Briefing Session on Analysis of Nitrate/Nitrite and ... - uri=govtlab.gov

CContents t t 

Nitrate/Nitrite Testing Methods 

Propionic acid Testing Methods 

Certified Reference Materials 

Proficiency Tests

Nitrate/Nitrite 

INS: 249, 250, 251, 252 

• Functional Uses: Antimicrobial preservative 

Colour l fixative f 

• 5 – 20 mg/kg of free HNO 2 redden meat 

• 50 mg/kg production of characteristic taste 

• > 100 mg/kg antimicrobial effects 

• Too much nitrosamines Carcinogenic g

Nitrate/Nitrite Background : 

PPreservatives ti in i Food F dR Regulation l ti (Cap (C 132BD) 

Item Column 1 

Specified food 

PART I 

ARTICLES OF FOOD WHICH MAY CONTAIN PRESERVATIVE AND THE NATURE 

AND PROPORTION OF PRESERVATIVE IN EACH CASE 

Column 2 

Permitted preservative 

1. Bacon Sodium nitrate 

Sodium nitrite 

Column 3 

Parts per million not 

exceeding 

500 

200 

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

Cheshire type cheese or soft 

cheese 

sodium nitrite 10 

44. Ham Sodium nitrate 

500 

Sodium nitrite 200 

54. Meat, pickled, cooked Sodium nitrate 

500 


200 

55. Meat, ,p pickled, , uncooked Sodium nitrate 500 

Sodium nitrite 200 

62. Pork, preserved Sodium nitrate 

500 


200 

72. Sausage, Chinese preserved Sodium nitrate 

500 


200

Sample Types

H 2 NO 2 S 

Nit Nitrate/Nitrite: t /Nit it Spectroscopic S t i Detection D t ti 

Sulfanilamide 

NO 3 - 

Cd 

/ Nitrate reductase + NADPH 

NO - 2 

NH 2 

Griess Reaction 

H 2 O 

N 

H 

H N 

N 

H2NO2S N 2NO2S N 

2+ H 

NH 2 

N-1-napthylethylenediamine (NED) 

Azo Compound (red colour) 

Concentration of NO 3 - = Concentration of NO2 - after reduction 

– Concentration of NO 2 - without reduction 

NH 2

Nitrate/Nitrite: International Standard Methods based on Griess Rection 

Standards 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 y 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 enzymatic 

reduction 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 and 

vegetable products after cadmium reduction 

BS EN ISO 14673 14673-1:2004 1:2004 Milk and milk products – Determination of nitrate and nitrite contents 

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 (Routine 

method) 

AOAC 976.14 Nitrate and Nitite in Cheese – Modified Jones Reduction Method 

AOAC 973.31 Nitrite in Cured Meat – Colorimetric Method 

AOAC 993.03 Nitrate in Baby Foods – Spectrophotometric method

Nitrate/Nitrite: International Standard Methods –Other techniques 

Standards Methods Techniques 

AOAC 935.48 Nitrates and Nitrites in Meat – Xylenol Method Covert nitrite to nitrate by KMnO4 Nitration of Xylenol 

MMeasure th the [NO - 

3 ] 

BS EN 12014-4: 

1997 

BS EN 12014-5: 

1997 

BS EN 12014-2: 

1997 

Foodstuffs – Determination of nitrate and/or nitrite content – 

Part 4: Ion-exchange chromatographic (IC) method for the 

determination of nitrate and nitrite content of meat products 


Part 5: Enzymatic determination of nitrate content of vegetablecontaining 

food for babies and infants 


Part 2: HPLC/IC method for the determination of nitrate content of 

vegetables and vegetable products 

Ion-chromatography with UV 

detector 

Enzymatic reduction by nitrate 

reductase and NADPH 

Measure the difference of 

[NADPH] 

Ion-chromatography with 

conductivity detector/ 

HPLC with UV detecter 

GB/T 5009.33- 5009.33 食品中亚硝酸盐与硝酸盐的测定 Cd reduction 

2003 

Measure [NO - 

2 ] by Griess 

method or conductivity

Nitrate/Nitrite: GL Methods 

(i) Flow Injection Analysis Method 

(ii) Ion Ion-chromatography chromatography Method

Nit Nitrate/Nitrite: t /Nit it GL Method M th d – FIA method th d 

Sample Extraction 

Meat and Meat Products Cheese Products 

Vegetable and 

Vegetable Products

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

1. Homogenize the sample 

g p 

2. Weigh 10 g of comminuted sample 

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

4. + 0.5 g activated charcoal.

Nit Nitrate/Nitrite: t /Nit it GL Testing T ti method th d for f Meat M t and d Meat M t Products P d t 

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

66. Allow to cool and add 2 mL Carrez Carrez Soluton Soluton I and II II. Swirl Swirl after each addition. addition 

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 g a Whatman No. 41 filter paper. p p 

•Carrez Solution I: 10.6 g potassium 

ferrocyanide (II) (K 4[Fe(CN) 6].3H 2O) in 

distilled water 

•Carrez Solution II: 22.0 g zinc acetate 

and 3 mL glacial acetic acid in distilled 

water and dilute to 100 mL. 

