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Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 90 

 
VALIDATION OF AN ENZYMATIC METHOD FOR NITRATES 

DETERMINATION FROM VEGETABLE PRODUCTS  
 

Monica CATANĂ1, Luminiţa CATANĂ1, Mioara NEGOIŢĂ1, Enuţa IORGA1,  
Alina BÂLEA1, Gabriela LILIOS2 

 
1National Institute of Research&Development for Food Bioresources – IBA Bucharest, 021102, Bucharest 2, 6 

Dinu Vintila Street, Romania, e-mail: mona.catana@bioresurse.ro 
2Ovidius University, 900527, Constanţa, 124 Mamaia Street, Romania, e-mail: liliosgabriela@yahoo.com 

 
 
 

Abstract: Nitrates and nitrites are natural components of soil, derived from mineralization of 
nitrogenous organic substances of vegetable and animal origin. Plants absorb nitrogen from soil, 
mainly as nitrates or ammonium forms. 
This paper presents the results of researches carried out to validate an enzymatic method for nitrates 
determination in vegetable products. Within this method, the nitrate is reduced by the reduced 
nicotinamide adenine dinucleotide phosphate (NADPH) to nitrite, in the presence of nitrate-reductase 
(NR). 
In the case of the enzymatic method for nitrates determination in vegetable products, the calibration 
curve achieved with 9 standard levels of nitrate ions (with three rehearsals for each one), in the range 
of concentrations 6 mg/L – 30 mg/L, beginning from origin and the linearity coefficient (r2), is 0.9999. 
For 10 vegetable samples grown in open field (egg plants, bell peppers, green peppers and onion) the 
average nitrate concentration of vegetable species varied in the range 27.91 mg NO3-/kg – 82.85 mg 
NO3-/kg, and repeatability was in the range 1.33 mg NO3-/kg – 1.50 mg NO3-/kg. 
The determination of nitrates in a cabbage sample, by three analysts, led to an average nitrate 
concentration of 107.42 mg NO3-/kg, relative standard deviation RSD(R), and the determined 
concentration is 0.81 %, and reproducibility is 2.45 mg NO3-/kg. 
The detection limit is 0.15 mg/L and quantification limit is 0.30 mg/L. 
In the case of onion sample grown in open field, the nitrates concentration was 55.5 mg NO3-/kg, and 
uncertainty was 0.68 mg NO3-/kg. 

 
Key words: linearity, repeatability, reproducibility, limit of detection, limit of quantification 
 
 
 

 
Introduction 

 
In growing and development of 
horticulture-wine-growing plants, the 
improvement of soil fertilization state and 
achievement of high and constant crops, 
organic or mineral fertilizers are applied in 
the soil [1]. 
Nitrates and nitrites are natural 
components of the soil from nitrogen 
mineralization of organic substances of 
plant and animal origin. Nitrogen  
 

mineralization takes place primarily 
through the existing micro-organisms in 
soil. In areas with temperate climate, this 
process is carried out with maximum 
intensity in hot season [2].  
Plants absorb nitrogen from soil, mainly as 
nitrates or ammonium forms. 
Vegetables and fruits can accumulate also 
nitrogen, as nitrate and nitrite forms. The 
mineral nitrogen amount in tissues of 
vegetable and fruit is much more in the 
case of species which the nitrates’ 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 91 

reduction happens in leaves and when light 
intensity and temperature are lower [3]. 
High concentration of nitrates in plants 
(especially in vegetables) means a hazard 
for human and animal body, for two 
reasons: the possibility of methemoglobin 
appearance at children and nitrates 
conversion to nitrites in saliva and 
formation of cancerigen nitrozamines in 
the intestinal tract [2]. 
In a normal diet, 54% of nitrates content 
comes through vegetables consumption. In 
the daily ingestion of vegetable products, 
especially in the case of vegans, the 
percentage can be much more, 75 – 80% 
[2]. 
Taking into consideration the cancerigen 
potential of nitrosamines, the high content 
of nitrates in vegetable products, presents a 
potential hazard. 
In order to determine nitrates within 
vegetable products, the following methods 
can be used: 
 Spectrophotometer methods 
 High performance liquid 
chromatography method, using UV 
detector 
 High performance ion 
chromatography, using electrical 
conductivity detector 
 Potentiometer method with 
selectively electrode 
 Enzymatic method 
 
This paper presents the results of the 
performed researches carried out to 
validate an enzymatic method for nitrates 
determination in vegetable products. This 
enzymatic method presents no risks for the 
analyst, eliminating the danger of using 
metallic cadmium and α – naphthyl amine 
(cancerous substance, which is one of the 
components of colour reagent) represent 
within classical spectrophotometer method. 
 
