知的財産権の出願．登録状況なし

C-4) 1 H-qNMR による ( 代用 ) 標準品の純度測定 RRF 及び RMS を算出するためには，測定対象

G. 知的財産権の出願．登録状況なし

)

C

₂₅

H

₃₀

O

₄

= 394.511 ( )

C

₂₄

H

₂₈

O

₄

= 380.484

4 13 8

Sudan II (S2)

C

₁₈

H

₁₆

N

₂

O = 276.339

2’ 3’

5’

Sudan I (S1)

C

₁₆

H

₁₂

N

₂

O = 248.285

10 9

8 4

2’

6’

Fig.1 Structures of main pigments in Annatto extract, bixin

(Bx) and norbixin (Nb), and appropriate internal standards

in this study, sudan I (S1) and sudan II (S2).

Fig. 2 The LC profiles of mixed standard solutions and PDA spectra of bixin, norbixin, sudan I and sudan II.

The mixed standard solutions, STD-Bx-LV3 and STD-Nb-LV3, were applied onto HPLC/PDA. The concentrations of bixin, norbixin, sudan I and sudan II are 30 µg/mL, 30 µg/mL, 60 µg/mL and 60 µg/mL,

respectively.

Flow 1mL/min Bixin+IS-std3-n1

10.00 20.00 30.00 40.00 50.00 60.00Time

0.0 2.5e-2 5.0e-2 7.5e-2 1.0e-1 1.25e-1 1.5e-1 1.75e-1 2.0e-1 2.25e-1

10.00 20.00 30.00 40.00 50.00 60.00

0.0 2.0e-2 4.0e-2 6.0e-2 8.0e-2 1.0e-1 1.2e-1

QmAnt451 2: Diode Array

460 Range: 1.215e-1 28.43

23.50

11.07

47.82

QmAnt452 2: Diode Array

Range: 2.735e-1460 11.07

23.50

47.82

Flow 1.4mL/min Bixin+IS-std3-n1

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00Time

0.0 2.5e-2 5.0e-2 7.5e-2 1.0e-1 1.25e-1 1.5e-1 1.75e-1 2.0e-1

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

0.0 1.0e-2 2.0e-2 3.0e-2 4.0e-2 5.0e-2 6.0e-2 7.0e-2 8.0e-2 9.0e-2 1.0e-1 1.1e-1

QmAnt464 2: Diode Array

460 Range: 1.113e-1 20.27

16.72

7.90

34.03

QmAnt465 2: Diode Array

Range: 2.449e-1460 7.88

16.70

34.05

STD-Bx-LV3

STD-Nb-LV3

STD-Bx-LV3

STD-Nb-LV3 (a) Flow rate 1.0 mL/min (b) Flow rate 1.4 mL/min

Nb S1

S1 S1

S2 S2

S2 Bx

Bx Bx

Bixin+IS-std3-n1

200 250 300 350 400 450 500 550 600nm

0.0 5.0e-2 1.0e-1 1.5e-1

200 250 300 350 400 450 500 550 600nm

0.0 2.0e-2 4.0e-2 6.0e-2 8.0e-2

QmAnt451 1700 (28.417) 2: Diode Array

9.715e-2 462.9

204.9216.9244.9

490.9

QmAnt452 659 (11.067) 2: Diode Array

2.464e-1 460.9

196.9 244.9

489.9

Bixin+IS-std3-n1

200 250 300 350 400 450 500 550 600nm

0.0 2.5e-2 5.0e-2 7.5e-2 1.0e-1 1.25e-1 1.5e-1

200 250 300 350 400 450 500 550 600nm

0.0 1.0e-2 2.0e-2 3.0e-2 4.0e-2 5.0e-2

QmAnt451 2864 (47.817) 2: Diode Array

5.567e-2 232.9

496.9

318.9

QmAnt451 1405 (23.500) 2: Diode Array

1.78e-1 230.9

481.9

313.9

Bixin

Norbixin

Sudan II

Sudan I

Fig. 3 T he

H-qNMR spectra of bixin, norbixin, sudan I and sudan II NMR solvent is pyridine-d

₅

(pyr) and qNMR reference is 1,4-BTMSB-d

₄

. The qNMR measurement conditions are shown in Table 2.

abundance 00.020.040.060.080.1

parts per Million

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

abundance 00.020.040.060.080.10.12

parts per Million

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

abundance 00.10.2

parts per Million8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

abundance 00.10.2

parts per Million8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

Bixin (Bx1)

Norbixin (Nb1)

Sudan (S1)

Sudan (S2)

8 Pyr

6’ 4’

8, 17, 4, 13 8, 4, 17, 13

1,4-BTMSB-d4

Pyr

1,4-BTMSB-d4

1,4-BTMSB-d₄

Fig. 4 The magnified views of the

H-qNMR spectra of bixin, norbixin, sudan I and sudan II.

