Methods Development Team
Industrial Hygiene Chemistry Division
OSHA Salt Lake Technical Center
Sandy UT 84070-6406
1. General Discussion
1.1. Background
1.1.1. History of Procedure.
Recently, the OSHA Analytical Laboratory received a set of field samples
requesting analysis for Acid Blue 9. The air samples had been collected on glass
fiber filters, at 1 Lpm for a total of about 90 liters air. This report describes the
analytical procedure developed and the preliminary validations of the sampling
method.
Acid Blue 9 is a widely used food dye. There has been many schemes proposed
for the qualitative analysis of food dyes, most of which depended on paper and
thin-layer chromatography. Less attention has been given to the quantitative
analysis of dyes. Some of the methods attempted were: (a) comparison of spot
intensities on TLC plates with those of a range of standards, (b)
spectrophotometric quantitation, (c) titration with titanous chloride solution, and
(d) electrophoresis on polyacrylamide gel. More recently, HPLC has been applied
for dye analysis, using anion-exchange columns or, more satisfactorily, by ion-
pairing. Paired-ion HPLC affords a means of separating a mixture of food dyes in
a single run (Ref. 5.2.). Preliminary search did not reveal an air sampling method
for Acid Blue 9. Judging from its physical properties, glass fiber filters may be a
suitable collection medium.
1.1.2. Toxic Effects.
(This section is for information only and should not be taken as the basis of
OSHA policy.)
Acid Blue 9 is carcinogenic in rats after its subcutaneous injection: it produced
fibrosarcomas following repeated injections. It also produced an increased
incidence of kidney tumors in mice after its oral administration (Ref. 5.1.).
1.1.3. Potential Workplace Exposure.
Acid Blue 9 is an FDA certified food dye and is used in such products as gelatin
desserts, ice cream and sherbets, carbonated beverages, dry drink powders,
candy and confectionary products when they do not contain oils and fats, bakery
products and cereal, puddings, aqueous drug solutions, tablets, capsules, bath
salts, and hair rinses (Ref. 5.3.). Acid Blue 9 has been produced in the U.S. for
over sixty years. In 1975, three U.S. companies produced 622,000 Kg of the
general dye grade, and another four companies produced 56,000 Kg of he food,
drug, and cosmetic grade (Ref. 5.1.). Preliminary literature searches did not
reveal any estimate on the extent of worker exposure.
1.1.4. Physical Properties
Color Index Names: Acid Blue 9, Food Blue 2
Color Index Number: 42090
Cas Reg. Number:2650-18-2 (3844-45-9 )
Chem. Abstr. Names:
N-Ethyl-N-(4[(4-(ethyl[(3-sulfophenyl)methyl]amino) phenyl)-(2-
sulfophenyl)methylene]-2, 5-cyclohexadien-1-ylidene)3-
sulfobenzenemethanaminium hydroxide inner salt, disodium salt; C.I. Acid Blue
9, disodium salt; D and C Blue No.1; D and C Blue No.4; ethyl(4-(p[ethyl ( m-
sulphobenzyl)amino]-α -(o-sulphophenyl)benzylidene)-2,5-cyclohexadiene-l-
ylidene)- (m-sulphobenzyl) ammonium hydroxide inner salt, disodium salt; FD
and C Blue 1; FD and C Blue. No.1; FDC Blue No.1; Acid Sky Blue A; Acilan
Turquoise Blue AE; A. F. Blue No.1; Aizen Brilliant Blue FCF; Aizen Food Blue
No.1; Alphazurine; Alphazurine FG; Alphazurine FGND; Amacid Blue FG; Amacid
Blue FG Conc; 1206 Blue, 11388 Blue; Blue Dye Number 1 food additive; Brilliant
Blue; Brilliant Blue FCF; Brilliant Blue Lake; Bucacid Azure Blue; Calcocid Blue EG;
Calcocid Blue 2G; Canacert Billiant Blue FCF, Cogilor Blue 512.12; Cosmetic Blue
Lake; Dispersed Blue 12195; Disulphine Lake Blue EG; Dolkwal Brilliant Blue;
Edicol Blue Cl 2; Edicol Supra Blue E6; Erioglaucine ; Erioglaucine A; Erioglaucine
E; Erioglaucine G; Eriosky Blue; Fenazo Blue XI; Fenazo Blue XR; Food Blue 1;
Hexacol Brilliant Blue A; Hidacid Azure Blue; Intracid Pure Blue L; Kjtoc Blue AR;
Kiton Pure Blue L; Maple Brilliant Blue FCF; Merantine Blue EG; Neptune Blue
BRA; Concentration; Patent Blue AE; Patent Blue 2Y; Peacock Blue X-1756;
Usacert Blue No.1; Xylene Blue VSG.
