Document Type: Original Articles


1 Department of Nutrition, School of Nutrition and Food Sciences, Shiraz University of Medical Sciences, Shiraz, Iran;

2 Department of Food Hygiene and Quality Control, School of Nutrition and Food Sciences, Nutrition and Food Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran


Background: Spinach is one of the vegetables with a high concentration of nitrate which is affected by various processes such as cooking and refrigeration before consumption. The present study aimed to investigate the effect of home cooking method, which has not been studied before, on the level of nitrate and nitrite in spinach. The effect of refrigeration was studied, as well. Methods: After removing non-edible parts of spinach, it was cut into 4-7 cm pieces. Then, it was washed with deionized water thrice and drained. A part of raw spinach was set aside to measure the nitrate and nitrite and the other part was heated in a closed container without adding water, until almost all the water of the spinach was evaporated and then refrigerated. HPLC method was used to measure the nitrite and nitrate levels. The collected data were analyzed using Wilcoxon test by SPSS. Results: The mean concentrations of nitrate and nitrite in the raw spinach were 336.54±182.2 and 26.49±10.07 mg/kg, respectively. Cooking process caused a significant increase in the level of nitrate, while refrigeration process led to a significant decrease in this regard. Besides, a slight reduction was observed in the nitrite level after cooking, but the change was not statistically significant. Refrigeration also had no significant effects on the nitrite level of spinach. Conclusion: Considering the non-significant changes in nitrite levels, it seems that home cooking without adding water, unlike refrigeration, is not a proper method to reduce the intake of nitrates and nitrites from spinach.


