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Colorimetric determination of nitrate and nitrite in milk and milk powders – Use of vanadium (III) reduction
Accepted Manuscript Colorimetric determination of nitrate and nitrite in milk and milk powders – Use of vanadium (III) reduction David C. Woollard, Harvey Indyk PII:
S0958-6946(13)00227-6
DOI:
10.1016/j.idairyj.2013.08.011
Reference:
INDA 3574
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.
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Colorimetric determination of nitrate and nitrite in milk and milk powders –
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Use of vanadium (III) reduction
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4 5 6 7 David C. Woollarda*, Harvey Indykb
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a
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Zealand
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New Zealand Laboratory Services, 35, O’Rorke Rd, Penrose, Auckland 1642, New
Fonterra, Main Road, Waitoa, New Zealand
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* Corresponding author. Tel.: +6495264514
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E-mail address: [email protected] (D. C. Woollard)
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Abstract
27 A manual method is described for the determination of nitrate and nitrite in milk
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and milk powders that is intended to provide an alternative to conventional manual
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methods accomplished by cadmium reduction. Reduction of nitrate is performed in
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solution utilising vanadium (III) and quantitation achieved by concurrent reaction with
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Griess reagent. Performance data are acceptable in terms of precision and accuracy,
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repeatability being about 6% and intermediate precision at 8% for both analytes, providing
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the limit of detection is not approached. Limit of quantitation is 0.1 mg kg-1 for both
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analytes.
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1.
Introduction
42 Nitrate (NO3-) exists throughout the biosphere from natural and man-made
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processes and is an important component of biological life-cycles; hence it is well-studied
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clinically (Ellis, Adatia, Yazdanpanah, & Makela, 1998). Bacterial action can reduce NO3-
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to NO2- (nitrite), which is minimised from entering the modern food chain because of
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associated health risks. Although there is inevitably a background level of NO3- , it is
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regularly monitored within the dairy industry as part of normal sanitary programs. Nitrite
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levels are also monitored, since microbiologically-contaminated water or post-secretory
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milk can contain bacteria with significant nitrate reductase activity. The widespread use of
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nitrogen-based fertilisers, combined with domestic, agricultural and industrial wastes,
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have increased the chances of NO3- and NO2- incorporation into manufactured dairy
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products (Indyk & Woollard, 2011). In addition, the use of nitric acid as a sanitiser within
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dairy plants represents a further risk of nitrate contamination. Although generally
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associated with increased risk of several pathologies, it is notable that recent studies
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indicate that health benefits may be derived from consumption of dietary NO3- and NO2- ,
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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
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rapid and reliable analytical methods for quantitation of the NO3- and NO2- content in
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foods and biological materials. There are many technology platforms for NOX- testing,
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each with benefits and disadvantages, but the well-studied Griess diazotisation reaction
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(Griess, 1879) involving NO2- is the most commonly employed detection chemistry (Fox,
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1979; Moorcroft, Davis, & Compton, 2001; Sun, Zhang, Broderick, & Fein, 2003; Woollard
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& Forrest, 1984). This typically involves two separate stages, the first to determine NO2-
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and a second for total NOX- after reduction of NO3- to NO2- , with the NO3- concentration
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determined by difference. Either manual or automated formats are commonly employed
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and the most frequently described assay utilises sulphanilamide and N-(1-naphthyl)
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ethylenediamine (NED) to create a sensitive chromophore with NO2- , although other
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amines are available (Tsikas, 2007).
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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
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very slowly with NO3- unless it is first coated with copper to give a good surface for
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electron transfer between solid and solution. Zinc has also been used in official methods
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as a replacement for cadmium to reduce toxicity of the analysis despite its increased
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thermodynamic opportunity to reduce NO2- to NO (Merino, Edberg, Fuchs, & Aman,
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2000). There is no separation of NO2- from other inorganic substances but this Griess
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reaction is considered specific enough for regular use at concentrations found in most
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dairy products. Enzymatic reduction can also be achieved by nitrate reductase from
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Aspergillus species (BS EN, 1997b; 2005a; 2008), although NADPH has been
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demonstrated to interfere with the subsequent Griess reaction. There are obvious
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advantages with this strategy, despite enzyme expense, particularly for biological samples
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that can use small reagent volumes (Bories & Bories, 1995; Jogben, Jobgen, Li,
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Meininger, & Wu, 2007).
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The reduction of NO3- to NO2- can be avoided using ion-chromatographic methods
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to separate NO2- and NO3- which are then measured individually by direct UV detection or
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conductivity (Jogben et al., 2007; Reece & Hird, 2000). Such methods have been
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successfully deployed and validated internationally for foods and biological samples,
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although there remains significant opportunity for interference because of the low
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wavelength necessary to detect NOX (BS EN, 1997a; 2005b; Jedličkova, Paluch, & Alušik,
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2002). Another ion-exchange approach has been to perform NO3- reduction in post-
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column mode to maintain the sensitive and selective detection modes available with the
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NO2- ion. For example, Lookabaugh and Krull (1988) used photochemical reduction and
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electrochemical re-oxidation of NO2- to improve detection, while Gapper, Fong, Otter,
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Indyk, and Woollard (2004) used cadmium reduction and post-column addition of Griess
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reagent to allow colorimetric detection at 540nm. Another HPLC technique has been
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reported to combine pre-column enzyme reduction and reaction of NO2- with 2, 3-
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diaminonapthalene to a stable fluorescent 2,3-napthotriazole (Jogben et al., 2007). A
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successful post-column reduction strategy involved addition of trivalent vanadium to the
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column eluent, which is followed by Griess reagent (Casanova, Gross, McMullen, &
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Schenck, 2006). The principal advantage of vanadium (III) reduction is that it occurs in
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the acidic solution compatible with the Griess reaction, the principle difficulty being the
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need for elevated temperatures and inert post-column equipment to prevent corrosion of
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stainless steel parts by hydrochloric acid.
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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
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samples including foods by reduction to nitric oxide (NO) and detection by highly sensitive
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chemiluminescence. Individual samples were placed into the reduction flask without prior
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clean-up, the liberated NO gas being removed by flow of helium to the detector. Similarly,
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Baskić, Jovanović, Jakovljević, Delibašić, & Aresenjević (2005) used vanadium (III) to
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measure NOX- but captured the NO2- as a diazo compound using Griess chemistry,
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avoiding the need for gas handling associated with chemiluminescence but enhancing the
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need to control inadvertent loss of the NO2- to NO. Miranda, Espey, and Wink (2001)
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successfully measured NO3- and NO2- in clinical samples using trivalent vanadium
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reduction and Griess reaction in microtitre wells and noted the better performance of HCl
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acidification compared to H3PO4. Beda and Nedospasov (2005) modified the microtitre
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assay to overcome problems associated of low NO3- in the presence of high NO2- , and
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described the preparation of trivalent vanadium from V2O5 and magnesium and the
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importance of leaving some tetravalent vanadium in the reaction mixture to improve
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analytical precision. The reaction was described by equation 1.
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2V3+ + NO3- + 2H+ = 2V4+ + NO2- + H2O
(1)
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Doane and Horwath (2003) reported a manual single-reagent procedure using
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trivalent vanadium for NO3- testing of water samples and extended to a number of other
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matrices. The aim of the present study was to evaluate the potential of vanadium (III)
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chemistry to analyse NOX- in dairy products using a manual, non-chromatographic
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technique. In this way, traditional reduction of NO3- with cadmium, zinc or enzymes can
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be avoided, thereby simplifying the analysis. The reduction and reaction procedures have
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been optimised, performance parameters estimated, and the method confirmed to be a
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useful routine alternative for compliance testing of NOX- in dairy laboratories.
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2.
Materials and methods
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2.1.
Equipment
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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)
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or polystyrene (PS) macro cuvettes (Brand GMBH, Wertheim, Germany), and also
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equipped with a sipper flow-through cell and a fibre-optic coupler. .
