TEST METHOD TM-OCS-101

STANDARDS AND TESTING DIVISION

CHEMISTRY LABORATORY
Page 1 of 6
ORGANIC CHEMISTRY SECTION
COLORIMETRIC DETERMINATION OF NITRITE IN MEAT AND Revision No.: 0
MEAT PRODUCTS 17 June 2020

1.0 PURPOSE
This method describes the procedure for the colorimetric determination of nitrite in
meat and meat products

2.0 SCOPE
This is a reference method applicable for meat and meat products

3.0 PRINCIPLE
Extraction of a test portion with hot water. In the presence of nitrite, development of
red colored solution by the addition of sulfanilamide and N-(1-naphthyl)
ethylenediamine∙2HCl to the filtrate and photometric measurement at 540 nm

4.0 TERMS AND DEFINITION

Colorimetry- technique that is used to determine the concentration of colored
compounds in solutions by the application of the Beer–Lambert law, which states that
the concentration of a solute is proportional to the absorbance
Nitrite (NO2 -)- is a multifunctional food additive, responsible for the characteristic
color and flavor associated with cured meats and at the same time providing
protection against growth and toxin formation by C. botulinum in cured meats

5.0 RESPONSIBILITIES
5.1 Organic Chemistry Section (OCS) analysts are responsible for the conduct of
this test method.
5.2 The OCS technical manager is responsible in ensuring the accuracy of test
results.

6.0 EQUIPMENT, CHEMICALS, AND SUPPLIES

6.1 EQUIPMENT
6.1.1 Analytical Balance
6.1.2 UV-VIS Spectrophotometer
6.1.3 Steam-bath
6.2 REAGENTS AND STANDARDS
6.2.1.1 Nitrite-free reagent water
6.2.1.2 15 % (v/v) Acetic acid (CH3COOH) solution
6.2.1.3 NED reagent- Dissolve 0.2 g N-(1-naphthyl
ethylenediamine∙2HCl in 150 mL 15 % (v/v) CH3COOH.

Prepared by: RCSalazar

Reviewed by: CCRamil
Approved by: MVParcon
Issued by STD. No part of this document may be reproduced, amended, or transmitted in any form or by any means
without prior written approval of the chief of STD
 TEST METHOD TM-OCS-101
STANDARDS AND TESTING DIVISION
CHEMISTRY LABORATORY
Page 2 of 6
ORGANIC CHEMISTRY SECTION
COLORIMETRIC DETERMINATION OF NITRITE IN MEAT AND Revision No.: 0
MEAT PRODUCTS 17 June 2020

Filter if necessary, and store in glass-stoppered brown

glass bottle.
6.2.1.4 Sulfanilamide reagent- Dissolve 0.5 g in 150 ml 15%
CH3COOH (v/v). Filter, if necessary and store in glass-
stoppered brown glass bottle
6.2.1.5 Nitrite standard solution
Stock Solution- 1000 ppm (ug/ml) NaNO2. Dissolve 1.000 g
NaNO2 in H2O and dilute to 1 (one) liter.
Alternatively, commercially available nitrite stock solution in
which SRM is traceable to NIST.
Intermediate Solution- Dilute 1 ml stock solution to 50 mL
with reagent water.
6.2.1.6 Filter paper- Test for nitrite contamination by analyzing 3-4
sheets, at random throughout box. Filter ca 40 ml reagent
water through each sheet. Add 4ml sulfanilamide reagent,
mix, let stand 5 min, add 4 ml NED reagent, mix and wait
15 min. If any sheets are positive, discard entire box.

6.3 GLASSWARE, SUPPLIES, AND APPARATUS

6.3.1 Volumetric Flask (50 ml, 500 ml)
6.3.2 Glass Funnel
6.3.3 Pipettor or Volumetric pipet (1ml)
6.3.4 Pipettor or Graduated pipet (10ml)
6.3.5 Beaker (50 ml)
6.3.6 Glass rod
6.3.7 Pipette tips (1ml, 10ml)
6.3.8 Spatula
6.3.9 Stopwatch
6.3.10 Filter paper
6.3.11 Aspirator

7.0 PROCEDURAL DETAILS

7.1 Sample Handling and Storage
7.1.1 For proper management and handling of samples follow the procedures
stated in SP-OCS-501, Management of Test Samples.
7.1.2 Preparation of test solution
7.1.2.1 Thoroughly grind samples repeatedly until homogeneous.
Store sample in polypropylene or polyethylene container
with proper labels.

