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Detailed Validation of a Method for the Determination of Nitrate in Water by

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DOI: 10.5740/jaoacint.12-007

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 Guimarães et al.: Journal of AOAC International 2014  1
RESIDUES AND TRACE ELEMENTS
Detailed Validation of a Method for the Determination of
Nitrate in Water by UV/Vis Spectroscopy
Vanessa Guimarães
University of Porto, Faculty of Sciences, CIQ-UP, Department of Chemistry and Biochemistry, Rua do Campo Alegre 687, 4169-
007 Porto, Portugal; IDIT—Institute for Development and Technological Innovation, Rua do IDIT–Espargo, 4520-102 Santa Maria
da Feira, Portugal
Helder Durão
IDIT—Institute for Development and Technological Innovation, Rua do IDIT–Espargo, 4520-102 Santa Maria da Feira, Portugal
Manuel Azenha
University of Porto, Faculty of Sciences, CIQ-UP, Department of Chemistry and Biochemistry, Rua do Campo Alegre 687, 4169-
007 Porto, Portugal
A spectroscopic method is reported for the
determination of nitrate in water samples based on
the reaction of nitrate and concentrated hydrochloric
acid. Nitrous acid is obtained with the subsequent
diazotization of sulfanilamide. In brief, the method
introduces a simpler, faster, and cheaper nitrate
reduction step, as compared to metal reduction.
Such described advantages prompted us to
undertake a validation and quality control study
aimed at the assessment of its fitness-for-use
as a routine laboratory procedure. The assessed
method exhibited a satisfactory performance under
indirect evaluation for drinking and wastewater
samples within the linear range between 0.5 to
5 mg/L nitrogen (N), sensitivity of 0.095 a.u. mg/L,
discrimination threshold at 0.06 mg/L N, LOD of
0.05 mg/L N (estimated in repeatability conditions),
LOQ of 0.5 mg/L N (estimated in intermediate
precision conditions), repeatability and intermediate
precision coefficients were estimated to be below
10%. In the case of wastewater samples, due to
the observed significant matrix effects, the original
procedure was modified. The dilution and simple
spike recovery test were included into the method in
order to decide the method’s applicability for each
sample. A recovery within 90–110% is considered
acceptable; while a recovery outside of that range
is not. Multiple standard addition quantification
recommended but not validated. Direct evaluation
of the method’s validation was accomplished by
successfully participating in a proficiency test and in
a comparison with a standard method. The combined
uncertainty was estimated at a 7% maximum.
The internal quality control provided positive
indications pointing to long-term stable behavior
of the procedure. Therefore, the proposed method
constitutes a reliable, simple, rapid, and inexpensive
procedure which may advantageously replace other
methods of nitrate determination in routine work.
Received January 5, 2012. Accepted by AK March 8, 2012.
Corresponding author’s e-mail: mazenha@fc.up.pt
DOI: 10.5740/jaoacint.12-007
N
itrate levels in water may pose concerns to human and
environmental health mainly due to its conversion to
nitrite, a hematoxic anion that compromises the oxygen-
transport function and that may also be implied in the generation
of N-nitroso compounds, some of which are considered as human
carcinogens (1–2). Nitrate admissible concentration values
(ACV) are thus established in the legislation for water pollution
control. In Portugal, the ACV for drinking water and wastewater
is 50 mg/L, the same as 11.3 mg/L nitrogen (N), which is in
agreement with the European Union legislation (3–4).
The determination of nitrate in water is a task performed
thousands of times daily in the world, mostly at routine
accredited laboratories employing validated analytical
methods. Comprehensive reviews (not limited to water matrixes
or specific analytical techniques) of methods for nitrate
determination can be found in the literature (5–6). A large
diversity of methods is compiled there, relying on spectroscopic
(UV/Vis absorption, fluorescence, chemiluminescence, IR,
Raman, and molecular cavity emission), electrochemical
(voltammetry and potentiometry) and chromatographic
(HPLC and ion chromatography), or electrophoretic analysis.
Chemiluminescent and chromatographic techniques are
envisaged as offering superior performance. However, the
instrumental complexity and cost required for their successful
implementation limit their widespread operation. The use of
spectroscopic analysis appears to be the method of choice due