 

Experimental 

This paper presents the results of the 
performed researches carried out to 
validate an enzymatic method for nitrates 
determination in vegetable products.   
Within this method, the nitrate is reduced 
by the reduced nicotinamide adenine 
dinucleotide phosphate (NADPH) to 
nitrite, in the presence of nitrate-reductase 
(NR) [4]. 
 

NR 
 NO3- + NADPH + H+        NO2- + NADP+ + 
H2O 
 
The amount of oxidized NADPH is 
stoechiometrically equal to the nitrate one.  
The decrease of NADPH amount is 
measured through absorbance at 340 = ג 
nm.  
 

 
  

Figure 1. Kit for nitrates determination through 
enzymatic method 

 
The objective of validation is to 
demonstrate that a defined analytic system 
leads to the obtaining of some precious and 
reproducible results, for a given property. 
In order to validate the method for nitrates 
determination in vegetable products, the 
following performance criteria were 
established: 
 linearity 
 accuracy 
 precision (repeatability and 
reproducibility) 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 92 

 sensitivity (limit of detection, 
limit of quantification) 
 measurement uncertainty  
In order to validate the enzymatic method 
for nitrates determination in vegetable 
products, we used as matrices the 
following vegetable species, cultivated in 
open field: egg plants, bell peppers, green 
peppers, cabbage and onion. 

 
Results and Discussion 

 
Linearity is defined as the ability of 
method to provide results proportional with 
level of measuring. 
Calibration curve has to be straight and to 
start from origin. Any deviations that 
appear must be known. In order to 
accomplish the calibration curve, minimum 
five concentrations are necessary, with 
three rehearsals for each of them.     
Linearity coefficient (r2) has to be of 
minimum 0.999, lower values may be 
accepted if there is a justification for this. 
In the case of the enzymatic method for 
nitrates determination in vegetable 
products, we achieved a calibration curve 
with 9 levels of nitrate ions standard (with 
three rehearsals for each one), in the 
concentrations range 6 mg/L – 30 mg/L, as 
following: 
 level 1 – 6 mg/L 
 level 2 – 9 mg/L 
 level 3 – 12 mg/L 
 level 4 – 15 mg/L 
 level 5 – 18 mg/L 
 level 6 – 21 mg/L 
 level 7 – 24 mg/L 
 level 8 – 27 mg/L 
 level 9 – 30 mg/L 
 
The achieved calibration curve starts from 
origin and linearity coefficient (r2) is 
0.9999. 

Calibration curve of ion nitrates 

y = 0,0335x - 0,0054
R2 = 0,9999

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 5 10 15 20 25 30 35
Concentration of ion nitrares (mg NO3-/L)

A
b

so
rb

an
ce

Linear

 
Figure 2. Calibration curve of ion nitrates 

 
   The equation of calibration curve is the 
following:  
    y = 0.0335 x – 0.0054        (1) 
of which: 
 y = absorbance measured at λ = 340 nm 
x =  concentration of nitrate ions, in mg 
NO3-/L 
       By equation (1), knowing absorbance 
value, we can directly calculate the 
concentration of nitrate ions in a test 
sample, using the following formula: 

   
0335.0

0054.0


y
x               (2) 

       Accuracy means the ability to make an 
analysis with low difference between real 
value and founded value.  
       In the case of enzymatic method for 
determination of nitrate ions in vegetable 
products, accuracy is expressed through 
recovery. 
 Recovery is calculated by the equation (3): 
 
 Recovery (%) = (cd/ct) x 100               (3) 
of which: 
ct – theoretical concentration of nitrate ion, 
in mg/L 
cd – determined concentration of nitrate 
ion, in mg/L 
 
      In order to determine recovery, in the 
case of enzymatic method for 
determination of nitrate ion, we used 9 
prepared solutions as in the case of the 
achievement of calibration curve. 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 93 

The concentration of nitrate ions of those 9 
nitrate solutions was determined in two 
ways: 
 calculated by equation (2) 
 using the enzymatic method 
 
