Accepted Manuscript Colorimetric determination of nitrate and nitrite in milk and milk powders – Use of vanadium (III) reduction David C. Woollard, Harvey Indyk PII:
To appear in:
International Dairy Journal
Received Date: 5 April 2013 Revised Date:
5 July 2013
Accepted Date: 22 August 2013
Please cite this article as: Woollard, D.C., Indyk, H., Colorimetric determination of nitrate and nitrite in milk and milk powders – Use of vanadium (III) reduction, International Dairy Journal (2013), doi: 10.1016/j.idairyj.2013.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Colorimetric determination of nitrate and nitrite in milk and milk powders –
Use of vanadium (III) reduction
4 5 6 7 David C. Woollarda*, Harvey Indykb
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10 11 12 13 14 15
New Zealand Laboratory Services, 35, O’Rorke Rd, Penrose, Auckland 1642, New
Fonterra, Main Road, Waitoa, New Zealand
* Corresponding author. Tel.: +6495264514
E-mail address: [email protected]
(D. C. Woollard)
27 A manual method is described for the determination of nitrate and nitrite in milk
and milk powders that is intended to provide an alternative to conventional manual
methods accomplished by cadmium reduction. Reduction of nitrate is performed in
solution utilising vanadium (III) and quantitation achieved by concurrent reaction with
Griess reagent. Performance data are acceptable in terms of precision and accuracy,
repeatability being about 6% and intermediate precision at 8% for both analytes, providing
the limit of detection is not approached. Limit of quantitation is 0.1 mg kg-1 for both
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42 Nitrate (NO3-) exists throughout the biosphere from natural and man-made
processes and is an important component of biological life-cycles; hence it is well-studied
clinically (Ellis, Adatia, Yazdanpanah, & Makela, 1998). Bacterial action can reduce NO3-
to NO2- (nitrite), which is minimised from entering the modern food chain because of
associated health risks. Although there is inevitably a background level of NO3- , it is
regularly monitored within the dairy industry as part of normal sanitary programs. Nitrite
levels are also monitored, since microbiologically-contaminated water or post-secretory
milk can contain bacteria with significant nitrate reductase activity. The widespread use of
nitrogen-based fertilisers, combined with domestic, agricultural and industrial wastes,
have increased the chances of NO3- and NO2- incorporation into manufactured dairy
products (Indyk & Woollard, 2011). In addition, the use of nitric acid as a sanitiser within
dairy plants represents a further risk of nitrate contamination. Although generally
associated with increased risk of several pathologies, it is notable that recent studies
indicate that health benefits may be derived from consumption of dietary NO3- and NO2- ,
through maintenance of systemic nitric oxide homeostasis (Hord, Tang, & Bryan, 2009).
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Health issues and subsequent regulatory requirements have created the need for
rapid and reliable analytical methods for quantitation of the NO3- and NO2- content in
foods and biological materials. There are many technology platforms for NOX- testing,
each with benefits and disadvantages, but the well-studied Griess diazotisation reaction
(Griess, 1879) involving NO2- is the most commonly employed detection chemistry (Fox,
1979; Moorcroft, Davis, & Compton, 2001; Sun, Zhang, Broderick, & Fein, 2003; Woollard
& Forrest, 1984). This typically involves two separate stages, the first to determine NO2-
and a second for total NOX- after reduction of NO3- to NO2- , with the NO3- concentration
determined by difference. Either manual or automated formats are commonly employed
and the most frequently described assay utilises sulphanilamide and N-(1-naphthyl)
ethylenediamine (NED) to create a sensitive chromophore with NO2- , although other
amines are available (Tsikas, 2007).
