Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

Davis, Carol, Gordon, Natasha, Murphy, Sinead , Singh, Ishwar, Kavanagh, Kevin, Carberry, Stephen and Doyle, Sean (2011) Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry. Analytical and Bioanalytical Chemistry, 401 (8). pp. 2519-2529. ISSN 1618-2642

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Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase-high-performance liquid chromatography (RP-HPLC), with absorbance detection (220-280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5'-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)(2)), of molecular mass 1103.931 Da ((M + H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)(2) also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (> 800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (> 530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean +/- SD = 3.55 +/- 0.07 mu g 100 mu L(-1); n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)(2) is also detectable (150 ng; 460 pmol) by thin-layer chromatography.

Keywords:Aspergillus fumigatu, Epipolythiodioxopiperazine, Gliotoxin, NRPS, Redox, Sporidesmin A}
Subjects:B Subjects allied to Medicine > B200 Pharmacology, Toxicology and Pharmacy
Divisions:College of Science > School of Pharmacy
ID Code:11293
Deposited On:25 Jul 2013 15:29

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