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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
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vials. Various temperatures were used, the chosen setting being at 60 oC ± 2 oC.
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Incubation at 25 oC or 40 oC within spectrophotometer cuvettes were performed in a
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Contherm Series 5 (Lower Hutt, New Zealand) convection oven.
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Centrifugation was performed at about 300 × g (~1000 rpm) with a Gerber
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Instruments (Effretikon, Switzerland) centrifuge fitted with a 28 mm radius rotor and
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buckets to carry 50 mL polypropylene tubes (Tarson 500040, Kolkata, India). Reagent
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and sample dispensing were performed using 1 mL and 5 mL variable-volume pipettors,
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sourced locally through Labserve (from Fisher Scientific, Vantaa, Finland). Nylon or
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cellulose acetate 0.45 µm high performance liquid chromatography filters, with 3 mL
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syringes were obtained from various for sample clarification. Some filters required a
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prewash with 0.1 M HCl before use to remove NOX- residues.
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2.2.
Reagents
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Incoming water was purified by commercial-scale deionisation units with separate
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cation- and anion-exchange resins. This water was then reduced to 18 MΩ resistivity by a
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Barnstead Epure system with in-line charcoal filter to ensure the absence of NOX-
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contamination.
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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
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to a 2 L volumetric flask and made to volume with water. All procedures were performed
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in a fume cupboard.
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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.
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The green solution was stable for many weeks under refrigerator as shown by its
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absorbance near 400 nm. Although some oxidation occurred over time, as revealed by a
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lighter colour, the reagent remained functional as it was prepared in excess
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concentration. For enhanced stability, however, the solution was purged with nitrogen
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gas. Vanadium trichloride and its solutions should be handled with care due to their
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corrosive nature
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Carrez solutions were prepared as follows from Merck reagents (Darmstadt,
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Germany):
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diluted to 200 mL (Carrez I). This solution was stable several months in the absence of
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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
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water and diluted to 200 mL (Carrez II). This was stable indefinitely, particularly under
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refrigeration. Individual Griess reagents were prepared separately from Merck reagents
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(Darmstadt, Germany) as follows: 5 g of sulfanilamide (Merck 1.08035) was dissolved in
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10 mL concentrated hydrochloric acid and made to 500 mL with water. This was stable
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for several months at 4 °C. 0.5 g N-1-naphthyl-eth ylenediamine dihydrochloride (Merck
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1.06237) was dissolved in water and diluted to 250 mL. This solution was stored up to
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one month in a darkened bottle, avoiding exposure to light. The combined Griess reagent
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was prepared just prior to use by mixing 100 mL sulfanilamide solution and 20 mL N-1-
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naphthyl-ethylenediamine dihydrochloride solutions.
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Stock standard solutions (1 mg mL-1) were prepared from pre-dried chemicals
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(110 o - 120 oC to constant weight) sourced from BDH Chemicals. Potassium nitrate
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(0.1629 g), containing 100 mg NO3-, was weighed quantitatively into a 100 mL volumetric
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flask and diluted with water. Similarly, 0.1850 g potassium nitrite, containing 100 mg NO2-
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, was weighed quantitatively into a 100 mL volumetric flask and diluted with water. These
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were stored in a refrigerator and replaced monthly. For longer storage, the stock
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solutions were subdivided and frozen (-20 oC). Alternatively, standards can be prepared
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from sodium salts using 0.1371 g and 0.1500 g of sodium nitrate and sodium nitrite,
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respectively.
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Intermediate NO3- and NO2- standards (10 µg mL-1) were prepared daily by dilution
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of 1.00 mL of each stock solution into separate 100 mL flasks and made to volume. These
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were further diluted in 10 mL volumetric flasks to generate six-point calibration curves,
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including blanks, in the range 0 – 1 µg mL-1 (ppm).
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2.3.
Sample preparation
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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
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added, the tubes capped and vortex mixed to dissolve. If necessary, the extracts were
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warmed to about 40 oC to achieve full dissolution. In the case of liquid milk, 15.0 mL was
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added to the centrifuge tube. A quality control milk powder sample with known NOX-
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concentrations was included to confirm run-to-run precision.
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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
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precipitation, and then centrifuged at about 300 × g to produce a clear supernatant. If a
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clear supernatant was not achieved, then a further 2 mL of each Carrez solution was
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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
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second filter. A duplicate blank was prepared similarly, by omitting the sample but
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including all reagents.
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2.4.
Nitrate determination (NO3- + NO2-)
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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
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chloride solution. The vials were capped tightly to prevent evaporation and incubated in a
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60 oC water bath for 40-45 min. Progress of the reaction could be seen by a developing
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red coloration.
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The vials were then mixed by inversion, cooled to room temperature in cold water
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and portions transferred to disposable 1 cm disposable cuvettes. The absorbances were
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read at 530 nm against water. For large sample numbers, a sipper cell was used with
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low- volume cell or a fibre-optic probe for the Cary 50 spectrophotometer.
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An alternative procedure was also evaluated whereby reduced volumes were pipetted directly into spectrophotometer cuvettes, with lower temperatures necessary
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during incubation to prevent loss of volume by evaporation. Thus, 1.0 mL sample extract,
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standard or blank, 1.0 mL mixed Griess reagent and 1.0 mL vanadium chloride solution
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were mixed in a capped cuvette and incubated at either 40 oC for 3 h, or overnight at 25
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Nitrite determination (NO2-)
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Nitrite levels were determined in the same way except the vanadium chloride
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solution was replaced with hydrochloric acid. In addition, since heating was not required,
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the reactions were conveniently performed directly within disposable cuvettes, namely 1.0
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mL of each sample filtrate, standard or blank, was mixed with 1.0 mL Griess reagent and
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1.0 mL hydrochloric acid. After 10-15 min, the absorbance could be measured at 530 nm
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against water.
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2.6. Calculations
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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:
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mg / kg =
[Absorbance of Sample − Sample Blank ] Slope of Calibration Graph
x
DF Weight Sample ( g )
(2)
Dilution factor DF, during sample dissolution, and clarification was usually 19 (ie
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15 mL water plus 2 mL of each Carrez solution). Dilutions during colorimetry were the
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same for standards and samples
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The concentrations of NO3- were determined by difference (with MW correction) of
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total NOX- and NO2- (equation 3)
mg NO3 kg
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=
mg NOX kg
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1.35
*
mg NO2 kg
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Optimisation
(3)
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The reactions with Griess reagents were well established from literature reports
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and previous experience. The reduction step by vanadium was optimised for reagent
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strength, time and temperature.
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Validation
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The optimised procedure was validated for intra-laboratory repeatability by
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replicate analysis of single samples. In addition, duplicate data of different samples over
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many months of testing yielded further robust intermediate precision data. Intermediate
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precision was also determined from control charts of three nitrite-positive samples
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obtained from six months of testing with multiple analysts. Reproducibility trials with other
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laboratories were not conducted.
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Accuracy was estimated by spiked recoveries and by comparison with
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conventional cadmium-reduction methods performed within a New Zealand inter-
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laboratory proficiency program.
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Limit-of-detection estimates were performed by dilution of standards until the
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absorbances were no longer distinguishable from spectrophotometer noise (3σ). In
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addition, samples with undetectable NOX- were repetitively analysed to determine data
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scatter around zero concentration.
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3.
Results and discussion
3.1.
Colorimetric Reaction
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To optimise the reduction of NO3- to NO2- a range of temperature conditions were
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evaluated. At room temperature (20 oC) the reduction was slow, whereas near-boiling
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temperatures facilitated a rapid reaction but also significant degradation of the coloured
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Griess azo product. A temperature of 60 oC was selected and provided an acceptable
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compromise between colour stability and reaction time. Fig. 1 illustrates the change in
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absorbance with time at 20 oC and 60 oC for a milk powder extract, and Table 1 gives the
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time to optimum signal at other temperatures. The maximum absorbance was achieved
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in about 30 min at 60 oC but took 18 -24 h at ambient temperature. It has been reported
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that the reduction of NO3- to NO2- by vanadium (III) is rate-limiting relative to NO2-
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detection, and that complete reduction is not required provided that calibration standards
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are included (Miranda et al., 2001).
