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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
Foodstuffs Determination of nitrate and/or nitrite content
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
Foodstuffs Determination of nitrate and/or nitrite content
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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