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
400
350
300
pH 2.0 Standard of 3-NPA and 3-NPOH Spiked Canadian MilkVetch Extract
250
pH 2.0 Standard of 3-NPA and 3-NPOH 200
3.199
150
100
50
0
1
2
3
4
5
6
7
min
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
300
200
pH 2.5 Standard of 3-NPA and 3-NPOH 100
0
1
2
3
4
5
6
7
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
S2 of S3
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
400
pH 3.0 standards of 3-NPA and 3-NPOH spiked Canadian milkvetch
300
pH 3.0 Canadian milkvetch Extract (CME)
100
2.9863.013 3.011
2.647
200
pH 3.0 standards of 3-NPA and 3-NPOH
0
1
2
3
4
5
6
min
7
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.
250
300
350
400
450
nm
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
250
S3 of S3
300
350
400
450
nm
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
500
pH 3.5 standards of 3-NPA and 3-NPOH spike Canadian milkvetch extract
400
300
pH 3.5 Canadian milkvetch extract
100
3.040 .0 33 .0 56 40 3.1 .19 81 4 3
2.177
200
pH 3.5 standards of 3-NPA and 3-NPOH
0
1
2
3
4
5
6
7
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
250
300
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400
450
nm
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.
250
300
350
400
450
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
S5 of S3
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
500
pH 4.0 standards of 3-NPA and 3-NPOH spike Canadian milkvetch extract
400
pH 4.0 Canadian milkvetch extract
pH 4.0 standards of 3-NPA and 3-NPOH 300
.0 33 .0 11 79 3.049 40 6 3.15
1.614
200
100
0
1
2
3
4
5
6
7
min
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.
250
300
350
400
450
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
250
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350
400
450
nm
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
800
700
600
pH 5.0 standards of 3-NPA and 3-NPOH spiked Canadian milkvetch extract 500
400
pH 5.0 Canadian milkvetch extract
300
3 3.0 .04 30 6
1.243
200
100
pH 5.0 standards of 3-NPA and 3-NPOH
0
1
2
3
4
5
6
7
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
250
300
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350
400
450
nm
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.
250
300
350
400
450
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
800
pH 6.0 standards of 3-NPA and 3-NPOH spiked Canadian milkveth extract
600
pH 6.0 standards of 3-NPA and 3-NPOH
1.175
400
pH 6.0 Canadian milkveth extract 3.0 .04 45 5 3
200
0
1
2
3
4
5
6
7
min
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.
250
300
350
400
450
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.
nm
Toxins 2017, 9, doi:10.3390/toxins9070204
250
300
S9 of S3
350
400
450
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.
nm
