International Dairy Journal 35 (2014) 88e94

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International Dairy Journal

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Colorimetric determination of nitrate and nitrite in milk and milk

powders e Use of vanadium (III) reduction
David C. Woollard a, *, Harvey E. Indyk b
a
Cawthron Institute, 98, Halifax Street East, The Woods, Nelson 7402, New Zealand
b
Fonterra Cooperative Group Ltd., Main Road, Waitoa, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: A manual method is described for the determination of nitrate and nitrite in milk and milk powders that
Received 5 April 2013 is intended to provide an alternative to conventional manual methods accomplished by cadmium
Received in revised form reduction. Reduction of nitrate is performed in solution utilising vanadium (III) and quantitation ach-
5 July 2013
ieved by concurrent reaction with Griess reagent. Performance data are acceptable in terms of precision
Accepted 22 August 2013
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 analytes.
Ó 2013 Published by Elsevier Ltd.

1. Introduction quantitation of the NO

Nitrate (NO
3 ) 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 NO
3 to
NO
2 (nitrite), which is minimised from entering the modern food
chain because of associated health risks. Although there is inevi-
tably a background level of NO
3 , 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 NO


3 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 NO


3 and NO2 , through
maintenance of systemic nitric oxide homoeostasis (Hord, Tang, &
Bryan, 2009).
Health issues and subsequent regulatory requirements have
created the need for rapid and reliable analytical methods for
* Corresponding author. Tel.: þ64 223134424.
E-mail addresses: david.woollard@nzlabs.co.nz, david.woollard@cawthron.org.nz
(D.C. Woollard).
3 and NO2 content in foods and biological
materials. There are many technology platforms for NO
x testing,
each with benefits and disadvantages, but the well-studied Griess
diazotisation reaction (Griess, 1879) involving NO
2 is the most
commonly employed detection chemistry (Fox, 1979; Moorcroft,
Davis, & Compton, 2001; Sun, Zhang, Broderick, & Fein, 2003;
Woollard & Forrest, 1980). This typically involves two separate
stages, the first to determine NO


2 and a second for total NOx after
reduction of NO
3 to NO

2 , with the NO

3 concentration determined
by difference. Either manual or automated formats are commonly
employed and the most frequently described assay utilises sul-
phanilamide and N-(1-naphthyl) ethylenediamine (NED) to create
a sensitive chromophore with NO
2 , although other amines are
available (Tsikas, 2007).
In many official methods, the NO
3 reduction is accomplished
by cadmium metal in manual (IDF, 2004a) or automated mode
(BS EN, 1998; IDF, 2004b, 2004c). Cadmium reacts very slowly
with NO
3 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 thermo-
dynamic opportunity to reduce NO
2 to NO (Merino, Edberg,
Fuchs, & Aman, 2000). There is no separation of NO
2 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 advan-
tages with this strategy, despite enzyme expense, particularly for

0958-6946/$ e see front matter Ó 2013 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.idairyj.2013.08.011
 D.C. Woollard, H.E. Indyk / International Dairy Journal 35 (2014) 88e94
biological samples that can use small reagent volumes (Bories &
Bories, 1995; Jobgen, Jobgen, Li, Meininger, & Wu, 2007).
The reduction of NO


3 to NO2 can be avoided using ion-
chromatographic methods to separate NO


2 and NO3 which are
then measured individually by direct UV detection or conductivity
(Jobgen 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; Jedli ckova, Paluch, & Alusik, 2002).
Another ion-exchange approach has been to perform NO
3 reduc-
tion in post-column mode to maintain the sensitive and selective
detection modes available with the NO
2 ion. For example,
Lookabaugh and Krull (1988) used photochemical reduction and
electrochemical re-oxidation of NO
2 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 540 nm. Another HPLC technique has
been reported to combine pre-column enzyme reduction and re-
action of NO
2 with 2, 3-diaminonapthalene to a stable fluorescent
2,3-napthotriazole (Jobgen 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 equip-
ment 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 NO
x 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,
Baskic, Jovanovic, Jakovljevi
c, Delibasi
c, and Aresenjevic (2005)
used vanadium (III) to measure NO


x 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 NO
2 to NO. Miranda, Espey,
and Wink (2001) successfully measured NO