  1. World Health Organization. Chemical fact sheets
  2. [internet]. In Guidelines for drinking-water quality,
  3. Volume 1: Recommendations. Third edition; 2008.
  4. p. 398-400. Available from:
  5. water_sanitation_health/dwq/gdwq3rev/en
  6. Machha A, Schechter AN. Dietary nitrite and nitrate:
  7. a review of potential mechanisms of cardiovascular
  8. benefits. Eur J Nutr 2011; 50(5): 293-303.
  9. Katan MB. Nitrate in foods: harmful or healthy?. The
  10. American Journal of Clinical Nutrition 2009; 90(1):
  11. -12.
  12. Yan PM, Xue WT, Tan SS, Zhang H, Chang XH. Effect
  13. of inoculating lactic acid bacteria starter cultures on
  14. the nitrite concentration of fermenting Chinese paocai.
  15. Food Control 2008; 19(1): 50-5.
  16. Sobhan Ardakani S, Shayesteh K, Afyouni M, Mahbobi
  17. Soufiani N. Nitrate Concentration in Some Plants in
  18. Isfahan. Journal of Environmental Studies 2005; 31(37): 69-76.
  19. Najafi N, Parsazadeh M. Effect of Nitrogen Form and
  20. pH of Nutrient Solution on the Shoot Concentration
  21. of Phosphorus, Nitrate, and Nitrogen of Spinach in
  22. Hydroponic Culture. ejgcst 2010; 1(1): 41-56.
  23. Sobko T, Marcus C, Govoni M, Kamiya S: Dietary
  24. nitrate in Japanese traditional foods lowers diastolic
  25. blood pressure in healthy volunteers. Nitric Oxide
  26. ; 22(2): 136-40.
  27. World Health Organization. Nitrate and nitrite in
  28. drinking-water, Background document for development
  29. of WHO Guidelines for Drinking-water Quality
  30. [internet]. Geneva; 2011. Available from: http://www.
  32. nitratenitrite2ndadd.pdf
  33. Wolff IA, Wasserman AE. Nitrates, nitrites and
  34. nitrosamines. Extensive research is needed to establish
  35. how great a food hazard these nitrogenous substances
  36. present. Science 1972; 177: 15-19.
  37. Hord NG, Tang Y, Bryan NS. Food sources of nitrates
  38. and nitrites: the physiologic context for potential health
  39. benefits. Am J Clin Nutr 2009; 90(1): 1-10.
  40. Food Standards Agency. 2004 UK Monitoring
  41. Programme for Nitrate in Lettuce and Spinach. Food
  42. Survey Information Sheet 74/05 [internet]. London;
  43. Available from:
  44. surveillance
  45. Tamme T, Reinik M, Roasto M, Juhkam K, Tenno
  46. T, Kiis A. Nitrates and nitrites in vegetables and
  47. vegetable-based products and their intakes by Estonian
  48. population. Food Additives and Contaminants 2006;
  49. (4): 355-61.
  50. Shahbazzadegan S, Hashemimajd K, Shahbazi B.
  51. Determination of Nitrate Concentration of Consumed
  52. Vegetables and Fruits in Ardabil. Journal of Ardabil
  53. University of Medical Sciences 1398; 10(1): 38-47.
  54. Hsu J, Arcot J, Lee NA. Nitrate and nitrite quantification
  55. from cured meat and vegetables and their estimated
  56. dietary intake in Australians. Food Chemistry 2009;
  57. : 334-9.
  58. Jaworska G. Nitrates, nitrites, and oxalates in products
  59. of spinach and New Zealand spinach Effect of
  60. technological measures and storage time on the level
  61. of nitrates, nitrites, and oxalates in frozen and canned
  62. products of spinach and New Zealand spinach. Food
  63. Chemistry 2005; 93: 395-401.
  64. Prasad S, Chetty AA. Nitrate-N determination in leafy
  65. vegetables: Study of the effects of cooking and freezing.
  66. Food Chemistry 2008; 106(2):772-80.
  67. Huarte-Mendicoa JC, Astiasaran I, Bello J. Nitrate
  68. and nitrite levels in fresh and frozen broccoli. Effect
  69. of freezing and cooking. Food Chemistry 1997;
  70. (1-2):39–42.
  71. Nabrzyski M, Gajewska R. The content of nitrates and
  72. nitrites in fruits, vegetables and other foodstuffs. Rocz
  73. Panstw Zakl Hig 1994; 45(3): 167-80.
  74. Abo Bakr TM, El-Iraqui SM, Huissen MH. Nitrate and Nitrite Contents of Some Fresh and Processed Egyptian
  75. Vegetables. Food Chemistry 1986; 19: 265-75.
  76. Chung JC, Chou SS and Hwang DF. Changes in nitrate
  77. and nitrite content of four vegetables during storage at
  78. refrigerated and ambient temperatures. Food Additives
  79. and Contaminants 2004; 21(4): 317-22.
  80. Chew SC, Yang Bao KNP, Ismail A. Changes in nitrate
  81. and nitrite levels of blanched amaranthus during
  82. refrigeration storage. Journal Sains Kesihatan Malaysia
  83. ; 9(1): 29-34.
  84. Pirsaheb M, Rahimian S, Pasdar Y. Nitrite and Nitrate
  85. content of fruits and vegetables in Kermanshah (2010).
  86. Journal of Kermansha University of Medical Sciences
  87. ; 16(1): 76-83.
  88. Joint FAO/WHO Expert Committee on Food Additives
  89. [Internet]. Available from:
  90. database/evaluations/chemical.aspx?chemID=711
  91. Chou SS, Chung JC and Hwang DF. A High Performance
  92. Liquid Chromatography Method for Determining
  93. Nitrate and Nitrite Levels in Vegetables. Journal of
  94. Food and Drug Analysis 2003; 11(3): 233-8.
  95. Bednar CM, Kies C, Carlson M. Nitrate-Nitrite levels in
  96. commercially processed and home processed beets and
  97. spinach. Plant Foods for Human Nutrition 1991; 41: 261-8.
  98. Gajda J, Karłowski K. The content of nitrates in
  99. vegetables and potatoes in 1987–1991. Roczniki Pan´
  100. stwowego Zakładu Higieny 1993; 44(4): 301-7.
  101. Petersen A, Stoltze S. Nitrate and nitrite in vegetables
  102. on the Danish market: content and intake. Food
  103. Additives and Contaminants 1999; 16(7): 291-9.
  104. Dennis M, Wilson LA. Nitrates and Nitrites. In
  105. Encyclopedia of Food Sciences and Nutrition. Second
  106. Edition; 2003. p. 4136-42.
  107. Tamme T, Reinik M, Püssa T, Roasto M, Meremäe K,
  108. Kiis A. Dynamics of nitrate and nitrite content during
  109. storage of home-made and small-scale industrially
  110. produced raw vegetable juices and their dietary intake.
  111. Food Addit Contam Part A Chem Anal Control Expo
  112. Risk Assess 2010; 27(4): 487-95.
  113. Iammarino M, Taranto AD, Cristino M. Endogenous
  114. levels of nitrites and nitrates in wide consumption
  115. foodstuffs: Results of five years of official controls
  116. and monitoring. Food Chemistry 2013; 140:763-71.
  117. Hunt J, Turner MK. A survey of nitrite concentrations in
  118. retail fresh vegetables. Food Additives & Contaminants
  119. ; 11(3): 327-32.
  120. Leszczynska T, Filipiak-Florkiewicz A, Cieslik
  121. E, Sikora E, Pisulewski PM. Effects of some
  122. processing methods on nitrate and nitrite changes in
  123. cruciferousvege tables. Journal of Food Composition
  124. and Analysis 2009; 22: 315-21.
  125. Bosch NB, Mata MG, Pefiuela MJ, Ruiz Galfin T, Lopez
  126. Ruiz B. Determination of nitrite levels in refrigerated
  127. and frozenspinach by ion chromatograph. Journal of
  128. Chromatography A 1995; 706: 221-8.
  129. Ferreira LC, Cataneo AC. Nitric oxide in plants: a brief
  130. discussion on this multifunctional molecule. Scientia
  131. Agricola 2010; 67(2): 236-43.
  132. Spoelstra SF. Nitrate in silage. Grass and Forage
  133. Science 1985; 40: l-11.