Supplementary Materials - MDPI

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DAD1 A, Sig=210,4 Ref=400,40 (HENRY\2017-06-06-02\201706060200003.D). pH 2.0 Standard of 3-NPA and 3-NPOH Spiked Canadian MilkVetch Extract.

Toxins 2017, 9, doi:10.3390/toxins9070204

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Supplementary Materials: A Simple and Fast Procedure to Determine 3-Nitropropanoic Acid and 3Nitropropanol in Freeze Dried Canadian Milkvetch (Astragalus canadensis) Huaizhi Liu, Suqin Shao and Mike Schellenberg 3.197

DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-06-02\201706060200002.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-06-02\201706060200003.D) mAU

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pH 2.0 Standard of 3-NPA and 3-NPOH Spiked Canadian MilkVetch Extract

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pH 2.0 Standard of 3-NPA and 3-NPOH 200

3.199

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Figure S1. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water and spiked Canadian milkvetch sample, with pH 2.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3NPOH was not separated. DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-06-01\201706060000002.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-06-01\201706060000003.D) mAU

pH 2.5 Standard of 3-NPA and 3-NPOH Spiked Canadian MilkVetch Extract 400

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pH 2.5 Standard of 3-NPA and 3-NPOH 100

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Figure S2. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water and spiked Canadian milkvetch sample, with pH 2.5 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3NPOH was not baseline separated.

min

Toxins 2017, 9, doi:10.3390/toxins9070204

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DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-12-03\201706120300002.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-12-03\201706120300003.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-12-03\201706120300004.D)

2.642

mAU

500

2.642

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pH 3.0 standards of 3-NPA and 3-NPOH spiked Canadian milkvetch

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pH 3.0 Canadian milkvetch Extract (CME)

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2.9863.013 3.011

2.647

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pH 3.0 standards of 3-NPA and 3-NPOH

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Figure S3. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water, Canadian milkvetch sample and spiked Canadian milkvetch samples, with pH 3.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3-NPOH was baseline separated.

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Figure S4. UV spectra of the peak of 3-NPA in Canadian milkvetch under the HPLC condition with pH 3.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

Toxins 2017, 9, doi:10.3390/toxins9070204

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Figure S5. UV spectra of the peak of 3-NPOH in Canadian milkvetch under the HPLC condition with pH 3.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

2.191

DAD1 A, Sig=210,4 Ref=400,40 (HENRY\2017-06-13-035\201706130350002.D) DAD1 A, Sig=210,4 Ref=400,40 (HENRY\2017-06-13-035\201706130350003.D) DAD1 A, Sig=210,4 Ref=400,40 (HENRY\2017-06-13-035\201706130350004.D) mAU

2.190

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pH 3.5 standards of 3-NPA and 3-NPOH spike Canadian milkvetch extract

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3.040 .0 33 .0 56 40 3.1 .19 81 4 3

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pH 3.5 standards of 3-NPA and 3-NPOH

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Figure S6. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water, Canadian milkvetch sample and spiked Canadian milkvetch samples, with pH 3.5 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3-NPOH was baseline separated.

min

Toxins 2017, 9, doi:10.3390/toxins9070204

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Figure S7. UV spectra of the peak of 3-NPA in Canadian milkvetch under the HPLC condition with pH 3.5 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

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Figure S8. UV spectra of the peak of 3-NPOH in Canadian milkvetch under the HPLC condition with pH 3.5 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

nm

Toxins 2017, 9, doi:10.3390/toxins9070204

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DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-040\201706130400002.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-040\201706130400003.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-040\201706130400004.D)

1.641

mAU

1.634

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pH 4.0 standards of 3-NPA and 3-NPOH spike Canadian milkvetch extract

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pH 4.0 Canadian milkvetch extract

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.0 33 .0 11 79 3.049 40 6 3.15

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Figure S9. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water, Canadian milkvetch sample and spiked Canadian milkvetch samples, with pH 4.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3-NPOH was baseline separated.

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Figure S10. UV spectra of the peak of 3-NPA in Canadian milkvetch under the HPLC condition with pH 4.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

nm

Toxins 2017, 9, doi:10.3390/toxins9070204

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Figure S11. UV spectra of the peak of 3-NPOH in Canadian milkvetch under the HPLC condition with pH 4.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

1.244

DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-050\201706130500002.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-050\201706130500003.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-050\201706130500004.D) mAU 900

1.243

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pH 5.0 standards of 3-NPA and 3-NPOH spiked Canadian milkvetch extract 500

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3 3.0 .04 30 6

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Figure S12. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water, Canadian milkvetch sample and spiked Canadian milkvetch samples, with pH 5.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3-NPOH was baseline separated.

min

Toxins 2017, 9, doi:10.3390/toxins9070204

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Figure S13. UV spectra of the peak of 3-NPA in Canadian milkvetch under the HPLC condition with pH 5.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

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Figure S14. UV spectra of the peak of 3-NPOH in Canadian milkvetch under the HPLC condition with pH 5.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

nm

Toxins 2017, 9, doi:10.3390/toxins9070204

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1.174

DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-060\201706130600003.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-060\201706130600004.D) DAD1 A, Si g=210,4 Ref=400,40 (HENRY\2017-06-13-060\201706130600005.D)

1.174

mAU

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pH 6.0 standards of 3-NPA and 3-NPOH spiked Canadian milkveth extract

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pH 6.0 standards of 3-NPA and 3-NPOH

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pH 6.0 Canadian milkveth extract 3.0 .04 45 5 3

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Figure S15. Overlaid HPLC chromatograms of mixture standards of 3-NPA and 3-NPOH at 10 ppm in water, Canadian milkvetch sample and spiked Canadian milkvetch samples, with pH 6.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column. All standard solution and Canadian milkvetch samples were recon at the corresponding pH mobile phase. 3-NPA and 3-NPOH was baseline separated.

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Figure S16. UV spectra of the peak of 3-NPA in Canadian milkvetch under the HPLC condition with pH 6.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

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Toxins 2017, 9, doi:10.3390/toxins9070204

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Figure S17. UV spectra of the peak of 3-NPOH in Canadian milkvetch under the HPLC condition with pH 6.0 12.5 mM ammonium phosphate buffer as moble phase on a Phenomenex Kinetex 2.6 µ F5 100Å column.

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