to the simplicity of the protocols and the wide availability of the
instrumentation involved.
For routine laboratory work, the ability to provide reliable
output with high-throughput and cost effectiveness is of utmost
importance to be competitive in today’s market. Reference
methods for the determination of nitrate, established by several
institutions [International Organization for Standardization,
(ISO); U.S. Environmental Protection Agency], assure the
potential reliability of such methods, which must be validated
within every individual laboratory. These methods are a
common choice in most labs, but any other method can be
used, provided that an acceptable validated performance can be
demonstrated. It is thus of great relevance for labs providing
analytical services to be aware of potentially advantageous novel
methods, but also to know in advance if these new proposals
comply with strict quality assurance checks. In this context, we
decided to carry out a validation study of a recently proposed
method (7) claiming to provide a simple, rapid, and inexpensive
2  Guimarães et al.: Journal of AOAC International 2014
procedure, and selective in the presence of nitrite, for the routine
determination of nitrate in the range between 0.5–10 mg/mL N,
in different aqueous samples. This is a spectroscopic method
based on the reaction of nitrate and concentrated HCl to obtain
nitrous acid (Equation 1), and the subsequent diazotization of
sulfanilamide (Equation  2). The diazo-compound reacts then
with N-1-(naphthyl)ethylenediamine dihydrochloride (NEDA)
(Equation  3) to create the diazo-dye compound which can
be analysed by UV/Vis spectroscopy, using a wavelength of
540 nm.
HNO3 (aq) + 2HCl (concn) → Cl2 (g) +
HNO2 (aq) + H2O (l) (1)
(2)
(3)
This method falls within the class of methods that require
reduction of nitrate to nitrite prior to analysis. Nitrite thus
becomes an obvious interferent that can be previously removed
by urea treatment (8). Basically, this proposed method introduces
a simpler, faster, and cheaper nitrate reduction step, as compared
with the most popular technique of metal reduction. The next
sections demonstrate, for the first time, that the proposed method
is in fact validated for the determination of nitrate in drinking
water samples. However, for wastewater samples, a sample-
dependent behavior was found, some possible interferents were
identified, and modifications were incorporated in the procedure
in order to reach an acceptable performance.
Experimental
Reagents and Solutions
All reagents used were of analytical grade purity, and included
sodium nitrite (Panreac, Cascais, Portugal), potassium nitrate
(Merck, Algés, Portugal), N-1-(naphthyl)ethylenediamine
dihydrochloride (Riedel-de Haën, through Sigma-Aldrich,
Sintra, Portugal), sulfanilamide (Himedia, Mumbai, India),
hydrochloric acid 37% (Panreac), and urea (Panreac). Distilled
water was used for making solutions, dilutions, and for material
washing.
Working solutions of nitrate and nitrite were made by diluting
stock solutions of 100 mg/L and 250 mg/L, respectively, in
distilled water. Both stock solutions were preserved with
0.2% (v/v) chloroform.
The sulfanilamide solution concentration was 0.5% (w/v)
sulfanilamide in 0.5% (v/v) hydrochloric acid. The NEDA
solution concentration was 1% (w/v) NEDA in 1% (v/v)
hydrochloric acid. The urea solution was 2% urea (w/v) in
distilled water.
The coloring reagent for nitrite determination was prepared
by diluting 100 mL 85% phosphoric acid and 10 g sulfanilamide
in 800 mL of distilled water. After complete dissolution, 1 g of
NEDA was added and then the solution was further diluted
to 1 L with distilled water. The coloring reagent is somewhat
unstable, so this solution was prepared every 15 days and was
always kept away from light.
Water samples from several different sources (cork industry,
metal finishing industry, car wash facilities, wood industry,
paint industry, textile industry, etc.) were collected, preserved,
and stored according to standard procedures. Before the nitrate
determinations, wastewater samples were diluted in the region
of the lower end of the working range so as to minimize
interferences while working above the LOQ. If required, these
samples were subjected to nitrite removal. Unless otherwise
stated, the experiments were carried out at ambient temperature.
Nitrite Determination
Nitrite was determined in each sample, prior to the
application of the nitrate procedure. In this process, 0.2 mL
of coloring reagent was added to 5 mL sample, or to a portion
diluted to 5 mL. Blanks and standards were treated using the