Prepared by: RCSalazar

Reviewed by: CCRamil
Approved by: MVParcon
Issued by STD. No part of this document may be reproduced, amended, or transmitted in any form or by any means
without prior written approval of the chief of STD
 TEST METHOD TM-OCS-101
STANDARDS AND TESTING DIVISION
CHEMISTRY LABORATORY
Page 3 of 6
ORGANIC CHEMISTRY SECTION
COLORIMETRIC DETERMINATION OF NITRITE IN MEAT AND Revision No.: 0
MEAT PRODUCTS 17 June 2020

7.1.2.2 Mix the sample thoroughly just before weighing by stirring

with a spatula.
7.1.2.3 Weigh 5 g finely comminuted and thoroughly mixed test
portion into 50 ml beaker. Add ca 40 ml reagent water
heated to 80 ºC.
7.1.2.4 Mix thoroughly with glass rod, taking care to break all
lumps, and transfer to 500 ml volumetric flask.
7.1.2.5 Thoroughly wash beaker and rod with successive portions
of the hot water, adding all washings to flask.
7.1.2.6 Add enough water to bring the volume to ca 300 ml,
transfer flask to steam bath and let it stand 2 h, shake
occasionally.
7.1.2.7 Cool to room temperature dilute to volume with reagent
water and remix. Filter
7.1.2.8 Centrifuge the solution if it remains turbid after filtration.
7.1.2.9 Pipet aliquot from sample stock solution into 50 ml flask
pipet. Add 2.5 ml sulfanilamide reagent. Mix
7.1.2.10 After 5 min, add 2.5 ml NED reagent, dilute to volume, mix
and let color develop for 15 mins.

7.1.3 Preparation of Working Standard Solution.

7.1.3.1 Pipet aliquots of appropriate volumes of intermediate
standard solution into 50 ml volumetric flask.
7.1.3.2 Add 2.5 ml sulfanilamide reagent. Mix.
7.1.3.3 After 5 min, add 2.5 ml NED reagent, dilute to volume, mix
and let color develop for 15 mins.
7.1.3.4 Prepare standard blank accordingly.
7.1.4 Instrumental Analysis
7.1.4.1 See spectrophotometer operational manual.
7.1.4.2 Set wavelength of the spectrophotometer to 540 nm. Use
cuvette with 1 cm or longer path length.
7.1.4.3 After all parameters have been set, accomplish the system
suitability checklist of the equipment used. Run the
standard blanks and working standards based on the
following recommended sequence (Table 3).

Prepared by: RCSalazar

Reviewed by: CCRamil
Approved by: MVParcon
Issued by STD. No part of this document may be reproduced, amended, or transmitted in any form or by any means
without prior written approval of the chief of STD
 TEST METHOD TM-OCS-101
STANDARDS AND TESTING DIVISION
CHEMISTRY LABORATORY
Page 4 of 6
ORGANIC CHEMISTRY SECTION
COLORIMETRIC DETERMINATION OF NITRITE IN MEAT AND Revision No.: 0
MEAT PRODUCTS 17 June 2020

Table 1. Recommended sequence of analysis

Order Solution Read as Purpose
01 Reagent Water AUTOZERO Calibration
Calibration and
02 Standard Blank BLK Checking for
Contamination
03 Working Standards STD Calibration
Checking for the
04 Sample Blank BLK Absence of
Contamination
CRM / PT Material/ QC Accuracy
05 UNK
QCM if available Check
06 Sample UNK Analysis
07 Spiked Sample UNK Analysis
08 Standard Blank BLK QC Drift Check
Calibration Verification
09 Check Solution UNK QC Drift Check
(CVCS)*
Checking for the
10 Sample Blank BLK Absence of
Contamination
11 Succeeding Samples UNK Analysis
12 Standard Blank BLK QC Drift Check
13 (CVCS)* UNK QC Drift Check
14 Reagent Water Cleaning (optional)
Legend: BLK – blank, STD – standard, UNK- unknown

