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Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

Muratovic AZ, Hagström T, Rosén J, Granelli K, Hellenäs KE - Toxins (Basel) (2015)

Bottom Line: The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g.The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach.Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

View Article: PubMed Central - PubMed

Affiliation: Science Department, National Food Agency, Box 622, Uppsala SE-751 26, Sweden. aida.zuberovic.muratovic@slv.se.

ABSTRACT
A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding (13)C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5-30 ng/g (R² = 0.92-0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%-30% and 5%-41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

No MeSH data available.


MRM-MS spectra from quantitative analysis of SEA in milk samples. The concentration of SEA is the nominal concentration. IS is the abbreviation for internal standard. No smoothing function was applied to the shown signals. The results from the analysis are presented in Table 6.
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toxins-07-03637-f002: MRM-MS spectra from quantitative analysis of SEA in milk samples. The concentration of SEA is the nominal concentration. IS is the abbreviation for internal standard. No smoothing function was applied to the shown signals. The results from the analysis are presented in Table 6.

Mentions: In another experiment, the performance of the method was investigated by comparative analysis of milk samples spiked with SEA at concentrations designed for analysis with MS. The samples were kindly provided by the EU reference laboratory (EU-RL) for coagulase positive staphylococci (CPS), (France). The samples were analyzed both at the National Food Agency (NFA, Sweden), using the present method and at the EU-RL using the EU approved immuno-affinity based method, ELISA [16]. The values obtained from the analyses of milk samples did not differ significantly between the two methods (Table 6) confirming the usefulness of the MS-based method in quantitative analysis of staphylococcal enterotoxins in food samples. The two additional test samples, on the other hand, consisting of the matrices for which matrix-matched standards were not used in the MS analysis, did not correlate very well. This indicates the importance of the use of matrix-matched calibration to compensate for signal differences mainly caused by matrix effects in the analysis of complex samples with ESI-MS. No SEA was detected in the corresponding blank samples, Figure 2. The performance of the method is also presented in an overview in Table 7. With this table, it is possible to compare several steps in the already existing LC-MS based methods, regarding matrix complexity, sample preparation comprehensiveness and the choice of standard for the identification confidence, in relation to the quantitative performance of the method. Except for the ideal, but comprehensive and high-cost associated work from Dupuis et al. [22], where full-length isotope labeled standards were used in combination with immunoaffinity based toxin extraction, the presented method offers detection and quantification of enterotoxins at low ppb level using relatively straight forward approach and at reasonable costs. This makes the new method more advantageous for frequent use in direct analysis of enterotoxins in foods in comparison to the previously published methods.


Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

Muratovic AZ, Hagström T, Rosén J, Granelli K, Hellenäs KE - Toxins (Basel) (2015)

MRM-MS spectra from quantitative analysis of SEA in milk samples. The concentration of SEA is the nominal concentration. IS is the abbreviation for internal standard. No smoothing function was applied to the shown signals. The results from the analysis are presented in Table 6.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591654&req=5

toxins-07-03637-f002: MRM-MS spectra from quantitative analysis of SEA in milk samples. The concentration of SEA is the nominal concentration. IS is the abbreviation for internal standard. No smoothing function was applied to the shown signals. The results from the analysis are presented in Table 6.
Mentions: In another experiment, the performance of the method was investigated by comparative analysis of milk samples spiked with SEA at concentrations designed for analysis with MS. The samples were kindly provided by the EU reference laboratory (EU-RL) for coagulase positive staphylococci (CPS), (France). The samples were analyzed both at the National Food Agency (NFA, Sweden), using the present method and at the EU-RL using the EU approved immuno-affinity based method, ELISA [16]. The values obtained from the analyses of milk samples did not differ significantly between the two methods (Table 6) confirming the usefulness of the MS-based method in quantitative analysis of staphylococcal enterotoxins in food samples. The two additional test samples, on the other hand, consisting of the matrices for which matrix-matched standards were not used in the MS analysis, did not correlate very well. This indicates the importance of the use of matrix-matched calibration to compensate for signal differences mainly caused by matrix effects in the analysis of complex samples with ESI-MS. No SEA was detected in the corresponding blank samples, Figure 2. The performance of the method is also presented in an overview in Table 7. With this table, it is possible to compare several steps in the already existing LC-MS based methods, regarding matrix complexity, sample preparation comprehensiveness and the choice of standard for the identification confidence, in relation to the quantitative performance of the method. Except for the ideal, but comprehensive and high-cost associated work from Dupuis et al. [22], where full-length isotope labeled standards were used in combination with immunoaffinity based toxin extraction, the presented method offers detection and quantification of enterotoxins at low ppb level using relatively straight forward approach and at reasonable costs. This makes the new method more advantageous for frequent use in direct analysis of enterotoxins in foods in comparison to the previously published methods.

Bottom Line: The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g.The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach.Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

View Article: PubMed Central - PubMed

Affiliation: Science Department, National Food Agency, Box 622, Uppsala SE-751 26, Sweden. aida.zuberovic.muratovic@slv.se.

ABSTRACT
A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding (13)C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5-30 ng/g (R² = 0.92-0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%-30% and 5%-41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.

No MeSH data available.