The concentrations of nitrate ions of those 
9 solutions, using the enzymatic method, 
were calculated by formula (4): 
Cnitrate = (1.01xV x MW / ε x d x V )xΔA  
in     
             [mg NO3-/L sample solution        
(4) 
of which: 
1.01 – correction factor of final volume of 
sample 
V – final volume of sample (3.05 mL) 
MW – molecular weight of nitrate ion (62  
            g/mol) 
ε – extinction coefficient of NADPH (at λ 
= 340 nm, ε = 6.3 l x mol-1 x cm-1) 
d – length of optical way (1 cm) 
V – sample volume, in mL 
ΔA – difference between variance of 
sample absorbance and blank absorbance 
Difference of absorbance for sample  (A1 
– A2)sample – 2x(A2 – A3)sample 
Difference of absorbance for blank  (A1 – 
A2)blank – 2x(A2 – A3)blank 
Anitrate  Asample – Ablank      (5) 
Changing these elements, we have 
obtained: 

        
V

A
x nitrat




316.30
     (6) 

The determination of concentrations of 
nitrate ions of 9 solutions of kalium nitrate 
was made by the formula (2) and recovery 
was in the range: 98.67 % - 100.23 %. 
 The determination of concentrations of 
nitrate ions of those 9 solutions of kalium 
nitrate was made by the formula (6), using 
the protocol of the enzymatic method, 
recovery was in the range: 97.00 % - 
102.67 %. 
 Precision is reverberated by the 
concordance degree between independent 

analytical results obtained under specified 
conditions.  
      Precision is a measure of results 
dispersion and is determined in conditions 
of repeatability and reproducibility. 
      Repeatability (r) – value below which 
the absolute difference between results of 
the two separated tests, achieved on the 
same test object, the same apparatus, the 
same executant and in the same short time 
spell, is below the specified probability 
limits (as a rule 95%).  The limit of 
repeatability is calculated by the equation 
(7): 
 

               r = 2.8 x SD(r)         (7) 
of which: 
r – limit of repeatability; 
SD(r) – standard deviation, calculated in 
the case of results obtained in repeatability 
conditions. 
      Relative standard deviation, RSD(r), 
obtained from the achieved results in 
repeatability conditions is calculated by 
equation (8): 
 
     RSD(r) = (SD(r)/ X )x 100     (8)              
of which: 
SD(r) – standard deviation, calculated in 
case of the obtained results in repeatability 
conditions; 
X – arithmetic average of determined 
concentrations in repeatability conditions. 
 
 Reproducibility (R) – value below which 
the absolute difference between results of a 
single test, obtained for an identical 
material, in two or more laboratories, by 
different operators, on different apparatus, 
is below the specified probability limits (as 
a rule 95 %). 
 Relative standard deviation, RSD(R), 
obtained from the achieved results in 
reproducibility conditions is calculated by 
the equation (9): 

    RSD(R) = (SD(R)/ X )x 100              (9) 
of which: 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 94 

SD(R) – standard deviation, calculated 
from the obtained results in reproducibility 
conditions; 
X – arithmetic average of the obtained 
results in reproducibility conditions. 
Standard deviation – measure dispersal 
tendency of data and is calculated by  
equation (10): 
                          

         
1

)( 2




 
n

XX
S iD             (10)                                                          

of which: 
SD – standard deviation; 
Xi – result of measurement; 
X – arithmetic average of measurement 
results; 
n – number of tests. 
In order to determine repeatability in the 
case of enzymatic method for 
determination of nitrates in vegetable 
products, we analysed 10 parallel samples 
of vegetables cultivated in field (egg 
plants, bell peppers, green peppers and 
onion) and we calculated for each 
vegetable species the following: 
 average concentration of nitrates 
 value of relative standard deviation 
    RSD(r), in repeatability conditions 
 limit of repeatability 
 