7.2 Calculations
7.2.1 The concentration of the analyte element, expressed in milligram per
kilogram sample as nitrite (NO2), is calculated using the formula:

Prepared by: RCSalazar

Reviewed by: CCRamil
Approved by: MVParcon
Issued by STD. No part of this document may be reproduced, amended, or transmitted in any form or by any means
without prior written approval of the chief of STD
 TEST METHOD TM-OCS-101
STANDARDS AND TESTING DIVISION
CHEMISTRY LABORATORY
Page 5 of 6
ORGANIC CHEMISTRY SECTION
COLORIMETRIC DETERMINATION OF NITRITE IN MEAT AND Revision No.: 0
MEAT PRODUCTS 17 June 2020

Where c the concentration of nitrite, in mg/L, read from the calibration curve, that
corresponds with the absorbance of the solution prepared from the test
portion.
Vf Total volume of the sample stock solution
Df Dilution factor (Final Volume/Initial Volume)
wtg Weight of sample in grams

7.3 Reporting Concentrations

7.3.1 For values greater than 999 ppm report as %w/w.
7.3.2 For values less or equal to 999 ppm report as mg / kg
7.3.3 For values greater than 1, report in three numerical digits.
7.3.4 For values less than 1, report in three decimal places.
7.3.5 Express mean result of replicates.
7.4 Reporting of Low Level Data
For values below the LOD, report as “None detected” and indicate the
LOD in the report of analysis.
7.5 Acceptance of Results
7.5.1 For the Calibration curve, the correlation coefficient for the linear
regression, r, must be ≥ 0.995.
7.5.2 CV or RSD should conform within acceptable limits for precision as
described in SP-OCS-601, Internal Laboratory Quality Control
Procedures.
7.5.3 Either one of the following criteria should be met:
7.5.3.1 Recovery of spiked samples should conform to the criteria
for recovery as described in SP-OCS-601, Internal
Laboratory Quality Control Procedures.
7.5.3.2 If available, analysis of Certified Reference Material /
Reference Material should conform to its reference value.

7.6 Computation of Measurement Uncertainty

Refer to SP-OCS-301, Measurement Uncertainty Estimation.

8.0 SAMPLE AND WASTE DISPOSAL

Prepared by: RCSalazar

Reviewed by: CCRamil
Approved by: MVParcon
Issued by STD. No part of this document may be reproduced, amended, or transmitted in any form or by any means
without prior written approval of the chief of STD
 TEST METHOD TM-OCS-101
STANDARDS AND TESTING DIVISION
CHEMISTRY LABORATORY
Page 6 of 6
ORGANIC CHEMISTRY SECTION
COLORIMETRIC DETERMINATION OF NITRITE IN MEAT AND Revision No.: 0
MEAT PRODUCTS 17 June 2020

8.1 For sample disposal refer to SP-OCS-502, Disposal of Test Samples.

8.2 For waste disposal refer to SP-CHL-002, Handling and Disposal of
Chemical Waste.

9.0 REFERENCES
9.1 AOAC International 201. Official Methods of Analysis – 20th ed., W.
Horwitz and G. Latimer Jr., ed. AOAC International, Maryland, U.S.A.

10.0 ATTACHMENTS
TM-OCS-XXX-F01 Colorimetric Determination of Nitrite in Meat and Meat
Products Analysis Worksheet

11.0 RECORDS

Identification
Collection and filling Filed by OCS personnel at the Food and Natural Products
Laboratory / Staff Room
Index TM-OCS-XXX-F01 Colorimetric Determination of Nitrite in Meat
and Meat Products Analysis Worksheet (TM-OCS-101-F01)

Access OCS personnel

Storage Food and Natural Products Laboratory / Staff Room (Room 206A)
Maintenance and Discarded by OCS personnel after 6 years
disposal

12.0 REVISION SUMMARY

Revision Date of Reason for revision Prepared by Approved by

No. Effectivity
0 Original Document RCSalazar/CCRamil MVParcon

Prepared by: RCSalazar

Reviewed by: CCRamil
Approved by: MVParcon
Issued by STD. No part of this document may be reproduced, amended, or transmitted in any form or by any means
without prior written approval of the chief of STD