Photometric Determination of Nitrite in Meat and Sausage Products Using the Griess Method After Extraction with Water

Section Overview

• Introduction
• Experimental
• Reagents, Instruments and Materials
• Analytical Procedure
• Calculation
• Analytical Quality Assurance
• Related Products

Introduction

Nitrites, primarily in the form of sodium nitrite (E250) and potassium nitrite (E249), are widely applied as curing agents in meat production due to their central role in maintaining product quality and safety. They contribute directly to the development of characteristic flavor, stabilization of cured color, and inhibition of lipid oxidation. In addition, nitrites exert strong antimicrobial effects, preventing the growth of spoilage organisms and foodborne pathogens, including Clostridium botulinum.

Despite these important functions, nitrites are associated with toxicological concerns. Under specific conditions, they may give rise to N-nitroso compounds, a class of substances classified by the International Agency for Research on Cancer (IARC) as probable human carcinogens.¹ Regulation of nitrite use, therefore, emphasizes maintaining a balance between microbiological safety and minimizing nitrosamine formation. This balance is reflected in strict maximum levels established by the European Union  [Regulation (EC) No 1333/2008 and its subsequent amendments],² and other international authorities which continue to regulate the use of nitrites in meat and meat products.

A variety of analytical techniques, including electrochemical and chromatographic methods, have been applied for nitrite determination, however, spectrophotometric approaches remain predominant owing to their simplicity, cost efficiency, and suitability for routine analysis.³ Among these, the photometric Griess nitrite test, where extracted nitrite is converted into a stable azo dye,³ provides a reliable reference procedure for both quality control and regulatory monitoring of nitrite in meat products.

Experimental

Griess Assay Principle

After aqueous extraction and Carrez clarification the nitrite ions react in acidic solution with sulfanilic acid to form a diazonium salt, which in turn reacts with N-(1-naphthyl) ethylenediamine dihydrochloride to form a red-violet azo dye. This dye is determined photometrically.

Measuring Range

Applicable Sample

Meat and sausage products

Reagents, Instruments and Materials

Reagent & Test Kits

• Spectroquant^® Nitrite Cell Test (1.14547) or
• Spectroquant^® Nitrite Test (1.14776)

Instrument(s) & Devices

For the measurement one of the following Spectroquant^® photometers is necessary

• Spectroquant^® VIS Spectrophotometer Prove 100 plus (1.73026)
• Spectroquant^® UV/VIS Spectrophotometer Prove 300 plus (1.73027)
• Spectroquant^® UV/VIS Spectrophotometer Prove 600 plus (1.73028)
• Spectroquant^® Colorimeter Move 100 (1.73632)

This application note pertains to the above listed photometers and all discontinued instruments from the Spectroquant^® Nova and Prove series.

Software for Data transfer

• Optional Spectroquant^® Prove Connect to LIMS software package (Y.11086) to transfer your data into an existing LIMS system.

Instrument Accessories

• Rectangular cells 10 mm (1.14946) or
• Rectangular cells 20 mm (1.14947) or
• Rectangular cells 50 mm (1.14944)

Note: Rectangular cells are only necessary if the Spectroquant^® Nitrite test (1.14776) is used.

Other Reagents and Accessories 

• MQuant^® pH-indicator strips pH 0 - 6.0 (1.09531)
• Carrez Clarification (1.10537)
• Sodium hydroxide solution (1.09137)
• Water for analysis (1.16754)
• Standard laboratory glassware (e.g. Erlenmeyer flasks) and pipettes 
• Analytical balance 
• Ultra Turrax
• pH-Meter
• Heating bath
• Folded filters

Analytical Procedure

Sample preparation

In an Erlenmeyer-flask weigh exactly 10 g of the sample and mix with about 80 mL water for analysis. With the Ultra-Turrax high speed blender homogenize the mixture for 60 seconds. With 50 mL hot water for analysis rinse the shaft of the homogenizer into the flask, adding it to the mixture. Then adjust the pH-value of the solution to 7 - 7.2 with sodium hydroxide solution 1 mol/L, using the pH-meter, and heat for 15 minutes in a bath of boiling water, while occasionally shaking. After cooling down to room temperature quantitatively transfer the prepared sample into a 200‑mL standard volumetric flask and successively mix it with 2 mL Carrez solution-1 and -2 at a time. With rich in connective tissue products use 4 mL Carrez solution-1 and -2 respectively. Shake after each addition. Then fill up to volume with water for analysis and, after mixing, filter through a folded filter. Discard the first filtrate and use the remaining, clear filtrate for the determination (=pretreated sample).

