Beer represents one of the most widelyconsumed beverages in the world.

Its annual worldwide production,measured in hundreds of millions ofhectoliters, is a testimony to its popu-larity. While there are many differenttypes of beer, all share one underlying

characteristic taste, bitterness. Beerderives its bitter taste from bitter acids

present in hops that undergo isomer-ization during the brewing process. Incontrast to old traditional processesmodern brewing techniques may usehop products such as extracts toachieve greater production volumesand improved batch consistency.

In this study, bitter acid composition

of different types of beers is presented.

The results are grouped by beer typeand geographical location. Beer tastedegradation with extended exposure to air is correlated to its reduction ofbitter acid content.

A reversed phase chromatography hasbeen used to separate the hop alphaacids from their isomerized products in beer, API 3000™ LC/MS/MSSystem triple quadrupole mass

spectrometer was then used to deter-mine the bitter acid contents. API3000 system mass spectrometer and

a high mass accuracy hybrid QqTOFinstrument, QSTAR® LC/MS/MSSystem, have been used to generate

fragmentation spectra for structural

elucidation of the acids.

IntroductionHop cones, a crop harvested from ahop plant, contain the bitter alpha andbeta acids; n-, co-, and ad- humulonesand lupulones respectively. The humu-lones in particular are widely used inthe brewing process to provide bitter-ness to beer. During traditionalbrewing, hops are added to a water-

extracted barley malt and the mixtureis boiled. This process causes the largely

insoluble alpha acids to convert tomore bitter and more soluble iso-alphaacids, the primary bittering com-pounds in beer. The next and last step

in the brewing process, fermentation,is different for ales and lagers. Lagersare produced by a process called bot-

tom fermentation requiring a lowertemperature and longer time, whereasales are fermented at room tempera-

ture requiring a shorter period of time.

Experimental and resultsA sample of hop pellets has beenobtained from a local home brewing

outlet. The pellets were then crushed

and the resulting powder extractedwith hexane. Beer samples wereextracted with equal volume of hexane

and spun to isolate the hexane compo-nent. In both cases hexane sampleswere evaporated and re-dissolved in

methanol for infusion or in mobilephase for LC work.

HPLC/MS/MS Analysis of Bitter Acids in Hops and Beer

Application Note Mass Spectrometry

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Since both the alpha acids and theirisomerized products have same molec-

ular weight, distinction can only bemade using their MS/MS fragmenta-tion patterns. MS and MS/MS spectrawere generated with 5 uL/min infusion of the respective samples.

Identification of the fragments has

been confirmed using the high massaccuracy of the MS/MS/TOF instru-ment, that allows determination ofelemental composition of the precursorand fragment ions. Isohumulones, asshown in the example of co-isohumulone,produce a unique fragment suitable formultiple reaction monitoring (MRM).

The base peak in fragmentation spec-tra of humulones is not unique asshown by the example of co-humuloneand co-isohumulone. Chromatographywas used to resolve the two groups. Anexample chromatogram lists the MRMtransitions used to detect the alpha andisomerized alpha acids.

LC conditions used in the separationwere as follows, Jones C 8 2.1 x

100 mm HPLC column with a mobilephase consisting of 2mM ammoniumacetate and methanol. An eight minutegradient run from 50% to 95%methanol was used with a flow rate of 300 µL/min.

A set of North American lagers wasopened and poured. While exposed

to air, the samples were extracted withhexane at a number of time points.The results were combined to form

a composite representative that illus-trates the reduction of the bitter acidswith exposure to air. Lack of commer-

cially available standards for the alphaand iso-alpha acids limits the analysisto only a relative comparison of the

acid concentrations.

MS of hops.

MS of beer.

MS/MS of co-humulone.

MS/MS of co-isohumulone.

Similarly a comparison of twenty different beers and ales is presented

by combining the results into groupscharacteristic of location or beer type.Each group is plotted as a single composite representative.

For a given group, the intensity of thebitter acids and their ratio likelydepends on hop variety, amount ofhops used and brewing process itself.

Where a greater amount of hops witha higher yield of alpha acids wouldproduce a higher intensity of the bitteracids, but the ratio between alpha andiso-alpha acids will depend on brewing

process factors. In a simple model theratio between the humulone and isohumulone columns of a given group is

a measure of the brewing efficiency inconverting the natural acids into theirisomerized form.

In order to investigate a possible application of this work to a high-throughput environment such as “on-line” quality control monitoringfor batch consistency, the samplepreparation step has been simplified by

replacing the hexane extraction with awater dilution step. A comparison ofthe two sample preparation methodshas been done by simultaneouslyextracting the same beer sample withhexane as one sample and diluting thebeer with distilled water as a secondsample. A same dilution step of 1:20has been used for both samples, wherethe sample evaporated from hexane

has been diluted in mobile phase.Comparison of the results shows thatwhile the intensity of alpha acids is thesame, the levels of iso-alpha acids arehigher for the water diluted sample.This reflects the higher solubility of

the iso-alpha acids in water. Bothpreparation methods provide samplessuitable for mass spectrometer analysis.

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MS/MS/TOF of hops.

MS/MS/TOF of beer.

LC/MS/MS of beer.

Based on the results from the waterdilution method a quantitation limitestimate is approximately beer diluted40,000 times.

While the water dilution provides sim-plification of the sample preparationand handling (lower CO2 contentafter dilution allows consistent sample

uptake with an autosampler syringe) itplaces greater emphasis on robustnessof the LC/MS interface. The API3000 interface has been tested with 12 hours of continuous injections (60) with results showing no loss in sensitivity.

ConclusionLC/MS/MS method for detection ofbitter acids in beer and hops has beenpresented. It offers a possible applica-tion in “on-line” quantitative analysisfor quality control as well as in

brewing process development. The

qualitative work presented could beextended to impurity identificationand structural elucidation of degrada-tion product present in hop products.

AuthorsPeter Kovarik, Yves Mouget,

Takeo Sakuma, MDS SCIEX

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Composite of North American lagers.

Comparison of beer groups.

Beer diluted in water.

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Printed in the USA, 10/04, P+s, Publication 114AP27-01

Beer extracted in hexane.