3 and NO2 in clinical
samples using trivalent vanadium reduction and Griess reaction in
microtitre wells and noted the better performance of HCl acidifi-
cation compared to H3PO4. Beda and Nedospasov (2005) modified
the microtitre assay to overcome problems associated of low NO
3 by a lighter colour, the reagent remained functional as it was pre-
in the presence of high NO
2 , 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 Equa-
tion (1).
2V3þ þ NO
þ
3 þ 2H ¼ 2V
4þ
þ NO

2 þ H2 O
Doane and Horwath (2003) reported a manual single-reagent
procedure using trivalent vanadium for NO
3 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 NO
x in dairy products using
a manual, non-chromatographic technique. In this way, tradi-
tional reduction of NO
3 with cadmium, zinc or enzymes can be
avoided, thereby simplifying the analysis. The reduction and
reaction procedures have been optimised, performance param-
eters estimated, and the method confirmed to be a useful routine
alternative for compliance testing of NO

x in dairy laboratories.
89
2. Materials and methods
2.1. Equipment
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 cu-
vettes (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  C  2  C.
Incubation at 25  C or 40  C within spectrophotometer cuvettes
were performed in a Contherm Series 5 (Lower Hutt, New Zealand)
convection oven.
Centrifugation was performed at about 300  g (w1000 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 mm high
performance liquid chromatography filters, with 3 mL syringes
were obtained from various vendors for sample clarification. Some
filters required a prewash with 0.1 M HCl before use to remove NO
x
residues.
2.2. Reagents
Incoming water was purified by commercial-scale deionisation
units with separate cation- and anion-exchange resins. This water
was then reduced to 18 MU resistivity by a Barnstead Epure system
with in-line charcoal filter to ensure the absence of NO
x
contamination.
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 of vanadium (III) chloride (Aldrich 20,827-2)
was dissolved in 100 mL 20% HCl. The green solution was stable for
many weeks under refrigeration as shown by its absorbance near
400 nm. Although some oxidation occurred over time, as revealed
pared 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 corrosive
nature.
Carrez solutions were prepared as follows from Merck reagents
(Darmstadt, Germany): 30.0 g potassium hexacyanoferrate (II) tri-
hydrate was dissolved in water and diluted to 200 mL (Carrez I).
(1)
This solution was stable several months in the absence of light,
preferably under refrigeration. Zinc acetate dihydrate (46.0 g) was
dissolved in 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-ethylenediamine 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
90 D.C. Woollard, H.E. Indyk / International Dairy Journal 35 (2014) 88e94
prior to use by mixing 100 mL sulfanilamide solution and 20 mL N-1-
naphthyl-ethylenediamine dihydrochloride solutions.
Stock standard solutions (1 mg mL
1) were prepared from pre-
dried chemicals (110 e120  C to constant weight) sourced from
BDH Chemicals. Potassium nitrate (0.1629 g), containing 100 mg
NO
3 , was weighed quantitatively into a 100 mL volumetric flask
and diluted with water. Similarly, 0.1850 g potassium nitrite, con-
taining 100 mg NO
2 , 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  C). Alternatively,
standards can be prepared from sodium salts using 0.1371 g and
0.1500 g of sodium nitrate and sodium nitrite, respectively.
Intermediate NO


1
3 and NO2 standards (10 mg mL ) were pre-
pared 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 0e1 mg mL
1.
2.3. Sample preparation
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  C 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 NO
x 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 15e
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.
The clear upper phase was filtered through a 0.45 mm 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.
2.4. Nitrate determination (NO


3 þ 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  C water bath for 40e45 min. Progress of the reaction could
be seen by a developing red colouration.
The vials were then mixed by inversion, cooled to room tem-
perature 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, stan-
dard 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  C for 3 h, or overnight at 25  C.
2.5. Nitrite determination (NO

