polar analyte and urine interference HPLC separation

[Edde]

I encounter a HPLC separation problem in a method to detect nitrite in urine. Nitrite spiked urine is ultrafiltrated through 3kDa centrifugal filter. The filtrate is then derivatized by reaction with N-acetyl-L-cysteine (NAC) in HCl (pH 2-3) to S-nitroso-N-acetylcysteine (SNAC). SNAC is then measured by HPLC-UV.

The analyte comes out at RT 3.5 min in an isocratic run with 50mM phosphate buffer (pH2.5) 15% acetonitrile/water, C18 column.

I identify SNAC by full UV spectrum (from 190 to 350nm). Then perform quantitation. However, there is noise background around the RT of SNAC which affects qualitative identification as well as quantitation. I tried 2 approaches to try to separate the noise from analyte:
1. Different columns (C8, HILIC).
2. SPE cleanup.

1. I tried using different ACN concentrations with HILIC column but even nitrite, NAC, and SNAC failed to be separated in aqueous standards. Using C8 column does not improve the background noise separation.

2. I tried Bond elut, HILIC, MCX by conditioning the SPE cartridge, then load the urine sample. The flow-through was collected for analysis. However, polar interferences remain and background noise is still there.

I am now thinking of 2 approaches to separate the interference from analyte:
1. try hexyl phenyl column.
2. changing the ACN conc in mobile phase in C18 run.

Am I right to try out other column type and then mobile phase organic conc.?
Or is there any SPE I can use to clean up polar interferences from inorganic anions?

I am grateful for any comments and directions.
Thanks.

[oscarBAL]

Hi Edde; I used to analyze Nitryte by LC using C8 and C18 by Ion Pair chromatography and UV detection; nitrite is well retained; I do not remember all the details of the method but my samples were very dirty.. you can type in google something like "Nitrite + Ion Pair + Cromtography + UV" and you could find more information.

You can also use Anion Exchange column instead of HILIC.

You say you useed MCX cartridges for clean up your sample, are you sure about that? MCX means Mixed Cation Excahge and you should use MAX or SAX.

Best Regards.

Oscar.

[Edde]

I used to analyze Nitryte by LC using C8 and C18 by Ion Pair chromatography and UV detection; nitrite is well retained

Yes, there is Ion pair method for nitrite. Since my HPLC analyzer is not dedicated for nitrite and ion pairing agent will affect other analysis, I did not use this method mode. In addition, the UV spectrum of nitrite itself is non-specific, so I choose to derivatize nitrite to give a more unique UV spectrum for identification.

You say you useed MCX cartridges for clean up your sample, are you sure about that? MCX means Mixed Cation Excahge and you should use MAX or SAX.

Yes, I used MCX. I am sorry to say that I just don't understand the rationale for MCX and MAX extraction. Could you please kindly explain?

Thanks a lot!

[oscarBAL]

Hi Edde; yes I think I can explain a little bit about about MAX as far as I can since english is not my lenguaje.
If you want to retain nitrite then you will need a cartridge able to retain anion; Strong Anion Excahnge involves a solid phase such as -RNH3+X- where the counter ion X- is exachnged by NO2-All the neutral and positive molecules will elute and thenitrite will remain in the stationary phase. Then you can elute using and stronger anion such as Cl-
If you use MCX you can retain cations so Nitre left the cartridge since you load your sample.
I found some information that can explain beter what I am trying to say.

http://chromatographyonline.findanalyti ... rticle.pdf

http://www.avantormaterials.com/search. ... t=AN%20482

http://www.weber.hu/PDFs/SPE/Tn103_SAX_SPE.pdf

May be I am wrong but I would not expect too much interferences analyzing Nitrite without derivatization if you optimize your SPE clean up and use an apropiate column.

I hope this help.

[Edde]

Many thanks to your explanation and references. They are very useful.

Previously, I loaded urine sample to SPE but I just wanted to hold the unwanted interferences such as non-polar compounds by C18 sorbent, or cations by MCX sorbent. The unheld nitrite or nitrate ions are collected directly in the flow-through liquid.

Now, I think I need to change to use MAX and try some generic SPE method as you suggested.

I have some questions. What is the aim of using Carrez solution? I can't find blank vegetable matrix for spiking. Is it a standard practice to use water as matrix for calibration curve in food testing?

I appreciate your help!

[oscarBAL]

Hi Edde; your approach was not wrong but you have more chane to improve your clean up retaining your analyte and doing selective waches to optimiza your method; for example passing diferent MeOH or NaCl concentration that help tu elute netral and weak anions.

The carrez is used basicly for decoloring your sample; I do not remember very well what impurity exactly remove, I think it was used in order to remove proteins and other compounds.

[Edde]

I even thought of using a sequential SPE approach, first C18 to remove non-polar and the flow-through would be further purified by another SPE. It seems that I shall try your suggested approach first.

Thank you very much for taking the time and sharing your valuable experience.