In many official methods, the NO3- reduction is accomplished by cadmium metal in manual (IDF, 2004a) or automated mode (BS EN, 1998; IDF, 2004b,c). Cadmium reacts
very slowly with NO3- unless it is first coated with copper to give a good surface for
electron transfer between solid and solution. Zinc has also been used in official methods
as a replacement for cadmium to reduce toxicity of the analysis despite its increased
thermodynamic opportunity to reduce NO2- to NO (Merino, Edberg, Fuchs, & Aman,
2000). There is no separation of NO2- from other inorganic substances but this Griess
reaction is considered specific enough for regular use at concentrations found in most
dairy products. Enzymatic reduction can also be achieved by nitrate reductase from
Aspergillus species (BS EN, 1997b; 2005a; 2008), although NADPH has been
demonstrated to interfere with the subsequent Griess reaction. There are obvious
advantages with this strategy, despite enzyme expense, particularly for biological samples
that can use small reagent volumes (Bories & Bories, 1995; Jogben, Jobgen, Li,
Meininger, & Wu, 2007).
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The reduction of NO3- to NO2- can be avoided using ion-chromatographic methods
to separate NO2- and NO3- which are then measured individually by direct UV detection or
conductivity (Jogben et al., 2007; Reece & Hird, 2000). Such methods have been
successfully deployed and validated internationally for foods and biological samples,
although there remains significant opportunity for interference because of the low
wavelength necessary to detect NOX (BS EN, 1997a; 2005b; Jedličkova, Paluch, & Alušik,
2002). Another ion-exchange approach has been to perform NO3- reduction in post-
column mode to maintain the sensitive and selective detection modes available with the
NO2- ion. For example, Lookabaugh and Krull (1988) used photochemical reduction and
electrochemical re-oxidation of NO2- to improve detection, while Gapper, Fong, Otter,
Indyk, and Woollard (2004) used cadmium reduction and post-column addition of Griess
reagent to allow colorimetric detection at 540nm. Another HPLC technique has been
reported to combine pre-column enzyme reduction and reaction of NO2- with 2, 3-
diaminonapthalene to a stable fluorescent 2,3-napthotriazole (Jogben et al., 2007). A
successful post-column reduction strategy involved addition of trivalent vanadium to the
column eluent, which is followed by Griess reagent (Casanova, Gross, McMullen, &
Schenck, 2006). The principal advantage of vanadium (III) reduction is that it occurs in
the acidic solution compatible with the Griess reaction, the principle difficulty being the
need for elevated temperatures and inert post-column equipment to prevent corrosion of
stainless steel parts by hydrochloric acid.
Vanadium (III) has also been exploited as reductant in non-chromatographic assay formats. Thus, Braman and Hendrix (1989) analysed NOX- in water and various biological
samples including foods by reduction to nitric oxide (NO) and detection by highly sensitive
chemiluminescence. Individual samples were placed into the reduction flask without prior
clean-up, the liberated NO gas being removed by flow of helium to the detector. Similarly,
Baskić, Jovanović, Jakovljević, Delibašić, & Aresenjević (2005) used vanadium (III) to
measure NOX- but captured the NO2- as a diazo compound using Griess chemistry,
avoiding the need for gas handling associated with chemiluminescence but enhancing the
need to control inadvertent loss of the NO2- to NO. Miranda, Espey, and Wink (2001)
successfully measured NO3- and NO2- in clinical samples using trivalent vanadium
reduction and Griess reaction in microtitre wells and noted the better performance of HCl
acidification compared to H3PO4. Beda and Nedospasov (2005) modified the microtitre
assay to overcome problems associated of low NO3- in the presence of high NO2- , and
described the preparation of trivalent vanadium from V2O5 and magnesium and the
importance of leaving some tetravalent vanadium in the reaction mixture to improve
analytical precision. The reaction was described by equation 1.