In the case of the 10 parallel samples of 
egg plants analysed to determine nitrates 
content, the average concentration of 
nitrates is 65.98 mg NO3-/kg, relative 
standard deviation RSD(r), for determined 
concentration is 0.77%, and the limit of 
repeatability is 1.43 mg NO3-/kg.  
In the case of the 10 parallel samples of 
bell peppers analysed to determine nitrates 
content, the average concentration of 
nitrates is 27.91 mg NO3-/kg, relative 
standard deviation RSD(r), for determined 
concentration is 1.90%, and limit of 
repeatability is 1.49 mg NO3-/kg. 
 In the case of 10 parallel samples of green 
peppers analysed to determine nitrates 

content, the average concentration of 
nitrates is 30.85 mg NO3-/kg, relative 
standard deviation RSD(r), for determined 
concentration is 1.54%, and limit of 
repeatability is 1.33 mg NO3-/kg. 
     In the case of 10 parallel samples of 
onion analysed to determine nitrates 
content, the average concentration of 
nitrates is 82.85 mg NO3-/kg, relative 
standard deviation RSD(r), for determined 
concentration is 0.65%, and limit of 
repeatability is 1.50 mg NO3-/kg. 
       In order to determine intralaboratory 
reproducibility in the case of the enzymatic 
method used in the determination of 
nitrates in vegetable products, we analysed 
by three analysts (analyst A – 3 parallel 
samples, analyst B – 3 parallel samples, 
analyst C – 3 parallel samples) one 
cabbage sample, carrots and onion and  we 
calculated: 
 average concentration of nitrates 
 value of relative standard deviation 
RSD(R), in reproducibility conditions 
 limit of reproducibility 
In the case of the analysed cabbage sample 
by analysts for determination of nitrates 
content (each analyst performed 3 parallel 
samples), the average nitrates 
concentration is 107.42 mg NO3-/kg, and 
relative standard deviation RSD(R), for 
determined concentration is 0.81% and 
limit of reproducibility is 2.45 mg NO3-
/kg. 
In the case of the analysed carrots sample 
by analysts for determination of nitrates 
content (each analyst performed 3 parallel 
samples), the average nitrates 
concentration is 92.27 mg NO3-/kg, relative 
standard deviation RSD(R), for determined 
concentration is 1.24%, and limit of 
reproducibility is 3.20 mg NO3-/kg. 
In the case of the analysed onion sample 
by analysts for determination of nitrates 
content (each analyst performed 3 parallel 
samples), the average nitrates 
concentration is 71.88 mg NO3-/kg, relative 
standard deviation RSD(R), for determined 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 95 

concentration is 1.22%, and limit of 
reproducibility is 2.45 mg NO3-/kg. 
Limit of detection of the method is 0.15 
mg/L and corresponds to a difference of 
absorbance between sample and blank 
(Anitrate) of 0.010 (absorbances being 
measured at λ = 340 nm) and a sample 
maximum volume V = 2 mL. 
Limit of quantification of the method is 
0.30 mg/L and corresponds to a difference 
of absorbance between sample and blank 
(Anitrate) of 0.020 (absorbances being 
measured at λ = 340 nm) and a sample 
maximum volume V = 2 mL. 
Uncertainty is a parameter associated with 
the result of measurement that 
characterises the dispersion of the values 
that could reasonably be attributed to the 
measurand.  
In order to estimate uncertainty the 
following steps should be taken: 
 specification of measurand 
 identification of the significant 
sources of uncertainty 
 estimation of combined standard 
uncertainty 
 estimation of extended standard 
uncertainty  
Combined uncertainty is calculated with 
equation (11): 

22
4

2
3

2
2

2
1 ..... nc uuuuuu     (11)              

of which: 
     uc – combined uncertainty 

       u1, u2, u3, u4, .....un – component 
uncertainties of the budget of uncertainties.  
       Extended uncertainty is calculated by 
equation (12): 
 
     U = k x uc                                         (12) 

 
of which: 
 U – extended uncertainty 
 uc – combined uncertainty 
  k – covering factor, for a confidence level 
of 95%, k = 2 
 

     Measurand = concentration of nitrates 
in vegetable products, expressed in mg 
NO3- /kg. 
     In the case of determination of nitrates 
in vegetable products, through enzymatic 
method, significant sources of uncertainty 
are: 
 measurement of volume of aqueous 
extract of sample 
 weighting of sample  
 measurement of absorbance of 
aqueous extract of sample 
 
 Relative standard uncertainty, within 
enzymatic method of nitrates 
determination, is calculated by equation 
(13) [5]: 
 

2.221 )()()
2

(
)(

A
u

m
uu

x
xu absmc


    (13) 

 
of which: 
uc1 – combined standard uncertainty at 
measurement of the aqueous extract of 
sample 
um – combined standard uncertainty at 
weighting of sample  
uabs. - combined standard uncertainty at 
measurement of the aqueous extract of 
sample 
m – weight of analysed sample, in g 
ΔA – difference between variance of 
absorbance of sample and blank 
x – nitrates concentration of sample, in 
mg/kg 
 

