Journal of Food Composition and Analysis 51 (2016) 93–105

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original research article

Nitrate and nitrite content of vegetables, fruits, grains, legumes, dairy

products, meats and processed meats
Zahra Bahadorana , Parvin Mirmirana , Sajad Jeddib , Fereidoun Azizic , Asghar Ghasemib,* ,
Farzad Hadaeghd
a
Nutrition and Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
b
Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
c
Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
d
Prevention of Metabolic Disorders Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences

A R T I C L E I N F O A B S T R A C T

Article history: Background and aim: The aim of this study was to investigate the contents of nitrate/nitrite in frequently
Received 3 March 2016 consumed food items among Iranians.
Received in revised form 13 June 2016 Methods: Stratified random sampling was conducted seasonally from November 2014 through January
Accepted 14 June 2016
2016 for samples collected from the main municipal markets of Tehran. To determine the nitrate/nitrite
Available online 23 June 2016
contents of foods, spectrophotometric methods, developed by International Organization for
Standardization (ISO), were used.
Keywords:
Results: Method limit of detection (LOD) was 0.61 mg 100 g
1 and 0.10 mg 100 g
1, for nitrate and nitrite,
Food composition
Food analysis
respectively, and method limit of quantification (LOQ) was 1.86 mg 100 g
1 and 0.16 mg 100 g
1 for
Nitrate nitrate and nitrite, respectively. A relatively high nitrate concentration was observed in breads (50.0 mg
Nitrite 100 g
1). Mean ranges of nitrate and nitrite in fruits were 7.50–46.8 mg 100 g
1 and 0.15–0.71 mg 100 g
1,
Food database respectively. Vegetables with the highest nitrate concentrations included radish (625 mg 100 g
1),
Vegetables beetroot (495 mg 100 g
1), tarragon (424 mg 100 g
1), lettuce (365 mg 100 g
1), mint (279 mg 100 g
1),
Legumes and celery (261 mg 100 g
1). Nitrite content of vegetables ranged 0.21–0.74 mg 100 g
1. In dairy products,
Grains mean nitrite and nitrate content ranged 0.14–0.45 mg 100 g
1 and 1.26–5.75 mg 100 g
1. Mean nitrate
Dairy
and nitrite concentrations in meats and processed meats were 5.56–19.4 mg 100 g
1 and 2.93–13.9 mg
Meats
100 g
1, respectively.
Conclusion: This database could provide opportunities to quantify nitrate and nitrite exposure of Iranians
and to evaluate risk-benefit of nitrate-nitrite exposure.
ã 2016 Elsevier Inc. All rights reserved.

1. Introduction nitrite-containing foods; more than 80-95 percent of dietary

Inorganic nitrate/nitrite are naturally occurring compounds in
foods, especially plant foods and vegetables, and are also used as
additives in industrially processed foods. Major sources of
exogenous nitrate exposure are vegetables and drinking water,
whereas processed meat and animal food products are major
Abbreviations: ELISA, enzyme-linked immunosorbent assay; FCT, food compo-
sition table; FFQ, food frequency questionnaire; HPLC, high performance liquid
chromatography; IC, ion chromatography; IQRs, inter-quartile ranges; ISO,
International Organization for Standardization; LOD, limit of detection; LOF,
lack-of-fit; LOQ, limit of quantification; USDA, United States Department of
Agriculture.
* Corresponding author at: No. 24, Shahid-Erabi Street, Yeman Street, Velenjak,
Tehran, Iran, PO Box 19395-4763.
E-mail address: ghasemi@endocrine.ac.ir (A. Ghasemi).
intake of nitrate is attributed to vegetables especially green leafy
vegetables including lettuce and spinach, cabbage, rocket, red
beetroot, and radish (Hord et al., 2009; Reinik et al., 2009). A wide
range of nitrate in vegetables from <1 to >1000 mg 100 g
1 has
been reported (Hord et al., 2009; Reinik et al., 2009), whereas most
levels of nitrate determined in fresh meats have been low (10–
50 mg kg
1) (Iammarino and Di Taranto, 2012; Walters, 1996).
Due to many biological and environmental factors including
cultivar type, composition of soil, light intensity, air temperature
and moister, growth density, duration of growth period, harvesting
time, storage time, edible plant portion and use of nitrogen
fertilizer, nitrate/nitrite concentrations of foods and especially
vegetables, reported in several studies, were extremely variable
and also varied from country to country and region to region
(Correia et al., 2010; Reinik et al., 2009; Tamme et al., 2006).

http://dx.doi.org/10.1016/j.jfca.2016.06.006
0889-1575/ã 2016 Elsevier Inc. All rights reserved.
94 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105
Increased exposure to nitrogen-containing compounds such as
nitrate/nitrite, due to increased usage of nitrogen fertilizers and
additives containing nitrite in processed food products is becoming
an important public health issue (Chan, 2011; Fewtrell, 2004;
WHO, 2003). The main concern regarding nitrate is its potential
endogenous conversion to nitrites and nitrosamines, and some
acute and chronic toxicities such as methemoglobinemia, thyroid
disorders and carcinogenesis (Bahadoran et al., 2015; Fewtrell,
2004; Gilchrist et al., 2010). On the other hand, several potential
therapeutic properties of nitrate/nitrite have been reported
recently, sparking a growing interest in its clinical applications
in several pathological conditions such as cardiovascular disease,
hypertension, diabetes, metabolic syndrome, and insulin resis-
tance (Ghasemi and Zahediasl, 2011, 2013; Kevil et al., 2011; Kevil
and Lefer, 2012; Lundberg et al., 2008; Machha and Schechter,
2011).
Studies also reveal the plausible health benefits and preventive
effects of high nitrate/nitrite foods against cardiovascular disease
(Gilchrist and Benjamin, 2010; Machha and Schechter, 2012).
Therefore, limit regulations on nitrate/nitrite intakes have already
been challenged, and it has been pointed out that the evidence for
adverse effects of nitrate is inconsistent and nitrate may actually be
beneficial (Butler and Feelisch, 2008; Katan, 2009).
Accordingly, the concept of risk-benefit of dietary exposure to
nitrate/nitrite has been highlighted (Gilchrist et al., 2010; van
Grinsven et al., 2006). To address this challenging issue, access to a
valid database of nitrate/nitrite food contents is essential, however
unlike other nutrients and food components, there is no valid
applicable and comprehensive database such as the United States
Department of Agriculture (USDA) food composition table (FCT) for
nitrate/nitrite. Substantial efforts have therefore been made during
two past decades in several countries to develop database (Ayaz
et al., 2007; Chung et al., 2003; Chung et al., 2011; Keeton et al.,
2009; Santamaria et al., 1999; Suh et al., 2013; Sušin et al., 2006);
beyond the accurate estimation of nitrate/nitrite exposure in
different populations, such studies could provide valuable
opportunities for testing novel hypotheses (Gilchrist and Benja-
min, 2010; Lundberg et al., 2006) and facilitate further insights in
to the health related outcomes of dietary exposure to nitrate/
nitrite in the framework of epidemiological studies and prospec-
tive cohorts.
Previous investigations in Iran were limited to nitrate/nitrite
measurements of a few numbers of vegetables and some meat
products (Rezaei et al., 2014; Taghipour et al., 2014; Ziarati and
Arbabi-Bidgoli, 2014), with substantial limitations in sample
collections and measurement methods. Currently, there is no
valid applicable and comprehensive database for nitrate/nitrite
contents of foods in Iran. Considering the lack of such data, the
main focus of this study was, therefore, to investigate the content
of nitrate and nitrite in frequently consumed food items among
Iranians. This database values could be used by Iranian food survey
to estimate dietary intake of nitrate and nitrite, and evaluate
related health and disease outcomes.
2. Material and methods
2.1. Reagents
All of the chemical materials and standards solutions were
purchased from Sigma-Aldrich (St. Louis, MO). All solutions were
prepared using double-distilled deionized water. All reagents,
including sodium tetraboratedecahydrate (Na2B4O710H2O),
Carrez reagent I (potassium hexacyanoferrate II trihydrate [K4Fe
(CN)63H2O]), and Carrez reagent II (zinc acetate solution; Zn
(CH3COO)22H2O) were prepared according to related
ISO6635:1984, ISO 3091:1975, ISO 2918:1975, and ISO 14673-
1:2004 (ISO, 1975a, 1975b, 1984, 2004). Reagents for color
development (Griess reagents) included Griess I (0.40 g of
sulfonamide in 20 mL hydrochloric acid and 180 mL water), Griess
II (0.10 g N-(1-naphtyl) ethylene diamine dihydrochloride
[C10H7NHCH2CH2NH22HCl] in 100 mL water) and hydrochloric
acid solution (445 mL hydrochloric acid in water up to 1000 mL).
Vanadium (III) chloride (VCl3) solution was prepared by dissolving
of 4.80 g VCl3 in 100 mL hydrochloric acid solution and water up to
volume 1000 mL. All chemical materials were weighed using
digital scale (Sartorius TE 124S, d = 0.1 mg).
2.2. Sampling
In this study, food items for measurement of nitrate and nitrite
were selected based on a 168-item food frequency questionnaire
(FFQ), previously developed and validated among a representative
Iranian population; this FFQ included more frequently consumed
food items with higher frequency of consumption among Iranian
adults (Mirmiran et al., 2010).
In this study, stratified random samplings were done from
November 2014 through January 2016. Due to different socio-
demographic status of the population and differences in quality of
supplied food products in different geographic regions, the Tehran
city map was divided into four distinct areas, that is, the south,
west, east, and north (Nazeri et al., 2010). In each region, main
municipal markets were selected and simple random samplings in
winter and summer were conducted at the three-week intervals
from the three vendors located in each municipal market. For dairy
products, processed meats, industrial tomato paste and sauce, and
packed fruit juices, random samplings were conducted from the
four popular brands, with a wide product distribution in the city, in
winter and summer at the three-week intervals. The sampling was
balanced across sampling locations (south, west, east, and north of
the Tehran), brands, and seasons. Totally, 1872 samples of 87 food
items were included in the final analysis.
2.3. Sample preparation
After sampling, fresh fruits and vegetables were cleaned, rinsed,
labeled and stored in a freezer at
20  C until analysis. All dairy
products, fresh and processed meat were also purchased from the
main municipal markets and kept at refrigeration temperature and
analyzed within 48 h. The samples which are usually used as
cooked or fried, were appropriately processed according to the
predominant traditional food culture. For the cooking process,
samples were cooked at approximately 100  C using distilled water
without using salt, in stainless-steel pots, until ready for
consumption (about 30 min), and then drained on absorbent
paper. For the frying processing, samples were fried at approxi-
mately 180  C using soybean oil for an average of 30 min and then
drained on absorbent paper. For breads, including both industrial
and traditional, samples were dried in an oven at 60  C for 24 h;
before and after heating, samples were weighed and the ratio of
two measurements were considered for final calculation of total
nitrate/nitrite content. Table 1 provides detailed descriptions of
food samples preparation.
2.4. Nitrate and nitrite determination
Sample extraction and preparation of filtrate were conducted
according to ISO methods (ISO, 1975a, 1975b, 1984, 2004); details
of the procedures are presented in Table 2. Briefly, aliquots 1–10 g
(Digital scale, A&D Co., LTD. Japan; d = 0.01 g) of the samples were
treated with tetraborate sodium (for plant foods and meat
products, only), Carrez solutions including potassium hexacyano-
ferrate (II) trihydrate (Carrez I) and zinc acetate solution (Carrez II);
 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105 95