Appearance: Reddish-violet powder or granules with a metallic luster.
Spectroscopy Data: λ max 630 nm.
Chemical Formula and Molecular Weight:
Solubility: Soluble in water and ethanol; insoluble in vegetable oils.
1.2. Limit Defining Parameters
1.2.1. Detection Limit of the Analytical Procedure
The detection limit of the analytical procedure is 0.83 ng Acid Blue 9 per
injection. This is the amount of analyte which will give a peak whose height is
approximately five times the amplitude of the baseline noise. See Figure 1.
1.2.2. Detection Limit of the Overall Procedure
The detection limit of the overall procedure is estimated to be 0.2 µg per sample
or 0.002 mg/cu m based on the recommended air volume, assuming 100%
recovery from the sampling device. The recovery test at this level has not been
performed.
1.2.3. Sensitivity
The sensitivity of the analytical procedure over a concentration range of 0.395 to
11.9 µg/mL is 19,280 area units per µg/mL of Acid Blue 9. The sensitivity is
determined by the slope of the calibration curve. See Figure 2.
1.3. Advantages
The analytical procedure is rapid, sensitive, and reproducible.
1.4. Disadvantages
The method has not been fully validated.
2. Sampling Procedure
2.1. Apparatus
2.1.1. An air sampling pump with a flow rate which can be calibrated to within
±5% of the recommended 1 Lpm flow rate while the sampler is in line.
2.1.2. Glass fiber filter, 37-mm diameter, Gelman Type A, or equivalent.
2.1.3. Filter holder for 37-mm filters, Millipore M000037AO, or equivalent.
2.2. Sampling Technique
2.2.1. Assemble the filter in the two-piece cassette holder and close firmly. The
filter is supported by a backup pad. Secure the cassette holder together with
tape.
2.2.2. Attach the outlet of the filter cassette to the personal sampling pump inlet
with flexible tubing.
2.2.3. Air being sampled should not pass through any hose or tubing before
entering the filter cassette.
2.2.4. A sample size of 100 liters is recommended. Sample at a flow rate of 1.0
liter/minute. The flow rate should be known with an accuracy of ±5%.
2.2.5. With each batch of samples, submit a blank filter from the same lot of
filters used for sample collection. This filter must be subjected to exactly the
same handling as the samples except that no air is drawn through it. Label this
filter as the blank.
2.2.6. The cassette should be shipped in a suitable container designed to prevent
damage in transit. The samples should be shipped to the laboratory as soon as
possible.
2.2.7. A sample of the bulk material should be submitted to the laboratory in a
glass container with a Polyseal cap. Never transport, mail, or ship the balk
sample in the same container as the sample or blank filter.
2.3. Retention Efficiency
Two glass fiber filters were spiked with 1.1.5 µg of Acid Blue 9. Humid air (87%
relative humidity) 140 liters was drawn through the filters at 1 Lpm. The average
recovery of the two filters was 101%.
Sample Spiked Amount Treatment Peak Height Recovery
YC5 41.5 µg on GFF 140 L humid air 138.0 mm 103.4%
YC6 41.5 µg on GFF 140 L humid air 131.5 mm 98.5%
YC7 41.5 µg; control none 131.0 mm ----
YC8 41.5 µg; control none 136.0 mm ----
Average recovery 100.9%
2.4. Extraction Efficiency
The average extraction efficiency from the glass fiber filters spiked with 41.5 µg
of Acid Blue 9 was 95.9%.
Sample Spiked Amount Peak Height Recovery
YC3 41.5 µg on GFF 126.5 mm 94.8%
YC4 41.5 µg on GFF 129.5 mm 97.0%
YC7 41.5 µg; control 131.0 mm ----
YC8 41.5 µg; control 136.0 mm ----
Average recovery 95.9%
2.5. Storage
Two glass fiber filters were spiked with 41.5 µg of Acid Blue 9 and stored at
room temperature in the dark for two days. The average recovery was 100.2%.
