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. 2022 Mar;414(8):2767-2774.
doi: 10.1007/s00216-022-03926-7. Epub 2022 Feb 2.

Rapid detection and quantitation of dipicolinic acid from Clostridium botulinum spores using mixed-mode liquid chromatography-tandem mass spectrometry

Benjamin W Redan  1 , Travis R Morrissey  2 , Catherine A Rolfe  2 , Viviana L Aguilar  3 , Guy E Skinner  4 , N Rukma Reddy  2
Affiliations

Rapid detection and quantitation of dipicolinic acid from Clostridium botulinum spores using mixed-mode liquid chromatography-tandem mass spectrometry

Benjamin W Redan et al. Anal Bioanal Chem. 2022 Mar.

Abstract

Analysis of the dipicolinic acid (DPA) released from Clostridium botulinum spores during thermal processing is crucial to obtaining a mechanistic understanding of the factors involved in spore heat resistance and related food safety applications. Here, we developed a novel mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of the DPA released from C. botulinum type A, nonproteolytic types B and F strains, and nonpathogenic surrogate Clostridium sporogenes PA3679 spores. DPA was retained on a mixed-mode C18/anion exchange column and was detected using electrospray ionization (ESI) positive mode within a 4-min analysis time. The intraday and interday precision (%CV) was 1.94-3.46% and 4.04-8.28%, respectively. Matrix effects were minimal across proteolytic type A Giorgio-A, nonproteolytic types QC-B and 202-F, and C. sporogenes PA3679 spore suspensions (90.1-114% of spiked DPA concentrations). DPA recovery in carrot juice and beef broth ranged from 105 to 118%, indicating limited matrix effects of these food products. Experiments that assessed the DPA released from Giorgio-A spores over the course of a 5-min thermal treatment at 108 °C found a significant correlation (R = 0.907; P < 0.05) between the log reduction of spores and amount of DPA released. This mixed-mode LC-MS/MS method provides a means for rapid detection of DPA released from C. botulinum spores during thermal processing and has the potential to be used for experiments in the field of food safety that assess the thermal resistance characteristics of various C. botulinum spore types.

Keywords: Botulism; Endospores; Food safety; LC–MS/MS; Pyridine-2,6-dicarboxylic acid; Thermal processing.

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Conflict of interest statement

Conflict of interest. The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Chemical structure of dipicolinic acid (DPA)
Figure. 2.
Figure. 2.
Representative LC-MS/MS chromatograms of (a) DPA standard and (b) DPA released from C. botulinum Giorgio-A spores after thermal treatment at 108 °C. DPA was detected at m/z 168 → 194. Data was collected in ESI positive mode
Figure 3.
Figure 3.
Linear correlation between log reduction of Giorgio-A spores and DPA release during thermal processing. C. botulinum Giorgio-A spores were subjected to thermal processing at 108 °C for up to 5 min. Thermally treated spore samples were divided into one aliquot for spore enumeration and a second aliquot for DPA analysis by LC-MS/MS. A significant (P<0.01) correlation was determined using Pearson’s correlation coefficient (R=0.907). Plotted values are from two replicate experiments
See this image and copyright information in PMC

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