same procedure. After a period of 10 min (but before 2 h),
absorbance was measured at 543 nm using quartz cuvettes with
a 1 cm pathlength (9). Beer’s law was obeyed up to 180 µg /L N.
Nitrite Removal
When nitrite concentration is greater than 200 µg /L N, it
must be removed with urea. This treatment protocol was
described previously for selective determination of nitrate
in the presence of nitrite (7). Each milliliter of nitrate sample
was treated with 0.1 mL urea solution and 0.2 mL HCl, and
incubated for 10 min in a boiling water bath. The nitrite- free
samples were, thereafter, processed for nitrate determination.
These samples were quantified against standards subjected to
the nitrite removal treatment just described.
Nitrate Determination
The method under study involves a sulfanilamide diazotization
process, which must be carried out at a high temperature. To
accomplish this, all samples and standards were immersed in a
boiling water bath, using heat-resistant 20 mL tubes. The tubes
were maintained in place using an improvised aluminum rack.
The water level in the water bath remained 2–3 cm above the
level of solution in the test tubes.
The experimental procedure was as follows: 0.2 mL
sulfanilamide solution and 2 mL HCl were added to each
milliliter of nitrite-free samples. The mixture was incubated
in a boiling water bath for 3 min, cooled with cold water to
room temperature, and 0.2 mL of coupling agent (NEDA)
was added. The absorbance of the solution was measured
 Guimarães et al.: Journal of aoaC international 2014 3
Parameter Value
Slope (b) 0,0952
Sb 0,0004
Origin (a)
Sa
0,0005
0,001
Sy/x 1,48x10-3
R 0.9999
Figure 1. Averaged (n = 9) calibration curve obtained with the
proposed method.
Figure 1.
at 540 nm after 30 min standing, at room temperature (7).
Quantification in drinking water samples was simply made by
calibration against external standards prepared in distilled water.
However, for wastewater samples the procedure was found not
to perform acceptably for every sample. Consequently, prior to
quantification, a recovery test was done with such wastewater
samples. The method was considered acceptable if sample
recoveries were between 90–110%. If recoveries were outside
of this range, the sample would then be analyzed for nitrate by
an alternative procedure.
Safety notes: Hydrochloric acid causes burns and is irritating
to respiratory system. Avoid contact with skin and eyes. 1 0.05 mg/L N, while the LOQ was determined according to the
Avoid inhalation of vapor or mist. NEDA is irritating to eyes,
respiratory system and skin. Avoid breathing dust and wear
suitable personal safety equipment.
Results and Discussion
Indirect Evaluation of the Method
The indirect evaluation involved the different assessments
of the calibration curve, such as operating range, linearity,
sensitivity, detection and quantification limits, precision
(repeatability, intermediate precision), accuracy, and selectivity.
Operating Range, Calibration Curve and Linearity
The intended working range, 0.5 to 5 mg/L N, was submitted
to a variance homogeneity test according to the ISO 8466-1
standard (10). The variances obtained for the signal from
independently prepared standards (n = 10) at the lower and
–5 2
upper range limits were 1.04 × 10 absorbance units (a.u.)
–5 2
and 1.69 × 10 (a.u.) , respectively. The corresponding F
value was 1.63, which is below the critical F (3.18) for 95%
degree of confidence; thus, warranting the applicability of the
non-weighed least-squares method for obtaining the calibration
equation.
The calibration curves were obtained from the signals
measured for six different concentration levels (0.5, 1.0, 2.0,
3.0, 4.0, 5.0 mg/L). Using linear regression, in accordance with
the ISO 8466-1 standard (10), the average (of 9 calibration
curves) regression parameters obtained are presented in Figure 1
(averaged calibration curve).
Although both residual analysis (not shown) and correlation
coefficients supported the acceptance of linearity for the
obtained calibration curve, the ISO 8466-1/2 standards (10, 11)
recommend testing linearity by taking the variances of the
2
residuals obtained when assuming the linear (S y/x) or the
2
non-linear (second order polynomial, S y2) models and then
2 2 2
calculating the difference ∆S = (N-2) * S y/x – (N-3) * S y/x.
2 2
This difference is then used for calculating F as ∆S /S y2 and
finally this “experimental” F value is compared with the critical
Snedecor/Fisher value, Fcrit. In this case, F is 0.08, which is
much smaller than Fcrit (7.7), thus the linearity is verified.
Sensitivity, LOD, and LOQ
Sensitivity, as given by the calibration slope, was