Sample Spiked Amount Storage Days Peak Height Recovery
YC3 41.5 µg 0 126.5 mm 94.8%
YC4 41.5 µg 0 129.5 mm 97.0%
YC7 41.5 µg control 131.0 mm ----
YC8 41.5 µg control 136.0 mm ----
YC1 41.5 µg 2 168.0 mm 105.0%
YC2 41.5 µg 2 157.0 mm 98.1%
YC7 41.5 µg control 162.0 mm ----
YC8 41.5 µg control 158.0 mm ----
2.6. Recommended Air Volume and Sampling Rate
2.6.1. The recommended air volume is 100 liters.
2.6.2. The recommended sampling rate is 1 Lpm.
2.7. Interferences
There are no known interferences associated with the sampling procedure.
2.8. Safety Precautions
2.8.1. Attach the sampling equipment to the worker in such a manner that it will
not interfere with work performance or safety.
2.8.2. Follow all safety practices that apply to the work area being sampled
3. Analytical Method
3.1. Apparatus
3.1.1. High performance liquid chromatograph equipped with pump, sample
injector, variable wavelength detector, chart recorder, and other necessary
hardware.
3.1.2. HPLC reverse phase C18 analytical column. Dupont Zorbax ODS column
was used for this study.
3.1.3. An electronic integrator or other suitable method to measure detector
response.
3.1.4. Microliter syringe or automatic sampling device for making sample
injections.
3.1.5. Volumetric flasks of convenient sizes for preparing standards.
3.1.6. Shaking device for extraction of samples.
3.2. Reagents
3.2.1. Acid Blue 9 (Erioglaucine)
3.2.2. Tetrabutylammonium phosphate, reagent grade
3.2.3. Methanol, HPLC grade
3.2.4. Water, HPLC grade
3.2.5. Phosphoric Acid
3.3. Sample Preparation
3.3.1. Remove the filter form the cassette clean tweezers and place it in a 20-mL
scintillation vial.
3.3.2. Add 5 mL of methanol/water (1:1) to the vial and cap it.
3.3.3. Shake the vials vigorously on a shaker for 30 minutes.
3.4. Standard Preparation
3.4.1. Standard of Acid Blue 9 is prepared by dissolving 8 to 12 mg (accurately
weighed) of Acid Blue 9 in water in a 10-mL volumetric flask and making it to
volume.
3.4.2. Dilute to the working range of 0.1 to 12 µg/mL with water.
3.4.3. Store standards in dark bottles under refrigeration.
3.5. Analysis
3.5.1. HPLC Conditions
Column: Zorbax ODS (25 cm x 4.6 mm)
55% methanol, 45% water, 0.005 M
Mobile phase:
tetrabutylammonium phosphate
Flow Rate: 1.0 mL/minute
Variable Wavelength
650 nm
Detector:
Injection Volume: 20 µL
Retention Time: 7.8 minutes
3.5.2. Chromatogram
See Figure 1.
3.5.3. Peak magnitude is measured by electronic integrator or other means.
3.5.4. An external standard procedure is used to prepare a calibration curve from
the analysis of at least three different concentrations from two separate
weighings.
3.5.5. Bracket the sample with analytical standards.
3.6. Interferences (Analytical)
3.6.1. Any collected compound that has the same LC retention time as analyte
and absorbs at 650 nm is an interference.
3.6.2. HPLC parameters may be varied to circumvent most interferences.
3.6.3. Retention time alone is not proof of a chemical identity. Confirmation by
other means should be sought when possible.
3.7. Calculations
3.7.1. The integrator value in area units for each standard is plotted against its
concentration in µg/mL and a calibration curve using the best fit straight line
through the points is obtained.
3.7.2. Sample concentration is calculated from the calibration curve.
3.7.3. The air concentration of Acid Blue 9 for a sample is calculated by the
following equation:
(µg/mL in sample)(extraction volume, mL)
mg/m =
3
(Air volume, L)
3.8. Safety Precautions
3.8.1. Confine the use of solvents to a fume hood.
3.8.2. Wear safety glasses in all laboratory areas.
4. Recommendations for Further Study
4.1. Preparation of pure standard
The commercially available Acid Blue 9 is not pure. The U.S. specification for the
food grade is 85% minimum. Purification of the standard should be attempted
either by preparative TLC or preparative HPLC.
