determinacion Nitrato

7/29/2019 determinacion Nitrato

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on en s

y Nitrate/Nitrite Testing Methodsy Propionic acid Testing Methods

y Certified Reference Materials

y Proficiency Tests


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INS: 249, 250, 251, 252

Functional Uses: Antimicrobial preservative

Colour fixative

5 20 mg/kg of free HNO2 redden meat50 mg/kg production of characteristic taste> 100 mg/kg antimicrobial effects

oo much nitrosamines Carcino enic


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Nitrate/NitriteBackground:reserva ves n oo egu a on ap

PARTIARTICLESOFFOODWHICHMAYCONTAINPRESERVATIVEANDTHENATURE

Item Column 1

Specified food

Column 2

Permitted preservative

Column 3

Parts per million not

1. Bacon Sodium nitrate

Sodium nitrite

500

200

8. Cheese, other than Cheddar or Sodium nitrate or 100

Cheshire type cheese or softcheese

sodium nitrite 10

44. Ham Sodium nitrate

500

o um n r e

54. Meat, pickled, cooked Sodium nitrate

Sodium nitrite

500

200

55. Meat ickled uncooked Sodium nitrate 500

Sodium nitrite 200

62. Pork, preserved Sodium nitrate

Sodium nitrite

500

200

72. Sausage, Chinese preserved Sodium nitrate

Sodium nitrite

500

200


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SampleTypes


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ra e r e: pec roscopc e ec onGriess Reaction

NO3-

/ Nitrate reductase + NADPH

NH

NH2

2

-

NH2

H2NO

2S

H2O

H2NO

2S N

2+ N

H

NH2

NH2NO2S N

N-1-napthylethylenediamine (NED)

Sulfanilamide

Azo Compound (red colour)

Concentration of NO3- = Concentration of NO2

- after reduction

Concentration of NO2- without reduction


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Nitrate/Nitrite:InternationalStandardMethodsbasedonGriess RectionStandards Methods

ISO 2918-1975 (E) Meat and meat products Determination of nitrite content

ISO 3091-1975 (E) Meat and meat products Determination of nitrite content

BS 1743-16.1 1983 Methods for Analysis of dried milk and dried milk products

Part 16: Determination of nitrate and nitrite contents

Section 16.1 Method for dried milk by cadmium reduction

BS 770-9:1985 Methods for Chemical analysis of cheese

Part 9: Determination of nitrate and nitrite content Cadmium reduction and photometry method

BS EN 12014-3:1997 Foodstuffs Determination of nitrate and/or nitrite content

Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymaticreduction of nitrate to nitrite

BS EN 12014-7:1997 Foodstuffs Determination of nitrate and/or nitrite content Part 7: Continuous flow method for the determination of nitrate content of vegetables andvegetable products after cadmium reduction

-

Part 1: Method using cadmium reduction and spectrometry

BS EN ISO 14673-2:2004 Milk and milk products Determination of nitrate and nitrite contents

Part 2: Method using segmented flow analysis (Routine method)

BS EN ISO 14673-3:2004 Milk and milk products Determination of nitrate and nitrite contents

Part 3: Method using cadmium reduction and flow injection analysis with in-line dialysis (Routinemethod)

AOAC 976.14 Nitrate and Nitite in Cheese Modified J ones Reduction Method

AOAC 973.31 Nitrite in Cured Meat Colorimetric Method

AOAC 993.03 Nitrate in Baby Foods Spectrophotometric method


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Nitrate/Nitrite:InternationalStandardMethods OthertechniquesStandards Methods Techniques

AOAC 935.48 Nitrates and Nitrites in Meat Xylenol Method Covert nitrite to nitrate by KMnO4Nitration of Xylenol

easure e 3-

BS EN 12014-4:1997

Foodstuffs Determination of nitrate and/or nitrite content

Part 4: Ion-exchange chromatographic (IC) method for thedetermination of nitrate and nitrite content of meat products

Ion-chromatography with UVdetector

BS EN 12014-5:1997


Part 5: Enzymatic determination of nitrate content of vegetable-

containing food for babies and infants

Enzymatic reduction by nitratereductase and NADPH

Measure the difference of[NADPH]

BS EN 12014-2:1997


Part 2: HPLC/IC method for the determination of nitrate content ofvegetables and vegetable products

Ion-chromatography withconductivity detector/

HPLC with UV detecter

GB T 5009.33- Cd reduction2003 Measure [NO2

-] by Griessmethod or conductivity


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Nitrate/Nitrite:GLMethods

(i) Flow Injection Analysis Method

-


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ra e r e: e o me o

SampleExtraction

Meat and Meat Products Cheese ProductsVegetable and

Ve etable Products


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Nitrate/Nitrite: GL Testing method for Meat and Meat Products

1. Homo enize the sam le

2. Weigh 10 g of comminuted sample

3. + 100 mL hot water + 5 mL borax solution

4. + 0.5 g activated charcoal.