Table 1
Individual instructions of food preparation.

Food Number of sample Preparation method

Total Season Regions

Winter Summer South West North East

Barley (Hordeum vulgare L.) 19 10 9 5 5 4 6 Weight 100 g of each purchased samples
Rice (Oryza sativa) 21 10 11 5 5 5 6 Cook at unsalted distilled water
Legumes (Soya, cowpea, chickpea, broad 169 83 86 40 42 43 44 Mix together thoroughly using blender
bean, red bean, white bean, lentil, Split
pea)
Industrial breads 31 16 15 7 8 8 8 Weight 100 g of each purchased samples
Traditional breads (Sangak, Taftoon, 82 40 42 20 21 19 22 Dried in an oven at 60  C for 24 h
Barbari, Lavash) Weight before and after heating
Powder and mix together using grinder mill (Moulinex, LM 30114A)
Pistachio (Pistacia vera) 19 11 8 5 4 5 5 Weight 100 g of each purchased samples
Almond (Prunus dulcis) 19 10 9 4 5 5 5 Remove hard shell, chop and mixed together using grinder mill
(Moulinex, LM 30114A)
Apple (Rosaceae) 21 11 10 6 5 5 5 One sample with a medium size and degree of ripeness
Apricot (Prunus armeniaca)* 19 - 19 4 5 5 5 Wash and drain on absorbent paper
Pears (Pyrus) * 17 - 17 4 5 5 3 Remove seeds
Peaches (Prunus persica) * 25 - 25 6 7 6 6 Chop and mix together using blender (Moulinex, LM 30114A)
Plums (Prunus domestica) 27
Cantaloupe (Cucumis melo) * 18 - 18 4 5 4 5 Weight 500 g of each purchased samples, with a medium degree of
Melon (Cucumis melo) * 19 - 19 5 5 5 4 ripeness, remove the skin and seeds
*
Watermelon (Citrullus lanatus) 27 - 27 7 7 6 7 Chop and mix together using blender (Moulinex, LM 30114A)
Cherries (Prunus avium) * 28 - 28 7 8 7 6 Combine 300 g of purchased samples, remove stalks
Grapes 19 10 9 5 5 5 4 Chop and remove seeds, mix together using blender (Moulinex, LM
30114A)
Lemons (Citrus Limon) 30 16 14 7 8 8 7 One sample with a medium size and degree of ripeness
Orange (Citrus sinensis) * 22 22 - 6 6 5 5 Remove the skin and seeds
Tangerine (Citrus tangerina) * 19 19 - 5 5 4 5 Chop and mix together using blender (Moulinex, LM 30114A)
*
Pomegranate (Punica granatum) 17 17 - 4 4 5 4 One sample with a medium size and degree of ripeness, remove the
outer skin, mix together using blender (Moulinex, LM 30114A)
Banana (Musa acuminate) 20 11 9 6 5 4 5 One sample with a medium size and degree of ripeness, remove the
Kiwi (Actinidia deliciosa) 19 19 - 4 5 5 5 outer skin, mix together using blender (Moulinex, LM 30114A)
Figs (Ficus carica) * 19 - 19 5 4 5 5 Combine 300 g of purchased samples, with a medium degree of
ripeness
Mash and mix together using hand blender (Moulinex, DD 100147)
Industrial fruit juice 19 9 10 5 5 5 4 One package, shake the bottles several times to mix the content
Green leafy vegetables (Basil, parsley, 233 113 120 60 55 57 61 Weight 100 g of each purchased sample, remove stalks below the
coriander, cress, dill, fenugreek, leek, leaves
mint, tarragon, spinach) Wash and drain on absorbent paper
Chop and mix together using grinder mill (Moulinex, LM 30114A)
Lettuce (Lactuca sativa) 41 19 22 9 10 10 12 Remove shriveled outer leaves or roots if any
Cabbage (Brassica oleracea capitata) 33 16 17 7 9 8 9 Wash and drain on absorbent paper
*
Cauliflower (Brassica oleracea botrytis) 19 19 - 5 4 5 5 Weight 100 g of each purchased lettuce, including both outer and
inner leaves
Chop and mix together using grinder mill (Moulinex, LM 30114A)
Celery (Apium graveolens) 21 12 9 5 5 5 6 Remove base of stalk, weight 100 g of each purchased sample
Wash and drain on absorbent paper
Chop and mix together using grinder mill (Moulinex, LM 30114A)
Radish (Raphanus sativus) 22 12 10 6 5 5 6 Combine 100 g of purchased samples, remove top, tail and leaves if
Scallion (Allium cepa) 19 10 9 5 5 5 4 any
Wash and drain on absorbent paper
Chop and mix together using grinder mill (Moulinex, LM 30114A)
Green Pea (Pisum sativum) * 19 - 19 4 5 5 5 Combine 100 g of purchased samples, remove outer skin
Carrot (Daucus carota sativus) 33 15 18 9 7 8 9 Cook at unsalted distilled water
Chop and mix together using hand blender (Moulinex, DD 100147)
Turnip (Brassica rapa)* 25 25 - 6 7 6 6 One sample with a medium size and degree of ripeness, remove top
Beetroot (Beta vulgaris)* 20 20 - 6 4 5 5 and tail
Wash and drain on absorbent paper
Cook at unsalted distilled water
Chop and mix together using blender (Moulinex, LM 30114A)
Potato (Solanum tuberosum) (Cooked or 28 15 13 7 7 6 8 One sample with a medium size, wash and drain on absorbent paper
fried) Peel and cook at unsalted distilled water (or fry in soy bean oil and
drain by oil-absorbent paper), Chop and mix together using hand
blender (Moulinex, DD 100147)
Tomato (Solanum lycopersicum) 39 19 20 9 11 9 10 One sample with a medium size, remove top
Wash and drain on absorbent paper
Chop and mix together using blender (Moulinex, LM 30114A)
Cucumber (Cucumis sativus) 41 20 21 11 11 9 10 One sample with a medium size, remove top and tail
Chop and mix together using blender (Moulinex, LM 30114A)
Zucchini (Cucurbita pepo cylindrica) 24 12 12 6 5 6 7 One sample with a medium size, wash and drain on absorbent paper
Eggplant (Solanum melongena) (Cooked 27 14 13 6 7 6 8 Cook at unsalted distilled water (or fry in soy bean oil and drain by oil-
or fried) absorbent paper), chop and mix together using hand blender
(Moulinex, DD 100147)
Mushrooms (Agaricus bisporus) 17 8 9 4 5 4 4
96 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105