Figure 1. Chromatogram of Acid Blue 9 at Target Concentration
and at Detection Limit.
Figure 2. Calibration of Acid Blue 9.
5. References
5.1. WHO, International Agency for Research on Cancer, IARC Monograph on
the Evaluation of the Carcinogenic Risk of Chemicals to Man. Some Aromatic
Amines and Related Nitro Compounds -- Hair Dyes, Colouring Agents and
Miscellaneous Industrial Chemicals. Vol. 16, pp. 171-86.
5.2. J. Chudy, N.T. Crosby, and I. Patel, J. Chromatogr., 154, (1978), p 306-312.
5.3. A Standen, ed., Kirk-Othmer Encyclopedia of Chemical Technology, Second
Ed., Vol. 5, pp. 865-66. Interscience Publishers, New York, N.Y., 1963.
DEVELOPMENT AND
VALIDATION OF A LIQUID
CHROMATOGRAPHIC METHOD
FOR ANALYSIS OF FOOD
COLOURS IN MULTIPLE FOOD
MATRICES
P O S T E D O N O C T O B E R 1 0 , 2 0 1 8 B Y ADMIN
INTRODUCTION
Food colours are added to different types of food commodities to
increase their visual appearance often to help consumer to find
them more appealing. The use of these additives is strictly
regulated in many countries worldwide. There is a growing
concern about the safety of some commonly used legal food
colorants and there is a trend to replace the synthetic forms with
natural products. Additionally, several dyes with known or
suspected carcinogenic properties have been shown to be added
illegally to foods. The most important food safety concerns in the
field of food colours are lack of uniform regulation concerning
legal food colours worldwide, possible link of artificial colours to
hyperactive behaviour, replacement of synthetic colours with
natural ones, and the presence of harmful illegal dyes—both
known but also new, emerging ones in food. To detect these
kinds of synthetic colour, a method has been developed by using
High Performance Liquid Chromatograph coupled with UV
detector.
PRINCIPLE
The colouring matter in food can be classified into two categories
namely water soluble and fat soluble. There are altogether 8
permitted coal-tar food colours which can be used in certain food
products under the provisions of Prevention of Food Adulteration
Act, 1954. They include 3 red shades, namely Carmoisine,
Ponceau 4R, Erythrosine, 2 yellow shades namely Tartrazine,
Sunset Yellow FCF, 2 blue shades i.e. Brilliant Blue FCF, Indigo
Carmine and 1 green shade i.e. Fast Green FCF.
The general scheme for identifying synthetic food colour in foods
normally involves extraction of the colour from the prepared
solutions of the food, separation of colours and identification and
quantification of the separated colour.
In present study, a High Performance Liquid chromatographic
method was developed for analysis of 7 water soluble synthetic
food dyes namely Carmoisine, Ponceau 4R, Erythrosine,
Tartrazine, Sunset Yellow FCF, Brilliant Blue FCF and Indigo
Carmine in food products like sugar boiled confectionary, Jelly,
Jams and fruit juices.
MATERIAL & METHODS
Standard preparation
Stock solution of about 1000 mg/L of each reference standard in
water was prepared individually. The standards were labelled
properly and stored at 2-8°C. From these standards intermediate
and working mixture standards were prepared by serial dilution.
The mixture standards were used for method validation.
Preparation of mobile phase
Mobile phase A (10 mM Ammonium acetate buffer): 0.7708 gm of
ammonium acetate was taken and finally volume made up to
1000mL with double distilled water. The solution was then filtered
through 0.45 µm membrane filter. Furthermore the solution was
sonicated for 15 minutes in an ultrasonic bath.
Mobile phase B: HPLC grade methanol was used as Mobile
phase B.
Preparation of calibration curve from mixtures of standard
A mixture stock solution (concentration of 100 mg/L) of 7 water
soluble colours was first prepared. Then the following dilutions
(Table-1) ranging from 1 mg/L to 20mg/L was prepared with
double distilled water as diluent.
