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Bioscience, Biotechnology, and Biochemistry ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage:
Bioscience, Biotechnology, and Biochemistry ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage:

Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Separation and Determination of Yellow and Red Safflower Pigments in Food by Capillary Electrophoresis

Toshiro Watanabe, Naoki Hasegawa, Akira Yamamoto, Shiro Nagai & Shigeru Terabe

To cite this article: Toshiro Watanabe, Naoki Hasegawa, Akira Yamamoto, Shiro Nagai & Shigeru Terabe (1997) Separation and Determination of Yellow and Red Safflower Pigments in Food by Capillary Electrophoresis, Bioscience, Biotechnology, and Biochemistry, 61:7, 1179-1183, DOI:

To link to this article: http://dx.doi.org/10.1271/bbb.61.1179

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Biosci. Biotech. Biochem., 61 (7), 1179~1183, 1997

Separation and Determination of Yellow and Red Safflower Pigments in Food by Capillary Electrophoresis

Toshiro WATANABE, Naoki HASEGAWA, Akira YAMAMOTO, Shiro NAGAI, and Shigeru TERABE*

Yaegaki Technology Development Laboratories, Yaegaki Bio-industry Inc., 681 Hayashida, Himeji, Hyogo 679-42, Japan *Faculty of Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan Received February 10, 1997

Capillary electrophoretic methods were developed for analyzing yellow and red safflower pigments in food. Yellow safflower pigment was successfully separated by capillary zone electrophoresis (CZE) with a 300 mM borate buffer (pH 9.0), but this method could not successfully separate the red safflower pigment. The red safflower pigment was discolored in an alkaline solution. Both the yellow and red pigments could be successfully separated by micellar electrokinetic chromatography (MEKC) with 2.0% butyl acrylate/butyl methacrylate/methacrylic acid copolymer sodium salts (BBMA), but neither pigment could be separated with 20 mM sodium dodecyl sulfate. The yellow safflower pigment was extracted from food samples (juice and candies) by solid-phase extraction cartridges and analyzed by the developed technique.

Key words:

capillary zone electrophoresis; micellar electrokinetic chromatography; safflower pigments

Safflower (Carthamus tinctorius L.) is indigenous to Egypt and is known as a herb. 1) Its flowers produce red and yellow pigments. Red safflower pigment was originally used as a cosmetic and textile dye, and today is also used as a food colorant. 2,3) The main component of the red pigment is called carthamin 4 ~8) (Fig. 1) and, due to its low solubility

in water, the red pigment is mainly used in colored chocolate. The yellow safflower pigment, on the other hand, has been used as a natural food colorant for a long time,9) mainly in colored juice, jello, and candy because of their water solubility. The yellow pigment has numerous compo-

nents,

main ones (Fig. 1). In recent years, natural pigments have drawn renewed attention from the viewpoint of safety, and an analysis method for natural pigments is thus im- portant. However, analyzing pigments extracted from food is very difficult because of their extremely low concentra- tions. Capillary electrophoresis (CE) is a relatively new separation technique that was introduced in late 1980 and has high separation efficiency, only requiring a small amount of a sample. Capillary zone electrophoresis (CZE) is the most popular separation mode for CE, where analytes are separated on the basis of their difference in electro- phoretic mobility, which depends on the charge and molecular size. Micellar electrokinetic chromatography (MEKC), in which an ionic micellar solution is employed as the running solution, is another popular separation mode for CE when small molecules are involve. With MEKC, neutral analytes can be separated by their different partitioning to the micelle. Although there are a few re- ports on the analysis of natural pigments by high-per- formance liquid chromatography (HPLC),2) to the best of our knowledge, there is no analytical report on using CEo This paper describes the extraction of safflower pigments from food samples and their analysis by CEo

Materials and Methods

9

13) saffiomin A and safflor yellow B being the

Materials. Yellow safflower pigment used commercially as a natural

coloring for food was obtained from Yaegaki Bio-industry (Himeji, Japan).