2)
Nitrite levels were determined in the same way except the va-
nadium 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 10e
15 min, the absorbance could be measured at 530 nm against water.
2.6. Calculations
From the two standard graphs, best linear fits were calculated
and used to determine the NO
x concentrations in each unknown
sample in mg kg
1 using Equation (2):
½Absorbance of Sample
Sample Blank
mg=kg ¼
Slope of Calibration Graph
DF
 (2)
Weight Sample ðgÞ
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 NO
3 were determined by difference (with
molecular weight correction) of total NO


x and NO2 (Equation (3))
 
mg mg mg
NO3 ¼ NOX
1:35* NO2 (3)
kg kg kg
2.7. Optimisation
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.
2.8. Validation
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 (3s). In addition, samples with un-
detectable NO
x were repetitively analysed to determine data
scatter around zero concentration.
3. Results and discussion
3.1. Colorimetric reaction
To optimise the reduction of NO


3 to NO2 a range of temperature
conditions were evaluated. At room temperature (20  C) 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  C was selected and pro-
vided an acceptable compromise between colour stability and re-
action time. Fig. 1 illustrates the change in absorbance with time at
20  C and 60  C for a milk powder extract, and Table 1 gives the
time to optimum signal at other temperatures. The maximum
 D.C. Woollard, H.E. Indyk / International Dairy Journal 35 (2014) 88e94 91

Fig. 1. Progress of nitrate reduction at ambient temperature and 60  C.

absorbance was achieved in about 30 min at 60  C but took 18e24 h

at ambient temperature. It has been reported that the reduction of
NO


3 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 NO
3 reduction and Griess
reaction directly within the spectrophotometer cuvettes, a proce-
dure similar to that described by Doane and Horwath (2003). It was
impractical to incubate at 60  C without losing volume, but incu-
bation at 40  C in capped cuvettes proved successful, with a reac-
tion time of 3 h. Alternatively, the reduction could proceed
overnight at 25  C, although this is not ideal for fast analytical
turnaround. Carrez solutions employed for protein removal did not Fig. 2. Changes in spectra during the reduction of trivalent vanadium V3þ and VOþ
(398 nm and 601 nm respectively) to tetravalent vanadium V4þ (at 765 nm).
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 filtrations. The strength of vanadium trichloride (z6 mM) was calculated to
During NO
3 reduction, vanadium changes oxidation state be stoichiometrically sufficient to reduce NO
3 contents in each
(III / IV), with both species being coloured. At the low pH used in cuvette (typically 1e10 mg). The Griess reagent was also well in
this method, V3þ exists largely as a green-grey solution with a excess of requirements to avoid its depletion in typical samples.
maximum absorbance near 400 nm. The spectrum of vanadium (III) Extracts with NO
3 levels above the calibration range, correspond-
is illustrated in Fig. 2 where the presence of the vanadyl ion VOþ is ing to 80e100 mg kg
1 in the milk powders, were generally
also evident at 600 nm. During the course of vanadium (III) retested using a smaller sample weight to avoid exceeding the
oxidation by NO
3 , the tetravalent V
4þ
ion is produced, as shown by linearity of the test.
the increased spectral absorption at 765 nm.
The absorption of the VOþ ion at 600 nm potentially interferes
3.2. Performance data
with the red colour of the Griess diazo compound, but the colour
was so intense that this was negligible. However, to alleviate
Since calibration graphs were run daily with each batch of
possible risk, the determinations were performed at 530 nm rather
samples, the between-run variation in slope was a good indication
than the wavelength maximum of 542 nm. Fig. 3 shows overlaid
of method performance. During eleven months of method devel-
spectral scans of a 1 mg mL
1 NO
3 solution over 6 h incubation at
opment and routine testing involving several technicians, the mean
ambient temperature. During the early part of the reaction the
linear regression slope for NO
3 was 0.7270 with a relative standard
vanadyl ion is seen as a shoulder, so it was expected to provide
deviation (RSD) of 4.7% (n ¼ 97). During the same period, the mean
some interference in samples with low NO
3 . However, this po-
slope for NO
2 was 1.0822 with an RSD of 5.3%. Assuming complete
tential concern was mitigated during quantitative work by blank
conversion of NO


