Beer represents one of the most widely consumed beverages in the world. Its annual worldwide production, measured in hundreds of millions of hectoliters, is a testimony to its popularity. While there are many different types of beer, all share one underlying characteristic taste, bitterness. Beer derives its bitter taste from bitter acids present in hops that undergo isomerization during the brewing process. In contrast to old traditional processes modern brewing techniques may use hop products such as extracts to achieve greater production volumes and improved batch consistency. In this study, bitter acid composition of different types of beers is presented. The results are grouped by beer type and geographical location. Beer taste degradation with extended exposure to air is correlated to its reduction of bitter acid content. A reversed phase chromatography has been used to separate the hop alpha acids from their isomerized products in beer, API 3000 LC/MS/MS System triple quadrupole mass spectrometer was then used to determine the bitter acid contents. API 3000 system mass spectrometer and a high mass accuracy hybrid QqTOF instrument, QSTAR LC/MS/MS System, have been used to generate fragmentation spectra for structural elucidation of the acids.
Hop cones, a crop harvested from a hop plant, contain the bitter alpha and beta acids; n-, co-, and ad- humulones and lupulones respectively. The humulones in particular are widely used in the brewing process to provide bitterness to beer. During traditional brewing, hops are added to a waterextracted barley malt and the mixture is boiled. This process causes the largely insolubl e alpha acids to convert to more bitter and more soluble iso-alpha acids, the primary bittering compounds in beer. The next and last step in the brewing process, fermentation, is different for ales and lagers. Lagers are produced by a process called bottom fermentation requiring a lower temperature and longer time, whereas ales are fermented at room temperature requiring a shorter period of time.
Experimental and results
A sample of hop pellets has been obtained from a local home brewing outlet. The pellets were then crushed and the resulting powder extracted with hexane. Beer samples were extracted with equal volume of hexane and spun to isolate the hexane component. In both cases hexane samples were evaporated and re-dissolved in methanol for infusion or in mobile phase for LC work.
Since both the alpha acids and their isomerized products have same molecular weight, distinction can only be made using their MS/MS fragmentation patterns. MS and MS/MS spectra were generated with 5 uL/min infusion of the respective samples. Identification of the fragments has been confirmed using the high mass accuracy of the MS/MS/TOF instrument, that allows determination of elemental composition of the precursor and fragment ions. Isohumulones, as shown in the example of co-isohumulone, produce a unique fragment suitable for multiple reaction monitoring (MRM). The base peak in fragmentation spectra of humulones is not unique as shown by the example of co-humulone and co-isohumulone. Chromatography was used to resolve the two groups. An example chromatogram lists the MRM transitions used to detect the alpha and isomerized alpha acids. LC conditions used in the separation were as follows, Jones C 8 2.1 x 100 mm HPLC column with a mobile phase consisting of 2mM ammonium acetate and methanol. An eight minute gradient run from 50% to 95% methanol was used with a flow rate of 300 L/min. A set of North American lagers was opened and poured. While exposed to air, the samples were extracted with hexane at a number of time points. The results were combined to form a composite representative that illustrates the reduction of the bitter acids with exposure to air. Lack of commercially available standards for the alpha and iso-alpha acids limits the analysis to only a relative comparison of the acid concentrations.
Similarly a comparison of twenty different beers and ales is presented by combining the results into groups characteristic of location or beer type. Each group is plotted as a single composite representative. For a given group, the intensity of the bitter acids and their ratio likely depends on hop variety, amount of hops used and brewing process itself. Where a greater amount of hops with a higher yield of alpha acids would produce a higher intensity of the bitter acids, but the ratio between alpha and iso-alpha acids will depend on brewing process factors. In a simple model the ratio between the humulone and iso humulone columns of a given group is a measure of the brewing efficiency in converting the natural acids into their isomerized form. In order to investigate a possible application of this work to a highthroughput environment such as on-line quality control monitoring for batch consistency, the sample preparation step has been simplified by replacing the hexane extraction with a water dilution step. A comparison of the two sample preparation methods has been done by simultaneously extracting the same beer sample with hexane as one sample and diluting the beer with distilled water as a second sample. A same dilution step of 1:20 has been used for both samples, where the sample evaporated from hexane has been diluted in mobile phase. Comparison of the results shows that while the intensity of alpha acids is the same, the levels of iso-alpha acids are higher for the water diluted sample. This reflects the higher solubility of the iso-alpha acids in water. Both preparation methods provide samples suitable for mass spectrometer analysis.
Based on the results from the water dilution method a quantitation limit estimate is approximately beer diluted 40,000 times. While the water dilution provides simplification of the sample preparation and handling (lower CO2 content after dilution allows consistent sample uptake with an autosampler syringe) it places greater emphasis on robustness of the LC/MS interface. The API 3000 interface has been tested with 12 hours of continuous injections (60) with results showing no loss in sensitivity.
LC/MS/MS method for detection of bitter acids in beer and hops has been presented. It offers a possible application in on-line quantitative analysis for quality control as well as in brewing process development. The qualitative work presented could be extended to impurity identification and structural elucidation of degradation product present in hop products.
Peter Kovarik, Yves Mouget, Takeo Sakuma, MDS SCIEX