estimated as 0.095 a.u. mg/L (taken from the average line of
9 calibration curves as shown in Figure 1). The discrimination
threshold (DT) is a useful parameter that provides the lowest
difference in analyte concentration that the method is able to
discriminate (12). It is closely related to sensitivity and residual
standard deviation, Sy/x. Admitting that the errors associated
with the concentrations within the working range are Gaussian,
then, to have statistical significance, two concentrations must
differ more than 3 * √2 * Sy/x/b (for a 99.7% confidence interval).
A DT of 0.06 mg/L N was thus obtained, meaning that with such
calibration curves it is possible to significantly discriminate
between 75 different concentration levels within the working
range.
The LOD was determined as 3.3 * Sy/x/b, corresponding to
International Union of Pure and Applied Chemistry criterion (13)
of taking LOQ as the lowest concentration at which acceptable
levels of precision and accuracy (<10%) are observable.
Ten replicated measurements performed under intermediate
precision conditions showed that a variation coefficient of 6.6%
and bias of 1% were obtained at a concentration of 0.5 mg/L N,
and so the lower limit of the working range was also considered
the method’s LOQ.
Precision
Precision was evaluated under repeatability and intermediate
precision conditions. Reproducibility, on the other hand, would
have required a between-lab scheme which unfortunately was
not possible to setup.
The repeatability study was carried out with eight real
samples [drinking water (DW) and wastewater (WW)] and
two standard solutions. Each sample was analyzed 10 times in
the same working session and, before proceeding any further,
the Grubbs’ test was applied so as to rule out possible outliers,
in compliance with standard ISO 5725-2 (14). After checking
for outliers, the repeatability standard deviation (Sr) and the
coefficient of variation (CVr) were computed according to the
standard ISO 5725-6 (15), and the corresponding values are
compiled in Table 1. Method repeatability, evaluated from
the CV values, is generally considered acceptable under 10%.
The CV values obtained lie at ≤5% (with one exception with
the nitrate at the LOQ concentration level), and therefore the
method under study has demonstrated acceptable repeatability
for real samples and standard solutions.
The intermediate precision may be ascertained by several
different ways (ISO 5725-3, 16). The approach selected here
4  Guimarães et al.: Journal of AOAC International 2014
Table  1.  Results from repeatability studies, using real (DW
and WW) samples and standard solutions)
Sample |Nitrate|/mg/L N Sr/mg/L N CVr, %
DW1 0.51 0.023
DW2 1.56 0.049
DW3 1.68 0.049
WW1 2.50 0.042
WW2 3.68 0.035
WW3 0.99 0.037
WW4 0.90 0.026
WW5 0.87 0.043
Standard solution 0.50 mg/L N 0.51 0.034
Standard solution 5.00 mg/L N 5.01 0.043
consisted on taking two standard solutions at concentration
levels located at the upper (5 mg/L N) and lower (0.5 mg/L
N) limits of the working range and subjecting them to
31 determinations performed over a three month period. Each
of the 31 determinations of nitrate relied on an independent
calibration prepared at the same session. In this manner, variance
contributions from sources over relatively long periods of time
(e.g., calibration curve parameters, preparation of new reagent
solutions, and others), may be accounted for (the operator was
kept constant). After outlier identification and rejection (using
the Grubbs test previously mentioned), intermediate precision
values of 0.033 mg/L N (CV 6.6%) and 0.037 mg/L N (CV
0.74%) were obtained for 0.5 and 5 mg/L N, respectively.
The corresponding variances were found statistically identical
(a = 0.05) which implies that the intermediate variance
appears to be sufficiently homogeneous over the working
range. Additionally, no statistically significant differences
were found between intermediate precision and repeatability,
which suggests that the time-dependent sources of variance
had no significant additional influence on the total variance.
We speculate that since a calibration curve was prepared each
day for each sample set, that this must have cancelled most
of such possible contributions. For example, when a reagent
solution was refreshed, the new solution was applied both to the
calibration solutions and the sample, and this source of variance
would be mostly cancelled.
Matrix Effects, Selectivity and Recovery
Although a good precision under repeatability conditions was
found for a set of different DW and WW samples (see Table 1),