NO - 

3 NO - 

2 

Cd 

Measure the NO - 

2 level

Nitrate/Nitrite: GL Testing method for Cheese Products 

Reference to BS EN ISO 14673-3:2004 

1. Grind or comminute the sample. 

2. Weigh accurately of 2.5 g comminuted samples to a 50- 50 

mL centrifuge tube. 

3. Add 24 mL of extraction buffer solution 

Buffer solution :26.6 g of NH4Cl + concentrated NH3 in 1L 

water (pH~8.5) (p ) 

4. Preheated to a temperature of 50oCto55oC. 5. Mix with the homogenizer for about 3 minutes until the 

test portion is well suspended. 

Milk and milk products – 

Determination of nitrate 

and nitrite contents 

Part 3: Method using 

cadmium reduction and 

flow injection j analysis y with 

in-line dialysis (Routine 

method)

Nitrate/Nitrite: GL Testing method for Cheese Products 

6. Centrifuge at 2000 rpm for 5 minutes. 

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

solution. 

8. Pipette p the de-fatted test solution from underneath the fat layer y in the centrifuge g tube. 

NO - 

3 NO - 

2 

Cd 

Measure the NO - 

2 level

Nitrate/Nitrite: Conversion of Nitrate to Nitrite 

In the past past…….. Now 

Flow Injection Analysis (FIA)

Nitrate/Nitrite: GL Testing method – Flow Injection Analysis 

Dialysis Cell 

From 

Dialysis Cell 

Sufanilamide + NED 

Sample 

Cd column

Nit Nitrate/Nitrite: t /Nit it Flow Fl Injection I j ti Analysis A l i Condition C diti 

Buffer / Carrier: Ammonium chloride buffer solution (Carrier and Buffer): 

1. 53.2 g NH4Cl + 1800 mL distilled deionized water in a 2 L volumetric flask. 

22. Adj Adjust pH H to 8.5 8 5 ± 00.1 1 with i h 15N sodium di hydroxide h d id solution l i ddropwise. i 

3. Make up to the mark with distilled deionized water. 

Sulphanilamide/NED colour reagent: 

1. 100 mL 85% phosphoric acid + 40.0 g sulphanilamide + 1.0 g of NED in about 1L 

distilled deionized water. 

2. Store in a dark bottle. 

Washbath 

Dissolve 1 g of Tween 20 in 4 L distilled deionized water. 

Buffer + sample 

Carrier 

Sulphanilamide/ 

NED colour reagent 

Dialysis Cell 

‐‐‐‐‐‐‐‐‐‐‐ 

Cd Column 

Waste 

Detector 

540nm

Nit Nitrate/Nitrite: t /Nit it Typical T i l Flow Fl Injection I j ti Analysis A l i of f Nitrite 

Nit it

Nitrate/Nitrite: Typical Flow Injection Analysis of Nitrate

Notes 

1. Use nitrate/nitrite free filter paper 

2. Pre-rinsed all glasswares, volumetric flasks 

with distilled deionized water before use 

3. Tween 20 is essential for getting good 

precision results 

44. Method blank is needed for correction correction. 

5. Check Cd column performance

Reporting Limits 

Nit Nitrite it (as ( sodium di nitrite) it it ) 5 mg/kg /k 

Nitrate (as sodium nitrate) 30 mg/kg

Alternative Technique q – Ion Chromatography g p y 

Reference to BS EN 12014-4:2005 

For those samples 

having concentration 

> legal limit 

Foodstuffs – Determination of 

nitrate and/or nitrite content 

- Part 4: Ion-exchange 

chromatographic h t hi (IC) method th d 

for the determination of nitrate 

and nitrite content of meat 

products

Ion Chromatography determination of Nitrate and Nitrite 

1. Weigh 10 g of comminuted sample and add about 50 mL 60 o Cwater 

2. Mix thoroughly g y after the addition. Swirl and sonicate the sample p 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. 

44. Allow to cool to room temperature temperature, make up to the mark with water water, mix and filter through a 

Whatman No. 41 filter paper 

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

Ion Chromatography with UV detector

Ion Chromatography condition of Nitrate and Nitrite 

C l W t IC P TM Column : Waters IC-Pac A i HC 150 4 6 

TM Anion HC 150 x 4.6 mm 

column 

Mobile phase : Lithium borate gluconate buffer in 

acetonitrile/water (pH~6.5) 

Flow rate : 1.0 mL/min. 

DDetector t t : UV 

Quantitation 

Wavelength g 

: 205 nm 

Injection volume : 40 µL

Ion Chromatogram 

Standard 

3 ppm NaNO2 5 ppm pp NaNO 3 

Sample Spike 

15 ppm NaNO2 30 ppm NaNO3 Sample Spike 

100 ppm NNaNO NO2 

250 ppm NaNO3

Method Comparison 

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

Leonardo Merino, Merino Ulla Edberg, Edberg Georg Fuchs Fuchs, Per Aman JAOAC2000 J AOAC 2000, 83 83, 22, 365 365. 