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5. Heat for 15 minutes on a boiling water bath.

ra e r e: es ng me o or ea an ea ro uc s

. . .

7. Add 5 mL borax solution and transfer to a 200-mL volumetric flask.

8. After standing for 30 minutes, make up to the mark with water, mix and filter

through a Whatman No. 41 filter paper.

-

Carrez Solution I: 10.6 g potassiumferrocyanide (II) (K4[Fe(CN)6].3H2O) indistilled waterCarrez Solution II: 22.0 g zinc acetate

3-

2-

Cd

and 3 mL glacial acetic acid in distilled

water and dilute to 100 mL.


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Nitrate/Nitrite: GL Testing method for Cheese Products

1. Grind or comminute the sample.

2. Wei h accuratel of 2.5 comminuted sam les to a 50-

Reference to BS EN ISO 14673-3:2004Milk and milk products Determination of nitrate

mL centrifuge tube.

3. Add 24 mL of extraction buffer solution

Buffer solution :26.6 g of NH4Cl + concentrated NH3 in 1Later H~8.5

Part 3: Method usingcadmium reduction andflow inection anal sis with

4. Preheated to a temperature of 50oC to 55oC.

5. Mix with the homogenizer for about 3 minutes until the

test portion is well suspended.

in-line dialysis (Routinemethod)


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6. Centrifuge at 2000 rpm for 5 minutes.

Nitrate/Nitrite: GL Testing method for Cheese Products

7. Place the centrifuge tube in a mixture of water and ice for 15 minutes to cool the test

solution.

8. Pi ette the de-fatted test solution from underneath the fat la er in the centrifu e tube.

-3-

2-

Cd


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Nitrate/Nitrite: Conversion of Nitrate to Nitrite

..

Flow Injection Analysis (FIA)


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Nitrate/Nitrite: GL Testing method Flow Injection Analysis

From

Sample

Sufanilamide + NED

Cd column


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ra e r e: ypca ow nec on nays s o r e


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Notes

1. Use nitrate nitrite free filter a er2. Pre-rinsed all glasswares, volumetric flasks

with distilled deionized water before use

3. ween 20 is essential for getting goodprecision results

. .

5. Check Cd column performance


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Reporting Limits

r e as so um n r e mg g

Nitrate (as sodium nitrate) 30 mg/kg


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Alternative Techni ue Ion Chromato ra h

Reference to BS EN 12014-4:2005

For those samples

nitrate and/or nitrite content- Part 4: Ion-exchange

> legal limitc roma ograp c me o

for the determination of nitrateand nitrite content of meatproducts


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1. Weigh 10 g of comminuted sample and add about 50 mL 60oC water

2. Mix thorou hl after the addition. Swirl and sonicate the sam le solution for 10 minutes.

3. Transfer the sample into a 200-mL volumetric flask. Add 50 mL of acetonitrile to the 200-mL

volumetric flask and mix gently.

. , ,

Whatman No. 41 filter paper

5. The solution is further filtered through a membrane filter of pore size of approximately 0.45m.

Ion Chromatography with UV detector


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Ion Chromatography condition of Nitrate and Nitrite

o umn : a ers - ac n on x . mmcolumn

Mobile phase : Lithium borate gluconate buffer inacetonitrile/water (pH~6.5)

Flow rate : 1.0 mL/min.

e ec or :

Quantitation

Wavelength: 205 nm

Injection volume : 40 L


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Ion Chromatogram

Standard

3 ppm NaNO25 ppm NaNO3

15 ppm NaNO230 ppm NaNO3

Sample Spike

ppm a 2250 ppm NaNO3


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Method ComparisonLiquid Chromatographic Determination of Residual Nitrite/nitrate in Foods: NMKL Collaborative Study

, , , , , , .