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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
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and Horvath (2003). It was impractical to incubate at 60 oC without losing volume, but
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incubation at 40 oC in capped cuvettes proved successful, with a reaction time of 3 hours.
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Alternatively, the reduction could proceed overnight at 25 oC, although this is not ideal for
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fast analytical turnaround. Carrez solutions employed for protein removal did not interfere
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with vanadium reduction and were generally efficient in sample clarification. In cases
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where insufficient clear supernatant was available further Carrez was required or multiple
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filtrations.
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During NO3- reduction, vanadium changes oxidation state (III → IV), with both
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species being coloured. At the low pH used in this method, V3+ exists largely as a green-
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grey solution with a maximum absorbance near 400 nm. The spectrum of vanadium (III)
315
is illustrated in Fig. 2 where the presence of the vanadyl ion VO+ is also evident at 600
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nm. During the course of vanadium (III) oxidation by NO3-, the tetravalent V4+ ion is
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produced, as shown by the increased spectral absorption at 765 nm.
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The absorption of the VO+ ion at 600 nm potentially interferes with the red colour
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of the Griess diazo compound, but the colour was so intense that this was negligible.
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However, to alleviate possible risk, the determinations were performed at 530 nm rather
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than the wavelength maximum of 542 nm. Fig. 3 shows overlaid spectral scans of a 1 µg
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mL-1 NO3- solution over 6 h incubation at ambient temperature. During the early part of
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the reaction the vanadyl ion is seen as a shoulder, so it was expected to provide some
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interference in samples with low NO3-. However, this potential concern was mitigated
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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) .
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The Griess reagent was also well in excess of requirements to avoid its depletion in
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typical samples. Extracts with NO3- levels above the calibration range, corresponding to
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80 – 100 mg kg-1 in the milk powders, were generally retested using a smaller sample
331
weight to avoid exceeding the linearity of the test.
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3.2.
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Performance data
Since calibration graphs were run daily with each batch of samples, the between-
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run variation in slope was a good indication of method performance. During eleven
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months of method development and routine testing involving several technicians, the
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mean linear regression slope for NO3- was 0.7270 with an RSD of 4.7% (n=97). During
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the same period, the mean slope for NO2- was 1.0822 with an RSD of 5.3%. Assuming
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complete conversion of NO3- to NO2-, the theoretical ratio of these slopes should equal the
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MW ratio of NO2-/NO3- , 0.741, thus, the observed ratio of 0.672 indicates an absolute
342
reduction efficiency of approximately 91%. However, the use of daily calibration graphs
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compensates for any reduction inefficiency so the relative recovery of NO3- was
344
quantitative as shown by spiked recoveries (91 – 103%, n= 14).
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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
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non-linearity above 5 mg L-1, possibly as a result of reagent depletion.
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Blanks were an important part of the test because analyte concentrations in
349
samples of interest were often trending towards zero concentration. Levels of NO2- in the
350
calibration blanks were always near zero (intercept/slope < 0.001) but small positive
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intercepts (typically 0.02 – 0.03) were common for the NO3- blanks. Potential NO3-
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sources in the calibrations were water, HCl and VCl3 but these reagents were also used at
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the same quantity in all samples, so intercepts were unimportant. Sample blanks
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compensated for contamination from all sources including Carrez solutions, disposable
355
equipment and the 0.45 µm nylon filters, and were typically 0.003 – 0.005 absorbance
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units for NO2- and 0.05 – 0.07 for NO3- . Filters presented a situation of variable blank
357
because a new one was used for each sample. Filters with low endogenous NO3- were
358
selected to avoid the need for each to be washed with HCl and air-dried prior to use. To
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obtain best precision, sample blanks were performed in replicate.
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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
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mg kg-1 NO2-, yielding average values of 4.29% (NO3-) and 6.62% (NO2-). Repeatability
363
estimates were also obtained using duplicate data across a wide range of samples of
364
varying NOX- thereby representing the real-life situation. Thirty-two (32) samples were
365
tested in duplicate where the pooled data for NO3- indicated an RSDr of 6.02% (1.38 –
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44.11 mg kg-1 NO3-). The data was fairly homoscedastic, because the NO3- rarely
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approaches zero in milk powders, thus, 6% represents a good RSDr estimation across all
368
samples.
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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
371
RSDr of 5.4% was observed but, below this concentration (0.01 – 0.47 mg kg-1), a 47%
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random error was encountered.
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To obtain an estimation of between-day (intermediate) precision, four milk powder
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samples were tested during many months as shown in Table 2. These samples had NOX-
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concentrations typically encountered in commercial milk powders, exhibiting about 8%
376
random error for NO3-, acceptable in terms of the expected random error for intermediate
377
precision (i.e., Horrat <1). NO2- was typically much lower in concentration although
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samples 3 and 4 were selected to allow meaningful error statistics, for which intermediate
379
precision was comparable to NO3-. Most milk powders have negligible NO2-, as found in
380
samples 1 and 2. In such cases the observed concentrations were scattered around
381
zero. Concentrations of 0.03 mg kg-1 in milk powders were therefore deemed at the limit
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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
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the New Zealand dairy industry the self-imposed limits for milk powders are 50 mg kg-1
385
NO3- and 1 mg kg-1 NO2-. The current method achieves these sensitivities and so is
386
adequate for routine quality control purposes and likely to meet the future requirements of
387
the milk and milk powder industries. As new-born infants are especially susceptible to
388
the detrimental effects of these contaminants, upper limits applied to the production of
389
milk-based infant formulas are generally lower at 25 - 40 mg kg-1 for NO3- and 0.5 mg kg-1
390
for NO2-. These requirements are also met by the current method
391
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As a consequence of analyte instability, no food-based reference materials are
392
available that contain certified nitrate or nitrite levels. The accuracy of the proposed
393
method has therefore been evaluated by comparison with conventional methods and
394
spiked recovery. Thus, over the course of two years, the proposed method was tested
15
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within a New Zealand national proficiency scheme (Fig 4). NO2- data was centrally
396
located among all participants because the chemistry of the proposed manual NO2-
397
method was equivalent to the automated conventional methods used by other
398
laboratories. However, NO3- results derived from the proposed V3+ reduction method
399
were typically higher than median data from conventional reduction methodology,
400
although the Z statistic was generally acceptable (<2). A UHT liquid milk sample and a
401
ready-to-feed infant formula, both in 250 mL tetrapak cartons, were spiked by pipetting
402
standard solutions to achieve NO2- and NO3- concentrations in representative ranges.
403
Both analytes returned acceptable recoveries as shown in Table 3, with no evidence of
404
positive bias for NO3-.
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405 406
4.
Conclusions
407 408
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
410
require the perfusion of extracts through cadmium columns. Nitrate is detected
411
colorimetrically with Griess reagent following reduction to nitrite with trivalent vanadium in
412
solution. The method avoids lengthy manipulative procedures involving excess glassware
413
and demonstrates figures of merit that are fit-for-purpose in dairy products. The
414
recommended reduction conditions are 60 oC for 30 min to meet high turnaround speeds.
415
The method relies on reaching a constant absorbance end-point but suits later automation
416
using kinetic measurements.
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Acknowledgements
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The authors wish to acknowledge the technical assistance of Mayson Kay of NZ Laboratory Services, Auckland, NZ.
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References
424 425
Baskić, D., Jovanović, P., Jakovljević, V., Delibašić, E, & Aresenjević, N. (2005). Spectrophotometric method for simultaneous detection of nitrate and nitrite,
427
Medicus, 6, 49-52.
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Beda, N., & Nedospasov, A. (2005). A spectrophotometric assay for nitrate in an excess of nitrite, Nitric Oxide, 13, 93-97.
