Improving sensitivity, robustness, and stability of beer flavor analysis by novel GC-MS/MS assays

In bioanalytics, the aim is to monitor or identify biological processes. The analytical tools employed depend on nature and concentration of the respective target analytes, as well as the matrix and system under investigation. In beer production, sometimes referred to as humankind’s oldest biotechnology, the goal is to produce a beverage with consistent flavor, which is the result of hundreds of volatiles present in the beverage. In order to quantify flavor compounds in beer and brewing samples, targeted HS SPME-GC-MS assays are most common. However, with the multiplicity of compounds present in brewing samples, the low concentration and reactivity of key aroma compounds, beer flavor analysis remains challenging. In order to improve beer flavor analysis, and in particular to increase sensitivity, robustness, and stability, three novel HS-SPME-GC-MS/MS assays were introduced and validated. Publication A “Analysis of Selected Hop Aroma Compounds in Commercial Lager and Craft Beers Using HS-SPME-GC-MS/MS” deals with the analysis of 16 selected hop aroma compounds covering the most relevant substance classes (terpenes, terpenoids, esters) commonly associated with hoppy beer flavor. With the industry standard HS SPME-GC-MS methods, sufficient extraction and enrichment of volatiles requires maximizing sample volume and extraction times that stresses fiber material and limits fiber lifetime. Using tandem-mass spectrometry, sample volumes can be decreased, which reduces the amount of volatiles adsorbed onto the fiber and consequently prolonged the fiber lifetime. This study indicated that the combination of HS-SPME-GC-MS/MS with suitable stable isotope labelled ISTDs provides an assay with excellent stability that is not affected by e.g. fiber-to-fiber variations. The method development focused on the individual steps of MRM optimization and discussed them in detail. Moreover, the advantages of MRM in the reduction of matrix effects were illustrated. In Publication B “Analysis of Selected Staling Aldehydes in Wort and Beer by GC EI MS/MS Using HS-SPME with On-Fiber Derivatization”, an assay to quantify 15 analytical markers for beer flavor (in)stability was developed. The list of analytes includes lipid oxidation products (e.g., (E) 2-nonenal), products of Strecker degradation (e.g., methional), and so-called heat indicators (e.g., furfural). While the established methodology HS-SPME-GC-MS combined to on fiber derivatization is a good approach regarding a fast sample preparation, the quantification of low abundant aldehydes in GC-MS must be performed in the lowest technically achievable level of quantification. The direct comparison of GC-EI-MS/MS and GC-EI-MS revealed that due to the lower selectivity of SIM mode the quantification of these low abundant aldehydes by MRM mode is more reproducible. As this method is designed to quantify and monitor staling aldehydes from both wort and fresh as well as aged beers, the assay covers a wide concentration range (0.01 1000 µg/L) and should also provide good long-term stability. Hence, the long-term stability, herein defined as peak area consistency of two ISTDs over 24 weeks, across different beer matrices was determined and evaluated as excellent. Publication C “Analysis of Hop-Derived Thiols in Beer Using On-Fiber Derivatization in Combination with HS-SPME and GC MS/MS” is the most complex of the three assays. This relates to the fact that thiols appear in ng/L levels and are very prone to oxidation. In order to achieve quantification via GC-MS, time-consuming multi-step sample preparation procedures, which in some cases involve the handling of mercury containing solvents, are used. By taking advantage of the noise filtering by MRM mode and by using an automated on-fiber derivatization approach, the sample preparation requires minimal manual handling, which improves analysis quality as shown by validation. Whilst in the two above-mentioned publications method development was performed in a stepwise procedure, this method development used a central composite design. Validation of the optimized assay proves its sensitivity (limits of quantification below the sensory threshold of 4MMP, 3MH, and 3MHA) while being more rapid than any of the previously published methods. In conclusion, the three developed HS-SPME-GC-MS/MS assays improved throughput, robustness, and sensitivity of beer flavor analysis. The improved detection sensitivity and selectivity offered by use of triple quadrupole GC-MS/MS resulted in prolonged fiber lifetimes and improved HS-SPME calibration consistency for hop aroma analysis, more reproducible results for low-abundant staling aldehydes as well as excellent long-term stability of the assay. Additionally, the required sensitivity for thiol quantification without extensive sample preparation was achieved. The publications demonstrate that the use of HS-SPME-GC-MS/MS in beer flavor analysis is highly beneficial, thus probably encouraging brewing chemists to adapt these published methodologies.