Table 1 (Continued)
Food Number of sample Preparation method

Total Season Regions

Winter Summer South West North East

Combine 100 g of purchased samples, wash and drain on absorbent
paper
Chop and mix together using hand blender (Moulinex, DD 100147)
Onions (Allium cepa) 34 16 18 8 8 9 9 Combine 100 g of purchased samples, peel of skin
Garlic (Allium sativum) (Raw or fried) 19 9 10 4 5 5 5 Chop and mix together using hand blender (Moulinex, DD 100147)
(for fried sample, chopped onions fry in soy bean oil and drain by oil-
absorbent paper, then mix together)
Green beans (Phaseolus vulgaris) 29 15 14 8 7 6 8 Combine 100 g of purchased samples, top and tail beans and remove
string
Cook at unsalted distilled water
Chop and mix together using hand blender (Moulinex, DD 100147)
Beef, lamb, ground mixed meat,chicken 81 39 42 20 21 19 21 Weight 200 g of purchased samples, remove skin or fat tissue
meat Cook lean meat at unsalted distilled water
Mix together using grinder mill (Moulinex, LM 30114A)
Fish 23 12 11 5 5 6 7 Weight 200 g of purchased samples, remove skin and scales
Fry in soy bean oil and drain by oil-absorbent paper
Mix together grinder mill (Moulinex, LM 30114A)

Summer: 20 March to 21 September; Winter: 22 September to 19 March.

*Samples were available only for the mentioned season.
Cooked at approximately 100  C using distilled water without using salt, in stainless-steel pots, until ready for consumption (about 30 min).
Fried at approximately 180  C using soybean oil for an average of 30 minutes.

to obtain a clear liquid sample, extraction was followed by
filtration through filter paper (Whatman, grade 41, Ashless,
12.5 cm). For dairy products, sample extractions were also
centrifuged at 1200g for 15 min and the supernatants were
removed for ultra-filtration.
The prepared filtrates were assessed for nitrate and nitrite
content using the spectrophotometric method after Griess
reaction; reduction of nitrate to nitrite was accomplished using
VCl3 (Braman and Hendrix, 1989; Woollard and Indyk, 2014)
(Table 2).
Briefly, to measure nitrite content of the samples, Griess reagent
I, hydrochloric acid solution (if needed), and Griess reagent II were
added to the sample extract, the solution was incubated at 25  C for
15 min away from light following which the absorbance was read
at 538 nm using the enzyme-linked immunosorbent assay (ELISA)
reader (BioTek, Power wave XS2).
To measure total nitrate/nitrite concentration of the sample,
after proportional dilution of filtrate (if needed), VCl3 and Griess
reagents were added to filtrate; the volumetric flask was incubated
at 37  C for 180 min and then the absorbance was read at 538 nm
using ELISA reader. To optimize the reaction of nitrate to nitrite
reduction, different amounts of VCl3, and a range of various time
and temperature conditions were evaluated. To consider maxi-
mum reduction, color stability and reaction time simultaneously, a
temperature of 37  C and a duration of 180 min, respectively were
selected (Woollard and Indyk, 2014).
To determine nitrate and nitrite content of the samples, several
calibration curves were prepared, according to ISO instructions,
during the method development and routine testing, using a series
of sodium nitrite or potassium nitrate solutions; a similar
procedure was used for development of Griess reaction; the

Table 2
Details of nitrate and nitrite assessment in food samples.

Sample extraction Nitrite assessment Total nitrate/nitrite assessment Calculations

Vegetables, 1–10 g of samples + 5 mL sodium 10 mL of extract+ 20 mL distilled 10 mL of extract+ 5 mL distilledNO2 ¼ m1  200DF
V1m0
fruits, tetraburate solution + 100 mL hot water + 5 mL Griess reagent I + 3 mL water + 15 mL VCl3 + 5 mL GriessNO3 ¼ 1:35  ðNOx
NO2Þ
grains, water (70–80  C) ! 15 min on the HCl + 1 mL Griess reagent reagent I + 3 mL HCl + 1 mL Griess
m 0 = weight of sample
legumes boiling water-bath ! 2 mL Carrez II + double distilled water up to m 1 = mass (mg) of nitrite from
reagent II + double distilled water
and by I + 2 mL Carrez II ! double distilled 50 mL ! incubate at 25  C for up to 50 mL ! incubate at 37  C for
calibration curve
products water up to 200 mL ! filtrate 15 min away from 180 min ! absorbance at 538 nm DF (dilution factor)=1 (if no dilution was
(ISO, 1984) through filter paper light ! absorbance at 538 nm carried out)
v1 = portion of filtrate (mL)
Dairy 1–10 g of samples + 50 mL hot water 0.5 mL of extract + 0.27 mL distilled 0.5 mL of extract + 0.27 mL NO2 ¼ m1  100DF
V1m0
products (70–80  C) ! 15 min on the boiling water + 0.25 mL Griess reagent VCl3 + 0.25 mL Griess reagent NO3 ¼ 1:35  ðNOx
NO2Þ
(ISO, water-bath ! 5 mL Carrez I + 5 mL I + 0.25 mL Griess reagent I + 0.25 mL Griess reagent m 0 = weight of sample
2004) Carrez II+ double distilled water up II ! incubate at 25  C for 15 min II ! incubate at 37  C for m1 = mass (mg mL
1) of sodium nitrite
to 100 mL ! filtrate through filter away from light ! absorbance at 180 min ! absorbance at 538 nm from calibration curve
paper ! centrifuge at 3000 rpm for 538 nm DF = 1 (if no dilution was carried out)
15 min v1 = portion of filtrate (mL)
Meats and 10 g of samples + 5 mL sodium 10 mL of extract+ 20 mL distilled 10 mL of extract+ 5 mL distilled NaNO2 ¼ m1  1000DF
processed tetraburate solution + 100 mL hot water + 5 mL Griess reagent I + 3 mL water + 15 mL VCl3 + 5 mL Griess
V1m0 
KNO3 ¼ 1:46  m1  10000DF
V1m0
NaNO2
meats water (70–80  C) !15 min on the HCl + 1 mL Griess reagent reagent I + 3 mL HCl + 1 mL Griess m 0 = weight of sample
(ISO, boiling water-bath ! 2 mL Carrez II + double distilled water up to reagent II + double distilled water m1 = mass (mg mL
1) of sodium nitrite
1975a, b) I + 2 mL Carrez II+ double distilled 50 mL ! incubate at 25  C for up to 50 mL ! incubate at 37  C for from calibration curve
water up to 200 mL ! filtrate 15 min away from 180 min ! absorbance at 538 nm DF = 1 (if no dilution was carried out)
through filter paper light ! absorbance at 538 nm v1 = portion of filtrate (mL)

NO2: Nitrite; NO3: Nitrate; NaNO2: Sodium nitrite; KNO3: Potassium nitrate.
 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105 97

Table 3
Characteristics of the analytical methods for nitrate and nitrite.

LOD (mg 100 g
1) LOQ (mg 100 g
1) Recovery values (%) RSD (%) HorRat values
(n = 10) (n = 10) (n = 4) (n = 10)

Nitrate Nitrite Nitrate Nitrite Nitrate Nitrite Nitrate Nitrite Nitrate Nitrite
Fruits and vegetables
Beetroot 0.732 0.192 4.75 6.40 81.7–99.5 98.9–118 6.09 12.3 1.20 2.43
Spinach 0.948 0.194 6.92 5.59 98.5–109 97.2–127 7.15 11.6 1.41 2.29
Carrot 0.846 0.173 5.90 4.48 95.6–98.7 94.4–108 10.6 11.2 1.70 1.78
Cucumber 0.102 0.174 7.56 4.61 98.8–104 99.6–105 5.58 11.6 0.89 1.85
Tomato 0.198 0.091 5.39 2.69 101–107 88.0–109 6.42 11.5 0.89 1.61
Potato 0.269 0.182 8.20 5.38 88.8–116 83.7–112 11.6 10.9 1.62 1.53

Dairy
Yogurt 0.061 0.032 0.245 1.30 96.2–111 92.5–106 10.2 12.6 1.27 1.58
Cheese 0.074 0.042 0.296 1.69 92.5–109 89.9–110 9.05 11.1 1.13 1.38

Meats
Ground mixed meat 0.374 0.247 1.64 3.74 91.3–103 88.5–103 10.7 11.6 1.50 1.62
Chicken meat 0.353 0.245 1.42 3.53 85.2–94.1 94.7–107 2.54 3.17 0.35 0.43

Processed meats
Sausages 0.343 0.244 1.32 3.43 97.6–106 97.3–110 4.00 4.69 0.56 0.65
Ham 0.511 0.262 3.01 5.11 86.7–120 86.1–106 3.97 4.55 0.55 0.63

RSD%: Relative standard deviation; LOD: Limit of detection; LOQ: Limit of quantification.
 