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Bories, P. N., & Bories, C. (1995). Nitrate determination in biological fluids by an
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enzymatic one-step assay with nitrate reductase, Clinical Chemistry, 41, 904-97.
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Braman, R. S., & Hendrix, S. A. (1989). Nanogram nitrite and nitrate determination in
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environmental and biological materials by vanadium (III) reduction with
437
chemiluminescence detection, Analytical Chemistry, 61, 2715-2718.
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BS EN (1997a). Foodstuffs: Determination of nitrate and/or nitrite content. HPLC/IC method for the determination of nitrate content of vegetables and vegetable
441
products. BS EN 12014-2:1997. Brussels, Belgium: European Committee of
442
Normalisation.
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447
Normalisation.
444 445
BS EN (1997b). Foodstuffs: Determination of nitrate and/or nitrite content. Enzymatic determination of nitrate content of vegetable-containing food for babies and infants. BS EN 12014-5:1997. Brussels, Belgium: European Committee of
448 449
BS EN (1998). Foodstuffs. Determination of nitrate and/or nitrite content. Continuous flow
450
method for the determination of nitrate content of vegetables and vegetable
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451
products after cadmium reduction. BS EN 12014-7:1998. Brussels, Belgium:
452
European Committee of Normalisation.
453 BS EN (2005a). Foodstuffs: Determination of nitrate and/or nitrite content- Part 3:
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454 455
Spectrophotometric determination of nitrate and nitrite content of meat products
456
after enzymatic reduction of nitrate to nitrite. BS EN 12014-3:2005. Brussels,
457
Belgium: European Committee of Normalisation.
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BS EN (2005b). Foodstuffs: Determination of nitrate and/or nitrite content. Ion-exchange
460
chromatographic (IC) method for the determination of nitrate and nitrite content of
461
meat products. BS EN 12014-4:2005. Brussels, Belgium: European Committee of
462
Normalisation.
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BS EN (2008). Milk and milk products: Determination of nitrate content – Method by enzymatic reduction and molecular-absorption spectrometry after Griess reaction.
466
BS EN ISO 20541:2008. Brussels, Belgium: European Committee of
467
Normalisation.
468
Casanova, J. A., Gross, L. K., McMullen, S. E., & Schenck, F. J. (2006). Use of Griess
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reagent containing Vanadium (III) for post-column derivatization and simultaneous
471
determination of nitrite and nitrate in baby food. Journal of AOAC International, 89,
472 473 474 475
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447-451.
Doane, T. A., & Horwath, (2003). Spectrophotometric determination of nitrate with a single reagent. Analytical letters, 36, 2713-2722.
476 477 478
Ellis, G., Adatia, I., Yazdanpanah, M., & Makela, S. K. (1998). Nitrite and nitrate analyses: a clinical biochemistry perspective. Clinical Biochemistry, 31, 195-220.
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Fox, J. B. (1979). Kinetics and mechanisms of the Griess reaction. Analytical Chemistry, 51, 1493-1502.
483
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482 Gapper, L. W., Fong, B. Y., Otter, D. E., Indyk, H. E., & Woollard, D. C. (2004).
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Determination of nitrite and nitrate in dairy products by ion exchange LC with
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spectrophotometric detection. International Dairy Journal, 14, 881-887.
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Griess, J. P. (1879). Bemerkungen zu der Abhandlung der H.H. Weselsky und Benedikt,
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ueber einige Azoverbindungen. Berichte der Deutsch. Chem. Ges., 12, 426-428.
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Hord, N. G., Tang, Y., & Bryan, N. S. (2009). Food sources of nitrates and nitrites: the
491
physiological context for potential health benefits. American Journal Clinical
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Nutrition, 90, 1-10.
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Indyk, H. E., and Woollard, D.C. (2011). Nitrates and nitrites as contaminants. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney, Encyclopaedia of dairy sciences, (2nd
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edn., Vol. 1, pp. 906-991): San Diego, CA, USA: Academic Press.
498 499 500 501 502
IDF. (2004a). Milk and milk products – Determination of nitrate and nitrite contents – Part 1: Method using cadmium reduction and spectrometry. ISO 14673-1:2004, IDF
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189-1:2004. Brussels, Belgium: International Dairy Federation.
IDF. (2004b). Milk and milk products – Determination of nitrate and nitrite contents – Part
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2: Method using segmented flow analysis (routine method). ISO 14673-2:2004,
504
IDF 189-1:2004 Brussels, Belgium: International Dairy Federation.
505
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506
IDF. (2004c). Milk and milk products – Determination of nitrate and nitrite contents – Part 3: Method using cadmium reduction and flow injection analysis with in-line dialysis
508
(routine method). ISO 14673-3:2004, IDF 189-1:2004. Brussels, Belgium:
509
International Dairy Federation.
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507
510
Jedličkova, V., Paluch, Z., & Alušik, S. (2002). Determination of nitrate and nitrite by high-
512
performance liquid chromatography in human plasma, Journal of Chromatography
513
B, 851, 51-70.
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511
514
Jobgen, W. S., Jobgen, S. C., Li, H., Meininger, C. J., & Wu, G. (2007). Analysis of nitrite
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and nitrate in biological samples using high-performance liquid chromatography.
517
Journal of Chromatography B, 851, 71-82.
518 519
Lookabaugh, M., & Krull, I. S. (1988), Determination of nitrite and nitrate by reversedphase high-performance liquid chromatography using on-line post-column
521
photolysis with ultraviolet absorbance and electrochemical detection, Journal of
522
Chromatography, 452, 295-308.
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520
523
Merino, L, Edberg, U, Fuchs, G., & Aman, P. (2000). Liquid chromatographic
EP
524
determination of residual nitrite/nitrate in foods: NMKL collaborative study. Journal
526
of AOAC International, 83, 365-375.
527 528 529
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Miranda, K. M., Espey, M. G., & Wink, D. A. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide, 5, 62-71.
530 531 532
Moorcroft, M. J., Davis, J., & Compton, R. G. (2001). Detection and determination of nitrate and nitrite: a review. Talanta, 54, 785-803.
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534
Reece, P., & Hird, H. (2000). Modification of the ion exchange HPLC procedure for the
535
detection of nitrate and nitrite in dairy products. Food Additives and Contaminants,
536
17, 219-222.
541
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537
542
Tsikas, D. (2007). Analysis of nitrite and nitrate in biological fluids by assays based on the
538
Sun, J., Zhang, X., Broderick, M., & Fein, H. (2003). Measurement of nitric oxide
production in biological systems by using Griess reaction assay, Sensors, 3, 276-
540
284.
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539
Griess reaction: Appraisal of the Griess reaction in the L-arginine/nitric oxide area
544
of research. Journal of Chromatography B, 851, 51-70.
545 546
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Woollard, D. C., & Forrest, L. J. (1980). Levels of nitrate and nitrite in casein products using an automated procedure, New Zealand Journal of Dairy Science and
548
Technology, 15, 83-90.
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Figure legends
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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).
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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
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Proficiency Studies.
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1
Table 1
2
Incubation times required for complete nitrate reduction at different temperatures.
3 Maximum absorbance
(oC)
(min)
10
>4800
20
1500
30
720
40
180
50
50
60
30
70
5
80
1
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Temperature
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5
7
Table 2
8
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
1.86 0.08
6.72 565
Powder 2 (N=21) Nitrate 34.70 Nitrite 0.02
2.68 0.07
7.73 352
Powder 3 (N=22) Nitrate 15.26 Nitrite 5.62
1.25 0.49
Powder 4 (N=15) Nitrate 7.15 Nitrite 6.48
0.56 0.56
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8.18 8.81
0.77 0.71
7.85 8.60
0.66 0.71
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0.32 20.7
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Sample
Mean (mg kg-1)
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6
a
HorRat = Observed RSD / Predicted RSD from Horwitz equation
12
b
N, number of observations
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Table 3
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Recovery estimates of nitrate and nitrite added to a UHT liquid milk and ready-to-drink (RTD) infant formulation.