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

NO 2 - +NO3 - added, 

mg/kg 

Found, mg/kg 

Spectrophotometry Liquid chromatography 

Carrez solution Acetonitrile Carrez solurion 

5 4.8 5.2 ND 

10 9.3 10.0 2.3 

30 33.7 31.2 21.5 

50 50.5 51.6 38.1 

100 108.5 103.4 88.4

Findings in 2007 

Nitrate/Nitrite total samples: 596 

Analytes No. of Concentration range 

Positive 

samples 

found 

Nitrite 126 5 ppm to 110 ppm 

Nitrate 194 30 ppm to 2,100 ppm 

No. of failed samples: 3 

Sodium nitrate concentration: 710 ppm to 2100 ppm

Propionic acid 

INS: 280, 281, 282, 283 

• Functional Uses: Antimicrobial preservative 

particularly i l l against i mold ld 

but no effect to yeast 

• Propionic acid can be found in nature during 

cheese fermentation. 

•Practically y 

nontoxic

Propionic acid Background : 

Preservatives in Food Regulation (Cap 132BD) 

Item Column 1 Column 2 Column 3 

Specified food Permitted 

preservative 

Parts per million 

not exceeding 

4. Bread Propionic p acid 3000 

(calculated on the 

weight of the flour) 

26 26. Flour confectionery Propionic acid 

or 

1000 

sorbic acid 

1000

Propionic acid: International Standard Methods 

Standards Methods 

AOAC 950.35 Acetic and Propionic acids in Bread - 

Chromatographic Method 

AOAC 950.38 950 38 Acetic and Propionic acids in Cake 

AOAC 970.36 Propionates in Food 

AOAC 965.24 Acids (Volatile) in Bread – 

Paper Chromatographic Method

GL Testing method for Propionic acid – GC method 

1. Comminute the chopped pp solid sample p 

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.

GL Testing method for Propionic acid 

4. Steam Distillation. 

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.

GL Testing method for Propionic acid 

7. Transfer to GC vials. 

8. Inject 5 uL into gas chromatograph.

Gas Chromatography Condition for Propionic acid Analysis 

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

CColumn l TTemperature: t 100 C i th l 

oC isothermal 

Injector Temperature: 200o j p 

C 

Detector Temperature: 200 o C 

Carrier gas: He at 7 to 8mL/min 

Detector : FID

Gas Chromatogram – Propionic acid 

Response_ 

Time 

230000 

220000 

210000 

200000 

190000 

180000 

170000 

160000 

150000 

140000 

130000 

Response_ 

Time 

240000 

230000 

220000 

210000 

200000 

190000 

180000 

170000 

160000 

150000 

140000 

130000 

Signal: STD3_1.D\FID1B.CH 

5.58 

5.58 – Butyric Acid 

8.61 – Propionic 8.61 Acid 

25 ppm standard t d d 

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 

5.58 – Butyric y 

Acid 

Signal: R1.D\FID1B.CH 

558 5.58 

8.61 – Propionic Acid 

8.61 

~600 ppm 

sample spike 

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

HPLC Condition for Propionic acid Analysis 

CColumn: l C18 C18, 5 5mm, 250 250mm x 44.6mm 6 id 

Mobile phase: 20mM KH 2PO 4 (aq) (pH = 2.8) 

Flow Rate: 15 1.5 mL/min 

Detector : UV or PDA detector 

Quantitation 

Wavelength: 214 nm

Liquid Chromatogram – Propionic acid 

mA U 

8 

6 

4 

2 

0 

-2 

mAU 

12 1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

-0.2 

-0.4 

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

DAD1 B, Sig=305,4 Ref=360,100 

11.075 - Propionic Acid 

0 2 4 6 8 10 12 14 

10.873 - Propionic Acid 

0 2 4 6 8 10 12 14 

min 

min 

20 ppm 

standard 

100 ppm pp 

sample spike

Reporting Limits 

Propionic acid 50 mg/kg

Findings in 2007 

Total samples: 1117 

No. of positive samples: 25 

Concentration Range: 50 ppm – 7200ppm 

Only y 1 sample p exceeds legal g 

limit

Certified Reference Materials 

Code Matrix Certified Value 

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

Code Matrix Certified Value 

SRM 3185 Nitrate Anion 

Standard Solution 

Nitrate: 0.9998 mg/g ±0.0023mg/g

Proficienc Proficiency Test 

FAPAS 

PProficiency fi i TTest t St Start t Date D t MMatrix t i AAnalytes l t 

1556 16/07/2008 Meat Nitrate 

Nitrite 

1557 23/09/2008 Spinach Puree Nitrate 



1559 08/12/2008 Cabbage Puree Nitrate 



http://www.fapas.com

Proficiency Test 

LGC Standards Proficiency Testing Group 

QMAS – Nitrate/Nitrite in Meat 

QUALITY IN MEAT ANALYSIS SCHEME 

•Next Dispatch Date will be on 09 Sept 2008 

http://www.lgcpt.com/

Briefing Session on Analysis of Nitrate/Nitrite and ... - uri=govtlab.gov

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