Pure water was prepared by purifying distilled water with a Milli-Q SP system (Millipore, Bedford, MA, U.S.A.) just before use. Sodium dodecyl sulfate (SDS) from Naca1ai Tesque (Kyoto, Japan) and butyl acrylate/butyl methacrylate/methacrylic acid copolymer sodium salts (BBMA) from Daiichi Kogyo Seiyaku (Kyoto, Japan) were used as anionic micellar pseudo~stationary phases for MEKC. All other chemicals and solvents were of analytical reagent grade and supplied by Wako (Osaka, Japan).

Apparatus. CE was performed with a BioFocus 3000 CE system (Bio-Rad, Richmond, California, U.S.A.), using a pre-packed cartridge of an uncoated fused silica capillary of 50 11.m i.d. and total length of 50 cm (46 cm to the detector). HPLC separation and purification were performed by a Beckman Gold HPLC system with a programmable 125 solvent module and programmable 166 detector module (Fullerton, California, U.S.A.), using an Inertsil ODS-2 packed column (4.6mm i.d. and 150mm length with a 5-.um particle size; GL Science, Tokyo, Japan). Mass spectra were measured with a JMS-DX303 mass spectrometer (JEOL, Tokyo, Japan) equipped with a fast atom bombardment (FAB) ionization device. Both negative and positive ions were measured. Glycerol was employed as a matrix for FAB ionization.

HPLC separation. Gradient elution was performed with distilled water containing 0.05% trifluoroacetic acid (TFA) at pH 2.5 (sol. A) and with 100% acetonitrile containing 0.05% TFA (sol. B). A linear~gradient program from 0% to 50% of sol. B in 30 min was employed at a flow rate of 1.0 ml/min. The wavelength of the detector was set at 400 nm for yellow pigment and at 500 nm for red pigment. The eluate from the HPLC column was collected when the relevant zones had been eluted, evaporated at 40°C, and used as standard pigments for the CE analysis.

Extraction of the pigments from food samples. Yellow safflower pigment was extracted by a solid-phase method from juice and candies. Carbonated juice was de-aerated for 2 min in an ultrasonic bath, and candies were dissolved in warm distilled water (60-80°C). A solid~phase extraction cartridge (SPE ODS-4, 500 mg/6 ml; Whatman, New Jersey, U.S.A.) was pre-conditioned with 10 m1 of methanol and then with 20 m1 of distilled water prior to use. A 10-ml sample solution was applied to the cartridge, and the cartridge was washed with 20 ml of distilled water. Yellow safflower pigment was eluted with 5 ml of methanol. The eluate was evaporated to dryness at 40°C, the resulting residue being dissolved in water and subjected to the CE analysis.

Capillary electrophoresis (CE). A borate buffer (300 mM at pH 6.0, 7.0, 8.0, or 9.0) was used for capillary zone electrophoresis (CZE). A 20mM SDS solution in a 50mM phosphate buffer (pH 7.0) and 2.0% BBMA (butyl acrylate/butyl methacrylate/methacrylic acid copolymer sodium

saJts)14) in a 20mM ammonium formate buffer (pH 7.0) were used for

micellar electrokinetic chromatography (MEKC). 15) A potential of 15 kV

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T. WATANABE et al.

HO

OH

OH

Carthamin

Safflomin A

H

H

OH

OH

H

OH

OH

OH

Safflor yellow B

Fig. 1. Structures of Red and Yellow Safflower Pigments (Carthamin, Safflomin A and Safflor Yellow B).

was applied, detection was performed by measuring the absorbance at 400 nm (yellow pigment) or at 500 nm (red pigment), and the capillary temperature was 20°C. Yellow safflower pigment was dissolved in distilled water, red pigment was dissolved in N,N-dimethylformamide, each solution then being passed through a disposable hydrophilic membrane filter (0.45,um pore size; Advantec Toyo, Tokyo, Japan) prior to use. Each sample was injected by pressure at 350 mbar for 1.0 s. The capillary was rinsed with a 1.0 M NaOH solution for 120 s and with distilled water for 120s before each run.

A

E

c

0

0

o:t

i

0

.0

«

B

0.120

0.080

0.040

0.000

0

5

10

15

Time (min)

Fig. 2.