The signals indicated by arrows were used for determining the purities.

abundance 00.030.060.090.120.150.18

parts per Million

8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7

(thousandths) 020.040.060.0

parts per Million

8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 (thousandths) 010.020.0

parts per Million

8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7

(thousandths) 010.020.030.0

parts per Million

8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7

Bixin (Bx1)

Norbixin (Nb1)

Sudan I (S1)

Sudan II (S2)

18 3

Pyr 10

11 2 19

Pyr

7 5 14

9 1216

18 3

15 10 11

6 7 5

14 9 12

16 2 19

2’

6’4

5 7 3‘5'

3 4’

2’ 4

57 6 3’

3 5’

Pyr Pyr

Fig. 5 Absolute calibration curves of bixin (Bx), norbixin (Nb), sudan I (S1) and sudan II (S2).

The HPLC conditions are shown in the experimental section. The absolute calibration curves were constructed by using the standard solutions LV0 to LV5 which are shown as closed circles. Open circles are shown for reference purpose only.

←LV6

² ²

Bixin (Bx)

Norbixin (Nb)

Sudan I (S1)

Sudan II (S2)

Bixin (Bx)

Norbixin (Nb)

Sudan I (S1)

Sudan II (S2)

a) Flow rate 1.0 mL/min b) Flow rate 1.4 mL/min

µg/mL

Fig. 6 Residual plots of bixin (Bx) and norbixin (Nb).

Bixin (Bx)

Norbixin (Nb)

Residual %Residual %

Bixin (Bx)

Norbixin (Nb)

a) Flow rate 1.0 mL/min b) Flow rate 1.4 mL/min

Residual %Residual %

µg/mL µg/mL

RRF, RMS Nb:S1:1.0

RRF, RMS Nb:S2:1.0

Abs. Calib. curve RRF, RMS Bx:S1:1.0

RRF, RMS Bx:S2:1.0

Abs. Calib. curve

RRF, RMS Bx:S1:1.4

RRF, RMS Bx:S2:1.4

Abs. Calib. curve

RRF, RMS Nb:S1:1.4

RRF, RMS Nb:S2:1.4

Abs. Calib. curve

0 10 20 30 40 50 60 70 80 90 100

Bx1 Bx2 Bx3 Bx4 Bx5 Bx6 Bx7 Bx8 Bx9 Nb1 Nb2 Nb3 Nb4 Nb5 Nb6 Nb7 Nb8 Nb9 Nb10Nb11Nb12Nb13Nb14Nb15Nb16Nb17Nb18

Fig. 7 Comparison of the total contents of bixin (Bx) and norbixin (Nb) in Annatto extract products determined by absolute calibration curves method (black) and color value measurement (white).

Table 1 Sample information of annatto extracts

Sample No. Voucher No.* Property Company Content (%)**

Bixin type annatto extract

Bx1 A1217 Powder A 96.9(LC area %)

Bx2 A1127 Liquid B - (color value 2300)

Bx3 A1146 Powder A

-Bx4 A1157 Liquid A 30 (Spectrophotometric)

Bx5 A1180 Powder C

-Bx6 B201 Powder A

-Bx7 B210 Powder D 69.3 (Spectrophotometric)

Bx8 A1295 Powder E 88.2 (-)

Bx9 A1296 Powder E 78.0 (-)

Norbixin type annatto extract

Nb1 A1218 Powder F 95 (LC area %)

Nb2 B202 Powder A

-Nb3 B207 Powder E - (color value 13500)

Nb4 B211 Powder D 71.4 (Spectrophotometric)

Nb5 B225 Powder G 35.5 (-)

Nb6 B227 Powder H 40.4 (-)

Nb7 B230 Powder I 34.2 (-)

Nb8 B242 Liquid J

-Nb9 A1291 Powder I - (color value 13200)

Nb10 A1292 Powder G 40.3 (-)

Nb11 A1293 Powder D 44 (-)

Nb12 A1294 Powder H 40 (-)

Nb13 A1297 Powder E 33.8 (-)

Nb14 A1298 Powder E 39.9 (-)

Nb15 A1299 Powder H 43 (-)

Nb16 A1302 Powder A 36.8 (-)

Nb17 A1303 Powder A 65.5 (-)

Nb18 A1304 Powder F - (color value 24300)

* The voucher No. is our idintifing number of the storing product. ** The measurement methods or color values on the labels are in parentheses. - means no information on the labels.

