matrix matched calibration problem

[Edde]

I am developing an HPLC method to detect nitrite through derivatization of nitrite with N-acetylcysteine and UV detection. The target sample is food which can be anything from vegetables, rice porridge with vegetable, vegetable soup sometimes with sugar and beans, etc.

My question is:
1. How can I cover the whole range of matrices in my method validation?
2. Can I just use vegetable as matrix, then generalize my validation with all other types of menu containing vegetable?
3. Vegetable contains varying amount of nitrite. I wonder if there is any methods to eliminate nitrite to make a nitrite-free matrix for spiking.

Please kindly comment.
Thanks.

[tom jupille]

Running in reverse order:

3. You should be able to oxidize nitrite to nitrate. The trick would be to subsequently remove the oxidizing agent and stabilize added nitrite in your calibrators.

2 & 1. As a pre-validation, you should look at as wide a variety of matrices as you can by getting rid of nitrite (per above) and then spiking with a known (single level). If the responses are fairly consistent, then you your validation with only 2 or 3 matrices, selecting the "most different".

[Edde]

Thank you very much.

I can oxidize nitrite to nitrate by hydrogen peroxide, but I don't know the way to inactivate the H2O2 afterwards.

Another problem is I also need to detect nitrate in the sample. My strategy is to reduce nitrate to nitrite by nitrate reductase. Nitrite is then derivatizated and measured. Therefore the matrix cannot contain nitrate, either.

Please comment.

[tom jupille]

One way to deal with the unavailablity of a blank matrix is to do the analysis by standard addition. The downside is multiple runs per sample.

[Edde]

Many thanks.

For standard addition experiment, is there a need to perform validation? Any guidelines available?

[tom jupille]

Same validation as any other method.

[Consumer Products Guy]

There are spectrophotometric test procedures for nitrite that have been used for years, can't you simply use one of those? See AOAC methods.

[Edde]

There are spectrophotometric test procedures for nitrite that have been used for years, can't you simply use one of those? See AOAC methods.

The Griess method also requires matrix matched calibration curve.

Besides, in urine analysis the Griess reaction is interfered by the antidote for nitrite toxicity, methylene blue. Therefore, this commonly used spectrophotometric method can't be used.

[Edde]

Same validation as any other method.

When I perform validation by standard addition to one matrix, say spinach, I only validate that particular matrix.

How about when I encounter a cauliflower matrix?

Thanks.

[tom jupille]

When I perform validation by standard addition to one matrix, say spinach, I only validate that particular matrix.

How about when I encounter a cauliflower matrix?

The real problem with validating *any* method where you can't get a blank matrix is demonstrating specificity. The only thing you can do is to compare the results with those you get from an independent method (in which case specificity and accuracy are determined at the same time).


On a different topic:

Besides, in urine analysis the Griess reaction is interfered by the antidote for nitrite toxicity, methylene blue.

I thought you were running vegetables?

And other thoughts:
- analyze for nitrate, then oxidize the sample with peroxide and re-analyze. The difference should represent the nitrite.?
- For the validation, run standard additions before, then oxidize, and re-run. The nitrite peak should be gone. ?

[Edde]

I thought you were running vegetables?

In fact, I have to validate in 3 matrices: serum, urine and vegetable.

- analyze for nitrate, then oxidize the sample with peroxide and re-analyze. The difference should represent the nitrite.?

Do you mean analyze for nitrite, then oxidize with peroxide and re-analyze. The difference represents nitrate?

- For the validation, run standard additions before, then oxidize, and re-run. The nitrite peak should be gone. ?

Is this a way to confirm/validate the specificity of standard addition method?
If I don't have a third party to compare my results against, can I just use this approach for validating specificity?

Thanks.

[tom jupille]

Do you mean analyze for nitrite, then oxidize with peroxide and re-analyze. The difference represents nitrate?

No, I meant what I said. Analyze for nitrate, then oxidize the sample so that any nitrite present is converted to nitrate, then re-analyze.

Is this a way to confirm/validate the specificity of standard addition method?
If I don't have a third party to compare my results against, can I just use this approach for validating specificity?

My understanding of specificity is that you have to demonstrate that you can differentiate your analyte from other compounds *which are likely to be present*, not necessarily from every other compound in the known universe. The problem with "messy" matrices is defining what is likely to be present.

Technically, you are demonstrating that you are quantitating a peak which has the same retention time as nitrite and is easily oxidized. If you also analyze for nitrate and show that the peak with the same retention time as nitrate increases, then you would have a fairly persuasive case for specificity (at least for me ).

[Edde]

Thanks.