跳转至内容
Merck
CN
HomeChemical Analysis for Food and Beverage TestingHydroxymethylfurfural Analysis of Beer by HPLC UV

Hydroxymethylfurfural (HMF) Analysis of Beer by HPLC-UV using a 4.6 mm ID Monolithic Silica Column

Anita Piper, Scientist
Merck

Hydroxymethylfurfural structure with a furan ring attached to methyl alcohol and carbaldehyde group at 2 and 5 positions on the furan ring.

Hydroxymethylfurfural (HMF)
(5-(hydroxymethyl)furfural)

Introduction

Hydroxymethylfurfural (HMF) is generated by thermal decomposition of carbohydrates or sugars. It can be detected in many heat-treated food and beverage samples. In the latter, low levels of HMF indicate its freshness and natural finish, while long-term storage or exposition to heat leads to high HMF content, caused by fructose decomposition. 

HMF is suspected to have negative health effects on animals and humans1 hence more attention is given to this by official institutions like the European Union2, where e.g. limit values for honey are defined.3

This application focusses on the analysis of HMF in the different beer samples, smoke beer, wheat beer dark, and dark beer by HPLC with UV detection using a matrix tolerant monolithic silica column, the Chromolith® HighResolution RP-18 endcapped. 

Experimental

Quantitation of HMF in three different beer samples was performed on a Chromolith® HighResolution RP-18 endcapped 100 x 4.6 mm column using UV detection without extensive sample preparation performed (Table 1). The samples were just degassed and then injected.

HPLC Conditions

Column:

Chromolith® HighResolution RP-18 endcapped 100x4.6mm (1.52022)

Mobile phase:

[A] 0.01% Trifluoroacetic acid in water;

[B] Acetonitrile

Gradient:

Time (min)

A%

B%

Flow (mL/min)

 

0

95

5

0.5

 

7

95

5

0.5

 

8

40

60

0.5

 

12

40

60

0.5

 

13

95

5

0.5

 

19

95

5

1.0

 

20

95

5

0.5

Flow rate:

See gradient table

Pressure drop:

15-34 bar (218-493 psi)

Column temp.:

25° C

Detection:

UV at 280 nm (micro flow cell; 1.4 µL/7 mm); 5 Hz

Injection:

5 µL

Samples:

Diluent:

Water

Standard solution (200 mg/L):

20 mg of homogenized HMF was weighed into a 100 mL volumetric flask, dissolved in diluent and filled up to mark with diluent. Standard solution needs to be prepared on the day of use.

Working solution (10 mg/L):

0.5 mL of the standard solution were pipetted into a 10 mL volumetric flask and filled up with diluent to mark. Working solution needs to be prepared on the day of use.

Sample preparation beer:

After degassing 1 L beer for 1 h in an ultrasonic bath the beer samples were filled into glass vials and injected directly.

Sample preparation spiked beer sample:

After degassing the beer samples were spiked with 20 mg/L of HMF. After the samples were filled into glass vials and injected directly.

Table 1.Conditions for HMF HPLC determination in beer

Results

A chromatogram representing the analysis of a working solution of Hydroxymethylfurfural (HMF) with a concentration of 10 mg/L. The x-axis, labeled "Retention Time (min)," ranges from 0 to 20 minutes, while the y-axis, labeled "Intensity (mAU)," ranges from -60 to 80. A black line graph displays the chromatographic peaks, with the baseline running horizontally along the retention time. Two peaks are labeled: Peak 1 appears as a small bump near the 4-minute mark, and Peak 2, representing the HMF compound, is a tall and narrow peak at approximately 6.5 minutes. The graph background is white, and the lines and labels are in black. The word "Working Solution" is displayed in black text in the top right corner of the plot.

Figure 1.HMF standard working solution 10 mg/L (Peak 2: HMF).

Peak no.

Compound

Retention time (min)

S/N

Area (mAU*min)

Tailing factor

1

Void volume

3.1

 

 

 

2

HMF

6.5

1930.2

13.7558

1.1

Table 2.Chromatographic data - Working solution (10 mg/L)

Calibration

An external calibration curve was established using 7 concentrations (Table 3 & Figure 2)

Conc. (mg/L)

Mean Area (mAU*min)

2.58

3.351

5.15

6.658

10.31

13.586

20.61

26.368

41.22

52.188

STEYEX

0.226

Slope

1.262

LOD (mg/L)

0.59

LOQ (mg/L)

1.79

R2

0.9999

Table 3.LOD, LOQ and linearity of external calibration for HMF
A calibration curve graph displaying the relationship between the concentration of Hydroxymethylfurfural (HMF) standards and the corresponding peak area. The x-axis, labeled "Concentration (mg/L)," ranges from 0 to 50, while the y-axis, labeled "Area (mAU*min)," ranges from 0 to 60. The graph shows a series of black data points plotted for five concentrations of HMF along a straight line that slopes upward to the right, indicating a positive correlation. A black dotted line connects the points, and a linear equation (y = 1.2617x + 0.2762) and a high coefficient of determination (R² = 0.9999) are displayed in black text above the trendline.

Figure 2.Calibration curve of HMF standards.