Table 2 ¹H-qNMR measurement conditions

NMR ECZ600 JEOL), ¹H : 600 MHz

Probe Varian-ColdProbe-CH

Spectral width -5 15 ppm

Data points 60,000 (resolution 0.25 Hz) Digital filter on

Flip angle 90 deg

Repetition time 64 s T₁×7 Sample spin off

Probe temp room temp.

Scan times 8

Table 3 The concentrations of mixed standard solutions.

Bixin (Bx) Norbixin (Nb) Sudan I (S1) Sudan II (S2)

STD-Bx-blank 0 0 0

STD-Bx-LV1 2 4 4

STD-Bx-LV2 10 20 20

STD-Bx-LV3 30 60 60

STD-Bx-LV4 70 140 140

STD-Bx-LV5 100 200 200

STD-Bx-LV6 400 800 800

STD-Nb-blank 0 0 0

STD-Nb-LV1 2 4 4

STD-Nb-LV2 10 20 20

STD-Nb-LV3 30 60 60

STD-Nb-LV4 70 140 140

STD-Nb-LV5 100 200 200

STD-Nb-LV6 400 800 800

Concentration (µg/mL) Standard solution

No.

The values are shown without the corrections of the concentrations by the purity of each compound.

Table 4 HPLC/PDA conditions.

HPLC Waters LC: Alliance 2695, PDA: 2996 photodiode array detector

Column Inertsil ODS-4 (4.6×250 mm, 5μm, GL Sciences) Column temp. 35˚C

Solvent isocratic 2% acetic acid aq.: CH₃CN (35 : 65) Flow rate 1.0 or 1.4 mL/min

PDA scan 190-600 nm,

PDA detect 460 nm (±4 nm), smoothing (±3 scans x 2 times) Sample temp. 10˚C

Injection volume 10 µL

Table 5 Color value measurement conditions.

Spectrophotometer V-650 (JASCO)

Wavelength range 200-700 nm, 1 nm interval Wavelength λ_max of 482-490 nm (bixin)

λ_max of 476-484 nm (norbixin)

Data scan 0.1 nm

Cell 1 cm quartz cell

Reference solvent acetone (bixin), 0.5w/v% KOH aq. (norbixin)

Table 6 Caliculated RRF and RMS values of bixin (Bx) and norbixin (Nb).

Flow rate 1.0 mL/min Flow rate 1.4 mL/min

Bixin (Bx) 7 - 70 24170.0 16985.5

Norbixin (Nb) 7 - 70 23737.3 16662.1

Sudan (S1) 20 - 200 4415.1 3110.9

Sudan (S2) 20 - 200 3642.2 2563.7

RRF [weight/weight]

Bx:S1 = Slope_Bx / Slope_S1 5.474 5.460

Bx:S2 = Slope_Bx / Slope_S2 6.636 6.625

Nb:S1 = Slope_Nb / Slope_S1 5.376 5.356

Nb:S2 = Slope_Nb / Slope_S2 6.517 6.499

RMS [mol/mol]

Bx:S1 = RRF_Bx:S1 / (MW_Bx / MW_S1) 3.445 3.436

Bx:S2 = RRF_Bx:S2 / (MW_Bx / MW_S2) 4.648 4.641

Nb:S1 = RRF_Nb:S1 / (MW_Nb / MW_S1) 3.508 3.495

Nb:S2 = RRF_Nb:S2 / (MW_Nb / MW_S2) 4.733 4.720

The concentrations of standard solutions used for development of calibration curves were corrected by absolute purities determined by ¹H-qNMR. All determination coefficients (R²) of calibration curves were not less than 0.999.

The molecular weights (MW) of bixin, norbixin, sudan I and sudan II are 394.511, 380.484, 248.285 and 276.339, respectively.

