 12.5 15 17.5

0

5

10

15

20

Before SPE Cleanup

After SPE Cleanup

Detection @ 285 nm

Sample : Cookie

2.5 mg/kg HMFoverestimates 80%

Figure. Chromatogram of a cookie sample. Chromatographic conditions; column Atlantis dC18(2.1 x 150 mm, 3 µm), mobile phase mixture of 10 mM formic acid and acetonitrile(95:5, v/v) at a flow rate of 0.2 ml/min at 25oC, detection 285 nm

Over-estimation of HMF

Figure. Chromatogram of a cookie sample. Chromatographic conditions; column Atlantis dC18(2.1 x 150 mm, 3 µm), mobile phase mixture of 10 mM formic acid and acetonitrile(95:5, v/v) at a flow rate of 0.2 ml/min at 25oC, detection SIM m/z 127

200000

300000

400000

500000

600000

700000

min6 7 8 9 10 11

55000

60000

65000

70000

75000

80000

85000

90000

95000

Before SPE Cleanup

After SPE Cleanup

Detection @ SIM m/z 127 nm

overestimates 45% Sample : Cookie

2.5 mg/kg HMF

Peak Purity Check after SPE

With SPE Cleanup

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

2

4

6

8

10

12

14

16

Figure. Chromatogram of a infant formulae with low HMF content (1.4 mg/kg). Chromatographicconditions; column Atlantis dC18 (2.1 x 150 mm, 3 µm), mobile phase mixture of 10mM formic acid and acetonitrile (95:5, v/v) at a flow rate of 0.2 ml/min at 25oC,detection 285 nm

LC/UV @ 285 nm

1 µg/ml

Sample

INFANT FORMULAE

1.4 mg/kg HMF

min6 7 8 9 10 11

25000

50000

75000

100000

125000

150000

175000

200000

With SPE Cleanup

Figure. Chromatogram of a infant formulae with low HMF content (1.4 mg/kg). Chromatographicconditions; column Atlantis dC18 (2.1 x 150 mm, 3 µm), mobile phase mixture of 10mM formic acid and acetonitrile (95:5, v/v) at a flow rate of 0.2 ml/min at 25oC, MSdetection at m/z 127 & m/z 109

LC/MS @ SIM m/z 127INFANT FORMULAE

1.4 mg/kg HMF

1 µg/ml

Sample

Reproducible SPE results

min7.5 8 8.5 9 9.5

mAU

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Figure. Chromatogram of a infant formulae with low HMF content (1.5 mg/kg). Chromatographicconditions; column Atlantis dC18 (2.1 x 150 mm, 3 µm), mobile phase mixture of 10mM formic acid and acetonitrile (95:5, v/v) at a flow rate of 0.2 ml/min at 25oC, MSdetection at m/z 127 & m/z 109

INFANT FORMULAE LC/UV @ 285 nm

LC/UV/MS

0

10

20

30

40

50

min0 2.5 5 7.5 10 12.5 15 17.5

50000

100000

150000

200000

LC/UV @ 285 nm

LC/MS @ SIM m/z 127

CRISP

BREAD

Figure. Chromatogram of crisp bread (24 mg/kg). Chromatographic conditions; column AtlantisdC18 (2.1 x 150 mm, 3 µm), mobile phase mixture of 10 mM formic acid andacetonitrile (95:5, v/v) at a flow rate of 0.2 ml/min at 25oC, MS detection at m/z 127 &m/z 109, and UV detection at 285 nm

Case Study-2

• Analysis of HMF with SPE

Portions of ground sample are

ready for analysis.

Further readings