Sample Measurements

As examples for beer samples, the results for the smoke beer unspiked and spiked (20 mg/L) are presented below (Figures 3 & 4, Tables 4 & 5). Reproducibility was determined by injecting each beer sample 5x (Table 6). In Table 7 an overview of the measure unspiked and spiked is displayed.

A chromatogram graph depicting the analysis of a smoke beer sample. The x-axis, labeled "Retention Time (min)," ranges from 0 to 20, while the y-axis, labeled "Intensity (mAU)," ranges from -200 to 1000. The chromatogram line, displayed in black, rises from the baseline at around 3.1 minutes, where a small peak labeled "1" appears, indicating the void volume. A larger peak, labeled "2," is observed at 6.4 minutes, representing HMF. The curve fluctuates with multiple smaller peaks before and after the main peak, indicating the presence of various compounds in the sample.

Figure 3.Injection of a smoke beer sample (Peak 2: HMF).

Peak no.

Compound

Retention time (min)

S/N

Area (mAU*min)

Tailing factor

1

Void volume

3.1

 

 

 

2

HMF

6.4

379.7

7.0099

0.9

Table 4.Chromatographic data - Smoke beer
A chromatogram graph representing the analysis of a spiked smoke beer sample containing 20 mg/L of HMF. The x-axis, labeled "Retention Time (min)," spans from 0 to 20 minutes, and the y-axis, labeled "Intensity (mAU)," ranges from -100 to 1000. A black line traces the chromatographic data, starting from the baseline and forming a small peak at 3.1 minutes, labeled "1," representing the void volume. A significantly larger peak, labeled "2," appears at 6.4 minutes, corresponding to the HMF. Additional smaller peaks are visible between these two labeled points, as well as fluctuations throughout the rest of the trace.

Figure 4.Injection of a spiked (20 mg/L HMF) smoke beer sample (Peak 2: HMF).

Peak no.

Compound

Retention time (min)

S/N

Area (mAU*min)

Tailing factor

1

Void volume

3.1

 

 

 

2

HMF

6.4

1997.7

30.2236

1.1

Table 5.Chromatographic data - Smoke beer spiked with 20 mg/L HMF

Peak no.

Smoke beer

Wheat beer dark

Dark beer

Sample 1

7.0791

3.4538

4.9731

Sample 2

7.0316

3.5016

4.9713

Sample 3

6.9511

3.5393

4.9973

Sample 4

7.0099

3.5446

4.9828

Sample 5

7.0695

3.4996

4.9704

Mean

7.0282

3.5078

4.9790

Standard deviation

0.0514

0.0366

0.0114

(%) RSD

0.7

1.0

0.2

Table 6.Sample repeatability of different unspiked beer samples in area (mAU*min)

Sample

Conc .HMF (mg/L)

Spike Recovery (%)

Smoke beer

5.34

 

Smoke beer spiked

23.74

92.0

Wheat beer dark

2.55

 

Wheat beer dark spiked

20.34

88.9

Dark beer

3.48

 

Dark beer spiked

22.29

94.0

Table 7.Overview of HMF amounts detected in unspiked and spiked (20 mg/L) beer samples using the external calibration

Conclusion

The developed simple HPLC-UV method allows for a 5-HMF test in three different beer samples on a Chromolith® HighResolution RP-18e 100x4.6 mm column with an LOD of 0.6 mg/L and LOQ of 1.8 mg/L. The recoveries for spiked beer samples were between 88.9% and 94%.

The bimodal pore structure of the monolithic silica Chromolith® HighResolution column provided the matrix tolerance that allowed us to reduce the sample preparation to a minimum (just degassing). By this, the workflow could be kept short, which otherwise typically requires more elaborate sample preparation steps like SPE to, e.g. avoid column clogging.

Another way of HMF determination can be performed by a rapid photometric test that can be found in our Manual on Analysis Methods for the Brewery Industry. This covers a range of other parameters for the brewery industry and is complemented by e.g. our application note on Sulfur Dioxide in Beer Photometric Determination with Ellmann’s Reagent.

More applications on Food & Beverage Testing

Related Products
Loading

REFERENCES

1.
Shapla UM, Solayman M, Alam N, Khalil MI, Gan SH. 2018. 5-Hydroxymethylfurfural (HMF) levels in honey and other food products: effects on bees and human health. Chemistry Central Journal. 12(1): https://doi.org/10.1186/s13065-018-0408-3
2.
Schrenk D, Bignami M, Bodin L, Chipman JK, del Mazo J, Grasl‐Kraupp B, Hogstrand C, Hoogenboom L(, Leblanc J, Stefano Nebbia C, et al. 2022. Evaluation of the risks for animal health related to the presence of hydroxymethylfurfural (HMF) in feed for honey bees. EFS2. 20(4): https://doi.org/10.2903/j.efsa.2022.7227
3.
European commission. Council Directive 2001/110/EC of 20 December 2001 relating to honey. . [Internet]. Available from: http://data.europa.eu/eli/dir/2001/110/2014-06-23j
登录以继续。

如要继续阅读,请登录或创建帐户。

暂无帐户?