Hydroxymethylfurfural (HMF) Analysis of Beer by HPLC-UV using a 4.6 mm ID Monolithic Silica Column
Anita Piper, Scientist
Merck

Hydroxymethylfurfural (HMF)
(5-(hydroxymethyl)furfural)
Section Overview
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.
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. |

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 |
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 |

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.

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 |

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 |
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 |
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 |
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
REFERENCES
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