Slope of the calibration curve Conc. Range for development of

calibration curve (ug/mL)*

Table 7 Contents of bixin (Bx) and norbixin (Nb) determined by RMS method and absoulte calibration curve method at the flow rate of 1.0 mL/min. Content of bixin (Bx) (%)Content of norbixin (Nb) (%)Content of bixin (Bx) (%)Content of norbixin (Nb) (%)Content of bixin (Bx) (%)Content of norbixin (Nb) (%) AVRSTDEVCV%AVRSTDEVCV%AVRSTDEVCV%AVRSTDEVCV%AVRSTDEVCV%AVRSTDEVCV% Bx1BixinPowder71.950.120.21.390.010.471.800.200.31.390.010.471.400.050.11.380.010.5 Bx2BixinLiquid6.620.020.20.120.000.86.570.020.30.120.000.76.720.010.10.120.000.7 Bx3BixinPowder92.400.550.61.720.021.091.880.730.81.710.021.393.750.540.61.750.010.6 Bx4(Bixin)Liquid2.580.020.70.030.001.42.570.020.90.030.001.62.610.031.30.030.001.7 Bx5BixinPowder14.450.070.50.330.039.714.400.070.50.330.039.614.680.080.50.340.039.6 Bx6BixinPowder3.860.041.00.040.001.73.860.041.00.040.001.73.930.041.00.040.001.7 Bx7BixinPowder8.890.080.91.910.168.68.880.101.11.900.168.69.080.091.01.950.178.7 Bx8BixinPowder83.270.981.21.270.000.383.090.991.21.270.000.382.360.780.91.260.000.3 Bx9BixinPowder70.710.630.90.690.033.870.670.610.90.690.033.770.120.480.70.690.033.8 Nb1NorbixinPowder1.260.118.471.660.470.71.250.118.471.320.380.51.250.108.471.290.530.7 Nb2NorbixinPowder0.010.008.416.350.120.70.010.008.516.290.110.70.010.008.616.520.100.6 Nb3NorbixinPowderND19.070.764.0ND19.020.764.0ND19.250.794.1 Nb4NorbixinPowderND38.290.280.7ND38.150.300.8ND38.540.511.3 Nb5NorbixinPowder0.020.002.023.661.014.30.020.001.923.541.034.40.020.001.924.041.054.4 Nb6NorbixinPowder0.040.0011.313.710.050.30.040.0011.313.650.050.30.040.0011.713.820.010.1 Nb7NorbixinPowderND19.440.191.0ND19.370.201.1ND19.580.180.9 Nb8(Norbixin)LiquidND2.020.010.5ND2.020.010.4ND2.050.021.0 Nb9NorbixinPowderND36.281.845.1ND36.131.865.1ND36.271.855.1 Nb10NorbixinPowder1.100.032.334.020.672.01.090.032.433.920.692.01.100.022.134.200.661.9 Nb11NorbixinPowderND33.020.130.4ND32.900.170.5ND33.320.040.1 Nb12(Norbixin)Powder0.920.000.333.490.170.50.910.000.533.400.110.30.910.000.433.370.160.5 Nb13NorbixinPowderND26.360.040.2ND26.300.030.1ND26.430.110.4 Nb14NorbixinPowderND32.310.240.7ND32.170.220.7ND32.150.300.9 Nb15(Norbixin)PowderND35.360.421.2ND35.170.501.4ND35.290.581.6 Nb16NorbixinPowder1.090.010.633.240.190.61.090.010.633.180.200.61.090.010.633.200.190.6 Nb17NorbixinPowder4.700.030.756.160.240.44.680.030.755.940.240.44.700.030.756.160.220.4 Nb18NorbixinPowder1.110.3127.971.420.570.81.100.3127.971.250.711.01.110.3128.071.770.490.7 RMS method with sudan I (S1)RMS method with sudan II (S2)Absolute calibration curve method Sample No.Labeled main componentProperty

ドキュメント内厚生労働科学研究費補助金（食品の安全確保推進研究事業） (ページ 68-81)

知的財産権の出願．登録状況 なし

C-4) 1 H-qNMR による ( 代用 ) 標準品の純度測定 RRF 及び RMS を算出するためには，測定対象

G. 知的財産権の出願．登録状況 なし

)

C

H

O

= 394.511 ( )

C

H

O

= 380.484

Sudan II (S2)

C

H

N

O = 276.339

Sudan I (S1)

C

H

N

O = 248.285

Fig.1 Structures of main pigments in Annatto extract, bixin

(Bx) and norbixin (Nb), and appropriate internal standards

in this study, sudan I (S1) and sudan II (S2).

Fig. 3 T he

H-qNMR spectra of bixin, norbixin, sudan I and sudan II NMR solvent is pyridine-d

(pyr) and qNMR reference is 1,4-BTMSB-d

. The qNMR measurement conditions are shown in Table 2.

Bixin (Bx1)

Norbixin (Nb1)

Sudan (S1)

Sudan (S2)

Fig. 4 The magnified views of the

H-qNMR spectra of bixin, norbixin, sudan I and sudan II.

The signals indicated by arrows were used for determining the purities.

Bixin (Bx1)

Norbixin (Nb1)

Sudan I (S1)

Sudan II (S2)

Fig. 5 Absolute calibration curves of bixin (Bx), norbixin (Nb), sudan I (S1) and sudan II (S2).

The HPLC conditions are shown in the experimental section. The absolute calibration curves were constructed by using the standard solutions LV0 to LV5 which are shown as closed circles. Open circles are shown for reference purpose only.

←LV6

Fig. 6 Residual plots of bixin (Bx) and norbixin (Nb).

Fig. 7 Comparison of the total contents of bixin (Bx) and norbixin (Nb) in Annatto extract products determined by absolute calibration curves method (black) and color value measurement (white).

G. 知的財産権の出願．登録状況なし