E

c

0

0

o:t

i

0

.0

«

C

0.120

0.080

0.040

0.000

5

0.120

E

c

0 0.080

0

o:t

i

0 0.040

Q

«

0.000

10

 

15

20

25

Time (min)

 

15

20

25

30

35

Time (min)

Separation of the Yellow Safflower Pigment by CZE.

20

30

40

A) Capillary, 50jlm Ld. x 50cm; running solution, 300mM borate buffer at pH 7.0; applied voltage, 15 kV; temperature, 20°C; detection, 400 nm. B) Running solution,

30 mM borate buffer at pH 8.0. The other conditions are the same as those in A. C) Running solution, 300 mM borate buffer at pH 9.0. The other conditions are the same as those in A.

2B). The highest peak was probably a major component of the yellow pigment of safflower. CZE separation of the red pigment of safflower was also attempted, but the red color faded in an alkaline solution, because the red pigment is only stable in acidic conditions.

MEKC analysis

MEKC with a SDS solution in a phosphate buffer was

not successful for separating the yellow pigment of safflower.

A 2.0% BBMA solution in 20 mM ammonium formate (pH

7.0) gave better resolution of the yellow pigment as shown

 

in

Fig. 3. Although the migration times are shorter in Fig.

Results and Discussion

3,

the electropherogram is similar to that obtained by CZE

CZE analysis

given in Fig. 2C. The highest peaks were probably the same

safflower was also successfully separated by MEKC with

Since the yellow pigment of safflower has several diol groups as shown in Fig. I, CZE was performed with a borate buffer at various alkaline pH values. Good separa-

component between Figs. 2 and 3. The red pigment of

the BBMA solution (Fig. 4). The main peak is presumed

tion was obtained with a 300 mM borate buffer at pH 9.0

to

have been carthamin, the principal ingredient of the red

(Fig. 2C), although it needed a long time to complete the

pigment of safflower. The red color of the solution did not

separation. At pH 7.0 and pH 8.0, several components of

the yellow pigment were not well separated (Figs. 2A and

fade under this condition.

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Capillary Electrophoresis of Safflower Pigments in Food

0.040

RUN 5

E

c:

0

0 oo::t

i

tI)

.c

<C

0.030

0.020

0.010

0.000

0.030,.-----------,

 

0.020

 

E

c

CI

CI

In

0

3

6

9

12

15

'lU

 

I/)

0.010

.CI

 

Time (min)

<C

Fig. 3.

Separation of the Yellow Safflower Pigment by MEKC.

E

c

CI

CI

In

'lU

I/)

.CI

<C

Running solution, 20mM ammonium formate buffer at pH 7.0 containing 2.0% BBMA. The other conditions are the same as those in Fig. 2A.

0.030

o.ooo

w

l

RUN 15

1181

0.030,--------,

0.020

0.010

0.000

---

E 0.020

c:

0

0

II)

i II)

0.010

.c

<C

 

0.000

I\.

Fig. 4.

5

7

9

11

Time (min)

13

15

Separation of the Red Safflower Pigment by MEKC with BBMA.

Running solution, 20 mM ammonium formate buffer at pH 7.0 containing 2.0% BBMA; detection, 500 nm. The other conditions are the same as those in Fig. 2A.

Repeatability

In order to check the reproducibility for separating the pigments by these methods, the red and yellow pigments of safflower were repeatedly analyzed fifteen times. The yellow pigment of safflower was analyzed by the CZE method with a borate buffer, and the red pigment by MEKC with the BBMA solution as already described. No significant difference in separation pattern and migration time was observed among the repeated runs as shown in Fig. 5 for the red pigment.

Standard curve

The principal ingredients of the yellow pigment of safflower isolated by HPLC were employed as standards for the yellow pigment by CEo The yellow pigment of safflower prepared by HPLC gave a single major peak (Fig. 6). The main component in Fig. 6 showed the same migration time as the highest peak in Fig. 2C, and it is

hence concluded to be the principal component of the yellow pigment of safflower. A standard curve obtained with this component from 50 to 1000 ppm showed a straight line

for the yellow pigment being

0.5 ppm. The yellow pigment component of safflower was

(r = 0.998), the detection limit

determined with this standard curve.

