LC/MS/MS Analysis of Aflatoxins in Hops After Solid Phase Extraction Cleanup

By: Olga I. Shimelis, Christine Dumas, Jennifer Claus, Reporter US Volume 34.1


Hops are widely used in brewing beer. Hops have unique composition containing bitter compounds in the form of α- and β-acids, essential oils, polyphenols and aroma compounds such as terpenes.1 Fungal contamination of the hops can result in the production of mycotoxins. Mycotoxins are a diverse group of compounds comprised of hundreds of secondary metabolic products from various fungal species. Mycotoxins are widely prevalent in many agricultural commodities and can form during growth, harvest, transportation, processing, or storage. Several mycotoxins show marked toxicity in humans. Aflatoxin B1, for example, is considered to be a Type 1 carcinogen by the International Agency for Research on Cancer (IARC).2 The sensitive and accurate detection of very low levels of aflatoxins is critical to identify contaminated products.

Chromatographic methods such as GC and HPLC are most commonly used in mycotoxin analysis, usually preceded by a number of operations such as sampling, sample preparation, extraction, and cleanup. The unique composition of hops makes it extremely difficult to apply sample cleanup methods commonly used for other commodities, such as grains. The methods may not produce good cleanup of the samples due to the complexity of the hops matrix. In this work, a sample preparation method was developed prior to LC/MS/MS analysis of aflatoxins using Supel™ Tox AflaZea SPE cartridges for cleanup of the hops extracts. Supel Tox cartridges are compatible directly with the extracts generated during mycotoxin analysis procedures. The Supel Tox AflaZea cartridges are based on the “interference removal” strategy that requires few processing steps and saves time by eliminating wash steps prior to elution of analytes.


Hops of the “US Golding” variety were purchased from a brewing supply store. A 0.5 g portion of the finely ground hop pellets was weighed out and added to a 50 mL centrifuge tube. A volume of 10 mL of the extraction solvent, acetonitrile: water (86:14), was added to the tube, and the suspension was mixed in a shaker for 30 minutes. The tube was centrifuged at 3,000 rpm for five minutes. A 2 mL aliquot of the supernatant was transferred to a different tube and spiked with the mixture of aflatoxins (Table 1). Subsequently, the resulting solution was passed through Supel Tox AflaZea SPE cartridge under vacuum. A portion of the collected cleaned sample, 200 μL, was diluted with 800 μL of water and injected into LC/MS/MS for analysis. No filtration of the samples was necessary at this stage as no precipitate was formed upon dilution of water. Photos of the hops samples before, during, and after cleanup are illustrated in Figure 1.

Matrix-matched calibration curves were constructed and ran along with solvent-based calibration curves to compare ionization effects and sample cleanliness.

Table 1. Spiking Levels for Aflatoxins in Hops

Compound Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Afaltoxin G2
Spiking Level 24.4 ppb 6.1 ppb 24.4 ppb 6.1 ppb
0.244 ng/mL 61 pg/mL 0.244 ng/mL 61 pg/mL


Hops Samples (a) Before cleanup, (b) On Supel Tox AflaZea SPE Cartridge, and (c) After cleanup

Figure 1. Photos of the Hops Samples (a) Before cleanup, (b) On Supel Tox AflaZea SPE Cartridge, and (c) After cleanup


Results and Discussion

The current study of aflatoxins was done using an Ascentis® Express Phenyl-Hexyl HPLC column (Figure 2). A previous study demonstrated the phenyl-hexyl stationary phase column produced increased retention of aflatoxin compounds while improving the separation of aflatoxins from co-extracted corn matrix.3

The hops matrix effects were investigated by comparing the calibration curves constructed in solvent versus those in extract (Figure 3). Significant ion suppression was present in hops samples, and the matrix-matched calibration curves were required for accurate quantitation. The matrix effects can be attributed to the complex hops composition and the limited capacity of the Supel Tox AflaZea SPE for removal of all of the components.

Recovery values from spiked extracts are presented in Table 3. They fall in the range of 97-119% with RSD below 15% for three replicates. Aflatoxins B2 and G2 also exhibited excellent recoveries with spiking as low as 6.1 ppb.

LC-MS/MS Chromatogram of Aflatoxins Spiked Into Hops Sample

Figure 2. LC-MS/MS Chromatogram of Aflatoxins Spiked Into Hops Sample at 24.4 ppb (Aflatoxin B1 and G1), and 6.1 ppb (Aflatoxin B2 and G2)


Comparison of Matrix-matched and Solvent Calibration Curves for Aflatoxins

Figure 3. Comparison of Matrix-matched and Solvent Calibration Curves for Aflatoxins


Table 2. LC-MS/MS Parameters for Aflatoxin Detection

  Retention Time Q1 Q2
Afaltoxin G2 3.174
331.1 189.0
Aflatoxin G1 3.472 329.1 243.0
Aflatoxin B2 3.579 315.9 259.0
Aflatoxin B1 3.862 313.1 241.0


Table 3. Percent Recovery for Aflatoxins from Hops* (n = 3)

  Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Afaltoxin G2
Recovery (%)
119 99 107 97
RSD% (n=3) 13 2 12 10

*Versus matrix-matched calibration curve.




  1. Belitz, H.-D.; Grosch, W.; Schieberle, P. Food Chemistry, 3rd Edition; Burghagen, M., Eds; Springer: Verlag Berlin Heidelberg, 2004.
  2. World Health Organization, Agents Classified by the IARC Monographs, Volumes 1-113 site.
  3. Shimelis, O.; Barrey, E.; Peres-Blanco, L. Selection of HPLC Stationary Phase for
    LC-MS/MS Analysis of Multiple Mycotoxins in Corn, Proceedings of 129-th AOAC
    Annual Meeting
    , Los Angeles, CA, September 27-30, 2015.


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