the matrix components may deleteriously affect the accuracy
of the determinations. This issue is especially relevant for
WW samples, which often exhibit quite complex and diverse
matrixes. To check for a significant influence of the matrix on
the accuracy of the results, 5 DW and 12 WW samples were
subjected to both the proposed method of determination (whose
quantification relies on an external calibration curve), and to
a modified method where the quantification was performed
by successive standard addition; an approach consensually
regarded as much less susceptible to matrix effects. The samples
were previously diluted with distilled water to the neighborhood
of the lower end of the working range, filtered (0.45 mm pore
size), and subjected to nitrite removal when necessary, as a
first step of minimization of potential matrix effects. For some
samples, their intense color/turbidity could interfere with the
standard addition procedure, even after being greatly reduced
4.6 by dilution and filtration. Therefore, the sample background
3.1 signal was systematically checked at 540 nm before NEDA
2.9 addition (Equation 3), to monitor any significant contribution
to the signal attributed to nitrate. We found that none of the
1.7
analyzed sample backgrounds contributed significantly (all
0.96
<0.003 a.u.), but we could not obviously encompass all possible
3.8 types of wastewater samples, so this check-up should not
2.9 be neglected. The results are presented in Table 2. All of the
4.9 DW samples appear not to induce matrix effects, but from the
6.6
12  WW samples tested, five produced significantly different
results (Student’s-t-test, a  =  0.05), by the two quantification
0.86
methods. These five samples (WWh to WWk) were sampled
from miscellaneous types of industries. Both colorless and
markedly colored, low and high organic contents, low and
high nitrogen contents, and low and high turbidity, were
characteristics found among the water samples exhibiting
matrix effects. None of the commonly known characteristics
of the samples seemed to be, by themselves, dictating the
appearance of a matrix effect. Nevertheless, an expedited way to
deal with the potential problem of significant matrix-effects in
wastewaters was necessary. It was decided, from this point on,
to include the previous dilution into the final method (referred
above plus a single standard addition check-up for recovery).
Recoveries with 90–110% limits were considered acceptable
and calibration curve quantification was performed. Otherwise,
samples were ruled-out from the method’s applicability and
a modified method, employing multiple standard addition
quantification, is suggested as a feasible alternative for handling
these samples. It is critical to notice that all figures of merit
presented above are not applicable to this alternative method,
which must be submitted to an independent validation process
which is not covered here. Therefore it must be emphasized that
all further data presented here relate to samples not ruled out by
the recovery check-up.
Aiming at the identification of some possible interferents when
using external calibration, it was decided to evaluate the effect of
typical concentrations of species commonly found in wastewater
samples. For inorganic species, we selected iron(II), sodium,
sulfate, ammonium, chloride, and zinc(II). For organic species,
in order to simulate chemical and biochemical oxygen demand
(COD and BOD) and also organic nitrogen, we chose potassium
hydrogen phthalate (KHP), glutamic acid (GTA)/glucose (GLU)
mixture, and glycine. Table 3 shows the recovery results for
0.50 mg/L N nitrate solutions containing different concentrations
of potential interferents. With the exception of iron(II), the
inorganic salts, tested at a relatively high concentration level
(1 g/L), did not interfere; iron (II) exhibited an interfering effect
above 100 mg/L, but a lower interfering threshold (>20 mg/L) is
probable. Concerning the effect of GTA/GLU, an abnormally high
recovery was observed at 3.5 + 3.5 g/L (corresponding to BOD
≈ 5000 mg O2/L), suggesting that high levels of organic acids
and/or sugars may present an interfering effect. KHP at 17 g/L
(COD = 20000 mg O2/L) was also an issue as far as interferences
are concerned, though such high values of COD are rare in real
wastewater samples. Nevertheless, GTA, GLU and KHP are
only surrogates for BOD and COD originating compounds, so
we cannot exclude the possibility that real matrices with lower
Table  2.  Results from matrix effect studies, using real (DW and WW) samples analysed by direct reading and by standard additions

