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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.


Summarizing flow chart of the sample preparation procedure and analysis of SEA and SEB.
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toxins-07-03637-f003: Summarizing flow chart of the sample preparation procedure and analysis of SEA and SEB.

Mentions: The sample was mixed with urea in 0.4 M NH4HCO3 so that the final urea concentration in the sample was 6 M, where after 10 μL of 45 mM DTT was added. The sample was kept at 50 °C for 15 min. After cooling to room temperature, 10 μL of 100 mM IAA was added and the sample was incubated for 15 min in darkness. Finally, trypsin was added to give a trypsin: protein ratio of approximately 2% (w/w) and the sample was incubated upon light shaking at 60 °C for 16 h. The digested sample was cooled to room temperature, acidified (to pH ≤ 4) with acetic acid (HAc) and cleaned with Isolute18 SPE columns. The SPE column was first washed with 5 × 1 mL 100% ACN and equilibrated with 5 × 1 mL 1% HAc. The tryptic peptides were adsorbed to the media using 5 repeated cycles of sample loading. The column was washed using 5 × 1 mL of 1% HAc and finally the peptides were eluted in 250 μL 50% ACN, 1% HAc. After the cleaning, the eluate was vacuum centrifuged to dryness and redissolved in a mixture of mobile phases, 80% of the mobile phase A and 20% of mobile phase B, (A 80%/B 20%) prior to the UPLC-ESI-MS/MS analysis. Additionally, standard proteins of SEA and SEB were mixed in Milli-Q water, after which the digestion, cleaning, drying and redissolving proceeded as described above to prepare a standard peptide mixture at a concentration of 10 ng/μL for each of the toxins after redissolving. Serial dilutions of this standard peptide mixture of SEA and SEB digest peptides were used for preparation of standard curves (2.5–30 ng/g) in mobile phase mixture at proportions described in Section 3.2 (these solutions of standard digest in mobile phase mixture are referred to as “standard digest in water” in the rest of the article). When a complete standard curve in water was not needed, at least one of the toxin digest solutions was continuously used to check the maintaining of the optimal experimental conditions for the analysis of enterotoxins in food samples. The experimental overview of the method set-up is shown by a flow chart in Figure 3.


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)

Summarizing flow chart of the sample preparation procedure and analysis of SEA and SEB.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-03637-f003: Summarizing flow chart of the sample preparation procedure and analysis of SEA and SEB.
Mentions: The sample was mixed with urea in 0.4 M NH4HCO3 so that the final urea concentration in the sample was 6 M, where after 10 μL of 45 mM DTT was added. The sample was kept at 50 °C for 15 min. After cooling to room temperature, 10 μL of 100 mM IAA was added and the sample was incubated for 15 min in darkness. Finally, trypsin was added to give a trypsin: protein ratio of approximately 2% (w/w) and the sample was incubated upon light shaking at 60 °C for 16 h. The digested sample was cooled to room temperature, acidified (to pH ≤ 4) with acetic acid (HAc) and cleaned with Isolute18 SPE columns. The SPE column was first washed with 5 × 1 mL 100% ACN and equilibrated with 5 × 1 mL 1% HAc. The tryptic peptides were adsorbed to the media using 5 repeated cycles of sample loading. The column was washed using 5 × 1 mL of 1% HAc and finally the peptides were eluted in 250 μL 50% ACN, 1% HAc. After the cleaning, the eluate was vacuum centrifuged to dryness and redissolved in a mixture of mobile phases, 80% of the mobile phase A and 20% of mobile phase B, (A 80%/B 20%) prior to the UPLC-ESI-MS/MS analysis. Additionally, standard proteins of SEA and SEB were mixed in Milli-Q water, after which the digestion, cleaning, drying and redissolving proceeded as described above to prepare a standard peptide mixture at a concentration of 10 ng/μL for each of the toxins after redissolving. Serial dilutions of this standard peptide mixture of SEA and SEB digest peptides were used for preparation of standard curves (2.5–30 ng/g) in mobile phase mixture at proportions described in Section 3.2 (these solutions of standard digest in mobile phase mixture are referred to as “standard digest in water” in the rest of the article). When a complete standard curve in water was not needed, at least one of the toxin digest solutions was continuously used to check the maintaining of the optimal experimental conditions for the analysis of enterotoxins in food samples. The experimental overview of the method set-up is shown by a flow chart in Figure 3.

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.