Accurate and Reliable Analysis of Beer Using Time-of-Flight Technology for Gas Chromatography

Alcoholic beverages such as beer are derived from natural plant products and contain many hundreds of organic components. The taste and aroma of different beers give a particular brand its subjective “signature,” but, unfortunately, many of the compounds attributable to a certain taste or smell often have very low olfactory thresholds. This means that the actual concentrations of signature compounds can be extremely low and therefore analytically challenging.

The screening of trace volatile organic compounds (VOCs) in complex matrices using conventional GC quadrupole MS or MS-MS generally requires operation under selected ion monitoring (SIM) conditions that preclude the gathering of spectral information and thus compromise compound identification. However, using the latest time-of-flight (TOF) technology, it is possible to record spectra at conventional SIM detection levels, yet still produce classical spectra that are compatible with both proprietary mass spectral libraries and commercial MS databases such as NIST. This article describes how one such high-sensitivity TOF-MS may be used in combination with GC and suitable sample preparation/introduction techniques for routine screening of trace compounds.

Solid-phase microextraction (SPME) and headspace have been widely used, but both techniques lack the required sensitivity. Sorptive extraction (which includes stir-bar sorptive extraction, SBSE) followed by thermal desorption (TD) is a sequence of techniques that show promise due to the improved sample enrichment factor.

An additional consideration for beer analysis is that not all of the components of interest are present at trace levels. Some are present at very high concentrations. It is therefore often necessary to analyze two samples under different sets of conditions to ensure that both high- and low-level compounds are measured within the dynamic range of the detector. If the complete analysis could be carried out on a single sample, it would save time/costs and eliminate a potential source of error.

This article describes how the combination of sorptive extraction/thermal desorption with a new generation of GC/TOF-MS allows extensive information on natural products to be gathered from a single extraction. The procedure merges the collection, then subsequent repeat analysis, of a proportion of the sample during thermal desorption, with the capability of the TOF to produce “classical” spectra for components across a very wide range of analyte concentrations.

Samples

A selection of popular supermarket bottled beers were used for the tests:

  • Beer 1 (750 mL), original German recipe
  • Beer 2 (750 mL), a modified Danish recipe, designed for the U.K. market
  • Beer 3 (750 mL), a U.S. recipe, designed for the U.K. market.

Double-distilled water was used as an analytical blank and for rinsing solid-phase extraction-thermal desorption (SPE-TD) cartridges after extractions. The commercial beer bottles were chilled to 5 °C prior to opening in an effort to limit the loss of volatile compounds. As soon as they were decapped, zero-headspace aliquots of each beer were collected from the bottles and stored in 20-mL screw-capped headspace vials.

Table 1    -    Analytical settings

Experimental

Sample preparation

  1. Sorptive extraction. A 20-mL headspace vial (Chromacol, Welwyn Garden City, U.K.) with a stainless steel screw cap was filled to zero headspace with the beer sample. A cleaned, glass-encapsulated magnetic stir-bar (10 mm) was introduced together with a preconditioned SPE-TD sorptive extraction cartridge (30 mm × 2.5 mm × 500 µm polydimethyl siloxane [PDMS] film thickness, MARKES International, Llantrisant, U.K.) before securing the vial cap. Other beer samples were also prepared at this time. The vials were then transferred in batches to a 10-position heated magnetic stirring platen (Ika-Werke GmbH, Staufen, Germany), where they were allowed to equilibrate for 30 min while stirred vigorously at 1100 rpm with a platen temperature of 45 °C.
  2. Transfer ofSPE-TDcartridge to thermal desorption tubes. After extraction, individual vials were sequentially opened and their SPE-TD cartridges transferred to clean vials (using tweezers), where they were rinsed twice in distilled water. Each cartridge was then dried with a separate lint-free KimTech laboratory wipe (Kimberly-Clark, Dallas, TX) to ensure that excess water was blotted away. Each dried cartridge was then placed in a precleaned glass TD tube and immobilized between the plug of captive deactivated glass wool (at the notched end of the tube) and the supplied stainless steel spring clip. Loaded TD tubes were immediately sealed with standard long-term storage caps (comprising metal fittings with combined PTFE ferrules) and stored until their subsequent analysis.

Thermal desorption

Figure 1 - Automated peak detection and identification of the 20 most abundant peaks.

Table 2    -    Top 20 components in Beer 1 (Figure 1)

A MARKES UNITY 2 thermal desorption system was interfaced to the host system, harnessing the electronic pneumatic control of the GC (see Table 1). The design of the thermal desorber ensures that contaminants picked up on the outsides of TD tubes cannot be desorbed with the sample. This enables operators to quickly load tubes by hand, without risking contamination of the samples.

Data processing

Method development was greatly simplified using BenchTOF’s real-time application, dx-Connect. dx-Connect is a chromatographic viewer that permits both raw and background-compensated total ion chromatogram (TIC) traces to be propagated simultaneously. Single-click library identifications are therefore possible—at run time—from trace-level peaks that would ordinarily be buried in background matrix. With this facility, it is possible to determine the retention times of many interesting compounds and assess the chromatography, before the end of the first test analysis.

Results

Introduction to SPE-TD

SPE-TD is a highly selective and sensitive technique for extracting organic compounds from aqueous matrices. The enrichment factor possible from thorough mixing of large volumes of original sample matrix with physically large sorbents, such as the MARKES SPE-TD cartridge, is the key to the sensitivity advantage. When compared to the very small quantity of sorbent material on a typical SPME fiber, it becomes evident that there will be an increased uptake onto the sorbent material. Further sample concentration via two-stage thermal desorption makes sub-ppt detection possible.

Figure 2 - Magnified TIC showing complexity of the SPE-TD beer extract.

Figure 3 - Small peak seen at RT 25.45 min.

Figure 4 - NIST search on small peak at RT 25.45 min.

The overall sample enrichment factor may pose a problem for many analytical detection systems, but the BenchTOF-dx can record high-quality mass spectra from extremes of concentration. There are many orders of magnitude of component concentrations in beer extracts. The beers were extracted with SPE-TD and analyzed by TD-GC/TOF-MS.

With the BenchTOF-dx, data were automatically processed using a dynamic background compensation (DBC) algorithm. This gave the ability to characterize low-level compounds with much greater confidence than traditional “peak detect then background-subtract” routines. Much smaller data files are generated and, as a result, it speeds up batch (re)processing. This is especially useful with complex chromatography, where there is a wide dynamic range within a given sample.

As can be seen in Figure 1, the TIC shows all peaks rising from the baseline. This is the advantage of DBC, which is applied on-the-fly during data acquisition, significantly simplifying peak detection and visual inspection.

Table 2 lists the top 20 components in Beer 1 (Figure 1) ranked by their abundance. The standard NIST05 mass spectral database was used. It is reasonable to assume that any beer manufacturer would have its own proprietary MS library, which could be substituted here to yield even higher-quality results.

Figure 2 depicts the baseline of the TIC magnified 25×. The complexity of the sample becomes evident. The red box in the figure shows the position of a minor component. An enlargement of this part of the chromatogram and a mass spectrum from the peak apex are shown in Figure 3. The area abundance of the peak is 22,000 counts, which is very small. Note also that the spectrum’s base peak (m/z 43) intensity is only about 2400 counts. Compare this spectrum with NIST (Figure 4). This is a very confident library hit from a standard commercial library. Most of the submitted NIST spectra have been acquired on quadrupole-type mass detectors, and BenchTOF is the first commercial TOF-MS for GC to generate truly classical spectra.

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