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Method Abstract
for Post-column Liquid Chromatography 121
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SAJI GEORGE, WENDY
RASMUSSEN, MARIA OFITSEROVA, PH.D.,
MICHAEL PICKERING, PH. D.
PICKERING LABORATORIES, INC., MOUNTAIN VIEW,
CALIFORNIA, USA
Nitrite and nitrate are added
to food to preserve the color and taste and to
prevent foods from becoming rancid. They are also
used in food for their anti-microbial properties.
Higher levels in vegetables and leafy greens are
possible from the use of nitrate fertilizers
and/or livestock manure. Nitrite levels in food
could also be produced by reduction of nitrate to
nitrite during processing.
AOAC Official method 993.031
for the analysis of nitrate involves reduction
using spongy Cadmium which is toxic and
carcinogenic. FDA improved on this method by using Vanadium(III) chloride and
heat2 for the post-column reduction of
nitrate to nitrite. Nitrite reacts with this
modified Griess reagent to produce a red
chromophore with maximal absorbance at 535nm.
METHOD
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LC
with a binary pump
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UV/VIS detector
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Pickering Laboratories single reagent
Pinnacle PCX post-column derivatization unit
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Pickering Laboratories anion exchange
column, 4.6 X 150mm (Cat. No. 0755150)
Sodium acetate
Vanadium(III)chloride
N-(1-Naphthyl)ethylenediamine dihydrochloride
m-Nitro aniline
20%(v/v) Hydrochloric acid
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LC COLUMN TEMPERATURE:
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40˚C |
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SAMPLE INJECTION VOLUME:
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10mL |
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LC FLOW RATE: |
1mL/min |
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MOBILE PHASE: |
15mM Sodium acetate
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Post-Column
Conditions |
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REACTOR VOLUME: |
0.5mL |
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REACTOR TEMPERATURE: |
100˚C |
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REAGENT FLOW RATE: |
0.1mL/min |
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REAGENT: |
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(i) |
1%
Vanadium(III)chloride in 20% HCl |
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(ii) |
1% m-Nitro
aniline in 20%HCl |
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(iii) |
1%
N-(1-Naphthyl) ethylenediamine
dihydrochloride in 20% HCl |
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Mix 50mL of
(i) and (ii), and 1.25mL of (iii)
and dilute to 250mL using 20%HCl |
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DETECTOR: |
UV/VIS, λmax = 535nm
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Sample Preparation
To 5g of baby food in a 50mL
centrifuge tube add 25mL of 5060˚C water (for
vegetables) or 15mM Sodium acetate (for fruits)
and shake for 10min. Add 12.5mL of acetonitrile
and make up the volume to 50mL using water (for
vegetables) and sodium acetate (for fruits).
Centrifuge the mixture for 15mins at 5000rpm.
Filter the supernatant through a 0.45μ nylon
filter and dilute to fall within the linear range.
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20ppm Nitrate and Nitrite Standard |
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Calibration Curve for Nitrite and Nitrate
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Sample |
Spiked Conc.
(mg/kg) |
Calc. Conc.
(mg/kg) |
Recovery (%)
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Nitrite
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Nitrate
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Nitrite
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Nitrate
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Nitrite
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Nitrate |
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Sweet Potato |
500 |
500 |
573 |
580 |
115 |
116 |
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2500 |
2500 |
2714 |
2667 |
109 |
107
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Pears
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500 |
500 |
544 |
550 |
109 |
110 |
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2500
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2500 |
2808 |
2710 |
112 |
108 |
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Apple Sauce |
500
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500 |
565 |
546 |
113 |
109 |
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2500
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2500 |
2839
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2657 |
114
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106
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NOTES:
Post-column
reagent solutions are stored in plastic or
Teflon containers. All solutions are filtered
through 0.45μ nylon filter before use.
Nitrate/Nitrite standards should be checked
prior to use for oxidation. Sample pH should
be checked to determine the choice of
extraction solution since acidic pH
facilitates the conversion of nitrite to
nitrate.
ACKNOWLEDGMENTS:
John A. Casanova,
Food and Drug Administration, 60 8th Street,
Atlanta, GA 30309
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REFERENCES:
AOAC- Official
Methods of Analysis of AOAC International
(2000) 17th Ed., Section 50.1.11. Use of
Griess Reagents Containing Vanadium (III) for
the Post-Column Derivatization and
Simultaneous Determination of Nitrite and
Nitrite in Baby Food. Casanova, John A.;
Gross, Lois K.; McMullen, Sarah E.; Schenck,
Frank, JAOAC International, 2006; 89(2),
447-451.
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