HorRat value, the ratio of relative standard deviation calculated from the measured data to the relative standard deviation predicted from the Horwitz equation 2ð1
0:5logcÞ ;),

1
(where c is dimensionless mass fraction of analyte) was used to evaluate repeatability values; mg 100 g of nitrate and nitrite were multiplied to 10
5 to
convert to mass fraction (Horwitz and Albert, 2006).

calibration curve was then constructed by plotting absorbance
versus concentration.
Duplicate blank tests for nitrate and nitrite were prepared
similarly by replacing the test portion by water in the presence of
all reagents. To avoid exceeding the linearity of the test, filtrates
were retested using a lower amount of sample if nitrate levels were
over the calibration range.
2.5. Method validation
2.5.1. Linearity test of calibration curve
To accurately evaluate linearity of calibration lines and
ascertain whether the chosen regression models adequately fits
the data (Joris Van Loco et al., 2002), lack-of-fit (LOF) test was used.
2.5.2. Limit of detection and quantification (LOD and LOQ)
Determining LOD of the method, a minimum level of analyte
could be reliably detected, 10 replicate measurements of the
samples with concentration 0.05 mg 100 g
1 were used. The
standard deviation of 10 replicates was obtained and method LOD
was calculated using the formula: [LOD = 3  standard deviation of
response]; minimum level of analyte quantified with acceptable
accuracy and precision, LOQ, was calculated as [LOQ = 10  stan-
dard deviation of response] (ICH, 1996; Magnusson, 2014; Walfish,
2006). Determining LOD and LOQ for the analytes in food samples,
standard deviation of 10 replicate measurements of the food
samples, in different matrices and different nitrate/nitrite levels of
the samples, were used (Table 3).
the uncertainty of this difference (Du = 1.24 and 1.33, for nitrate
and nitrite, respectively) caused by the uncertainty of the Certified
Standard Solution (uref) and the uncertainty of the measured mean
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
p
values (um), and calculated by: Du = (k  u2 ref þ ðu2 m= nÞ),
where k was coverage factor and n was number of measurements
(Haesselbarth, 2006).
Method precision was determined by analyzing results from an
interday repeatability of the known concentrations of the standard
solutions of nitrate and nitrite procedure; the relative standard
deviations% = 100  SD/x, were 2.2 and 3.6% for nitrate and nitrite,
respectively.
To obtain more representative values for the between-day
intermediate repeatability precision, simultaneous replicates at
different times, on different matrices including plant foods (at
different nitrate/nitrite concentrations including spinach and
beetroot as high, cucumber and carrot as medium, and tomato
and apple as low), dairy products and meats, were performed
during the study period (Horwitz, 2002). HorRat value, the ratio of
relative standard deviation calculated from the measured data to
the relative standard deviation predicted from the Horwitz
equationð2ð1
0:5logcÞ ), where c is dimensionless mass fraction of
analyte, was used to evaluate repeatability values; mg 100 g
1
were multiplied to 10
5 to convert to mass fraction, for nitrate and
nitrite (Horwitz and Albert, 2006).
Repeatability relative standard deviation in the intra-day
analyses, determined by several food samples with a wide range
of nitrate-nitrite concentrations, resulted in average values of 2.7
and 5.8% for nitrate and nitrite, respectively.

2.5.3. Method trueness and precision

Trueness of measurement procedure was evaluated by analyz-
ing results from 6 repeated measurements of Certified Standard
Solution of nitrate and nitrite were purchased from Sigma-Aldrich
(prepared by FLUKA1 Analytical; no.74246, and Lot: BCBP0387 V
for nitrate, 67276 and Lot: BCBP4288 V for nitrite; purity 99.5%).
Absolute values of the difference between the Certified Standard


Solution and the mean measured values jDj ¼ jxm
xref j = 0.98
and 1.19 for nitrate and nitrite, respectively) were compatible with
2.5.4. Recoveries
The accuracy of the procedures was assessed by performing
spike recovery studies. The known concentrations of commercial
Certified Standard Solution of nitrate and nitrite (10% nitrate/
nitrite content of the samples) were used to fortify the samples of
vegetables, fruits, dairy, meat and processed meat products. To
assess the reliability of the outcome across the whole analytical
range, spiked samples (for fruits and vegetables) were in three
categories of nitrate/nitrite content (spinach and beetroot as high,
98 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105

cucumber and carrot as medium, and tomato and apple as low).
The experimental recovery was calculated as follows: [Recovery
% = (expected concentration of analyte  concentration of analyte
in spiked sample)  100].
2.5.5. Calculations
In the study, analyses were run in duplicate for all samples and
then the average values for all samples were considered for
calculation of nitrate/nitrite content of foods. Linear regressions of
the mean values of the absorbance at 538 nm for each standard
curves minus the blank values were used to determine the total
nitrate/nitrite and nitrite concentrations in the samples; the
respective formulas for calculation of nitrate/nitrite contents of
different food groups are provided in Table 2.
2.5.6. Statistical analysis
Mean and standard deviation, as well as median and inter-
quartile ranges (IQR) of nitrate and nitrite in the food samples were
calculated. Mean nitrate and nitrite values of the samples, collected
in winter and summer, were compared using the independent
sample t test. To evaluate linearity of the calibration lines, LOF test
was conducted using the general linear model. All statistical
analysis were conducted using SPSS (Version 16.0; Chicago, IL), and
P values < 0.05 were considered significant.
3. Results
3.2. Limit of detection and quantification
Method LOD was 0.61 mg 100 g
1 and 0.10 mg 100 g
1, for
nitrate and nitrite, respectively, and method LOQ was 1.86 mg
100 g
1 and 0.16 mg 100 g
1 for nitrate and nitrite, respectively.
Concentrations of both nitrate and nitrite in all samples were
higher than the method LOD levels. Higher LOD levels of nitrate/
nitrite in different samples are reported in Table 3. Lower LOD
levels of nitrate and nitrite were observed for yogurt and cheese
(0.06 and 0.07 mg 100 g
1 for nitrate, and 0.032 and 0.042 mg
100 g
1 for nitrite). Among vegetables, lower and higher levels of
LOD for nitrate were observed for tomato (0.20 mg 100 g
1) and
cucumber (1.02 mg 100 g
1), respectively. LOD levels of nitrate and
nitrite for meats and processed meats were 0.34–0.51 mg 100 g
1
and 0.24–0.26 mg 100 g
1.
3.3. Method trueness and precision
Relative standard deviations of the samples are shown in
Table 3. Repeatability estimations using pooled data of the samples
indicated a relative standard deviation of 5.2 and 12.7% for nitrate
and nitrite, respectively. The random errors of different nitrate
containing-foods were within the acceptable values for repeat-
ability (HorRat values=0.5-2); in the case of low-nitrate containing
foods as well as medium- and low-nitrite containing foods, higher
HorRat values were observed (Table 3).

3.1. Method Validation 3.4. Recoveries

3.1.1. Calibration curve and linearity test
Four calibration curves were fitted to estimate nitrate-nitrite
contents of plant-based foods, dairy and meat products. The
determination coefficients (r2) of calibration graphs were within
an acceptable range (r2 = 0.974–0.999). The result of LOF test
confirm linearity of calibration lines and fitness of the regression
models (Plant-based foods: F = 1.78, P = 0.15; meat and processed
meats: F = 1.62, P = 0.24; dairy products: F = 1.84 and 1.96, P = 0.11
and 0.13, for nitrite and nitrate).
3.4.1. Nitrate and nitrite content of grains, legumes and nuts
Nitrate and nitrite content of frequently consumed grains,
legumes and nuts are reported in Table 4. Our analysis determined
a mean 0.6 mg 100 g
1 for nitrite content and 24.3 and 59.6 mg
100 g
1 mean nitrate levels for rice and barley, respectively. The
inter-quartile ranges (IQRs) of nitrate and nitrite were 39.1–
67.4 mg 100 g
1 and 0.19–0.34 mg 100 g
1 in industrial breads, and
were 29.3–64.7 mg 100 g
1 and 0.17–0.39 mg 100 g
1 in traditional
breads.

Table 4
Nitrate and nitrite content of commonly consumed grains, legumes and nuts.