Significant
|NO3–|/mg/L Nmg/L N |NO3–|/mg/L Nmg/L N Dilution (α = 0.05)
a
Sample Calibration curve (*) Standard addition (*) Recovery, % factor difference (Y/N)** Observations*

DWa 0.47 0.46 102 4 N Source: cork industry (well water).

DWb 0.56 0.53 106 30 N Source: cork industry.

DWc 0.47 0.50 94 16.7 N Source: metal finishing industry.

DWd 0.68 0.65 105 20 N Source: car wash.

DWe 0.85 0.85 100 10 N Source: cork industry.

WWa 0.79 0.79 100 50 N Source: wood industry; after microbiological treatment; yellow color; COD < 4 mg O2/L, BOD < 0.4 mg O2/L.

WWb 0.38 0.39 97 50 N Source: paint industry; light yellow color; COD < 4 mg O2/L, BOD < 0.4 mg O2/L.

WWc 0.50 0.51 98 6 N Source: textile industry; brown color.

WWd 0.49 0.51 96 20 N Source: cork industry; light brown color; COD < 2 mg O2/L.

WWe 0.55 0.56 98 40 N Source: aluminum processing industry; light yellow color; COD < 4 mg O2/L, BOD < 0.4 mg O2/L.

WWf 0.70 0.72 97 50 N Source: paint industry; light yellow color; COD < 4 mg O2/L.

WWg 0.50 0.58 86 20 Y Source: cork industry; colorless, COD < 2 mg O2/L, BOD < 0.4 mg O2/L.

WWh 0.66 0.74 89 10 Y Source: municipal wastewater treatment plant; colorless, COD < 2 mg O2/L, BOD < 0.4 mg O2/L.

WWi 0.23 (<LOD) 0.56 41 40 Y Source: waste management (oils and fats); yellow color (colorless after dilution), foaming, intense smell, COD
625 mg O2/L, containing oils and fats
WWj 0.58 0.46 126 10 Y Source: metal ware industry; colorless.

WWk 0.56 0.44 127 10 Y Source: wood industry; dark-brown color, COD 1650 mg O2/L, ammonia nitrogen 23 mg/L.
a –
 
(*) = |NO3 | in the diluted sample. COD (Chemical Oxygen Demand) and BOD (Biochemical Oxygen Demand) values after sample dilution. ** = Student’s t-test comparison
Guimarães et al.: Journal of AOAC International 2014  5
6 Guimarães et al.: Journal of aoaC international 2014

Table 3. Recoveries obtained for 0.5 mg/L N nitrate 0.81. In the case of the WW sample, z-score/En values could
solutions in the presence of potential interferents not be calculated since the reference value is indeterminate
(<1.51 mg/L). However, it should be noted that the analyzed
Potential interferent Concentration, g/L Recovery, %
concentration was also indeterminate (<0.50 mg/L) and that the
FeCl2 0.010 104 two results are therefore compatible. The method under study
0.020 102 satisfactorily passed this proficiency test, which was based on a
0.10 140 single z-score/En set. Further tests, such as the comparison with
a standard method, are thus required to ultimately determine the
1.0 29
adequacy of this method.
Na2SO4 1 107
NH4Cl 1 98 Standard Method Comparative Study
ZnCl2 1 98
Glycine 0.35 113 Five samples, two DW and three WW, analyzed by the
3.5 94 current method, were sent to an accredited lab (GBA, Hamburg,
Glutamic acid + Glucose
a
0.07+0.07(BOD ≈100) 109
Germany) for nitrate determination using an accredited standard
method based on ion chromatography (18). Table 5 shows the
0.35+0.35(BOD≈500) 109
obtained results. A very close agreement (relative deviation
3.5+3.5(BOD≈5000) 193 ≤7%) between the results was observed for this set of samples.
a
Potassium hydrogen phthalate 0.85 (COD ≈1000) 105 The most statistically meaningful comparison would be
8.5 (COD≈10000) 105 achieved through a metrological compatibility test (19) but for
17 (COD≈20000) 128 that the uncertainties for each individual measurement needed
a
to be known, and unfortunately the contracted laboratory
COD (Chemical Oxygen Demand) and BOD (Biochemical Oxygen
Demand); Values in mg/L O2.
only provided a general estimate for this (uncertainty = 4%).
Alternatively, the results of the two methods were found to be
statistically (a = 0.05) indistinguishable, both by the paired
BOD or COD values may originate an interfering effect. Glycine, t-test and regression analysis (slope not statistically different
a compound containing nitrogen, appears not to interfere up to from 1). It is reasonable to consider then that the method under
653 mg/L N but this does not necessarily imply that other amino- study passed this comparative test, which supports its ability to
compounds do not interfere at lower concentrations. provide true results.

