How the ENO-30 Detects Nitrate and Nitrite

How the ENO-30 Detects Nitrate and Nitrite

An overview of the ENO-30, which can simultaneously detect nitrate and nitrite in biological and other samples.

The ENO-30 is a uniquely designed system that combines HPLC with a colorimetric assay optimized for fast and highly sensitive quantitative analysis of nitrate and nitrite. By utilizing the ENO-30, you can get accurate data in 10 minutes from a single injection. Combined with laboratory automation using an autosampler, you can run hundreds of samples overnight.

Let’s take a look inside to see how it works: The ENO-30 has an upper and lower unit. The lower unit contains the HPLC pumps optimized at just the right pumping speeds for the proper mixing of reagents.

Samples are injected with either a manual injector as shown or with an autosampler. Both nitrate and nitrite are first separated by retention time with an HPLC column. After separation, nitrate is reduced to nitrite. The system utilizes the Griess reaction, in which each nitrite forms an azo dye and is detected as the pink color develops and is measured by absorbance detection at 540 nm.

The design of the entire system has been optimized for highly reproducible results. It’s simple and easy to use. Here at Amuza, it’s important that instruments we provide work just as well for any given end-user as they do for us here at our own facility. That’s why we offer installation and training, as well as product support for all our products.

Thank you for watching. Feel free to contact us if you would like to learn more about running nitrate and nitrite samples.

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Benefits of using the ENO-30 for Nitrate and Nitrite Detection

Benefits of using the ENO-30 for Nitrate and Nitrite Detection

Today, I’d like to talk about a system capable of quantitative analysis of nitrate and nitrite from biological samples in minutes, called the ENO-30. The ENO-30 is a fast and highly sensitive system down to 0.1 picomoles.

Let me walk you through how easy it is to use. First, you get your sample ready for analysis. Sample prep is easy. In our manual, we’ve outlined a simple sample preparation protocol for tissue homogenate, blood, cell culture, urine, saliva, and microdialysis samples. Samples are injected into the ENO-30 with a manual injector or an autosampler for laboratory automation.

Once injected, nitrate and nitrite are separated by an HPLC separation column. Once separated, nitrate is reduced to nitrite, forming 2 separated nitrite compounds. Then, the separated nitrites are mixed with a Greiss reagent to form an azo dye, which is then detected by absorbance at 540 nm. In 10 minutes, you will be able to detect both nitrate and nitrite from a single injection.

The ENO-30 combines HPLC with colorimetric analysis. It’s like having a chemist and HPLC expert right on your benchtop. We’ve optimized the conditions to take away any guesswork and made the system straightforward and easy to use.

At Amuza, your success is our priority. It’s important that our products work for you just as well as they do for us here at our facility. When you acquire one of our machines, not only will it include installation and training, you’ll have access to our self-help support center as well as support from our knowledgeable support staff.

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Measurement of Nitrate and Nitrite in Biopsy-Sized Muscle Samples

Measurement of Nitrate and Nitrite in Biopsy-Sized Muscle Samples

Researchers at  Indiana University Purdue University Indianapolis recently presented greatly improved methodology using the ENO-30 to determine nitrite and nitrate levels in human skeletal muscle at the American College of Sports Medicine annual meeting. Muscle tissue has been determined to be an important reservoir of nitrite and nitrate and thus may serve as an endogenous source for nitric oxide production in the body. However, analysis of human muscle biopsy samples for NOx has until now been greatly limited by their small size. A recent study1 reported that nitrite was below the limit of detection by chemiluminescent analysis even when assaying up to 40 mg of tissue.

Assoc. Prof. Andrew Coggan’s lab validated a method for reproducibly determining nitrite and nitrate in samples as small as 5 mg using the ENO-30. The levels of NOx so determined were consistent with literature results from much larger muscle tissue samples analyzed by other methods.

Results of the study include:

  • The ENO-30 HPLC proved to be ideally suited for the present application, with excellent baseline stability (Fig. 1), sensitivity (limit of detection = 0.06±0.01 pmol; limit of quantification = 0.20±0.03 pmol; n=6 standard curves) (Fig. 2), and linearity of response (Fig. 3). A representative sample chromatogram is shown in Fig. 4.

Figure 2. Sensitivity of HPLC (0.3 pmol NO2- standard).

 

  • Pulverization of tissue at liquid N2 temperature followed by extraction in 50 uL methanol + 0.5% Triton X-100 resulted in the highest NO3- and lowest NO2- contents and the least variability. (Fig. 7)

Figure 7. Reproducibility of extraction methods.

 

  • NO2- content of tissue extracts stored at -80°C increased over time, suggesting residual xanthine oxidoreductase (XOR) activity (Fig. 8). Inclusion of 0.1 mmol/L oxypurinol in the extraction medium completed blocked this increase.

Researchers at  Indiana University Purdue University Indianapolis recently presented greatly improved methodology

They further determined that the method may be applicable for nitrate analysis in samples as small as 5 ng.

The Coggan lab’s method should become useful in studies of NOx metabolism in muscle tissue in response to exercise and dietary interventions – and make participation in these studies much less painful!

The poster can be viewed at the Amuza Neuroscience website:
https://www.eicomusa.com/nox-analyzer-eno-30/

 

1Nyakayiru et al. J Appl Physiol 2017; 123:637-644