Food items Nitrite (mg 100 g
1) Nitrate (mg 100 g
1)

N Mean SD Median IQR Mean SD Median IQR

Grains
Barley 19 0.61 0.35 0.59 0.24–0.95 59.6 32.5 59.5 32.0–71.6
Rice 21 0.63 0.34 0.68 0.27–0.86 24.3 11.9 22.7 14.4–30.6
Industrial breads 31 0.39 0.33 0.23 0.19–0.34 51.3 21.5 53.3 39.1–67.4
Traditional breads 82 0.32 0.24 0.23 0.17–0.39 51.2 27.4 49.7 29.3–64.7
Lavash 20 0.29 0.21 0.23 0.16–0.28 46.8 16.3 45.6 31.0–60.2
Barbari 21 0.32 0.26 0.22 0.17–0.35 49.3 23.8 56.3 24.8–62.2
Sangak 19 0.29 0.24 0.17 0.15–0.34 56.3 33.6 45.3 38.6–87.2
Taftoon 22 0.39 0.24 0.31 0.25–0.40 51.0 32.4 56.8 21.9–67.8

Legumes
Soya 21 0.72 0.23 0.68 0.48–0.96 10.3 6.53 8.17 4.50–14.6
Cowpea 19 0.46 0.31 0.43 0.35–0.46 10.6 0.63 10.4 10.0–11.4
Red bean 18 0.46 0.32 0.38 0.22–0.73 15.7 7.80 14.6 9.01–20.9
White bean 20 0.69 0.21 0.73 0.54–0.84 18.5 4.53 18.3 13.5–21.9
Chickpea 27 0.55 0.34 0.51 0.19–0.91 32.2 29.0 18.9 10.7–45.6
Lentil 24 0.51 0.34 0.42 0.22–0.75 11.3 5.17 12.4 6.40–15.0
Split pea 17 0.73 0.37 0.81 0.33–0.95 37.5 18.1 32.3 23.8–55.6
Broad beans 23 0.77 0.25 0.76 0.68–0.98 35.4 24.1 25.2 21.6–40.8

Nuts
Pistachio 19 0.34 0.22 0.31 0.21–0.34 59.1 44.2 45.6 25.2–112
Almond 19 0.17 0.02 0.16 0.14–0.19 28.5 21.0 16.3 13.2–46.7

IQR: Inter-quartile range.

 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105 99

Compared to samples collected in winter, a significant higher
nitrate concentration was observed in breads samples collected in
summer including Barbari (63.3 vs. 40.6 mg 100 g
1, P < 0.01),
Sangak (64.2 vs. 50.0 mg 100 g
1, P < 0.05), Lavash (53.7 vs. 41.4 mg
100 g
1, P < 0.05), Taftoon (62.8 vs. 41.8 mg 100 g
1, P < 0.05), and
Industrial breads (55.0 vs. 47.7 mg 100 g
1, P < 0.05). Among
legumes, soybean and cowpeas had the lowest nitrate values
(10.3 and 10.6 mg 100 g
1, respectively), whereas split pea and
broad beans contained the highest levels (37.5 and 35.4 mg 100 g
1,
respectively); mean nitrite concentrations in legumes ranged from
0.46–0.77 mg 100 g
1.
3.6. Nitrate and nitrite content of meats, processed meats, and dairy
products
Nitrate and nitrite content of meats, processed meats, and dairy
products are presented in Table 7. The range of nitrite and nitrate
content of natural meats (without preservatives) including beef,
lamb, ground mixed meat, and fish meat was 3.39–4.96 mg 100 g
1
and 5.56–13.3 mg 100 g
1, respectively. The range of nitrite and
nitrate concentrations in processed meat products including
canned fish, ham, sausages, and hamburgers were 2.75-13.9 mg
100 g
1 and 6.09–19.4 mg 100 g
1, respectively.

4. Discussion
3.5. Nitrate and nitrite content of vegetables, fruits and byproducts
In this study, 1872 samples of 87 food items including grains,
As shown in Table 5, the range of nitrite in our fruit samples
legumes, fruits and vegetables, dairy products, meats and
varied from 0.15 mg 100 g
1 in pomegranate to 0.71 mg 100 g
1 in
processed meats collected from different regions of Tehran, were
grapes. Lowest mean nitrate levels was observed in peaches
analyzed for nitrate and nitrite concentrations. The results of study
(7.49 mg 100 g
1), kiwi (9.65 mg 100 g
1), pomegranate (9.74 mg
are summarized in Fig. 1.
100 g
1), and apple (10.3 mg 100 g
1), whereas cantaloupe
(46.8 mg 100 g
1), banana (44.4 mg 100 g
1), and tangerine
4.1. Method validation
(32.6 mg 100 g
1) had the highest nitrate concentrations; other
fruits had a mean nitrate content between 10.0–25.0 mg 100 g
1.
Our method LOD was sufficiently low for the determination of
Analysis of common brands of commercial fruit juices showed
nitrate and nitrite in the samples. In previous studies, the LOD of
mean nitrate and nitrite concentrations of 12.5 mg 100 mL
1 and
methods used to analyse nitrate in food samples, varied between 1
0.44 mg 100 mL
1, respectively.
and 50.0 mg 100 g
1; 60% of these analytical results were reported
Nitrate and nitrite levels in different type of vegetables are
with a LOD <0.5 mg 100 g1 and less than 2% with a LOD
shown in Table 6. Vegetables with the highest nitrate concen-
>100 mg kg
1 (Alexander et al., 2008). To consider confounding
trations included radish (626 mg 100 g
1), beetroot (495 mg
effects of food matrices, LOD and LOQ were also calculated for the
100 g
1), tarragon (424 mg 100 g
1), lettuce (365 mg 100 g
1),
analytes in original samples with different food matrices and
mint (279 mg 100 g
1), celery (261 mg 100 g
1). As seen in the
different levels of the analytes. LOD levels of nitrate and nitrite in
table, the lowest average nitrate values were documented for
different samples were comparable with previous reports.
tomato (17.5 mg 100 g
1), potato (37.6 mg 100 g
1), garlic (35.0 mg
Recovery values from various food samples were mainly within
100 g
1), corn (27.9 mg 100 g
1), green beans (46.6 mg 100 g
1),and
the acceptable range (81.7–116% and 83.7–127% for nitrate and
carrot (50.3 mg 100 g
1). Compared to samples collected in winter,
nitrite, respectively). In agreement with previous studies, a higher
a significant higher nitrate concentration was observed in summer
variability of recovery values was observed for spiked samples of
samples of some vegetables, including leek (227 vs. 147 mg 100 g
1,
the vegetables (Prasad and Chetty, 2008). Recommended values for
P < 0.01), mint (322 vs. 194 mg 100 g
1, P < 0.01), spinach (268 vs.
recovery of nitrate is dependent to concentration levels of nitrate,
137 mg 100 g
1, P < 0.01), green beans (61.7 vs. 36.0 mg 100 g
1,
and is reported 60–120% for vegetables with a nitrate content
P < 0.01), potato (43.3 vs. 30.0 mg 100 g
1, P < 0.05), and onions
<50.0 mg 100 g
1 and 90-110% for vegetables with a nitrate
(86.7 vs. 44.9 mg 100 g
1, P < 0.05).
content 50.0 mg 100 g
1 (EC, 2006).

Table 5
Nitrate and nitrite content of commonly consumed fruits.

Food items Nitrite (mg 100 g
1) Nitrate (mg 100 g
1)

N Mean SD Median IQR Mean SD Median IQR

Apple 21 0.51 0.27 0.16 0.15–0.19 10.3 0.638 16.2 13.1–46.7
Apricot 19 0.46 0.24 0.33 0.25–0.62 17.8 9.20 16.6 12.2–20.2
Pears 17 0.43 0.18 0.41 0.28–0.54 17.3 10.3 16.8 8.31–23.8
Peaches 25 0.54 0.18 0.54 0.39–0.67 7.49 3.80 7.79 3.48–10.3
Cantaloupe 18 0.54 0.31 0.47 0.25–0.85 46.8 45.4 26.5 10.9–107
Melon 19 0.42 0.32 0.32 0.16–0.58 22.3 15.6 18.3 11.5–25.5
Watermelon 27 0.48 0.23 0.52 0.31–0.66 11.0 8.16 9.07 5.02–16.3
Cherries 28 0.42 0.35 0.22 0.15–0.62 21.2 12.5 17.7 11.0–26.1
Grapes 19 0.71 0.31 0.72 0.44–0.98 16.0 7.63 16.9 8.90–22.4
Kiwi 19 0.55 0.22 0.52 0.38–0.68 9.65 5.14 9.5 6.99–11.2
Lemons 30 0.58 0.23 0.57 0.46–0.66 24.8 9.31 27.2 22.4–30.4
Orange 22 0.45 0.24 0.43 0.25–0.70 13.2 11.2 9.89 6.98–14.7
Tangerine 19 0.53 0.26 0.54 0.35–0.63 32.6 24.8 27.3 17.5–39.8
Plums 27 0.68 0.18 0.67 0.59–0.81 12.7 8.40 8.9 6.61–19.6
Banana 20 0.52 0.28 0.56 0.35–0.61 44.4 16.1 42.5 36.7–51.9
Figs 19 0.51 0.37 0.64 0.13–0.83 24.1 2.53 22.3 21.4–26.3
Pomegranate 17 0.15 0.01 0.14 0.13–0.15 9.74 1.32 9.79 8.78–10.4
Industrial fruit juice 19 0.44 0.12 0.44 0.35–0.49 125 11.1 6.1 4.02–24.2

IQR: Inter-quartile range.