-1
RTD
Nitrite (mg L ) Found NO3-
Recovery (%)
Added NO2-
0
4.91
-
0
10.00 20.00
15.13 24.66
102.2 98.8
1.00 3.00
1.69 3.45
107.0 94.3
40.00 60.00
44.01 65.43
97.8 100.9
5.00 10.00
5.62 11.12
100.0 105.0
0 10.00
6.62 16.12
95.0
0 1.00
0.22 1.20
98.0
20.00 40.00
25.98 44.99
96.8 95.9
3.00 5.00
3.34 5.04
104.0 96.4
60.00
66.57
99.9
10.00
10.12
99.0
Average =
98.4
Average =
100.5
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Found NO20.62
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Added NO3UHT
-1
Nitrate (mg L )
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Drink type
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Recovery (%) -
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S0958-6946(13)00227-6
DOI:
10.1016/j.idairyj.2013.08.011
Reference:
INDA 3574
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.
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Colorimetric determination of nitrate and nitrite in milk and milk powders –
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Use of vanadium (III) reduction
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4 5 6 7 David C. Woollarda*, Harvey Indykb
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a
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Zealand
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b
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New Zealand Laboratory Services, 35, O’Rorke Rd, Penrose, Auckland 1642, New
Fonterra, Main Road, Waitoa, New Zealand
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* Corresponding author. Tel.: +6495264514
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E-mail address: [email protected] (D. C. Woollard)
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_______________________________________________________________________
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Abstract
27 A manual method is described for the determination of nitrate and nitrite in milk
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and milk powders that is intended to provide an alternative to conventional manual
30
methods accomplished by cadmium reduction. Reduction of nitrate is performed in
31
solution utilising vanadium (III) and quantitation achieved by concurrent reaction with
32
Griess reagent. Performance data are acceptable in terms of precision and accuracy,
33
repeatability being about 6% and intermediate precision at 8% for both analytes, providing
34
the limit of detection is not approached. Limit of quantitation is 0.1 mg kg-1 for both
35
analytes.
36
_______________________________________________________________________
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40 41
1.
Introduction
42 Nitrate (NO3-) exists throughout the biosphere from natural and man-made
44
processes and is an important component of biological life-cycles; hence it is well-studied
45
clinically (Ellis, Adatia, Yazdanpanah, & Makela, 1998). Bacterial action can reduce NO3-
46
to NO2- (nitrite), which is minimised from entering the modern food chain because of
47
associated health risks. Although there is inevitably a background level of NO3- , it is
48
regularly monitored within the dairy industry as part of normal sanitary programs. Nitrite
49
levels are also monitored, since microbiologically-contaminated water or post-secretory
50
milk can contain bacteria with significant nitrate reductase activity. The widespread use of
51
nitrogen-based fertilisers, combined with domestic, agricultural and industrial wastes,
52
have increased the chances of NO3- and NO2- incorporation into manufactured dairy
53
products (Indyk & Woollard, 2011). In addition, the use of nitric acid as a sanitiser within
54
dairy plants represents a further risk of nitrate contamination. Although generally
55
associated with increased risk of several pathologies, it is notable that recent studies
56
indicate that health benefits may be derived from consumption of dietary NO3- and NO2- ,
57
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
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rapid and reliable analytical methods for quantitation of the NO3- and NO2- content in
60
foods and biological materials. There are many technology platforms for NOX- testing,
61
each with benefits and disadvantages, but the well-studied Griess diazotisation reaction
62
(Griess, 1879) involving NO2- is the most commonly employed detection chemistry (Fox,
63
1979; Moorcroft, Davis, & Compton, 2001; Sun, Zhang, Broderick, & Fein, 2003; Woollard
64
& Forrest, 1984). This typically involves two separate stages, the first to determine NO2-
65
and a second for total NOX- after reduction of NO3- to NO2- , with the NO3- concentration
66
determined by difference. Either manual or automated formats are commonly employed
67
and the most frequently described assay utilises sulphanilamide and N-(1-naphthyl)
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68
ethylenediamine (NED) to create a sensitive chromophore with NO2- , although other
69
amines are available (Tsikas, 2007).
70
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
72
very slowly with NO3- unless it is first coated with copper to give a good surface for
73
electron transfer between solid and solution. Zinc has also been used in official methods
74
as a replacement for cadmium to reduce toxicity of the analysis despite its increased
75
thermodynamic opportunity to reduce NO2- to NO (Merino, Edberg, Fuchs, & Aman,
76
2000). There is no separation of NO2- from other inorganic substances but this Griess
77
reaction is considered specific enough for regular use at concentrations found in most
78
dairy products. Enzymatic reduction can also be achieved by nitrate reductase from
79
Aspergillus species (BS EN, 1997b; 2005a; 2008), although NADPH has been
80
demonstrated to interfere with the subsequent Griess reaction. There are obvious
81
advantages with this strategy, despite enzyme expense, particularly for biological samples
82
that can use small reagent volumes (Bories & Bories, 1995; Jogben, Jobgen, Li,
83
Meininger, & Wu, 2007).
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The reduction of NO3- to NO2- can be avoided using ion-chromatographic methods
85
to separate NO2- and NO3- which are then measured individually by direct UV detection or
86
conductivity (Jogben et al., 2007; Reece & Hird, 2000). Such methods have been
87
successfully deployed and validated internationally for foods and biological samples,
88
although there remains significant opportunity for interference because of the low
89
wavelength necessary to detect NOX (BS EN, 1997a; 2005b; Jedličkova, Paluch, & Alušik,
90
2002). Another ion-exchange approach has been to perform NO3- reduction in post-
91
column mode to maintain the sensitive and selective detection modes available with the
92
NO2- ion. For example, Lookabaugh and Krull (1988) used photochemical reduction and
93
electrochemical re-oxidation of NO2- to improve detection, while Gapper, Fong, Otter,
94
Indyk, and Woollard (2004) used cadmium reduction and post-column addition of Griess
95
reagent to allow colorimetric detection at 540nm. Another HPLC technique has been
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reported to combine pre-column enzyme reduction and reaction of NO2- with 2, 3-
97
diaminonapthalene to a stable fluorescent 2,3-napthotriazole (Jogben et al., 2007). A
98
successful post-column reduction strategy involved addition of trivalent vanadium to the
99
column eluent, which is followed by Griess reagent (Casanova, Gross, McMullen, &
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Schenck, 2006). The principal advantage of vanadium (III) reduction is that it occurs in
101
the acidic solution compatible with the Griess reaction, the principle difficulty being the
102
need for elevated temperatures and inert post-column equipment to prevent corrosion of
103
stainless steel parts by hydrochloric acid.
104
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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
106
samples including foods by reduction to nitric oxide (NO) and detection by highly sensitive
107
chemiluminescence. Individual samples were placed into the reduction flask without prior
108
clean-up, the liberated NO gas being removed by flow of helium to the detector. Similarly,
109
Baskić, Jovanović, Jakovljević, Delibašić, & Aresenjević (2005) used vanadium (III) to
110
measure NOX- but captured the NO2- as a diazo compound using Griess chemistry,
111
avoiding the need for gas handling associated with chemiluminescence but enhancing the
112
need to control inadvertent loss of the NO2- to NO. Miranda, Espey, and Wink (2001)
113
successfully measured NO3- and NO2- in clinical samples using trivalent vanadium
114
reduction and Griess reaction in microtitre wells and noted the better performance of HCl
115
acidification compared to H3PO4. Beda and Nedospasov (2005) modified the microtitre
116
assay to overcome problems associated of low NO3- in the presence of high NO2- , and
117
described the preparation of trivalent vanadium from V2O5 and magnesium and the
118
importance of leaving some tetravalent vanadium in the reaction mixture to improve
119
analytical precision. The reaction was described by equation 1.