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2V3+ + NO3- + 2H+ = 2V4+ + NO2- + H2O
Doane and Horwath (2003) reported a manual single-reagent procedure using
trivalent vanadium for NO3- testing of water samples and extended to a number of other
matrices. The aim of the present study was to evaluate the potential of vanadium (III)
chemistry to analyse NOX- in dairy products using a manual, non-chromatographic
technique. In this way, traditional reduction of NO3- with cadmium, zinc or enzymes can
be avoided, thereby simplifying the analysis. The reduction and reaction procedures have
been optimised, performance parameters estimated, and the method confirmed to be a
useful routine alternative for compliance testing of NOX- in dairy laboratories.
Materials and methods
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Spectral scans and absorbance readings were taken with Varian Cary 50 spectrophotometer (Varian, Palo Alto, USA) with 1.0 cm polymethyl methacrylate (PMMA)
or polystyrene (PS) macro cuvettes (Brand GMBH, Wertheim, Germany), and also
equipped with a sipper flow-through cell and a fibre-optic coupler. .
Reactions were performed in a Grant circulating water-bath with GD100 controller (Chelmsford, UK) and suitable stainless steel rack to take 20 mL (approx. volume) glass
vials. Various temperatures were used, the chosen setting being at 60 oC ± 2 oC.
Incubation at 25 oC or 40 oC within spectrophotometer cuvettes were performed in a
Contherm Series 5 (Lower Hutt, New Zealand) convection oven.
Centrifugation was performed at about 300 × g (~1000 rpm) with a Gerber
Instruments (Effretikon, Switzerland) centrifuge fitted with a 28 mm radius rotor and
buckets to carry 50 mL polypropylene tubes (Tarson 500040, Kolkata, India). Reagent
and sample dispensing were performed using 1 mL and 5 mL variable-volume pipettors,
sourced locally through Labserve (from Fisher Scientific, Vantaa, Finland). Nylon or
cellulose acetate 0.45 µm high performance liquid chromatography filters, with 3 mL
syringes were obtained from various for sample clarification. Some filters required a
prewash with 0.1 M HCl before use to remove NOX- residues.
154 155 156
Incoming water was purified by commercial-scale deionisation units with separate
cation- and anion-exchange resins. This water was then reduced to 18 MΩ resistivity by a
Barnstead Epure system with in-line charcoal filter to ensure the absence of NOX-
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Hydrochloric acid, 20%, was prepared by careful measurement, in measuring cylinders, of 1080 mL of concentrated hydrochloric acid (37%, w/w). This was transferred
to a 2 L volumetric flask and made to volume with water. All procedures were performed
in a fume cupboard.
To prepare 0.1%, w/v, vanadium trichloride (VCl3) solution, approximately 0.10 g (~100 mg) of vanadium (III) chloride (Aldrich 20,827-2) was dissolved in 100 mL 20% HCl.
The green solution was stable for many weeks under refrigerator as shown by its
absorbance near 400 nm. Although some oxidation occurred over time, as revealed by a
lighter colour, the reagent remained functional as it was prepared in excess
concentration. For enhanced stability, however, the solution was purged with nitrogen
gas. Vanadium trichloride and its solutions should be handled with care due to their
Carrez solutions were prepared as follows from Merck reagents (Darmstadt,
diluted to 200 mL (Carrez I). This solution was stable several months in the absence of
light, preferably under refrigeration. Zinc acetate dihydrate (46.0 g) was dissolved in
30.0 g potassium hexacyanoferrate (II) trihydrate was dissolved in water and
water and diluted to 200 mL (Carrez II). This was stable indefinitely, particularly under
refrigeration. Individual Griess reagents were prepared separately from Merck reagents
(Darmstadt, Germany) as follows: 5 g of sulfanilamide (Merck 1.08035) was dissolved in
10 mL concentrated hydrochloric acid and made to 500 mL with water. This was stable
for several months at 4 °C. 0.5 g N-1-naphthyl-eth ylenediamine dihydrochloride (Merck
1.06237) was dissolved in water and diluted to 250 mL. This solution was stored up to
one month in a darkened bottle, avoiding exposure to light. The combined Griess reagent
was prepared just prior to use by mixing 100 mL sulfanilamide solution and 20 mL N-1-
naphthyl-ethylenediamine dihydrochloride solutions.