Nitrate/nitrite contents were determined in row samples of fruits.
100 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105

Table 6
Nitrate and nitrite content of commonly consumed vegetables.

Food items Nitrite (mg 100 g
1) Nitrate (mg 100 g
1)

N Mean SD Median IQR Mean SD Median IQR

Leafy vegetables
Basil 19 0.67 0.32 0.72 0.44–0.96 236 91.9 258 154–281
Parsley 30 0.36 0.19 0.36 0.20–0.43 171 182 129 79.7–196
Coriander 26 0.42 0.19 0.39 0.25–0.49 237 101 196 146–337
Cress 19 0.37 0.22 0.39 0.19–0.43 225 168 173 118–249
Dill 20 0.48 0.26 0.4 0.28–0.78 183 140 161 86.9–259
Fenugreek 21 0.60 0.32 0.57 0.27–0.89 656 41.8 55.2 45.9–71.4
Leek 21 0.61 0.28 0.65 0.35–0.81 177 96.5 161 87.5–277
Mint 21 0.67 0.37 0.79 0.23–0.11 279 248 182 82.4–396
Tarragon 19 0.55 0.27 0.53 0.29–0.78 424 298 355 230–496
Spinach 37 0.43 0.29 0.36 0.23–0.65 183 124 142 73.4–242
Lettuce 41 0.54 0.31 0.51 0.28–0.84 365 232 313 210–468
Celery 21 0.43 0.29 0.41 0.19–0.63 261 160 206 139–337
Cabbage 33 0.54 0.24 0.54 0.38–0.69 198 146 155 101–249

Roots
Radish 22 0.49 0.40 0.24 0.16–0.92 626 318 509 393–898
Turnip1 25 0.38 0.28 0.36 0.18–0.49 223 285 120 97.6–210
Beetroot1 20 0.56 0.37 0.48 0.20–0.91 495 127 508 413–581
Carrot 33 0.48 0.28 0.39 0.24–0.66 50.3 29.7 38.7 25.4–73.8

Starchy vegetables
Green Pea 19 0.52 0.31 0.61 0.18–0.89 88.2 61.4 68.2 22.5–143
Potato1,2 28 0.49 0.33 0.42 0.12–0.77 37.6 17.1 33.2 25.7–48.5
Corn 19 0.53 0.32 0.46 0.22–0.91 27.9 12.7 31.4 12.9–39.4

Other vegetables
Tomato 39 0.55 0.26 0.56 0.36–0.77 17.5 11.6 15.4 8.40–26.1
Cucumber 41 0.57 0.31 0.46 0.33–0.81 87.7 70.1 57.1 44.6–103
Scallion 19 0.48 0.34 0.39 0.21–0.76 101 55.6 72.8 55.4–151
Zucchini1,2 24 0.53 0.31 0.4 0.30–0.76 108 64.4 96.6 60.0–160
Mushrooms 17 0.26 0.17 0.24 0.13–0.29 65.4 33.4 57.9 50.9–60.3
Eggplant1,2 27 0.72 0.28 0.71 0.51–0.93 110 106 57.4 31.6–174
Cauliflower 19 0.39 0.23 0.36 0.21–0.52 102 92.2 64.2 25.7–190
Onions2 34 0.49 0.30 0.45 0.23–0.64 60.9 79.5 34.9 15.5–59.3
Garlic2 19 0.21 0.08 0.18 0.16–0.26 35.0 5.89 35.2 30.4–39.9
Green beans1 29 0.38 0.34 0.28 0.08–0.73 46.6 36.9 49.2 6.30–75.1
Tomato paste/sauce 19 0.56 0.36 0.48 0.33–0.74 36.2 56.9 9.07 5.03–16.3
Dill Pickles 15 0.74 0.38 0.95 0.18–0.1.0 20.4 5.54 21.5 14.5–27.1
Potato chips 16 0.39 0.21 0.27 0.21–0.63 41.4 7.83 45.8 32.3–48.2

IQR: Inter-quartile range.

Nitrate/nitrite contents were determined in row samples unless stated otherwise.
1
Cooked at approximately 100  C using distilled water without using salt, in stainless-steel pots, until ready for consumption (about 30 min).
2
Fried at approximately 180  C using soybean oil for an average of 30 min.

Table 7
Nitrate and nitrite content of commonly consumed meats, processed meats and dairy products.

Food items Nitrite (mg 100 g
1) Nitrate (mg 100 g
1)

N Mean SD Median IQR Mean SD Median IQR

Beef1 23 3.87 1.19 3.7 2.84–4.84 8.35 8.32 7.16 3.44–8.60
Lamb1 18 4.96 1.73 4.4 3.87–5.85 7.43 4.79 6.18 4.56–10.5
Ground mixed meat1 13 3.72 1.16 3.59 2.71–4.85 12.4 7.27 9.38 6.75–19.6
Chicken meat1 27 4.18 1.75 4.14 2.79–5.00 13.3 13.1 8.57 7.02–15.7
Fish2 23 3.39 1.67 2.82 2.21–4.14 5.56 3.27 5.17 2.58–8.62
Canned fish 18 2.93 0.52 2.97 2.51–3.36 6.09 5.03 4.49 2.94–7.23
Ham 32 5.76 3.20 4.52 3.55–7.40 19.4 22.6 11.4 8.60–16.5
Sausages2 45 13.9 7.40 13.9 9.72–17.2 18.8 20.3 14.9 10.8–17.7
Hamburger2 28 2.75 0.58 2.64 2.29–3.02 10.7 3.48 9.87 8.08–12.1
Low-fat milk 20 0.14 0.06 0.12 0.11–0.17 1.26 0.91 1.28 0.28–2.09
High-fat milk 24 0.15 0.05 0.13 0.11–0.18 1.32 1.32 1.02 0.79–1.52
Low-fat Yogurt 25 0.45 0.21 0.39 0.29–0.52 5.75 2.96 4.21 2.85–8.46
High-fat Yogurt 26 0.21 0.11 0.24 0.08–0.28 2.86 2.27 2.79 0.90–3.80
Cheese 29 0.34 0.15 0.34 0.26–0.41 2.96 3.72 1.36 0.14–4.58
Dough 18 0.17 0.08 0.16 0.10–0.20 1.46 0.57 1.74 0.82–1.86

IQR: Inter-quartile range.