3 to NO2 , the theoretical ratio of these slopes
subtraction.
should equal the molecular weight ratio of NO


2 /NO3 , 0.741, thus,
the observed ratio of 0.672 indicates an absolute reduction effi-
Table 1
ciency of approximately 91%. However, the use of daily calibration
Incubation times required for complete nitrate reduction at different
temperatures. graphs compensates for any reduction inefficiency so the relative
recovery of NO
3 was quantitative as shown by spiked recoveries
Temperature ( C) Maximum absorbance (min)
(91e103%, n ¼ 14).
10 >4800 The calibration graphs were invariably linear (r2 > 0.997) within
20 1500
the scope of the method (from 0 to 1 mg L
1) for both NO


3 and NO2 .
30 720
1
40 180 However, the curves showed some non-linearity above 5 mg L ,
50 50 possibly as a result of reagent depletion.
60 30 Blanks were an important part of the test because analyte
70 5 concentrations in samples of interest were often trending towards
80 1
zero concentration. Levels of NO
2 in the calibration blanks were
92 D.C. Woollard, H.E. Indyk / International Dairy Journal 35 (2014) 88e94

with 10e20 mg kg
1 NO
3 and 0.8e1.5 mg kg

1
NO

2 , yielding
average values of 4.29% (NO
3 ) and 6.62% (NO

2 ). Repeatability es-
timates were also obtained using duplicate data across a wide range
of samples of varying NO
x thereby representing the real-life situ-
ation. Thirty-two (32) samples were tested in duplicate where the
pooled data for NO
3 indicated an RSDr of 6.02% (1.38e
44.11 mg kg
1 NO
3 ). The data was fairly homoscedastic, because
the NO
3 rarely approaches zero in milk powders, thus, 6% repre-
sents a good RSDr estimation across all samples.
In the case of NO
2 , many results challenged the sensitivity of the
test so a single estimate of repeatability was not feasible. Above
0.5 mg kg
1 (0.68e5.31 mg kg
1), an RSDr of 5.4% was observed
but, below this concentration (0.01e0.47 mg kg
1), a 47% random
error was encountered.
To obtain an estimation of between-day intermediate precision
(RSD), four milk powder samples were tested during many months
as shown in Table 2. These samples had NO
x concentrations typi-
cally encountered in commercial milk powders, exhibiting about 8%
random error for NO
3 , acceptable in terms of the expected random
error for intermediate precision (i.e., Horrat <1). NO
2 was typically
much lower in concentration although samples 3 and 4 were
selected to allow meaningful error statistics, for which intermedi-
ate precision was comparable to NO
3 . Most milk powders have
negligible NO
2 , as found in samples 1 and 2. In such cases the
observed concentrations were scattered around zero.

Fig. 3. Formation of Griess diazo colour at 542 nm during 6 h of reduction of nitrate to

nitrite at ambient temperature.

always near zero (intercept/slope < 0.001) but small positive in-
tercepts (typically 0.02e0.03) were common for the NO
3 blanks.
Potential NO
3 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 compen-
sated for contamination from all sources including Carrez solutions,
disposable equipment and the 0.45 mm nylon filters, and were
typically 0.003e0.005 absorbance units for NO
2 and 0.05e0.07 for
NO
3 . Filters presented a situation of variable blank because a new
one was used for each sample. Filters with low endogenous NO
3
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 same-day analyses (n ¼ 6) of various milk powders

Table 2
Intermediate precision estimates (RSDiR) for four typical milk powders.