Relative  s tandard uncertainty ,at nitates 
determ ination, in the cas e of onion sam ple 

0 0,002 0,004 0,006 0,008

V

m

∆A

u(x)/x

Relative  s tandard uncertainty 

Series1

 
Figure 3. Relative standard uncertainty, at 
nitrates determination, in the case of onion 

sample 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 
Year IX, No. 4 - 2010 

 
 

 96 

In the case of the analysed onion sample,  
by changing the suitable data,  we 
obtained: 

0062.0
)(


x
xu

 

x = 55.5 mg NO3-/kg sample 
 

34.0)( xu  mg NO3-/kg sample 
 U(x) = k x u(x) 
  of which: 
  U(x) – extended uncertainty 
  k – covering factor for a confidence level 
of 95%, k = 2 
 U(x) = 0.68 mg NO3-/kg sample  
        Therefore, result can be expressed in 
this way: x = 55.5 ± 0.68 mg NO3-/kg 
sample 

 
Conclusion 

 
1. This paper presents the results of the 
performed researches carried out to 
validate an enzymatic method for nitrates 
determination in vegetable products. 
  2. In the case of the enzymatic method for 
nitrates determination in vegetable 
products, the calibration curve achieved 
with 9 standard levels of nitrate ions (with 
three rehearsals for each one), in the range 
of concentrations 6 mg/L – 30 mg/L, starts 
from origin, and the linearity coefficient 
(r2) is 0.9999. 
3. For 10 vegetable samples grown in open 
field (egg plants, bell peppers, green 
peppers and onion) the average nitrate 
concentration of vegetable species varied 
in the range 27.91 mg NO3-/kg – 82.85 mg 
NO3-/kg, and repeatability was in the range 
1.33 mg NO3-/kg – 1.50 mg NO3-/kg. 
4. The determination of nitrates in a 
cabbage sample, by three analysts, led to 
an average nitrate concentration of 107.42 
mg NO3-/kg, relative standard deviation 
RSD(R), for determined concentration is 
0.81%, and reproducibility is 2.45 mg NO3-
/kg. 
5. In the case of carrots sample analysed by 
analysts to determine nitrates content (each 

analyst performed 3 parallel samples), 
average nitrates concentration is 92.27 mg 
NO3-/kg, relative standard deviation 
RSD(R), for determined concentration is 
1.24%, and limit of reproducibility is 3.20 
mg NO3-/kg. 
6. The detection limit of method is 0.15 
mg/L and corresponds to a difference of 
absorbance between sample and blank 
(Anitrate) of 0.010 (absorbances being 
measured at λ = 340 nm) and a maximum 
volume of sample V = 2 mL. 
7. The quantification limit of method is 
0.30 mg/L and corresponds to a difference 
of absorbance between sample and blank 
(Anitrate) of 0.020 (absorbances being 
measured at λ = 340 nm) and a maximum 
volume of sample V = 2 mL. 
8. In the case of onion sample grown in 
open field, nitrates concentration was 55.5 
mg NO3-/kg, and uncertainty was 0.68 mg  
NO3-/kg. 
 
Acknowledgments 

 
The experiments were performed within 
the contract no. 51-050/18.09.2007, 
financed through Programme 4 
“Partnerships in priority S&T Domains” 
2007 – 2013 – National Centre for Projects 
Management. 

 
References 
 
1. Luchian Viorica, Legumicultură generală şi 
specială, Ed. Elisavaros, Bucureşti, 2007 
2. Bibicu Miruna, Cercetări metodologice privind 
determinarea nitraţilor şi nitriţilor din ţesuturi 
vegetale şi nivelului de acumulare în produsele 
horticole, Bucureşti, 1994 
3. Gherghi A., Burzo I.,  Miruna Bibicu, Liana 
Mărgineanu, Liliana Bădulescu, şi colab., 
Biochimia şi fiziologia legumelor şi fructelor, Ed. 
Academiei Române, Bucureşti, 2001 
4. .*** Protocol de lucru pentru metoda enzimatică 
- UV de determinare a nitraţilor din produse 
alimentare, R – Biopharm, 2008 
5. ***  CITAC / EURACHEM GUIDE - 
Quantifying Uncertainty in Analytical 
Measurement, 2000