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120 121
2V3+ + NO3- + 2H+ = 2V4+ + NO2- + H2O
(1)
122
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Doane and Horwath (2003) reported a manual single-reagent procedure using
124
trivalent vanadium for NO3- testing of water samples and extended to a number of other
125
matrices. The aim of the present study was to evaluate the potential of vanadium (III)
127
chemistry to analyse NOX- in dairy products using a manual, non-chromatographic
128
technique. In this way, traditional reduction of NO3- with cadmium, zinc or enzymes can
129
be avoided, thereby simplifying the analysis. The reduction and reaction procedures have
130
been optimised, performance parameters estimated, and the method confirmed to be a
131
useful routine alternative for compliance testing of NOX- in dairy laboratories.
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133
2.
Materials and methods
134 135
2.1.
Equipment
136 137
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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)
139
or polystyrene (PS) macro cuvettes (Brand GMBH, Wertheim, Germany), and also
140
equipped with a sipper flow-through cell and a fibre-optic coupler. .
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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
143
vials. Various temperatures were used, the chosen setting being at 60 oC ± 2 oC.
144
Incubation at 25 oC or 40 oC within spectrophotometer cuvettes were performed in a
145
Contherm Series 5 (Lower Hutt, New Zealand) convection oven.
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Centrifugation was performed at about 300 × g (~1000 rpm) with a Gerber
147
Instruments (Effretikon, Switzerland) centrifuge fitted with a 28 mm radius rotor and
148
buckets to carry 50 mL polypropylene tubes (Tarson 500040, Kolkata, India). Reagent
149
and sample dispensing were performed using 1 mL and 5 mL variable-volume pipettors,
150
sourced locally through Labserve (from Fisher Scientific, Vantaa, Finland). Nylon or
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cellulose acetate 0.45 µm high performance liquid chromatography filters, with 3 mL
152
syringes were obtained from various for sample clarification. Some filters required a
153
prewash with 0.1 M HCl before use to remove NOX- residues.
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2.2.
Reagents
157
Incoming water was purified by commercial-scale deionisation units with separate
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cation- and anion-exchange resins. This water was then reduced to 18 MΩ resistivity by a
160
Barnstead Epure system with in-line charcoal filter to ensure the absence of NOX-
161
contamination.
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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
164
to a 2 L volumetric flask and made to volume with water. All procedures were performed
165
in a fume cupboard.
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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.
168
The green solution was stable for many weeks under refrigerator as shown by its
169
absorbance near 400 nm. Although some oxidation occurred over time, as revealed by a
170
lighter colour, the reagent remained functional as it was prepared in excess
171
concentration. For enhanced stability, however, the solution was purged with nitrogen
172
gas. Vanadium trichloride and its solutions should be handled with care due to their
173
corrosive nature
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Carrez solutions were prepared as follows from Merck reagents (Darmstadt,
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Germany):
176
diluted to 200 mL (Carrez I). This solution was stable several months in the absence of
177
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
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water and diluted to 200 mL (Carrez II). This was stable indefinitely, particularly under
179
refrigeration. Individual Griess reagents were prepared separately from Merck reagents
181
(Darmstadt, Germany) as follows: 5 g of sulfanilamide (Merck 1.08035) was dissolved in
182
10 mL concentrated hydrochloric acid and made to 500 mL with water. This was stable
183
for several months at 4 °C. 0.5 g N-1-naphthyl-eth ylenediamine dihydrochloride (Merck
184
1.06237) was dissolved in water and diluted to 250 mL. This solution was stored up to
185
one month in a darkened bottle, avoiding exposure to light. The combined Griess reagent
186
was prepared just prior to use by mixing 100 mL sulfanilamide solution and 20 mL N-1-
187
naphthyl-ethylenediamine dihydrochloride solutions.
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Stock standard solutions (1 mg mL-1) were prepared from pre-dried chemicals
189
(110 o - 120 oC to constant weight) sourced from BDH Chemicals. Potassium nitrate
190
(0.1629 g), containing 100 mg NO3-, was weighed quantitatively into a 100 mL volumetric
191
flask and diluted with water. Similarly, 0.1850 g potassium nitrite, containing 100 mg NO2-
192
, was weighed quantitatively into a 100 mL volumetric flask and diluted with water. These
193
were stored in a refrigerator and replaced monthly. For longer storage, the stock
194
solutions were subdivided and frozen (-20 oC). Alternatively, standards can be prepared
195
from sodium salts using 0.1371 g and 0.1500 g of sodium nitrate and sodium nitrite,
196
respectively.
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Intermediate NO3- and NO2- standards (10 µg mL-1) were prepared daily by dilution
198
of 1.00 mL of each stock solution into separate 100 mL flasks and made to volume. These
199
were further diluted in 10 mL volumetric flasks to generate six-point calibration curves,
200
including blanks, in the range 0 – 1 µg mL-1 (ppm).
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201 202 203
2.3.
Sample preparation
204
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205
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
207
added, the tubes capped and vortex mixed to dissolve. If necessary, the extracts were
208
warmed to about 40 oC to achieve full dissolution. In the case of liquid milk, 15.0 mL was
209
added to the centrifuge tube. A quality control milk powder sample with known NOX-
210
concentrations was included to confirm run-to-run precision.
211
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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
213
precipitation, and then centrifuged at about 300 × g to produce a clear supernatant. If a
214
clear supernatant was not achieved, then a further 2 mL of each Carrez solution was
215
added and centrifugation repeated.
216
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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
218
second filter. A duplicate blank was prepared similarly, by omitting the sample but
219
including all reagents.
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220 221 222
2.4.
Nitrate determination (NO3- + NO2-)
224
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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
226
chloride solution. The vials were capped tightly to prevent evaporation and incubated in a
227
60 oC water bath for 40-45 min. Progress of the reaction could be seen by a developing
228
red coloration.
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229
The vials were then mixed by inversion, cooled to room temperature in cold water
230
and portions transferred to disposable 1 cm disposable cuvettes. The absorbances were
231
read at 530 nm against water. For large sample numbers, a sipper cell was used with
232
low- volume cell or a fibre-optic probe for the Cary 50 spectrophotometer.
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233
An alternative procedure was also evaluated whereby reduced volumes were pipetted directly into spectrophotometer cuvettes, with lower temperatures necessary
235
during incubation to prevent loss of volume by evaporation. Thus, 1.0 mL sample extract,
236
standard or blank, 1.0 mL mixed Griess reagent and 1.0 mL vanadium chloride solution
237
were mixed in a capped cuvette and incubated at either 40 oC for 3 h, or overnight at 25
238
o
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234
C.
239 2.5.
Nitrite determination (NO2-)
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240 241
Nitrite levels were determined in the same way except the vanadium chloride
243
solution was replaced with hydrochloric acid. In addition, since heating was not required,
244
the reactions were conveniently performed directly within disposable cuvettes, namely 1.0
245
mL of each sample filtrate, standard or blank, was mixed with 1.0 mL Griess reagent and
246
1.0 mL hydrochloric acid. After 10-15 min, the absorbance could be measured at 530 nm
247
against water.
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248 249
2.6. Calculations
250
253 254 255 256
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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:
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mg / kg =
[Absorbance of Sample − Sample Blank ] Slope of Calibration Graph
x
DF Weight Sample ( g )
(2)
Dilution factor DF, during sample dissolution, and clarification was usually 19 (ie
257
15 mL water plus 2 mL of each Carrez solution). Dilutions during colorimetry were the
258
same for standards and samples
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The concentrations of NO3- were determined by difference (with MW correction) of
259 260
total NOX- and NO2- (equation 3)
mg NO3 kg
262
=
mg NOX kg
−
1.35
*
mg NO2 kg
263 264 2.7.
Optimisation
(3)
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266 267
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The reactions with Griess reagents were well established from literature reports
268
and previous experience. The reduction step by vanadium was optimised for reagent
269
strength, time and temperature.
