Nitrate in Vegetables

Reflectometric determination after reduction to nitrite and reaction with Griess reagent.

Introduction

Nitrates are naturally occurring compounds that are found in the air, water, soil, and plants, forming part of the nitrogen cycle. Humans consume nitrate from a variety of sources, but plant matter in the form of vegetables is usually the biggest contributor unless the nitrate content of drinking water is exceptionally high.¹ A number of factors can influence nitrate levels in vegetables including the type/part of the vegetable, cultivation frequency, weather conditions, soil quality, food production processes, and the type/amount of fertilizers.²

Nitrates themselves are relatively non-toxic, but metabolites produced in the body such as nitrite, nitric oxide, and N-nitroso compounds are of concern because they have been linked to both cancer and a blood condition called methaemoglobinaemia.³ As a result, it is important to monitor nitrate levels in vegetables in order to avoid these potential adverse health effects. Here, we outline a general method for determination of nitrate levels in vegetables using reflectometry.

Experimental Method

Nitrate ions are reduced to nitrite ions by a reducing agent. In the presence of an acidic buffer, these nitrite ions react with an aromatic amine to form a diazonium salt, which in turn reacts with N-(1-naphthyl)-ethylenediamine to form a red-violet azo dye that is determined reflectometrically.

Reagents, Instruments and Materials

Test Kits

• Nitrate Test Method: reflectometric with test strips 5 – 225 mg/l NO₃^- Reflectoquant^® (1.16971) or
• Nitrate Test Method: reflectometric with test strips 3 - 90 mg/l NO₃^- Reflectoquant^® (1.16995)

Instruments

• Reflectometer RQflex^® 20 Reflectoquant^® (1.17246)

Note: This application note pertains to RQflex^® 20 and all discontinued instruments (RQflex^® 10, RQflex^® plus).

Sample preparation

• Homogenize the sample in a blender (e.g., Ultra-Turrax^®).
• Weigh a representative sample into a beaker and add distilled water.
• Cover the beaker with a watch glass and boil for ca. 15 minutes.
• Allow to cool, transfer to a volumetric flask and make up to the final volume with distilled water.

Recommended sample quantities and volumes for several vegetables are given in below sample preparation table. As the concentrations of nitrate can vary substantially from vegetable to vegetable, the dilution factor should be appropriately adapted.

Instrumental Analysis

• Press the reflectometer START key and simultaneously dip the test strip into the sample (15 - 30 °C) for ca. 2 seconds, ensuring that both reaction zones are immersed.
• Allow excess liquid to run off via the long edge of the strip on to an absorbent paper towel.
• Wait for 60 seconds and then measure the strip in the reflectometer.

The value [mg/l] will be stored automatically. (Please refer to the RQflex^® operating instructions and the instruction for use of the Reflectoquant^® Nitrate Tests.)

Calculation

Nitrate content [mg/kg] = Measured value [mg/l] x Volume of water [ml] ÷ Weight of sample [g]

Nitrate content [mg/kg] = Measured value x factor

Table 1. Dilution overview

Sample	Weight [g]	Water added [ml]	Final volume [ml]	Factor
Broccoli	10	50	100	10
Chicory	10	40	50	5
Endive lettuce	10	50	100	10
Lamb's lettuce	5	50	100	20
Cucumber	10	50	100	10
Carrot	10	40	50	5
Kohlrabi	5	50	100	20
Pepper	25	20	25	1
Radicchio	10	40	50	5
Radish	10	50	100	10
Sauerkraut	10	40	50	5
Tomato	25	40	50	2
White cabbage	10	40	50	5
Zucchini	10	50	100	10

Results

Table 2. Comparison with enzymatic method

Sample	Reflectoquant® [mg/kg]	Enzymatic [mg/kg]
Brokkoli	606	464
Chicory 1	< 15	< 25
Chicory 2	57	53
Endive 1	4967	4900
Endive 2	376	390
Lamb's lettuce	4240	4270
Carrot	74	74
Kohlrabi	1616	1639
Red pepper	< 3	< 5
Yellow pepper	6	8
Radicchio	63	55
Radish	94	91
Sauerkraut	59	51
Tomato	< 7	< 12
White cabbage	196	107
Zucchini 1	524	737
Zucchini 2	1185	1170

References

1. 2016. WHO. Nitrate and Nitrite in Drinking-Water. Background Document for Development of WHO Guidelines for Drinking-Water Quality. Geneva, Switzerland. [Internet].

2. Salehzadeh H, Maleki A, Rezaee R, Shahmoradi B, Ponnet K. The nitrate content of fresh and cooked vegetables and their health-related risks. PLoS ONE. 15(1):e0227551. https://doi.org/10.1371/journal.pone.0227551

3. 2008. Authority EFS. Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission to perform a scientific risk assessment on nitrate in vegetables. The EFSA Journal.689:1-79.