Table-1. Details for preparation of Working mixture standards
Volume of Volume of Final
Stock Conc. Final Conc.
Stock Diluent Volume Name
(mg/L) (mg/L)
(mL) (mL) (mL)
10 0.100 0.900 1 1 CC-1
10 0.200 0.800 1 2 CC-2
10 0.500 0.500 1 5 CC-3
100 0.100 0.900 1 10 CC-4
100 0.200 0.800 1 20 CC-5
Sample preparation
About 5 gm of sample was taken in a 50 mL centrifuge tube and
10 mL of water was added to it. The tube was then degassed in
an ultrasonic bath for 15 minutes. After that volume of the solution
was made up to 25 mL. The mixture was filtered through 0.45 µm
membrane filter and aqueous extract was injected into HPLC
system for determination.
Instrumental Condition
LC system: Shimadzu LC-2030
Column: Shim-pack GWS C-18, 5 µm, 250X4.6 mm
Injection Volume: 10 µL
Absorbance wavelength: 420nm, 510nm
Flow rate: 1mL/minute
Column temperature: 35 ºC
Gradient Programming:
Time (minutes) % of A % of B
0.01 90 10
2.00 90 10
4.00 70 30
6.00 50 50
8.00 30 70
10.00 10 90
12.00 30 70
14.00 50 50
16.00 90 10
RESULTS AND DISCUSSION
All the 7 food colours viz. Carmoisine, Ponceau 4R, Erythrosine,
Tartrazine, Sunset Yellow FCF, Brilliant Blue FCF and Indigo
Carmine were validated in various matrices like sugar boiled
confectionary, Jelly, Jams and fruit juices at two different spiking
levels. Various Performance characteristics like Specificity, Limit
of Detection, Linearity, Matrix Effect, Accuracy, Precision, Limit of
detection, Limit of Quantification and Robustness were assessed.
The method was found to be specific for the analytes and matrix
combinations. The response of the detector was found to be
linear within 1-20 mg/L (Figures. 1-7). Spiked recoveries were
performed by two analysts on two different days at two different
concentration levels to evaluate the accuracy and precision of the
method. A typical chromatogram of Reference Standard mixture,
Chromatogram of Blank Control Sample and Control Sample
fortified with Reference Standards are presented in Figure. 8, 9
and 10. At LOQ level (2 mg/Kg) the mean recoveries obtained
were between 93.92 – 101.59 % with relative standard deviation
ranging between 1.65 – 10.35 % whereas at the higher
fortification level (10 mg/kg) the recovery ranged between a 90.81
– 102.90 % with relative standard deviation of up to 3.21%. LOQ
was fixed at the level of 2 mg/kg.
CONCLUSIONS
The analytical method described in this study was optimised for
determination of 7 water soluble synthetic food dyes namely
Carmoisine, Ponceau 4R, Erythrosine, Tartrazine, Sunset Yellow
FCF, Brilliant Blue FCF and Indigo Carmine in food products like
sugar boiled confectionary, Jelly, Jams and fruit juices by HPLC.
The UV detection and quantification after the separation of the
colours through HPLC method provides the specificity of the
analysis. This method can be used to determine the analytes in
food products as mentioned above at extremely low concentration
(~ 1.6 mg/Kg). The Limit of Quantification of the method is found
to fall within the range of 1.61 mg/Kg to 9.72 mg/Kg. The
calibration curve was found to be linear. This was apparent from
the square of the correlation coefficient (r2) value which ranged
between 0.9996 to 0.9999. All the method validation parameters
were within acceptable limits. This method found to be cheap,
easy, effective and quite rugged for the analytes and matrix
combination. In conclusion the method was found to be fit for its
intended purpose.
Figure 1: CC for Indigo carmine
Figure 2: CC for Ponceau 4R
Figure 3: CC for Sunset yellow
Figure 4: CC for Carmoisine
Figure 5: CC for Erythrosine
Figure 6: CC for Tartrazine
Figure 7: CC for Brilliant blue
Figure 8: Chromatogram
of Reference Standard mixture
Figure 9: Chromatogram of
Blank Control Sample
Figure 10: Chromatogram
of Control Sample fortified with Reference Standards
Conducted by Rituparn