Direct Evaluation of the Method Estimation of Combined Uncertainty

This kind of evaluation aims primarily at the direct
determination of the accuracy of the method by resorting to
certified reference materials, proficiency tests or standard
methods. Herein we present results of a proficiency test and
comparative results against a standard method.
Proficiency Test
Two water samples (DW and WW) from the Association
of Portuguese Accredited Labs (RELACRE) wastewater
proficiency test were subjected to the method under scrutiny
in 2011. The results are shown in Table 4. A z-score between
–2 and 2 or |En|<1 (normalized error) are generally considered
indexes of satisfactory performance of a laboratory (17). For
the DW sample, the reference and sample concentrations were
The estimation of combined uncertainty followed Eurachem’s
guidelines (20). Standard uncertainties are denoted, u, while
relative standard uncertainties are denoted u′.
The uncertainty associated with precision, u′p, was estimated
at the two ends of the working concentration range (0.500 and
5.000 mg/L N). The data previously acquired for the intermediate
precision (Sip) was used for the purpose. The values of u′p were
computed simply as u′= Sip/|NO3–| and the obtained values were
0.07 (0.50 mg/L N) and 0.007 (5.00 mg/L N).
The uncertainty associated with accuracy , u′t, was estimated
from the information collected when comparing against the
reference method (Table 5). Estimated uncertainty attributed
to recovery (defined as R=Ccurrent method/Creference method) was first
calculated as
 S ip2   u(C ref.method ) 
2

u(R m ) = R m ⋅  + 
almost identical, corresponding to a z-score of 1.3 and to En =  n ⋅ C curr .method 2   
   C ref.method 

Table 4. Results obtained in the RELACRE Wastewater Proficiency Test 2011

Sample Reference concn/mg/L NO3– Sref/mg/L NO3– Analyzed concn/mg/L NO3– z-scorea Enb

DW 10.1 0.4 10.6 1.3 0.81

WW < 1.51 0.51 < 0.50 — —
a
z = (Vlab-Vref)/S; where S stands for the standard deviation associated to the reference value (Vref) and Vlab: the value obtained by the participating
laboratory.

b
where Ulab and Uref are the expanded uncertainties associated to Vlab and Vref, respectively. Ulab was considered 0.14 (see Table 6)
 Guimarães et al.: Journal of AOAC International 2014  7

Table  5.  Comparison of nitrate concentration obtained by 0,1040

a standard method and by the proposed method 0,1020

Standard method Current method Relative 0,1000

Sample ID concn/mg/L N concn/mg/L N deviation, %
0,0980 +3s

DW I 1.90 1.84 3

Slope / L/mg N
+2s
0,0960
DW II 2.70 2.89 7
0,0940
WW I 2.00 1.92 4 +2s
+3s
0,0920
WW II 2.70 2.82 4
0,0900
WW III 0.75 0.77 2
0,0880