Mass spectrometric analysis

Mass spectra for the principal component of the yellow

5

7

9

11

13

Time (min)

15

5

7

9

11

13

Time (min)

15

Fig. 5.

BBMA (5th and 15th Runs).

The conditions are the same as those in Fig. 4.

Repeated Separation of Red Safflower Pigment by MEKC with

0.040 ,--------------------,

E

c:

0

0 0.020

"It

i

II)

.Q

<C

0.000

10

1

15

1

.,.,.,-

20

"

25

Time (min)

30

35

40

Fig. 6. Separation of the Principal Ingredient of Yellow Safflower Pigment by CZE.

The conditions are the same as those in Fig. 2C.

safflower pigment isolated by HPLC were measured in both the positive and negative ion modes by FABMS. The molecular mass of the principal component was determined to be 612 from the m/z 613 peak for M + 1 in the positive ion mode and m/z 611 peak for M 1 in the negative ion mode (Fig. 7). The molecular mass strongly suggests that

the

safflomin A as suggested in earlier reports. 9 ,16)

was

principal

component

of the

yellow

pigments

Analysis of the pigments extracted from food samples

Pigments were extracted from candies as described in Materials and Methods, and juice with a dark color was analyzed without any pretreatment. The recovery of the pigments by solid-phase extraction was 75%. An elec- tropherogram of the pigments extracted from candies (Fig. 8B) was similar to that of the yellow pigment (Fig. 2C). In the electropherogram (Fig. 8A) of the pigment extracted from juice, many peaks appeared, among which the peak at about 21 min was easily identified as safflomin A.

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T. WATANABE et al.

A A 183~5-' 1 0.040 E c 256 c C "It 0.020 611 I 'S
A
A
183~5-'
1
0.040
E
c
256
c
C
"It
0.020
611
I 'S
III
Q
C(
551 591
703
0.000
100
200
300
400
500
600
700
m/e
B
B
553
ji ~1361r 737
0.120
500
2~3
IIt.m,'it'b"t,yj';
Gi
: 258
369
X 10
I
~ 0.080
a:
20
. 294~28
t
8
"It
407442 461
'S
O~
~~
w~~~~.-----~~--~~
100
200
300
400
500
600
700
!
0.040

m/e

C(

10

15

20

25

Time (min)

30

35

40

Fig. 7.

FABMS Spectrum of the Principal Ingredient of Yellow Safflower

Pigment.

A, negative ions; B, positive ions.

Fig. 8.

10

15

20

25

Time (min)

30

35

40

CZE Separation of Pigments Extracted from Food Samples.

A, yellow safflower pigment extracted from juice; B, yellow safflower pigment extracted from candies. The conditions are the same as those in Fig. 2C.

Table

Analytical Results for the Yellow Pigment Extracted from Food Samples

Species

Standard

n Migration time

(Intra-day)

Mean (min)

C.V. (%)

Determination

C.V. (%)

Safflower yellow*

Yellow pigment extracted from food samples

21.62

0.16

1380

1.87

From juice*

3

21.33

0.26

427

2.36

From candies*

3

21.17

0.45

1253

0.94

* Safflomin A.

However in Fig. 8A, a number of extra peaks are also apparent other than those in the yellow safflower pigment (Fig. 2C), suggesting that some pigments other than that of safflower pigments had also been added to the juice. The coefficient of variation (C.V.) of the migration times was less than 0.5% and that for quantification was less than 2.5% (Table). In conclusion, CE was found to be a useful technique for analyzing food pigments. The amount of the sample required for the analysis was very small, and the repeat- ability of migration times and quantification was high even for real samples, probably due to the absence of any packing material inside the capillary. Another advantage

of the CE method is its low running cost. The CE method will also be widely applicable to analyzing several different food additives.

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1)

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Capillary Electrophoresis of Safflower Pigments in Food

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