270 271 2.8.
Validation
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The optimised procedure was validated for intra-laboratory repeatability by
275
replicate analysis of single samples. In addition, duplicate data of different samples over
276
many months of testing yielded further robust intermediate precision data. Intermediate
277
precision was also determined from control charts of three nitrite-positive samples
278
obtained from six months of testing with multiple analysts. Reproducibility trials with other
279
laboratories were not conducted.
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Accuracy was estimated by spiked recoveries and by comparison with
281
conventional cadmium-reduction methods performed within a New Zealand inter-
282
laboratory proficiency program.
283
Limit-of-detection estimates were performed by dilution of standards until the
284
absorbances were no longer distinguishable from spectrophotometer noise (3σ). In
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285
addition, samples with undetectable NOX- were repetitively analysed to determine data
286
scatter around zero concentration.
287
289
3.
Results and discussion
3.1.
Colorimetric Reaction
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290
292
To optimise the reduction of NO3- to NO2- a range of temperature conditions were
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291
evaluated. At room temperature (20 oC) the reduction was slow, whereas near-boiling
294
temperatures facilitated a rapid reaction but also significant degradation of the coloured
295
Griess azo product. A temperature of 60 oC was selected and provided an acceptable
296
compromise between colour stability and reaction time. Fig. 1 illustrates the change in
297
absorbance with time at 20 oC and 60 oC for a milk powder extract, and Table 1 gives the
298
time to optimum signal at other temperatures. The maximum absorbance was achieved
299
in about 30 min at 60 oC but took 18 -24 h at ambient temperature. It has been reported
300
that the reduction of NO3- to NO2- by vanadium (III) is rate-limiting relative to NO2-
301
detection, and that complete reduction is not required provided that calibration standards
302
are included (Miranda et al., 2001).
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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
305
and Horvath (2003). It was impractical to incubate at 60 oC without losing volume, but
306
incubation at 40 oC in capped cuvettes proved successful, with a reaction time of 3 hours.
307
Alternatively, the reduction could proceed overnight at 25 oC, although this is not ideal for
308
fast analytical turnaround. Carrez solutions employed for protein removal did not interfere
309
with vanadium reduction and were generally efficient in sample clarification. In cases
310
where insufficient clear supernatant was available further Carrez was required or multiple
311
filtrations.
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During NO3- reduction, vanadium changes oxidation state (III → IV), with both
312
species being coloured. At the low pH used in this method, V3+ exists largely as a green-
314
grey solution with a maximum absorbance near 400 nm. The spectrum of vanadium (III)
315
is illustrated in Fig. 2 where the presence of the vanadyl ion VO+ is also evident at 600
316
nm. During the course of vanadium (III) oxidation by NO3-, the tetravalent V4+ ion is
317
produced, as shown by the increased spectral absorption at 765 nm.
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The absorption of the VO+ ion at 600 nm potentially interferes with the red colour
318
of the Griess diazo compound, but the colour was so intense that this was negligible.
320
However, to alleviate possible risk, the determinations were performed at 530 nm rather
321
than the wavelength maximum of 542 nm. Fig. 3 shows overlaid spectral scans of a 1 µg
322
mL-1 NO3- solution over 6 h incubation at ambient temperature. During the early part of
323
the reaction the vanadyl ion is seen as a shoulder, so it was expected to provide some
324
interference in samples with low NO3-. However, this potential concern was mitigated
325
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) .
328
The Griess reagent was also well in excess of requirements to avoid its depletion in
329
typical samples. Extracts with NO3- levels above the calibration range, corresponding to
330
80 – 100 mg kg-1 in the milk powders, were generally retested using a smaller sample
331
weight to avoid exceeding the linearity of the test.
332 333 334 335
3.2.
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Performance data
Since calibration graphs were run daily with each batch of samples, the between-
336
run variation in slope was a good indication of method performance. During eleven
337
months of method development and routine testing involving several technicians, the
338
mean linear regression slope for NO3- was 0.7270 with an RSD of 4.7% (n=97). During
339
the same period, the mean slope for NO2- was 1.0822 with an RSD of 5.3%. Assuming
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complete conversion of NO3- to NO2-, the theoretical ratio of these slopes should equal the
341
MW ratio of NO2-/NO3- , 0.741, thus, the observed ratio of 0.672 indicates an absolute
342
reduction efficiency of approximately 91%. However, the use of daily calibration graphs
343
compensates for any reduction inefficiency so the relative recovery of NO3- was
344
quantitative as shown by spiked recoveries (91 – 103%, n= 14).
345
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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
347
non-linearity above 5 mg L-1, possibly as a result of reagent depletion.
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Blanks were an important part of the test because analyte concentrations in
349
samples of interest were often trending towards zero concentration. Levels of NO2- in the
350
calibration blanks were always near zero (intercept/slope < 0.001) but small positive
351
intercepts (typically 0.02 – 0.03) were common for the NO3- blanks. Potential NO3-
352
sources in the calibrations were water, HCl and VCl3 but these reagents were also used at
353
the same quantity in all samples, so intercepts were unimportant. Sample blanks
354
compensated for contamination from all sources including Carrez solutions, disposable
355
equipment and the 0.45 µm nylon filters, and were typically 0.003 – 0.005 absorbance
356
units for NO2- and 0.05 – 0.07 for NO3- . Filters presented a situation of variable blank
357
because a new one was used for each sample. Filters with low endogenous NO3- were
358
selected to avoid the need for each to be washed with HCl and air-dried prior to use. To
359
obtain best precision, sample blanks were performed in replicate.
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Repeatability relative standard deviation (RSDr) estimates were determined by
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361
same-day analyses (n=6) of various milk powders with 10-20 mg kg-1 NO3- and 0.8 – 1.5
362
mg kg-1 NO2-, yielding average values of 4.29% (NO3-) and 6.62% (NO2-). Repeatability
363
estimates were also obtained using duplicate data across a wide range of samples of
364
varying NOX- thereby representing the real-life situation. Thirty-two (32) samples were
365
tested in duplicate where the pooled data for NO3- indicated an RSDr of 6.02% (1.38 –
366
44.11 mg kg-1 NO3-). The data was fairly homoscedastic, because the NO3- rarely
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367
approaches zero in milk powders, thus, 6% represents a good RSDr estimation across all
368
samples.
369
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
371
RSDr of 5.4% was observed but, below this concentration (0.01 – 0.47 mg kg-1), a 47%
372
random error was encountered.
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To obtain an estimation of between-day (intermediate) precision, four milk powder
374
samples were tested during many months as shown in Table 2. These samples had NOX-
375
concentrations typically encountered in commercial milk powders, exhibiting about 8%
376
random error for NO3-, acceptable in terms of the expected random error for intermediate
377
precision (i.e., Horrat <1). NO2- was typically much lower in concentration although
378
samples 3 and 4 were selected to allow meaningful error statistics, for which intermediate
379
precision was comparable to NO3-. Most milk powders have negligible NO2-, as found in
380
samples 1 and 2. In such cases the observed concentrations were scattered around
381
zero. Concentrations of 0.03 mg kg-1 in milk powders were therefore deemed at the limit
382
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
384
the New Zealand dairy industry the self-imposed limits for milk powders are 50 mg kg-1
385
NO3- and 1 mg kg-1 NO2-. The current method achieves these sensitivities and so is
386
adequate for routine quality control purposes and likely to meet the future requirements of
387
the milk and milk powder industries. As new-born infants are especially susceptible to
388
the detrimental effects of these contaminants, upper limits applied to the production of
389
milk-based infant formulas are generally lower at 25 - 40 mg kg-1 for NO3- and 0.5 mg kg-1
390
for NO2-. These requirements are also met by the current method
391
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As a consequence of analyte instability, no food-based reference materials are
392
available that contain certified nitrate or nitrite levels. The accuracy of the proposed
393
method has therefore been evaluated by comparison with conventional methods and
394
spiked recovery. Thus, over the course of two years, the proposed method was tested
15
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within a New Zealand national proficiency scheme (Fig 4). NO2- data was centrally
396
located among all participants because the chemistry of the proposed manual NO2-
397
method was equivalent to the automated conventional methods used by other
398
laboratories. However, NO3- results derived from the proposed V3+ reduction method
399
were typically higher than median data from conventional reduction methodology,
400
although the Z statistic was generally acceptable (<2). A UHT liquid milk sample and a
401
ready-to-feed infant formula, both in 250 mL tetrapak cartons, were spiked by pipetting
402
standard solutions to achieve NO2- and NO3- concentrations in representative ranges.