Sample Mean (mg kg
1) SD (mg kg
1) RSDiR (%) HorRata

b
Powder 1 (N ¼ 18 )
Nitrate 68.32 1.86 6.72 0.32
Nitrite 0.03 0.08 565 20.7
Powder 2 (N ¼ 21)
Nitrate 34.70 2.68 7.73 0.82
Nitrite 0.02 0.07 352 12.2
Powder 3 (N ¼ 22)
Nitrate 15.26 1.25 8.18 0.77
Nitrite 5.62 0.49 8.81 0.71
Powder 4 (N ¼ 15)
Nitrate 7.15 0.56 7.85 0.66
Nitrite 6.48 0.56 8.60 0.71
a
HorRat ¼ Observed RSD/Predicted RSD from Horwitz equation. Fig. 4. Comparison of nitrite (A) and nitrate (B) data with median results from New
b
N, number of observations. Zealand Proficiency Studies.
 D.C. Woollard, H.E. Indyk / International Dairy Journal 35 (2014) 88e94 93

Table 3 Acknowledgements
Recovery estimates of nitrate and nitrite added to a UHT liquid milk and ready-to-
drink (RTD) infant formulation.
The authors wish to acknowledge the technical assistance of
Drink Nitrate (mg L
1) Nitrite (mg L
1) Mayson Kay of NZ Laboratory Services, Auckland, NZ.
type
Added Found Recovery Added Found Recovery
NO

3 NO
3 (%) NO

2 NO
2 (%) References
UHT 0 4.91 e 0 0.62 e
10.00 15.13 102.2 1.00 1.69 107.0 Baski c, D., Jovanovi c, P., Jakovljevic, V., Delibasi
c, E., & Aresenjevi
c, N. (2005).
20.00 24.66 98.8 3.00 3.45 94.3 Spectrophotometric method for simultaneous detection of nitrate and nitrite.
40.00 44.01 97.8 5.00 5.62 100.0 Medicus, 6, 49e52.
Beda, N., & Nedospasov, A. (2005). A spectrophotometric assay for nitrate in an
60.00 65.43 100.9 10.00 11.12 105.0
excess of nitrite. Nitric Oxide, 13, 93e97.
RTD 0 6.62 e 0 0.22 e
Bories, P. N., & Bories, C. (1995). Nitrate determination in biological fluids by an
10.00 16.12 95.0 1.00 1.20 98.0 enzymatic one-step assay with nitrate reductase. Clinical Chemistry, 41, 904e
20.00 25.98 96.8 3.00 3.34 104.0 997.
40.00 44.99 95.9 5.00 5.04 96.4 Braman, R. S., & Hendrix, S. A. (1989). Nanogram nitrite and nitrate determination in
60.00 66.57 99.9 10.00 10.12 99.0 environmental and biological materials by vanadium (III) reduction with
Average ¼ 98.4 Average ¼ 100.5 chemiluminescence detection. Analytical Chemistry, 61, 2715e2718.
BS EN. (1997a). Foodstuffs: Determination of nitrate and/or nitrite content. HPLC/IC
method for the determination of nitrate content of vegetables and vegetable
products. BS EN 12014-2. Brussels, Belgium: European Committee of
Concentrations of 0.03 mg kg
1 in milk powders were therefore Normalisation.
deemed at the limit of detection, yielding a limit of quantitation of BS EN. (1997b). Foodstuffs: Determination of nitrate and/or nitrite content. Enzy-
matic determination of nitrate content of vegetable-containing food for babies
0.1 mg kg
1. and infants. BS EN 12014-5. Brussels, Belgium: European Committee of
Maximum contamination levels (MCLs) vary between country Normalisation.
and matrix but for the New Zealand dairy industry the self-imposed BS EN. (1998). Foodstuffs. Determination of nitrate and/or nitrite content. Continuous
flow method for the determination of nitrate content of vegetables and vegetable
limits for milk powders are 50 mg kg
1 NO
3 and 1 mg kg