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Stock standard solutions (1 mg mL-1) were prepared from pre-dried chemicals
(110 o - 120 oC to constant weight) sourced from BDH Chemicals. Potassium nitrate
(0.1629 g), containing 100 mg NO3-, was weighed quantitatively into a 100 mL volumetric
flask and diluted with water. Similarly, 0.1850 g potassium nitrite, containing 100 mg NO2-
, was weighed quantitatively into a 100 mL volumetric flask and diluted with water. These
were stored in a refrigerator and replaced monthly. For longer storage, the stock
solutions were subdivided and frozen (-20 oC). Alternatively, standards can be prepared
from sodium salts using 0.1371 g and 0.1500 g of sodium nitrate and sodium nitrite,
Intermediate NO3- and NO2- standards (10 µg mL-1) were prepared daily by dilution
of 1.00 mL of each stock solution into separate 100 mL flasks and made to volume. These
were further diluted in 10 mL volumetric flasks to generate six-point calibration curves,
including blanks, in the range 0 – 1 µg mL-1 (ppm).
201 202 203
Milk powder samples, approximately 1 g, were weighed to four decimal places into 50 mL polypropylene centrifuge tubes. Fifteen millilitres NO3-free deionised water was
added, the tubes capped and vortex mixed to dissolve. If necessary, the extracts were
warmed to about 40 oC to achieve full dissolution. In the case of liquid milk, 15.0 mL was
added to the centrifuge tube. A quality control milk powder sample with known NOX-
concentrations was included to confirm run-to-run precision.
Two mL Carrez I was added to the sample followed by 2.0 mL Carrez II to precipitate protein. The well-mixed extract was left 15 – 30 min to facilitate full
precipitation, and then centrifuged at about 300 × g to produce a clear supernatant. If a
clear supernatant was not achieved, then a further 2 mL of each Carrez solution was
added and centrifugation repeated.
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The clear upper phase was filtered through a 0.45 µm membrane into a suitable glass vial or test-tube ensuring sufficient volume was collected, sometimes requiring a
second filter. A duplicate blank was prepared similarly, by omitting the sample but
including all reagents.
220 221 222
Nitrate determination (NO3- + NO2-)
Two mL of each sample extract, standard and blank were pipetted into separate glass vials. To each was added 2.0 mL of Griess reagent, followed by 2.0 mL of vanadium
chloride solution. The vials were capped tightly to prevent evaporation and incubated in a
60 oC water bath for 40-45 min. Progress of the reaction could be seen by a developing
The vials were then mixed by inversion, cooled to room temperature in cold water
and portions transferred to disposable 1 cm disposable cuvettes. The absorbances were
read at 530 nm against water. For large sample numbers, a sipper cell was used with
low- volume cell or a fibre-optic probe for the Cary 50 spectrophotometer.
An alternative procedure was also evaluated whereby reduced volumes were pipetted directly into spectrophotometer cuvettes, with lower temperatures necessary
during incubation to prevent loss of volume by evaporation. Thus, 1.0 mL sample extract,
standard or blank, 1.0 mL mixed Griess reagent and 1.0 mL vanadium chloride solution
were mixed in a capped cuvette and incubated at either 40 oC for 3 h, or overnight at 25
Nitrite determination (NO2-)
Nitrite levels were determined in the same way except the vanadium chloride
solution was replaced with hydrochloric acid. In addition, since heating was not required,
the reactions were conveniently performed directly within disposable cuvettes, namely 1.0
mL of each sample filtrate, standard or blank, was mixed with 1.0 mL Griess reagent and
1.0 mL hydrochloric acid. After 10-15 min, the absorbance could be measured at 530 nm
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253 254 255 256
From the two standard graphs, best linear fits were calculated and used to determine the NOX- concentrations in each unknown sample in mg kg-1 using equation 2:
mg / kg =
[Absorbance of Sample − Sample Blank ] Slope of Calibration Graph
DF Weight Sample ( g )
Dilution factor DF, during sample dissolution, and clarification was usually 19 (ie
15 mL water plus 2 mL of each Carrez solution). Dilutions during colorimetry were the
same for standards and samples
The concentrations of NO3- were determined by difference (with MW correction) of
total NOX- and NO2- (equation 3)
mg NO3 kg
mg NOX kg
mg NO2 kg
263 264 2.7.