Using 1.14547: Procedure and measurement

For more information on the measurement see the packaging insert of the test.

Procedure

• Pipette 5.0 mL pretreated sample into a reaction cell, close the cell tightly, and shake vigorously until the reagent is completely dissolved.
• Leave to stand for 10 min (reaction time), then measure the sample in the photometer.

Measurement

• It is recommended to zero the method each new working day. To do this, open the method, either by manually selecting the method or by inserting a barcoded cell. Tap the <Settings> button and select the <ZERO ADJUSTMENT> menu item. After prompting, insert the 16-mm zero cell through the corresponding opening. The zero adjustment is performed automatically. Confirm the performance of the zero-adjustment procedure by clicking on <OK>.
• After the zero has been performed, insert the barcoded Spectroquant^® round cell through the corresponding opening, ensuring that the white position mark on the cell is aligned with the positioning mark on the spectrophotometer. The measurement starts automatically.
• Read off the result in mg/L from the display.

Hint: The above written measurement description is only valid for the Spectroquant^® Prove (plus) series photometer. If a Nova 60A or a Move 100 is used, please consult the corresponding instrument manual for more details on how to perform the measurement.

Using 1.14776: Procedure and measurement

For more information on the measurement see the packaging insert of the test.

Procedure

• Pipette 5.0 mL pretreated sample into a test tube.
• Add 1 level microspoon (in the cap of the NO₂-1 bottle) and shake vigorously for 1 min until the reagent is almost completely dissolved. The pH must be in the range of pH 2.0 – 2.5. Check with MQuant^® pH-indicator strips. Adjust, If necessary, with sodium hydroxide solution or sulfuric acid.
• Leave to stand for 10 min (reaction time), then fill the sample into the cell, and measure the sample in the photometer.

Note: For measurement in the 50-mm cell both the sample volume as well as the quantity of reagent NO₂-1 must be doubled. Alternatively, the semi-microcell Cat. No. 1.73502 can be used. It is recommended to measure against an own prepared blank sample (preparation as per measurement sample, but with distilled water instead of sample) to increase the accuracy. Configure the photometer for blank measurement.

Measurement

• It is recommended to zero the method for each new working day. To do this, open the method by inserting the barcode, tap the <Settings> button and select the <ZERO ADJUSTMENT> menu item. Fill same cell which will be used for the sample measurement with distilled water. After prompting, insert the filled rectangular cell into the cell compartment. The zero adjustment is performed automatically. Confirm the performance of the zero-adjustment procedure by clicking on <OK>.
• If a 50 mm cell is used, it is recommended to perform the reagent blank one time for each measurement series. To do this tap the <Settings> button and select the <REAGENT BLANK> menu item. Fill the corresponding rectangular cell with the reagent blank and insert the cell into the cell compartment. The measurement is performed automatically. Accept the reagent blank by activating the <User RB> field and confirm with <OK>.
• After the reagent blank has been measured, fill the measurement sample into the same or a matched rectangular cell and insert the cell into the cell compartment. The measurement starts automatically.
• Read off the result in mg/L from the display.

Hint: The above written measurement description is only valid for the Spectroquant^® Prove (plus) series photometer. If a Nova 60A or a Move 100 is used, please consult the corresponding instrument manual for more details on how to perform the measurement.

Calculation

Nitrite content in mg/kg NO₂^- = analysis value in mg/L NO₂^- x 20

Nitrite content in mg/kg NaNO₂ = analysis value in mg/L NO₂^- x 30

Analytical quality assurance

Analytical quality assurance (AQA) is recommended before each measurement series.

To check the photometric measurement system (test reagent, measurement device, handling) and the mode of working, the nitrite standard solution (see section 5 of the respective test kit instruction) can be used.

Sample-dependent interferences (matrix effects) can be determined by means of standard addition.