0,0860
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

where u(Rm) is the uncertainty of the mean recovery Rm and n Time / days

the number of recovery tests (5 in this case). The value used as
u(Cref.method)/Cref.method) = 0.04 was provided by the contracted
lab. A Student’s-t test showed that Rm is not significantly
(α  =  0.05) different from 1. In such case, the uncertainty
u′t is simply equivalent to the uncertainty of recovery, i.e.:
u′t = u′(Rm) = u(Rm)/∙Rm. The average uncertainty of recovery
value of 0.03 (see Table 6), was then taken as an estimate of u′t
for the whole working range of concentrations.
The combined (u′c) and expanded [u′c, (95%) confidence
interval], uncertainties were finally estimated for the two
ends of the working range, according to the relationship;
u′c = ((u′p)2+(u′t)2)1/2 and U′c(95%) = k.u′c, where k stands for the
coverage factor (20). The results are presented in Table 6. The
estimated values locate the expanded uncertainty of the method
between 6% (upper end of the working range; uncertainty
dominated by accuracy ) and 14% (lower end of the working
Figure  2.  Individual control chart for calibration slope.
Figure 2.
chart was inspected at each new point according to the criteria
recommended in the ISO 8258 standard (21). Abnormally high
values were observed at several points for the first twenty days.
The cause for these anomalies was found to be related to the
short-term stability of the colorant solution, which prompted a
change to the method of a maximum stability period of 15 days
for this solution (protected from light ). Another individual
value of the ICC (47th day) fell off-limits but then the slope
returned to the acceptable region within the prescribed control
limits until the end of the study. It can finally be stated that,
as long as the previously mentioned corrective measure was
maintained, the calibration remained under statistical control
during the remaining enclosed period.
2

range; uncertainty dominated by precision). These values
may be considered quite satisfactory in the light of the main
purpose of such a method which is to verify the compliance
with a regulatory upper limit of 11.3 mg/L N in water. In this
case, uncertainty in the upper working range, due to a question
of eventual proximity to the decision threshold (the ACV), is
especially relevant and the value estimated is adequately low.
Internal Quality Control
Both indirect and direct evaluation of the method as well
as the respective expanded uncertainty estimation provided
satisfactory outputs that allowed for assuming the overall
reliability of the method. It was further decided to advance
to the internal quality control stage, which can assure that the
method performance remains valid throughout a relatively
long period of operation. An individual control-chart (ICC) of
calibration slope and a range control-chart (RCC) of triplicate
determinations were constructed in the course of 2.5 months.
The control line of the RCC (Figure 3) is located at range 3.27
Rm (Rm stands for the mean range observed from a previous
set of 9 triplicate determinations). The triplicate determinations
were performed under repeatability conditions on incoming
wastewater samples. Throughout the whole period the range
remained below the control line and therefore the short-term
precision was under statistical control, despite the much
diversified provenance of the wastewater samples analyzed.
Conclusions
The assessed method exhibited a satisfactory performance
under indirect evaluation (operating range, calibration curve,
linearity, sensitivity, LOD and LOQ, precision, selectivity, and
recovery), for drinking and wastewater samples. In the case of
wastewater samples, due to the possibility of significant matrix
0,3

Control and warning upper and lower limits of the ICC 0,25
3.27 Rm*

(Figure 2) were obtained from the mean and standard

deviation of an initial set of 9 calibrations, after passing the 0,2
Kolmogorov-Smirnov test for a normal distribution. The
Amplitude

0,15

0,1
Table  6.  Combined and expanded uncertainties estimated
for the proposed method
0,05
U′c (95%) = k∙u′c
Concn level/mg/L N u′p u′t =u′ (Rm) u′c (k = 2) 0
0 2 4 6 8 10 12 14 16
0.500 0.07 0.03 0.07 0.14
Sample number
5.000 0.007 0.03 0.03 0.06
Figure  3. * Range control chart for triplicate determinations.
Rm stands for the mean range observed from a previous set of nine triplicate
determinations.

Figure 3
8  Guimarães et al.: Journal of AOAC International 2014
effects, dilution and a simple spike recovery test were included
in order to decide the method’s applicability to each sample;
a recovery within 90–110% is acceptable; while a recovery
outside of that range is not and a multiple standard additions
quantification would be recommended but unable to be
validated). Results from a proficiency test and comparison with a
standard method provided a direct evaluation of the method, and
further emphasized the fitness-for-use of the proposed method.
Acceptable estimated values of the combined uncertainty were
obtained, and a 2.5 month period study of the internal quality
control of the procedure provided positive indications pointing
to long-term stable behavior of the procedure. Therefore, the
proposed method constitutes a reliable, simple, rapid, and
inexpensive procedure which may advantageously replace other
methods of nitrate determination in routine work.
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