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The reactions with Griess reagents were well established from literature reports
and previous experience. The reduction step by vanadium was optimised for reagent
strength, time and temperature.
270 271 2.8.
The optimised procedure was validated for intra-laboratory repeatability by
replicate analysis of single samples. In addition, duplicate data of different samples over
many months of testing yielded further robust intermediate precision data. Intermediate
precision was also determined from control charts of three nitrite-positive samples
obtained from six months of testing with multiple analysts. Reproducibility trials with other
laboratories were not conducted.
Accuracy was estimated by spiked recoveries and by comparison with
conventional cadmium-reduction methods performed within a New Zealand inter-
laboratory proficiency program.
Limit-of-detection estimates were performed by dilution of standards until the
absorbances were no longer distinguishable from spectrophotometer noise (3σ). In
addition, samples with undetectable NOX- were repetitively analysed to determine data
scatter around zero concentration.
Results and discussion
To optimise the reduction of NO3- to NO2- a range of temperature conditions were
evaluated. At room temperature (20 oC) the reduction was slow, whereas near-boiling
temperatures facilitated a rapid reaction but also significant degradation of the coloured
Griess azo product. A temperature of 60 oC was selected and provided an acceptable
compromise between colour stability and reaction time. Fig. 1 illustrates the change in
absorbance with time at 20 oC and 60 oC for a milk powder extract, and Table 1 gives the
time to optimum signal at other temperatures. The maximum absorbance was achieved
in about 30 min at 60 oC but took 18 -24 h at ambient temperature. It has been reported
that the reduction of NO3- to NO2- by vanadium (III) is rate-limiting relative to NO2-
detection, and that complete reduction is not required provided that calibration standards
are included (Miranda et al., 2001).
Further studies involved coupling NO3- reduction and Griess reaction directly
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within the spectrophotometer cuvettes, a procedure similar to that described by Doane
and Horvath (2003). It was impractical to incubate at 60 oC without losing volume, but
incubation at 40 oC in capped cuvettes proved successful, with a reaction time of 3 hours.
Alternatively, the reduction could proceed overnight at 25 oC, although this is not ideal for
fast analytical turnaround. Carrez solutions employed for protein removal did not interfere
with vanadium reduction and were generally efficient in sample clarification. In cases
where insufficient clear supernatant was available further Carrez was required or multiple
During NO3- reduction, vanadium changes oxidation state (III → IV), with both
species being coloured. At the low pH used in this method, V3+ exists largely as a green-
grey solution with a maximum absorbance near 400 nm. The spectrum of vanadium (III)
is illustrated in Fig. 2 where the presence of the vanadyl ion VO+ is also evident at 600
nm. During the course of vanadium (III) oxidation by NO3-, the tetravalent V4+ ion is
produced, as shown by the increased spectral absorption at 765 nm.
The absorption of the VO+ ion at 600 nm potentially interferes with the red colour
of the Griess diazo compound, but the colour was so intense that this was negligible.
However, to alleviate possible risk, the determinations were performed at 530 nm rather
than the wavelength maximum of 542 nm. Fig. 3 shows overlaid spectral scans of a 1 µg
mL-1 NO3- solution over 6 h incubation at ambient temperature. During the early part of
the reaction the vanadyl ion is seen as a shoulder, so it was expected to provide some
interference in samples with low NO3-. However, this potential concern was mitigated
during quantitative work by blank subtraction.
