The ENO-20 is an essential system for nitric oxide (NO) research. The combination of liquid chromatography with the Griess reaction enables high sensitivity detection of nitrite and nitrate (NOx) in biological liquid samples. For one sample injection, nitrite (NO2-) and nitrate (NO3-) levels are analysed in less than 10 minutes.
- Sensitivity at 0.1 pmol for nitrite and nitrate
- Clear separation of nitrite and nitrate from other compounds
- Single injection to measure both nitrite and nitrate
- Analysis time is less than 10 min
- Easy sample preparation (see end of page for details)
- Capability for fully automated analyses with an autosampler
The data shown is a typical profile obtained from a human plasma sample using Eicom ENO-20, NOx Analyzer. The peaks correspond to 110 nM nitrite and 73 µM nitrate in 10 µl applied sample.
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The role of nitrite
If detection of NO is important to your research, have you thought of detecting nitrite, which can bring you more insight? The detection of NO in biological liquid samples is extremely difficult due to the transient half-life of NO molecules. Some report the biological lifetime to be in the order of milliseconds. Once you remove the sample from the animal or other source, it is almost impossible to directly detect nitric oxide. Thus, detection of nitrite and nitrate, considered to be the major metabolites of NO, can be used to determine the NO levels. Moreover, if you evaluate the NO level with an in-vivo sensing procedure, it shows only one aspect of the NO profile. Monitoring nitrite level has become increasingly important in recent years. Studies have shown nitrite to be indirectly related to physiological activity and it is also a signaling molecule. The specific biological activity of NO is explained here.
NOx assay kits are not sensitive enough
The reported range of nitrite levels in the plasma is as low as 50 to 300 nM. On the market, fluorometric or colorimetric reagent kits to detect nitrite, and indirectly nitrate, are available following enzymatic reduction. The typical detection limit is 20 pmol which is equivalent to 1-10 µM with actual sample volumes. This lower limit of detection is not robust enough to detect nitrite in biological fluids. Moreover, those reagents are relatively specific to nitrite, but still there can be cross talks with other compounds. The ENO-20 easily provides quantitative data with enhanced specificity and sensitivity for nitrite and nitrate.
Principle of the ENO-20: HPLC + diazo coupling,
The ENO-20’s high sensitivity and specificity are accomplished with the combination of a dizao coupling method and chromatography. Nitrite and nitrate are separated from other substances on a unique separation column and mobile phase. Nitrite then reacts with a compound called Griess reagent and generates diazo compounds which have a red color. The level of nitrite can be monitored with peak height or area with a retention time of 4.5 min from the injection of the sample. Nitrate is reduced to nitrite using a cadmium reduced copper column which reacts to the Griess reagent as well. The nitrate peak has an 8 min retention time. The level of diazo compound is measured by absorbance at 540 nm using a visible detector. The separation column is robust as well as the entire ENO-20. Normal lifetime of the column is at least 3 months with regular use. All separation and detection technologies are provided by Eicom for the ENO-20 including mobile phase ingredients, Griess reagent, separation columns, and reduction columns. You can relax and detect nitrite and nitrate with 10 nM sensitivity (0.1 pmol).
How does the ENO-20 attain the high sensitivity?
The system configuration and separation technology of the ENO-20 are the reasons for the enhanced sensitivity. The column oven of the ENO-20 includes the detector cell, which improves the stability. The eight microliter replacement volume of the binary pump is also important in eliminating any mixing noise from the Griess reagent. If you were to use the same method on a normal HPLC system, you may not be able to obtain this sensitivity and the HPLC could be damaged. In fact, the level of hydrochloride in the Griess reagent is harmful to most HPLC systems but the ENO-20’s pump system has no problem tolerating it.
What is the advantage of the ENO-20 compared to chemiluminescence procedures?
NO and ozone react in the gaseous phase to produce chemical luminescence. It is possible to detect nitrite in the liquid phase using this procedure but it is not as simple as using the ENO-20. What types of samples require the nitrite assay? Usually the sample matrix is liquid; the ENO-20 is the perfect device for this. In the process of reducing nitrite/nitrate, the system is heated which results in condensation on the inner surface of the glassware after cooling down. The condensation dissolves the nitrite/nitrate and will have an effect on carry-over when the condensation evaporates during the next process. This is a common reason for variation in the detected values of nitrite using the chemiluminescense procedure. As explained above, the ENO-20 installed with an autosampler yields hands free analysis. You do not need to clean the glassware of the detection system following the analysis of several samples or following the sample conversion from liquid to gas.
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