1
Cooked at approximately 100  C using distilled water without using salt, in stainless-steel pots, until ready for consumption (about 30 min).
2
Fried at approximately 180  C using soybean oil for an average of 30 min.
 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105
Fig. 1. Mean (minimum-maximum) nitrate and nitrite contents of the samples in each food group.
4.2. Nitrate and nitrite content of grains, legumes and nuts
Our samples of white rice, barley and wheat flour by products,
as staple foods of grains in Iranian food culture, indicated a high
nitrate/nitrite levels compared to Asian reports; a relatively high
nitrate concentration was observed in both traditional and
industrial breads (50.0 mg 100 g
1) whereas nitrite levels were
comparable with previous reports ( 0.3 mg 100 g
1). Considering
the high proportion of breads (320 g/d) in dietary pattern of Iranian
population (Bahreynian and Esmaillzadeh, 2012), nitrate exposure
from this food group may be considerable.
Limited data are available regarding nitrate/nitrite content of
breads and cereal products; previous studies report a relatively low
level of nitrate and nitrite in breads (mean=1.77–2.50 and 0.13–
0.17 mg 100 g
1,for nitrate and nitrite, respectively) and wheat
flour (range = 0.10–1.98 and 0.10–0.46 mg 100 g
1 for nitrate and
nitrite, respectively) (Jakszyn et al., 2004; Nabrzyski et al., 1990).
Nitrate and nitrite contents of traditional breads in Turkey also
ranged 0.71-8.54 and 0.04-1.54 mg 100 g
1 (Ayaz et al., 2007). In a
recent study in Iran, mean nitrate levels in grains and breads were
reported 33.6-42.0 mg 100 g
1 (Taghipour et al., 2014). Mean
nitrate and nitrite concentrations of rice in Japan were 2.10 and
0.014 mg 100 g
1, respectively (Himeno et al., 2003). Mean nitrate
and nitrite levels in different rice varieties were reported 17.0-94.0
and 0.006-0.011 mg 100 g
1 in Nigeria (Ezeagu, 2006).
4.3. Nitrate and nitrite content of vegetables and fruits
Mean nitrite contents fruit samples were relatively high,
compared to nitrite contents of most fruits were <0.05 mg
100 g
1 reported from other countries (Reinik et al., 2009). The
highest content of nitrate among fruit samples was observed in
cantaloupe and banana (46.8 and 44.4 mg 100 g
1), which was 4-5
times of fruits such as apple, peaches, watermelon, kiwi,
pomegranate, plums and orange. A wide range of nitrate and
nitrite levels, 0.05-13.7 and 0.05-6.5 mg 100 mL
1, have been
reported in packed fruit juices by an Iranian study (Rezaei et al.,
2014), whereas the range of nitrate and nitrite in fruit juices were
lower in other reports (0.31-0.96 and 0.97-2.02 mg 100 mL
1, for
nitrite and nitrate, respectively) (Okafor and Ogbonna, 2003).
Overall, a wide range of nitrate levels were observed in our
vegetables. Levels of nitrate reported in vegetables ranges from
<1 mg kg
1 to >1000 mg 100 g1, depending on factors including
genotype, light intensity, soil composition, growth conditions,
fertilizers, harvesting and storage conditions (Hord et al., 2009). It
has been reported that dark green leafy vegetables mostly contain
extremely large amounts of nitrate and nitrite, compared to other
vegetables (Himeno et al., 2003; Hord et al., 2009). In this study,
among roots vegetables, radish had a highest nitrate levels (10,
101
2.5 and 1.3 times of carrot, turnip and beetroot). Among green leafy
vegetables, tarragon had a highest nitrate level (2 times of basil,
coriander, cress, dill and leek).
Our findings on nitrate levels of vegetables were consistent
with the proposed classification for nitrate-containing vegetables
as low- (<10 mg 100 g
1), medium- (10–100 mg 100 g
1) and high-
nitrate (>100 mg 100 g
1) (IARC, 2010). In another classification,
nitrate-containing vegetables were categorized as very low, low,
middle, high, and very high (<20, 20-50, 50-100, 100-250, and >
250 mg nitrate 100 g
1) (Hord et al., 2009); accordingly, in our
study, tomato is classified as very low, corn, garlic, potato, green
beans, as low, carrot, onions, mushroom, fenugreek, cucumber,
green pea as medium, scallion, cauliflower, zucchini, eggplant,
parsley, leek, dill, spinach, cabbage, turnip, cress, basil, coriander as
high, and celery, mint, lettuce, tarragon, beetroot, and radish are
considered as very high nitrate-containing vegetables (Fig. 2).
In our study, mean nitrate levels in 68.3% of lettuce, 92.5% of
potato, 90.9% of radish, and 51.0% of cabbage samples exceeded the
maximum limits legislated by European countries for trade of
some vegetables (250.0 mg 100 g
1 for lettuce, 20.0 for potato mg
100 g
1, 150.0 for cabbage mg 100 g
1, 350.0 mg 100 g
1 for radish)
(Maynard and Barker, 1979; Santamaria, 2006).
Nitrite concentrations of vegetables were generally low, with
average <1 mg kg
1 in most reports, however in some studies
higher levels of nitrite with a wide range have been reported
(Table 8). Nitrite contents of vegetables in our study (0.17-0.74 mg
100 g
1) were comparable to Japan, Turkey, Korea, and some
European countries but were higher than ranges (0.01-0.25 mg
100 g
1) reported from Finland, Slovenia and Poland (Reinik et al.,
2009). Correia et al. in an analysis of different varieties of
vegetables, found nitrite concentrations ranged 0.11-5.4 mg
100 g
1 (Correia et al., 2010); Mor et al. also reported a wide
range of nitrite levels in vegetables especially in lettuce (0.29-
0.88 mg 100 g
1), spinach (0.38-1.27 mg 100 g
1) and radish (0.24-
1.42 mg 100 g
1) (Mor et al., 2010). Similarly, nitrite content of
different vegetables in Denmark and Germany ranged 0.015-1.10
and <0.01-1.96 mg 100 g
1, respectively (Reinik et al., 2009);
nitrite concentration of vegetables in a survey in Korea were also
reported to range from 0.01 to 63.5 mg 100 g
1 (Suh et al., 2013).
Increased use of nitrogen-containing fertilizers, and ground-
water nitrate pollutions in some parts of Iran (Mahvi et al., 2005)
may be an explanation for higher nitrate/nitrite concentrations in
some vegetables and breads samples.
4.4. Nitrate and nitrite content of meats, processed meats, and dairy
In our cured and fresh meat samples, sodium nitrite content
ranged from 3.39 to 13.9 mg 100 g
1, and potassium nitrate content
ranged from 5.56–19.4 mg 100 g
1. In previous studies a wide range
102 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105
Fig. 2. Classification of the vegetable samples of the current study according to
nitrate contents; A) Very high and high nitrate-containing vegetables (>250 and
100–250 mg nitrate 100 g
1); B) Medium nitrate-containing vegetables (50–100 mg
nitrate 100 g
1); C) Low and very low 20–50 and <20 mg nitrate 100 g
1).
of nitrite and nitrate content 0.02–19.5 and 0.37 to 0.14–44.5 mg
100 g
1 was reported (Hsu et al., 2009; Ysart et al., 1999).
According to maximum permitted levels of nitrate and nitrite in
processed meat products vary from country to country; according
to United States Food and Drug Administration (FDA) regulations,
level of sodium nitrate and sodium nitrite does not exceed 500 and
200 ppm in the finished product (Sindelar and Milkowski, 2012).
According to Australia and New Zealand food standard, nitrate and
nitrite are permitted to be added to processed meat products to a
maximum level of 50.0 and 12.5 mg 100 g
1 (Thomson et al., 2007).
Permitted levels of nitrate and nitrite in processed meats in Iran is
500 and 120 ppm, respectively, wheraese there is no determined
levels for agriculture products (Sadeghi et al., 2014).
Compared to other foods such as vegetables and meat products,
total nitrate/nitrite contents of dairy products are low. Main
residues of nitrate and nitrite in dairy products are related to oral
administration of potassium nitrate to dairy cows, added water
with high nitrate content, and use of nitrate/nitrite containing food
additives; increased nitric oxide production during endotoxin-
induced mastitis also leads to higher nitrate/nitrite levels in dairy
products (Reinik et al., 2009). Nitrite and nitrate content of our
dairy products was relatively low; previous studies reported a
mean nitrate and nitrite concentration 0.08-4.48 and 0.07-
2.88 mg kg
1 (Lima et al., 2006; Merino, 2009).
4.5. Study limitations
Although use of high performance liquid chromatography
(HPLC) and ion chromatography (IC), has gained more popularity in
recent years, some advantages of spectrophotometric methods
including convenience, feasibility and inexpensive equipments
make them as appropriate choices for measurement of nitrate/
nitrite in food matrices (Reinik et al., 2009). Moreover, an
acceptable agreement has been reported between these methods
in several previous studies (Alonso et al., 1992; Bianchi et al., 1995;
Schuster and Lee, 1987).
There was some limitations in our study should be considered
in interpretation of the findings. Due to lack of matrix matched
food control materials, potential confounding effects of food
matrices on color development during Griess reaction were not
determined in our study. It has been suggested that interferences
occurred in different food matrices, such as vitamin C, flavonoids,
magnesium, iron and calcium as natural interferences, as well as
sulphate and chloride as added additives interferences could affect
spectrophotometric determination of nitrate and nitrite in foods
(Lopez-Moreno et al., 2016; Moorcroft et al., 2001).
Moreover, buffering effect of organic acids which could affect
the absorbance of azo dye, or any component is able to compete
with sulphanamide reagent for nitrite during the color develop-
ment is also lead to reduced recovery (Walters, 1996). Theoreti-
cally, antioxidative components of foods such as ascorbate and
sulphydryl thiols can destroy nitrous acid and lead to a reduction in
the recovery of nitrite (Moorcroft et al., 2001); however, different
concentrations of ascorbic acid has not interfered over nitrite
spectrophotometric analyses in processed meat samples, and a
little (<5–15%) or no effect of different matrices on final
determination of nitrate/nitrite content of food samples has been
reported in previous investigations (Casanova et al., 2006; Lopez-
Moreno et al., 2016; Reis et al., 2009). The interference of anions
(phosphate, sulfate, Iodide, oxalate, acetate) and cations (calcium,
magnesium, potassium, zinc, iron, manganese, and copper) on
spectrophotometric determination of nitrate was also reported
<5% (Fakhre and Qader, 2013). On the other hand a good
agreement of the results obtained by colorimetry compared to
HPLC method, where confounding effects of food matrices is less
 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105 103

Table 8
Nitrate and nitrite content (mg kg
1) of commonly consumed vegetables, reported in previously published results from different countries.

