Supplementary materials

Title Novel high-throughput and widely-targeted liquid chromatography–time of flight mass spectrometry method for D-amino acids in foods

Author Yutaka Konya, Moyu Taniguchi, and Eiichiro Fukusaki

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan　　　

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Supplementary Figures

Fig. S1. LC-MS chromatograms of Water calibration sample (1000 nmol/mL in water) using CR-I(-) column (extracted ion of [M+H]+ ± 5 mDa).

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Fig. S2. LC-MS chromatograms of calibration samples and food samples using CR-I(+) column (extracted ion of [M+H]+ ± 5 mDa).(WC) Water calibration sample (100 nmol/mL in water)(MC) Milk calibration sample (100 nmol/mL in milk)(MB) Milk blank(VC) Vinegar calibration sample (100 nmol/mL in grain vinegar)(VB) Vinegar blank(BV) Black vinegar (AEON)

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Fig. S2.Continued

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Fig. S2.Continued

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Fig. S3. LC-MS chromatograms of foods using CR-I(+) column (extracted ion of [M+H]+ ± 5 mDa), (A) Kimchi (Ichioshi), (B) Yogurt (LB81).

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Supplementary Tables

Table S1. Peak area ratios of D-form to L-form in mixed standard solutions (DL-20mix 1 and 10 nmol/mL in the mobile phase) using CR-I(+) and CR-I(-) columns.

　　 CR-I(+) CR-I(-)

Index Name 1 nmol/mL 10 nmol/mL 1 nmol/mL 10 nmol/mL

1 Ala 　　 0.95 0.95 1.10 1.06

2 Ser 　　 1.00 0.97 1.00 1.03

3 Val 　　 1.02 0.99 1.03 1.01

4 Thr 　　 1.00 1.00 0.96 0.97

5 Cys 　　 0.98 0.97 1.01 1.04

6 Ile + allo-Ile a 　　 0.99 0.97 1.00 1.02

7 Leu 　　 0.99 0.92 1.02 1.08

8 Asn 　　 0.99 0.87 0.98 1.09

9 Asp 　　 0.99 0.99 1.00 1.02

10 Gln 　　 0.96 1.03 1.05 1.00

11 Lys 　　 1.00 0.95 1.04 1.03

12 Glu 　　 0.96 1.00 1.08 1.03

13 Met 　　 1.00 0.98 1.02 1.03

14 His 　　 0.99 0.92 1.00 1.10

15 Phe 　　 0.98 0.91 0.97 1.05

16 Arg 　　 1.06 1.06 0.93 0.93

17 Tyr 　　 1.00 0.91 1.01 1.09

18 Trp 　　 0.95 0.90 0.93 1.00

a [(D-Ile area) + (D-allo-Ile area)] / [(L-Ile area) + (L-allo-Ile area)]

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Table S2. Summary of the parameters of calibration curves for D-amino acids.

Recovery rate (%) (nmol/mL)Milk Vinegar 　 rc 　 LODd LLOQe ULOQf

Index Name 　 Avga RSDb 　 Avga RSDb 　Water Milk Vinegar 　 Water 　Water Milk Vinegar 　All-Matrices 　

1 D-Ala 101.5 1.2 103.5 1.7 0.997 0.989 0.993 10 10 40 40 1000

2 D-Ser 99.9 3.2 95.3 2.6 0.992 0.991 0.997 4 4 10 10 1000

3 D-Val 102.5 3.0 98.7 2.1 0.997 0.997 0.997 4 4 4 4 1000

4 D-Thr 99.6 2.9 93.5 2.7 0.993 0.985 0.996 1 4 4 4 1000

5 D-Cys 94.9 4.1 90.7 2.5 0.998 0.996 0.996 4 4 4 4 1000

6 D-Ile + D-allo-Ile 98.8 2.1 98.4 3.0 0.998 0.994 0.997 4 4 4 1 1000

7 D-Leu 101.9 3.1 100.1 1.7 0.996 0.994 0.995 1 4 4 1 1000

8 D-Asn 97.8 4.4 94.0 2.4 0.995 0.994 0.967 4 4 4 4 1000

9 D-Asp 85.4 3.4 95.8 2.1 0.996 0.996 0.995 4 4 4 10 1000

10 D-Gln 99.8 4.3 89.9 2.4 0.998 0.996 0.974 4 4 4 4 1000

11 D-Lys 72.1 6.6 107.3 2.4 0.998 0.992 0.997 40 40 10 10 1000

12 D-Glu 97.0 2.8 93.9 2.2 0.996 0.999 0.995 4 4 10 10 1000

13 D-Met 100.9 3.0 95.5 1.6 0.998 0.995 0.995 4 4 4 4 1000

14 D-His 85.7 3.0 94.1 3.7 0.994 0.998 0.994 4 4 4 10 1000

15 D-Phe 101.4 2.7 98.3 2.2 0.990 0.995 0.994 1 4 4 4 1000

16 D-Arg 79.8 4.6 93.8 4.4 0.992 0.981 0.998 1 4 4 4 1000

17 D-Tyr 100.6 3.5 96.1 1.6 0.994 0.993 0.990 1 1 4 4 1000

18 D-Trp 　 99.9 2.6 93.9 1.7 　0.997 0.997 0.993 　 1 1 1 1 1000 　a Average of accuracy (Avg) (n=3) (%), b Relative standard deviation (RSD) (%), c Correlation coefficient (r),d Limit of detection (LOD), e Lower limit of quantification (LLOQ), f Upper limit of quantification (ULOQ)

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Table S3. Accuracy, LLOQ and LOD of Water calibration curves for D-amino acids

　 Concentration of calibration curve (nmol/mL) 　

　 1 4 10 40 100 　 400 　 1000 　 (nmol/mL)

Index Name 　 Avga RSDb Avga RSDb Avga RSDb Avga RSDb Avga RSDb 　 Avga RSDb 　 Avga RSDb 　LODc LLOQd

1 D-Ala 98.7 14.1 104.0 2.1 103.2 4.4 99.6 4.4 94.4 3.4 10 10

2 D-Ser 93.9 16.7 115.0 9.2 102.2 9.3 98.5 3.8 97.8 0.9 92.6 4.8 4 4

3 D-Val 97.2 15.4 106.6 2.8 101.9 2.3 100.3 1.8 100.5 2.0 93.6 3.8 4 4

4 D-Thr 98.0 28.1 106.3 7.4 104.5 1.8 98.3 5.7 97.5 3.3 99.6 3.1 95.7 5.0 1 4

5 D-Cys 100.0 12.0 99.4 3.7 103.0 5.5 99.2 0.8 101.9 1.3 96.6 4.1 4 4

6 D-Ile + D-allo-Ile 98.6 6.4 102.5 2.4 103.3 6.7 101.6 2.8 101.2 2.4 92.8 4.5 4 4

7 D-Leu 98.3 16.1 103.2 3.6 102.6 2.1 104.3 2.0 100.7 0.9 90.8 4.5 4 4

8 D-Asn 96.6 12.1 106.5 1.9 106.9 1.9 104.5 3.4 98.1 3.0 87.4 1.8 4 4

9 D-Asp 98.7 14.1 102.1 7.1 104.2 7.4 102.2 4.2 99.6 2.0 93.2 3.9 4 4

10 D-Gln 97.4 6.2 106.5 5.2 100.4 6.7 100.3 3.6 101.0 3.2 94.5 4.7 4 4

11 D-Lys 101.4 5.2 96.1 6.2 101.3 2.8 101.2 2.9 40 40

12 D-Glu 99.7 9.1 101.5 14.2 97.2 7.8 97.7 5.5 103.9 2.1 100.0 4.0 4 4

13 D-Met 98.0 8.9 104.4 4.0 101.9 7.2 99.9 4.1 99.3 2.2 96.5 3.4 4 4

14 D-His 105.2 5.0 88.1 13.9 94.8 7.2 101.2 7.0 108.7 3.8 102.0 4.0 4 4

15 D-Phe 95.6 16.7 113.6 17.1 109.4 0.7 101.9 3.6 100.7 3.8 94.8 1.7 83.9 4.9 1 4

16 D-Arg 102.3 17.9 93.8 19.0 93.8 9.2 94.0 2.2 99.9 6.4 108.7 5.2 107.6 4.5 1 4

17 D-Tyr 96.9 3.9 108.3 7.4 108.8 7.3 106.0 5.3 102.1 4.0 95.8 2.5 82.2 4.6 1 1

18 D-Trp 99.8 2.5 97.5 4.3 107.6 7.8 103.5 5.1 102.4 4.1 99.4 1.4 89.7 4.9 1 1a Average of accuracy (Avg) (n=3) (%), b Relative standard deviation (RSD) (%) c Limit of detection (LOD), d Lower limit of quantification (LLOQ)

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Table S4. Accuracy and LLOQ of Milk calibration curves for D-amino acids

　 Concentration of calibration curve (nmol/mL) 　

　 1 4 10 40 100 　 400 　 1000 　 (nmol/mL)

Index Name 　 Avga RSDb Avga RSDb Avga RSDb Avga RSDb Avga RSDb 　 Avga RSDb 　 Avga RSDb 　 LLOQc

1 D-Ala 　 100.5 27.5 96.1 15.2 104.7 4.3 101.9 5.7 96.9 1.2 40

2 D-Ser 　 96.5 23.9 108.9 13.9 98.4 7.0 102.4 4.1 99.1 4.8 94.6 3.2 10

3 D-Val 　 97.0 5.7 107.4 1.5 99.5 10.5 102.6 2.2 99.6 2.9 93.8 3.1 4

4 D-Thr 　 101.1 43.6 96.3 4.7 98.6 11.8 96.3 11.4 103.0 3.3 104.8 3.8 99.9 2.9 4

5 D-Cys 　 103.6 9.0 92.0 2.0 94.5 10.0 103.2 2.5 105.4 4.1 101.4 3.9 4

6 D-Ile + D-allo-Ile 98.7 26.8 104.8 4.6 101.3 6.0 99.5 8.7 101.9 3.0 99.2 4.1 94.7 2.2 4

7 D-Leu 　 98.9 25.7 102.5 5.3 104.0 5.1 100.4 6.6 103.7 2.7 100.2 3.5 90.2 3.2 4

8 D-Asn 　 101.2 17.4 96.0 6.5 101.5 12.5 106.4 3.2 103.6 3.1 91.2 4.3 4

9 D-Asp 　 122.8 19.3 99.4 10.3 101.9 14.3 97.3 5.6 102.3 3.5 103.2 2.7 95.8 2.9 4

10 D-Gln 　 101.0 13.2 98.0 8.1 96.7 10.0 103.8 5.1 101.6 3.1 98.9 4.3 4

11 D-Lys 　 103.3 11.6 90.3 1.4 89.2 2.6 104.8 4.8 112.4 4.7 10

12 D-Glu 　 180.2 3.8 99.9 7.2 100.0 6.6 100.9 4.1 101.5 2.6 97.8 2.7 10

13 D-Met 　 99.5 13.7 102.4 11.5 93.5 10.4 103.4 4.9 102.2 3.9 98.9 3.0 4

14 D-His 　 122.3 44.5 103.3 4.8 93.0 2.0 94.4 1.5 100.5 2.2 105.6 2.7 103.2 2.6 4

15 D-Phe 　 60.7 104.6 100.7 15.9 97.5 0.1 100.7 8.9 107.6 3.8 102.5 4.4 91.1 2.8 4

16 D-Arg 　 101.4 39.2 102.3 6.1 83.4 3.7 87.5 6.9 97.2 4.4 112.3 6.4 116.0 3.7 4

17 D-Tyr 　 98.6 20.8 105.6 8.0 99.6 1.8 98.9 10.0 108.3 4.4 101.9 3.5 87.1 3.6 4

18 D-Trp 　 99.4 6.5 102.9 9.9 99.0 6.2 95.8 10.7 105.8 5.1 103.5 3.4 93.6 2.6 1a Average of accuracy (Avg) (n=3) (%), b Relative standard deviation (RSD) (%) c Lower limit of quantification (LLOQ)

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Table S5. Accuracy and LLOQ of Vinegar calibration curves for D-amino acids

　 Concentration of calibration curve (nmol/mL) 　

　 1 4 10 40 100 　 400 　 1000 　 (nmol/mL)

Index Compound 　 Avga RSDb Avga RSDb Avga RSDb Avga RSDb Avga RSDb 　 Avga RSDb 　 Avga RSDb 　 LLOQc

1 D-Ala 　 35.3 111.1 100.8 23.5 95.6 7.6 103.3 1.9 100.2 1.3 100.2 1.9 40

2 D-Ser 　 57.4 51.9 98.1 8.5 106.4 3.0 103.7 5.2 99.2 4.9 92.6 2.5 10

3 D-Val 　 97.0 8.8 105.9 7.9 106.1 2.4 101.5 3.3 96.6 5.6 92.8 2.1 4

4 D-Thr 　 134.9 16.6 98.9 14.7 101.1 3.0 106.4 3.9 101.1 4.0 98.1 7.1 94.4 2.5 4

5 D-Cys 　 101.7 14.9 94.4 9.6 105.2 5.3 100.4 1.9 101.0 4.1 97.3 2.3 4

6 D-Ile + D-allo-Ile　 99.1 6.9 102.8 9.5 100.5 3.4 107.4 7.7 101.5 5.2 96.7 4.0 92.0 3.0 1

7 D-Leu 　 98.3 17.1 103.7 5.7 105.7 0.5 107.0 6.7 101.4 2.1 94.9 3.9 88.9 1.8 1

8 D-Asn 　 98.7 65.6 99.6 10.9 111.5 5.1 111.3 5.5 102.7 4.9 93.0 7.1 83.3 2.3 4

9 D-Asp 　 44.2 103.7 97.6 9.5 108.6 9.6 103.4 5.5 99.2 6.6 91.1 2.1 10

10 D-Gln 　 97.8 61.5 107.8 4.5 100.9 6.6 107.3 7.9 99.1 6.4 95.3 7.6 91.8 2.2 4

11 D-Lys 　 124.0 7.9 99.5 12.6 102.0 7.4 100.6 2.9 99.3 2.9 98.7 2.5 10

12 D-Glu 　 217.8 8.1 87.3 36.1 99.3 15.1 103.1 10.3 99.6 4.0 101.6 9.5 96.5 2.1 10

13 D-Met 　 100.1 17.4 97.8 7.1 107.2 6.0 101.7 3.2 98.7 6.6 94.6 1.5 4

14 D-His 　 139.5 3.1 97.2 14.2 105.1 9.5 107.7 7.3 100.6 5.2 97.9 7.1 91.5 3.4 10

15 D-Phe 　 31.7 65.7 98.7 13.1 100.1 8.2 111.8 6.2 103.5 3.0 97.4 4.2 88.5 2.2 4

16 D-Arg 　 149.9 18.0 99.3 2.6 100.4 2.1 105.4 7.7 100.7 5.4 99.3 7.1 95.0 4.1 4

17 D-Tyr 　 60.3 18.5 96.8 11.3 103.7 6.3 116.2 5.5 104.6 6.0 95.0 6.3 83.8 1.6 4

18 D-Trp 　 97.2 19.9 109.3 4.0 102.3 2.1 109.4 7.7 100.8 5.0 94.8 7.2 86.2 1.6 1a Average of accuracy (Avg) (n=3) (%), b Relative standard deviation (RSD) (%) c Lower limit of quantification (LLOQ)

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Table S6. Recovery rates (%) of D-amino acids in foods

Black vinegar Kimchi Yogurt

Index Name AEON JHS Mizkan Amakuchi 　 Ichioshi 　　GS Bifidus LB81 Gasseri

1 D-Ala 103.1 103.1 103.5 111.0 102.0 104.4 103.1 104.2 104.1

2 D-Ser 103.4 103.4 110.6 101.5 103.6 96.1 94.3 97.7 96.7

3 D-Val 105.3 94.2 103.7 102.9 107.1 102.6 101.4 104.0 103.5

4 D-Thr 103.4 89.6 100.1 100.2 103.6 97.6 95.9 98.6 98.4

5 D-Cys 93.5 84.6 77.3 44.5 8.7 12.5 102.3 108.8 104.5

6 D-Ile + D-allo-Ile 103.1 93.5 102.0 102.9 106.1 100.8 96.7 99.5 99.9

7 D-Leu 107.1 102.7 110.2 106.7 113.9 109.2 103.3 104.5 105.2

8 D-Asn 97.9 91.0 97.9 99.8 101.7 95.7 91.3 94.8 95.5

9 D-Asp 94.8 99.1 100.5 77.1 82.1 80.8 38.5 46.1 47.5

10 D-Gln 89.5 71.6 84.5 101.0 103.6 97.0 96.0 98.8 100.6

11 D-Lys 108.0 113.8 113.2 94.2 62.9 46.8 81.8 90.1 66.8

12 D-Glu 100.9 102.5 109.1 98.7 100.1 92.5 83.0 89.2 87.8

13 D-Met 104.9 102.7 110.6 105.9 112.3 103.3 103.2 105.4 105.2

14 D-His 93.2 88.2 86.6 104.3 113.1 96.9 71.7 75.1 74.2

15 D-Phe 105.9 100.0 107.8 106.3 112.7 107.3 103.4 105.1 105.6

16 D-Arg 106.9 92.1 103.2 96.9 82.6 65.6 75.5 81.1 76.2

17 D-Tyr 102.7 98.5 104.6 105.1 110.9 104.7 101.9 103.8 105.6

18 D-Trp 102.4 99.3 104.6 102.5 108.5 101.1 99.3 101.8 102.9

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