I thought spinach was good for you…

I thought spinach was good for you…

It was a wonderful day at my friend’s house.  After several years, they were finally going to welcome a baby into the world.  They were excited but also nervous about the new responsibilities they had.  They asked me about something they had read in a book about not giving spinach to their child and why that was a problem.

After doing some research on the topic, nitrates and nitrites are present in vegetables including spinach, and these compounds have positive health benefits but can also have negative effects when consumed in large enough quantities by very young infants, where an excess of nitrates and/or nitrites can cause methemoglobinemia or blue baby syndrome.  We do not claim to be experts in this field, but we mention this only to point out that there is an interest in measuring nitrates and nitrites in spinach and other vegetables as well as in processed meats where nitrates are added as a preservative and antimicrobial.

So, how do we test to see if something has nitrates or nitrites in it?  To do this experiment I needed some way to extract these two substances.  Also, I needed a way to test and separate out the compounds.  I found a paper online¹ that outlines a sample preparation method as well as an HPLC method, so we decided to use our Lucidity SimplePREP along with our Lucidity LC-UV to perform the sample prep and analysis.

For the study, about 1.5 g of spinach was added to a centrifuge tube by manually ripping the spinach into small bits.  Then, a pancake magnet was added.  The solvent used was 1:1 Methanol:Water solution.  The extraction was carried out on the Lucidity SimplePREP with the following parameters:

Temperature (°C) 50
Solvent amount (mL) 30
Agitation On
Extraction Time (min:sec) 25:00
Settling Time (min:sec) 5:00
Filtering On

Here you see me as I introduce the spinach samples into the SimplePREP in 50mL centrifuge tubes, and when the system is done my samples have been extracted and filtered into 2mL HPLC / GC vials that are ready for analysis.

To be sure that I knew where the nitrates and nitrites were eluting, I ran Sodium Nitrate and Sodium Nitrite standards and then created a 1000 ppm mixed standard. 

The standards and samples were run in the Lucidity LC-UV using the following method:

Mobile Phase 90:10 0.01 M octylamine:methanol
Column Restek ARC-18 150 x 4.6 mm, 5.0 µm
Flow 1.5 mL/min
Temperature 30 °C
Type Isocratic 15 minutes
Wavelength 210 ±4 nm

The chromatogram of the standard shows great separation between the Sodium Nitrite and the Sodium Nitrate. 

The samples were run using the same method.  While the retention times were the same, the amount of the nitrate varied a bit due to each sample being made from different spinach leaves but were still in a reasonable range of each other.  This could probably be tightened up by homogenizing a large mass of spinach and then using the mixture of leaves to do the extraction.

Using this method we have a fairly straightforward method that allows for the determination of nitrate and nitrite content in spinach leaves and potentially other foods as well.  For the scope of this study we only ran one concentration level of the standard (1000 ppm) instead of running multiple levels so that we could create a calibration curve and accurately quantitate the amount of these compounds in the sample.  It’s also encouraging that although we analyzed several different samples from within the same bag of spinach leaves the amount of nitrates and nitrites present is within a reasonable range as determined by peak area and as can be seen from the chromatogram.  The spinach leaves we examined also seem to contain quite a bit more nitrates than nitrites.

More studies are need to fully quantify the amounts of these compounds present, test repeatability and robustness of the overall method, and to compare measured amounts of the compounds present versus labelled amounts for different samples, but this is a promising start.


Study by:

Daniel Iversen

R&D Chemist



Najdenkoska, A. (2016), Development of HPLC Method for Analysis of Nitrite and Nitrate in Vegetable, Journal of Agricultural, Food, and Environmental Sciences, Vol 67, pgs. 33-39.

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