(a)

Food items Turkey (Ayaz et al., 2007; Japan China (Chung et al., Korea (Chung et al., UK (Meah et al., Germany (Reinik Italy(De Martin and
Mor et al., 2010) (Himeno 2011; Zhou et al., 2003; Reinik et al., 1994; Ysart et al., et al., 2009) Restani 2003;
et al., 2003; 2000) 2009 1999) Santamaria et al.,
Reinik et al., 1999)
2009)

NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2
Lettuce 1439 949 11.0 6.9 1226 10.1 580-1452 0. 1-0.4 2430 0.6 2330 0.6 750-5500 – 1089 –
914 380 0.8 0.4 340-2700 ND-5.9 33-3944 0.3-129 1051 – 1489 – 672-1745 –
Cabbage 510 250 2.25 0.95 1040 0.7 1200 – 725 0.4 712 0.8 3100 – 400 –
1100 0.5 480-2900 ND-1.2 1740 ND-25.0 338 – ND-3270 –
222-1366 26-1523 – 8-929 –
390 ND
Spinach 1132 281 7.2 2.9 3560 7.0 3700-6300 0.9-2.6 4259 1.0 2470 3.8 900-5400 – 1845 –
1456 658 2.3 2.6 1100-4700 ND-5.7 17.6-6719 ND-246 1631 – ND-3720 –
190-2500 ND-1.2 266-2834 ND 547-3350 –
Turnip 43.4 0.8 2127 ND 380-970 –
Parsley 1070 612 0.7 0.4 2679 2.3 63.3-4626 ND-1635 780 1150 –
1513 804 1.8 1.4
Radish 3428 3172 6.4 4.3 1060 0.4 1400-2078 < 0.8 1878 0.8 2600 0.3 780-2400 – 2067 –
2725 6.4 414-1622 ND-0.3 ND-3486 ND-48.5 1117-2993 –
Tomato 11.1 13.3 0.4 0.3 1.55 0.07 ND-180 ND-0.9 17 ND 50.0 –
ND-71.0 ND-1.3 1-131 ND-0.2 4-42
Celery 8200 2.6 119-1589 0.1-0.6 639 – ND-150 ND-19.0 1678 –
2090 – 101-6302 – 1009-2167 –
Leek 40.0 11.0 0.7 0.3 547 0.6 53.0 – 1631
480
Beetroot 1600-4100 3.1-8.9 1211 – 1630 – 1727 –
1520 – 224-1877 ND-71.0 1023-2414 –
Carrot 190 131 0.6 0.3 193 4.1 43-490 ND-1.1 316 0.1-178 97 195 –
ND-2800 ND-1.8 1031 220 0.8-1.1 ND-1005 11-566 28-394 –
– 457
Potato 713 4.6 100-270 ND-1.7 452 155 2.0-60 81.0 –
199 346 0.8 ND-640 3-1077 ND-179 –
75-283
Onion 124 10.0 5.0-36.0 ND-1.1 436 ND-3.4 80-210 410 –
566 42.0-8921 ND-14 6.1-1319 13-192 69-1046 –

(b) Nitrite and nitrite content of commonly consumed vegetables, reported in previously published results from different countries.

Food items USA (Keeton et al., Greek (Fytianos and Tunisia( France (Menard et al., Slovenia(Sušin Jordan (Amr and Iran (Rezaei et al., 2014;
2009; Sciences Zarogiannis 1999; Razgallaha et al., 2008) et al., 2006) Hadidi 2001) Ziarati and
1981;) Siomos and Dogras 2016) Arbabi
Bidgoli, 2014)
2000; Sušin et al.,
2006)

NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2
Lettuce 850 0.6 282 – 1079 1973-5600 0.1-6.2 1074 0.2 4.6-96.0 ND- 3359 0.8
90-13000 – 8-808 – 795-1200 21-3986 ND-1.4 2.5 1123 –
1100-1400 0.3-0.4 222 – 360 230* 0.54 0.31*
Cabbage ND-2700 – 209 243 – 1963 498-1855 ND-18.6 881 0.2 1.3-53.7 ND- 1272 1.4
418 0.1 19.6-414 – 1842-2160 112- ND- 2.6 376 –
780 0.5 208 – 1864 0.4 190 140* 0.54 0.24*
Spinach 2797 8.0 1250 4.0 1681-8700 10.1- 2.5-55.0 ND- 3440 6.2
65-8000 ND-137 454-3760 0.8- 18.8 2.5 1770 –
2220 375 – 8.5 180 120* 0.43 0.29*
0.7 0.3
Turnip 3460 358 4.4 3.0 137-851 – 2970 656-2128 ND-2.0 10.7- ND-3.1 58.8 –
2460- 41.0 220 280* 0.38 0.28*
3600
Parsley ND-4100 202-937 – 1980 1980-4300 ND-2.0 2246 ––
720-2730 214 221 0.36 0.19*
170 180*
Radish 60-9000 – 100-663 – 2400 1860- ND-3.4 584 91.0 4.2 0.3
2400-3000 0.2 1800-2800 5000 620 320* 0.49 0.40*
Tomato ND-170 – 34.2 19.4 – 57.0 17.6-157 ND-2.0 4.3 0.5 1.4-24.1 ND-1.0 7.8 1.8
8.2-54.6 – 25.0-91.0 2.0-12.0 ND-1.5 12.8 2.5 2.9 0.4
62.0 – 17.0 12.0* 0.55 0.26*
Celery 1495 0.1 250 242 – 1240- ND-2.0 3632 3.3
50-5300 0.4-0.5 43-692 – 2500 2960 –
1600-2680 – 95.0-290 – 260 160* 4.3 2.9*
Leek 132 124 409-2860 ND-2.5 550 ––
42.6-333 235 219 0.61 0.28*
170 96.0*
Beetroot 100-4500 – 6.0 4.6 – 543 644-1144 –
3010 688 – 443-981 – 490-610
104 Z. Bahadoran et al. / Journal of Food Composition and Analysis 51 (2016) 93–105

Table 8 (Continued)
(b) Nitrite and nitrite content of commonly consumed vegetables, reported in previously published results from different countries.

Food items USA (Keeton et al., Greek (Fytianos and Tunisia( France (Menard et al., Slovenia(Sušin Jordan (Amr and Iran (Rezaei et al., 2014;
2009; Sciences Zarogiannis 1999; Razgallaha et al., 2008) et al., 2006) Hadidi 2001) Ziarati and
1981;) Siomos and Dogras 2016) Arbabi
Bidgoli, 2014)
2000; Sušin et al.,
2006)

NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2 NO3 NO2
3045 0.55-1.4
2255-3646 –
490 130* 0.56 0.37*
Carrot ND-2800 – 6.0 0.5 – 890 120-861 ND-4.1 264 0.2 333-797 ND-1.1
72.0 56.0 – 87.0 – 440-1800 7-1042 ND- 50.0 30.0* 0.48 0.28*
0.4
Potato 57-1000 – 32.0 – 225 191-730 ND-10.5 158 1.2 6.6-54.0 ND-1.3 2520 0.9
11-59 – 474 717 – 2-704 ND-7.6 347 45 –
215-1080 88.0 61.0* 0.52 0.31*
Cucumber 17-570 – 157 188 – 875 191-1800 – 63 0.2 42.7 9.0
24.0 0.5 19.5-576 – 800-959 4-245 ND- 12.3-88.6 2.8-18.5
0.8 88.0 70.0* 0.57 0.31*
Onion 128 0.4 127 84 – 310 52.0-210 ND-11.0 999 4.0
19.8-240 – 270-350 712-129 1.1-9.4
61.0 79.0* 0.49 0.30*

*Data of the current study (mg 100 g
1 for nitrate and nitrite).
ND: Non detectable.

expected, indicates that this is not a considerable limitation
(Walters, 1996).
Lack of Certified Standard Solution for assessment of inorganic
nitrate and nitrite in food samples, to confirm consistency of the
measurement process (Castanheira et al., 2004), was another
limitation of the current study. However, use of the experimental
spike recovery in our study partially address the issue of validation
of accuracy for different matrices; since, the recovery values
observed for the spiked samples of different food matrices were
within the acceptable ranges, the sample matrices could be
considered valid for the assay procedures.
4.6. Conclusion
To the best of our knowledge, this study was the first effort to
develop a database for nitrate/nitrite content of more commonly
consumed food items by Iranians. Our data provides a basis for
overall estimation of dietary nitrate/nitrite exposure and monitor-
ing of related health outcomes among Iranian populations. In
conclusion, mean nitrate/nitrite levels of major food samples, were
comparable to other countries, however, nitrate/nitrite contents of
some food items including breads, some vegetables and fresh
meats were higher than the previous reports.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study, as part of Ph.D. thesis of Ms. Zahra Bahadoran, was
supported by the Research Institute for Endocrine Sciences of
Shahid Beheshti University of Medical Sciences (grant No. 753).
The authors wish to acknowledge Ms. Niloofar Shiva for critical
editing of English grammar and syntax of the manuscript and Dr.
M. Hedayati for his laboratory advisory.
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