Comparision of residual nitrite levels in minced meat as determined by 2 different methods

Found, mg/kg

Spectrophotometry Liquid chromatography

NO2

-+NO3

- added,

mg/kg

Carrez solution Acetonitrile Carrez solurion

5 4.8 5.2 ND

10 9.3 10.0 2.3

30 33.7 31.2 21.550 50.5 51.6 38.1

100 108.5 103.4 88.4


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y Nitrate/Nitrite total samples: 596

Analytes No. of Concentration range

Positive

samples

found

Nitrate 194 30 ppm to 2,100 ppm

No. of failed samples: 3Sodium nitrate concentration: 710 ppmto 2100 ppm


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INS: 280, 281, 282, 283

Functional Uses: Antimicrobial preservativepart cu ar y aga nst mo

but no effect to yeast

Propionic acid can be found in nature duringcheese fermentation.

Practicall nontoxic


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PreservativesinFoodRegulation(Cap132BD)

Item Column 1 Column 2 Column 3Specified food Permitted

preservative

Parts per million

not exceeding

4. Bread Pro ionic acid 3000

(calculated on theweight of the flour)

.

or

sorbic acid 1000


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Propionic acid:InternationalStandardMethods

Standards MethodsAOAC 950.35 Acetic and Propionic acids in Bread -

Chromatographic Method

.

AOAC 970.36 Propionates in Food

AOAC 965.24 Acids (Volatile) in Bread

Paper Chromatographic Method


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GL Testing method for Propionic acid GC method1. Comminute the chopped solid sample

2. Weigh 10g well-blended sample in a distillation flask.

3. Add 2 mL distilled water, 10 mL of 0.5M sulphuric acid, 10 mL of

phosphotungstic acid, mix by swirling.


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4. Steam Distillation.

GL Testing method for Propionic acid

5. Collect about 220 mL into 250 mL v-flask.

6. Add 2 mL formic acid and 1 mL buytric acid ( IS ), then make-up.


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7. Transfer to GC vials.

GL Testing method for Propionic acid

8. Inject 5 uL into gas chromatograph.


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Gas Chromatography Condition for Propionic acid Analysis

Column: Nukol, 0.53mm id, 30 m long, 0.5m thick

o umn emperature: o sot erma

Injector Temperature: 200oC

Detector Temperature: 200oC

Carrier gas: He at 7 to 8mL/min

Detector : FID


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230000

Response_

Signal: STD3_1.D\FID1B.CH

190000

200000

210000

220000

5.58

8.61

5.58 Butyric Acid

8.61 Propionic Acid

140000

150000

160000

170000ppms an ar

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

130000

Time

Response_

210000

220000

230000

240000

Signal: R1.D\FID1B.CH

8.618.61 Propionic Acid

160000

170000

180000

190000

200000

5.58 Butyric Acid

~600 ppmsample spike

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

130000

140000

150000

Time

.


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HPLC Condition for Propionic acid Analysis

o umn: , mm, mm x . mm

Mobile phase: 20mM KH2PO4 (aq) (pH = 2.8)

.

Detector : UV or PDA detector

Wavelength: 214 nm


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DAD1 B, Sig=305,4 Ref=360,100 (P080502\P0000016.D)

4

6

811.075 - Propionic Ac id

20 ppm

0

2standard

mAU

DAD1 B, S ig=305,4 Ref=360,100

min0 2 4 6 8 10 12 14

-2

0.4

0.6

0.8

1

.

10.873 - P ropionic Acid

100 m

-0.4

-0.2

0

0.2 sample spike

min0 2 4 6 8 10 12 14


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Findings in 2007

o a samp es:

y No. of positive samples: 25

y

Concentration Range: 50 ppm 7200ppmy Onl 1 sam le exceeds le al limit


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Certified Reference Materials

Code Matrix Certified Value

BB501a Processed Meat Nitrate: 0.209 g/kg (certified)

SRM 3185 Nitrate Anion

Standard Solution

Nitrate: 0.9998 mg/g 0.0023mg/g


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yFAPAS

ro c ency es ar a e a r x na y es

1556 16/07/2008 Meat NitrateNitrite

1557 23/09/2008 Spinach Puree Nitrate

1558 08/10/2008 Meat Nitrate

Nitrite

1559 08/12/2008 Cabbage Puree Nitrate

1560 18/02/2008 Meat NitrateNitrite

http://www.fapas.com


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ProficiencyTestLGC Standards Proficiency Testing Group

QUALITY IN MEAT ANALYSIS SCHEME

Next Dispatch Date will be on 09 Sept 2008

http://www.lgcpt.com/


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