1
NO
2. products after cadmium reduction. BS EN 12014-7. Brussels, Belgium: European
The current method achieves these sensitivities and so is adequate Committee of Normalisation.
for routine quality control purposes and is likely to meet the future BS EN. (2005a). Foodstuffs: Determination of nitrate and/or nitrite content e Part 3:
Spectrophotometric determination of nitrate and nitrite content of meat products
requirements of the milk and milk powder industries. As new-born after enzymatic reduction of nitrate to nitrite. BS EN 12014-3. Brussels, Belgium:
infants are especially susceptible to the detrimental effects of these European Committee of Normalisation.
contaminants, upper limits applied to the production of milk-based BS EN. (2005b). Foodstuffs: Determination of nitrate and/or nitrite content. Ion-
infant formulae are generally lower at 25e40 mg kg
1 for NO

exchange chromatographic (IC) method for the determination of nitrate and ni-
3 and trite content of meat products. BS EN 12014-4. Brussels, Belgium: European
0.5 mg kg
1 for NO
2 . These requirements are also met by the cur- Committee of Normalisation.
rent method. BS EN. (2008). Milk and milk products: Determination of nitrate content e Method by
enzymatic reduction and molecular-absorption spectrometry after Griess reaction.
As a consequence of analyte instability, no food-based reference
BS EN ISO 20541. Brussels, Belgium: European Committee of Normalisation.
materials are available that contain certified nitrate or nitrite levels. Casanova, J. A., Gross, L. K., McMullen, S. E., & Schenck, F. J. (2006). Use of Griess
The accuracy of the proposed method has therefore been evaluated reagent containing vanadium (III) for post-column derivatization and simulta-
by comparison with conventional methods and spiked recovery. neous determination of nitrite and nitrate in baby food. Journal of AOAC Inter-
national, 89, 447e451.
Thus, over the course of two years, the proposed method was tested Doane, T. A., & Horwath. (2003). Spectrophotometric determination of nitrate with
within a New Zealand national proficiency scheme (Fig. 4). NO
2 a single reagent. Analytical Letters, 36, 2713e2722.
data was centrally located among all participants because the Ellis, G., Adatia, I., Yazdanpanah, M., & Makela, S. K. (1998). Nitrite and nitrate an-
alyses: a clinical biochemistry perspective. Clinical Biochemistry, 31, 195e220.
chemistry of the proposed manual NO
2 method was equivalent to Fox, J. B. (1979). Kinetics and mechanisms of the Griess reaction. Analytical Chem-
the automated conventional methods used by other laboratories. istry, 51, 1493e1502.
However, NO
3 results derived from the proposed V
3þ
reduction Gapper, L. W., Fong, B. Y., Otter, D. E., Indyk, H. E., & Woollard, D. C. (2004).
Determination of nitrite and nitrate in dairy products by ion exchange LC with
method were typically higher than median data from conventional spectrophotometric detection. International Dairy Journal, 14, 881e887.
reduction methodology, although the Z statistic was generally Griess, J. P. (1879). Bemerkungen zu der Abhandlung der H.H. Weselsky und Ben-
acceptable (<2). A UHT liquid milk sample and a ready-to-feed edikt, ueber einige Azoverbindungen. Berichte der Deutschen Chemistry Gesell-
schaft, 12, 426e428.
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2 and NO3 concentra- physiological context for potential health benefits. American Journal Clinical
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recoveries as shown in Table 3, with no evidence of positive bias
Part 1: Method using cadmium reduction and spectrometry. ISO 14673-1:2004,
for NO
3. IDF 189-1. Brussels, Belgium: International Dairy Federation.
IDF. (2004b). Milk and milk products e Determination of nitrate and nitrite contents e
Part 2: Method using segmented flow analysis (routine method). ISO 14673-2:
4. Conclusions 2004, IDF 189-1. Brussels, Belgium: International Dairy Federation.
IDF. (2004c). Milk and milk products e Determination of nitrate and nitrite contents e
Part 3: Method using cadmium reduction and flow injection analysis with in-line
The manual method for determination of nitrate and nitrite in dialysis (routine method). ISO 14673-3:2004, IDF 189-1. Brussels, Belgium: In-
milk and milk powders has been developed with sufficient benefit ternational Dairy Federation.
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