Fingerprinting Analysis of Different Types of Beer Using Comprehensive 2D-LC

Significance of the Topic

Beer is a widely consumed alcoholic beverage whose complex molecular composition influences flavor, stability and quality. Comprehensive profiling of hop-derived bitter acids and polyphenols is crucial for quality control, authenticity verification and understanding processing impacts. Advanced techniques such as comprehensive two-dimensional liquid chromatography (2D-LC) provide the resolution and peak capacity required to dissect beer’s intricate chemical fingerprint.

Study Objectives and Overview

This study demonstrates the development and application of the Agilent 1290 Infinity 2D-LC solution to fingerprint and classify various beer styles including German pils, weizen, Irish stout, American lager and alcohol-free weizen. Objectives include:

• Maximizing orthogonality using C18 separations at alkaline and acidic pH.
• Identifying key bitter compounds (iso-alpha-acids and reduced tetrahydro derivatives) by UV and mass spectrometric detection.
• Implementing nontargeted multisample analysis combined with principal component analysis (PCA) for robust classification.

Methodology and Instrumentation Used

Instrumentation:

• Agilent 1290 Infinity 2D-LC Solution comprising binary pumps, thermostat-controlled autosampler, column compartment and valve drive
• C18 columns: Extend-C18 2.1×100 mm, 3.5 µm (1D) at pH 9.95; Poroshell HPH-C18 4.6×50 mm, 2.7 µm (2D) at acidic pH
• Agilent 6530 Accurate-Mass Q-TOF MS with Jet Stream ESI and diode array detector
• Software suite: OpenLAB CDS, 2D-LC Acquisition, MassHunter Workstation, LCxLC data analysis

Sample Preparation: Degassing by stirring and sonication followed by 0.45 µm filtration.
Chromatographic Conditions:

• 1D gradient from 2 to 95% ACN/EtOH (alkaline pH) at 0.075 mL/min
• 2D gradient cycling every 21 s from 5 to 92% ACN with 0.25% formic acid at 4.0 mL/min

Main Results and Discussion

Orthogonality was confirmed by two-dimensional contour plots showing broad peak distribution across weizen, pils and lager samples. UV detection at 270 nm and accurate-mass spectra identified iso-alpha-acids and tetrahydroiso-alpha-acids with mass errors below 1 ppm. Nontargeted feature matching across triplicates enabled quantitation of percent response for each 2D peak region. PCA explained ~88% of variance: F1 separated American lager and Irish stout from German beers; F2 distinguished alcohol-free weizen; F3 separated weizen from pils variants.

Benefits and Practical Applications

• High-resolution fingerprinting supports routine quality control and authenticity checks
• Comprehensive profiling aids product development and process optimization
• Rapid modulation cycles enable high-throughput analysis of complex matrices

Future Trends and Potential Applications

Further integration of 2D-LC with high-resolution MS and automated chemometrics will enhance fingerprinting workflows. Prospective applications include real-time monitoring of fermentation and maturation, extension to other beverages and foods, and predictive quality modeling.

Conclusion

The Agilent 1290 Infinity 2D-LC solution delivers robust, reproducible separations and accurate identification of beer’s bitter and polyphenolic components. Combined with advanced data analysis, this workflow provides a powerful tool for comprehensive fingerprinting and quality control in the brewing industry.

References

1. Araujo A S et al., Electrospray ionization mass spectrometry fingerprinting of beer, Analyst, 2005, 130, 884–889
2. Vanhoenacker G et al., Analysis of iso-alpha-acids and reduced iso-alpha-acids in beer, J Chromatogr A, 2004, 1035, 53–61
3. Intelmann D et al., LC-MS/MS quantitation of hop-derived bitter compounds in beer, J Agric Food Chem, 2009, 57, 1172–1182
4. Ceslova L et al., Characterization of prenylflavonoids and hop bitter acids, J Chromatogr A, 2009, 1216, 7249–7257
5. Reichenbach S E & Naegele E, 2D-LC comparison of ingredients, Agilent Application Note, 5991-1455EN, 2013
6. Naegele E, Performance evaluation of Agilent 1290 Infinity 2D-LC Solution, Agilent Application Note, 5991-0138EN, 2012