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The strength of vanadium trichloride (≈ 6 mM) was calculated to be stoichiometrically sufficient to reduce NO3- contents in each cuvette (typically 1 – 10 µg) .
The Griess reagent was also well in excess of requirements to avoid its depletion in
typical samples. Extracts with NO3- levels above the calibration range, corresponding to
80 – 100 mg kg-1 in the milk powders, were generally retested using a smaller sample
weight to avoid exceeding the linearity of the test.
332 333 334 335
Since calibration graphs were run daily with each batch of samples, the between-
run variation in slope was a good indication of method performance. During eleven
months of method development and routine testing involving several technicians, the
mean linear regression slope for NO3- was 0.7270 with an RSD of 4.7% (n=97). During
the same period, the mean slope for NO2- was 1.0822 with an RSD of 5.3%. Assuming
complete conversion of NO3- to NO2-, the theoretical ratio of these slopes should equal the
MW ratio of NO2-/NO3- , 0.741, thus, the observed ratio of 0.672 indicates an absolute
reduction efficiency of approximately 91%. However, the use of daily calibration graphs
compensates for any reduction inefficiency so the relative recovery of NO3- was
quantitative as shown by spiked recoveries (91 – 103%, n= 14).
The calibration graphs were invariably linear (r2 >0.997) within the scope of the
method (from 0 to 1 mg L-1) for both NO3- and NO2- . However, the curves showed some
non-linearity above 5 mg L-1, possibly as a result of reagent depletion.
Blanks were an important part of the test because analyte concentrations in
samples of interest were often trending towards zero concentration. Levels of NO2- in the
calibration blanks were always near zero (intercept/slope < 0.001) but small positive
intercepts (typically 0.02 – 0.03) were common for the NO3- blanks. Potential NO3-
sources in the calibrations were water, HCl and VCl3 but these reagents were also used at
the same quantity in all samples, so intercepts were unimportant. Sample blanks
compensated for contamination from all sources including Carrez solutions, disposable
equipment and the 0.45 µm nylon filters, and were typically 0.003 – 0.005 absorbance
units for NO2- and 0.05 – 0.07 for NO3- . Filters presented a situation of variable blank
because a new one was used for each sample. Filters with low endogenous NO3- were
selected to avoid the need for each to be washed with HCl and air-dried prior to use. To
obtain best precision, sample blanks were performed in replicate.
Repeatability relative standard deviation (RSDr) estimates were determined by
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same-day analyses (n=6) of various milk powders with 10-20 mg kg-1 NO3- and 0.8 – 1.5
mg kg-1 NO2-, yielding average values of 4.29% (NO3-) and 6.62% (NO2-). Repeatability
estimates were also obtained using duplicate data across a wide range of samples of
varying NOX- thereby representing the real-life situation. Thirty-two (32) samples were
tested in duplicate where the pooled data for NO3- indicated an RSDr of 6.02% (1.38 –
44.11 mg kg-1 NO3-). The data was fairly homoscedastic, because the NO3- rarely
approaches zero in milk powders, thus, 6% represents a good RSDr estimation across all
In the case of NO2- , many results challenged the sensitivity of the test so a single estimate of repeatability was not feasible. Above 0.5 mg kg-1 (0.68 – 5.31 mg kg-1), an
RSDr of 5.4% was observed but, below this concentration (0.01 – 0.47 mg kg-1), a 47%
random error was encountered.
To obtain an estimation of between-day (intermediate) precision, four milk powder
samples were tested during many months as shown in Table 2. These samples had NOX-
concentrations typically encountered in commercial milk powders, exhibiting about 8%
random error for NO3-, acceptable in terms of the expected random error for intermediate
precision (i.e., Horrat <1). NO2- was typically much lower in concentration although
samples 3 and 4 were selected to allow meaningful error statistics, for which intermediate
precision was comparable to NO3-. Most milk powders have negligible NO2-, as found in
samples 1 and 2. In such cases the observed concentrations were scattered around
zero. Concentrations of 0.03 mg kg-1 in milk powders were therefore deemed at the limit
of detection, yielding a limit of quantitation of 0.1 mg kg-1.
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Maximum contamination levels (MCLs) vary between country and matrix but for
the New Zealand dairy industry the self-imposed limits for milk powders are 50 mg kg-1
NO3- and 1 mg kg-1 NO2-. The current method achieves these sensitivities and so is
adequate for routine quality control purposes and likely to meet the future requirements of
the milk and milk powder industries. As new-born infants are especially susceptible to
the detrimental effects of these contaminants, upper limits applied to the production of
milk-based infant formulas are generally lower at 25 - 40 mg kg-1 for NO3- and 0.5 mg kg-1
for NO2-. These requirements are also met by the current method
As a consequence of analyte instability, no food-based reference materials are
available that contain certified nitrate or nitrite levels. The accuracy of the proposed
method has therefore been evaluated by comparison with conventional methods and
spiked recovery. Thus, over the course of two years, the proposed method was tested
within a New Zealand national proficiency scheme (Fig 4). NO2- data was centrally
located among all participants because the chemistry of the proposed manual NO2-
method was equivalent to the automated conventional methods used by other
laboratories. However, NO3- results derived from the proposed V3+ reduction method
were typically higher than median data from conventional reduction methodology,
although the Z statistic was generally acceptable (<2). A UHT liquid milk sample and a
ready-to-feed infant formula, both in 250 mL tetrapak cartons, were spiked by pipetting
standard solutions to achieve NO2- and NO3- concentrations in representative ranges.
Both analytes returned acceptable recoveries as shown in Table 3, with no evidence of
positive bias for NO3-.
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The manual method for determination of nitrate and nitrite in milk and milk powders has been developed with sufficient benefit to replace traditional methods that
require the perfusion of extracts through cadmium columns. Nitrate is detected
colorimetrically with Griess reagent following reduction to nitrite with trivalent vanadium in
solution. The method avoids lengthy manipulative procedures involving excess glassware
and demonstrates figures of merit that are fit-for-purpose in dairy products. The
recommended reduction conditions are 60 oC for 30 min to meet high turnaround speeds.
The method relies on reaching a constant absorbance end-point but suits later automation
using kinetic measurements.
419 420 421
The authors wish to acknowledge the technical assistance of Mayson Kay of NZ Laboratory Services, Auckland, NZ.
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Fig. 1. Progress of nitrate reduction at ambient temperature and 60 oC.
Fig. 2. Changes in spectra during the reduction of trivalent vanadium V3+ and VO+ (398 nm and 601 nm respectively) to tetravalent vanadium V4+ (at 765 nm).
Fig. 3. Formation of Griess Diazo colour at 542 nm during 6 h of reduction of nitrate to nitrite
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at ambient temperature.
Fig. 4. Comparison of nitrite (A) and nitrate (B) data with median results from New Zealand
Incubation times required for complete nitrate reduction at different temperatures.
3 Maximum absorbance
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Intermediate precision estimates (RSDiR) for four typical milk powders.
9 SD (mg kg-1)
RSDiR (%) HorRat a
Powder 1 (N=18 b) Nitrate 68.32 Nitrite 0.03
Powder 2 (N=21) Nitrate 34.70 Nitrite 0.02
Powder 3 (N=22) Nitrate 15.26 Nitrite 5.62
Powder 4 (N=15) Nitrate 7.15 Nitrite 6.48
SC 0.82 12.2
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Mean (mg kg-1)
HorRat = Observed RSD / Predicted RSD from Horwitz equation
N, number of observations
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Recovery estimates of nitrate and nitrite added to a UHT liquid milk and ready-to-drink (RTD) infant formulation.
Nitrite (mg L ) Found NO3-
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Nitrate (mg L )
Recovery (%) -
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