403
Both analytes returned acceptable recoveries as shown in Table 3, with no evidence of
404
positive bias for NO3-.
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405 406
4.
Conclusions
407 408
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
410
require the perfusion of extracts through cadmium columns. Nitrate is detected
411
colorimetrically with Griess reagent following reduction to nitrite with trivalent vanadium in
412
solution. The method avoids lengthy manipulative procedures involving excess glassware
413
and demonstrates figures of merit that are fit-for-purpose in dairy products. The
414
recommended reduction conditions are 60 oC for 30 min to meet high turnaround speeds.
415
The method relies on reaching a constant absorbance end-point but suits later automation
416
using kinetic measurements.
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Acknowledgements
419 420 421
The authors wish to acknowledge the technical assistance of Mayson Kay of NZ Laboratory Services, Auckland, NZ.
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References
424 425
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Medicus, 6, 49-52.
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Beda, N., & Nedospasov, A. (2005). A spectrophotometric assay for nitrate in an excess of nitrite, Nitric Oxide, 13, 93-97.
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Bories, P. N., & Bories, C. (1995). Nitrate determination in biological fluids by an
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Braman, R. S., & Hendrix, S. A. (1989). Nanogram nitrite and nitrate determination in
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441
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BS EN (1998). Foodstuffs. Determination of nitrate and/or nitrite content. Continuous flow
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method for the determination of nitrate content of vegetables and vegetable
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453 BS EN (2005a). Foodstuffs: Determination of nitrate and/or nitrite content- Part 3:
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454 455
Spectrophotometric determination of nitrate and nitrite content of meat products
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BS EN (2005b). Foodstuffs: Determination of nitrate and/or nitrite content. Ion-exchange
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BS EN (2008). Milk and milk products: Determination of nitrate content – Method by enzymatic reduction and molecular-absorption spectrometry after Griess reaction.
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BS EN ISO 20541:2008. Brussels, Belgium: European Committee of
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Casanova, J. A., Gross, L. K., McMullen, S. E., & Schenck, F. J. (2006). Use of Griess
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reagent containing Vanadium (III) for post-column derivatization and simultaneous
471
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Doane, T. A., & Horwath, (2003). Spectrophotometric determination of nitrate with a single reagent. Analytical letters, 36, 2713-2722.
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Ellis, G., Adatia, I., Yazdanpanah, M., & Makela, S. K. (1998). Nitrite and nitrate analyses: a clinical biochemistry perspective. Clinical Biochemistry, 31, 195-220.
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spectrophotometric detection. International Dairy Journal, 14, 881-887.
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Griess, J. P. (1879). Bemerkungen zu der Abhandlung der H.H. Weselsky und Benedikt,
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Hord, N. G., Tang, Y., & Bryan, N. S. (2009). Food sources of nitrates and nitrites: the
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Indyk, H. E., and Woollard, D.C. (2011). Nitrates and nitrites as contaminants. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney, Encyclopaedia of dairy sciences, (2nd
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IDF 189-1:2004 Brussels, Belgium: International Dairy Federation.
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IDF. (2004c). Milk and milk products – Determination of nitrate and nitrite contents – Part 3: Method using cadmium reduction and flow injection analysis with in-line dialysis
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(routine method). ISO 14673-3:2004, IDF 189-1:2004. Brussels, Belgium:
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International Dairy Federation.
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Jedličkova, V., Paluch, Z., & Alušik, S. (2002). Determination of nitrate and nitrite by high-
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performance liquid chromatography in human plasma, Journal of Chromatography
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B, 851, 51-70.
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Jobgen, W. S., Jobgen, S. C., Li, H., Meininger, C. J., & Wu, G. (2007). Analysis of nitrite
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Journal of Chromatography B, 851, 71-82.
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Lookabaugh, M., & Krull, I. S. (1988), Determination of nitrite and nitrate by reversedphase high-performance liquid chromatography using on-line post-column
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photolysis with ultraviolet absorbance and electrochemical detection, Journal of
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Chromatography, 452, 295-308.
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Merino, L, Edberg, U, Fuchs, G., & Aman, P. (2000). Liquid chromatographic
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determination of residual nitrite/nitrate in foods: NMKL collaborative study. Journal
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of AOAC International, 83, 365-375.
527 528 529
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Miranda, K. M., Espey, M. G., & Wink, D. A. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide, 5, 62-71.
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Moorcroft, M. J., Davis, J., & Compton, R. G. (2001). Detection and determination of nitrate and nitrite: a review. Talanta, 54, 785-803.
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Reece, P., & Hird, H. (2000). Modification of the ion exchange HPLC procedure for the
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detection of nitrate and nitrite in dairy products. Food Additives and Contaminants,
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Tsikas, D. (2007). Analysis of nitrite and nitrate in biological fluids by assays based on the
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Sun, J., Zhang, X., Broderick, M., & Fein, H. (2003). Measurement of nitric oxide
production in biological systems by using Griess reaction assay, Sensors, 3, 276-
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284.
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Griess reaction: Appraisal of the Griess reaction in the L-arginine/nitric oxide area
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of research. Journal of Chromatography B, 851, 51-70.
545 546
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Woollard, D. C., & Forrest, L. J. (1980). Levels of nitrate and nitrite in casein products using an automated procedure, New Zealand Journal of Dairy Science and
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Technology, 15, 83-90.
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Figure legends
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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).
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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
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Proficiency Studies.
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1
Table 1
2
Incubation times required for complete nitrate reduction at different temperatures.
3 Maximum absorbance
(oC)
(min)
10
>4800
20
1500
30
720
40
180
50
50
60
30
70
5
80
1
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Temperature
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5
7
Table 2
8
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
1.86 0.08
6.72 565
Powder 2 (N=21) Nitrate 34.70 Nitrite 0.02
2.68 0.07
7.73 352
Powder 3 (N=22) Nitrate 15.26 Nitrite 5.62
1.25 0.49
Powder 4 (N=15) Nitrate 7.15 Nitrite 6.48
0.56 0.56
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8.18 8.81
0.77 0.71
7.85 8.60
0.66 0.71
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0.32 20.7
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Sample
Mean (mg kg-1)
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a
HorRat = Observed RSD / Predicted RSD from Horwitz equation
12
b
N, number of observations
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Table 3
16
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Recovery estimates of nitrate and nitrite added to a UHT liquid milk and ready-to-drink (RTD) infant formulation.
-1
RTD
Nitrite (mg L ) Found NO3-
Recovery (%)
Added NO2-
0
4.91
-
0
10.00 20.00
15.13 24.66
102.2 98.8
1.00 3.00
1.69 3.45
107.0 94.3
40.00 60.00
44.01 65.43
97.8 100.9
5.00 10.00
5.62 11.12
100.0 105.0
0 10.00
6.62 16.12
95.0
0 1.00
0.22 1.20
98.0
20.00 40.00
25.98 44.99
96.8 95.9
3.00 5.00
3.34 5.04
104.0 96.4
60.00
66.57
99.9
10.00
10.12
99.0
Average =
98.4
Average =
100.5
AC C
19
EP
18
Found NO20.62
M AN U
Added NO3UHT
-1
Nitrate (mg L )
TE D
Drink type
SC
17
Recovery (%) -
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT