hplc of a basic compound

[sonal]

I have a basic compound with a pKa of 9.0 and need to analyse it in plasma, for which I will be using a reversed internal phase column by . It has a pH range of 2.5-7.5 and an organic content limit of 25%. I tried using mobile phases of lower pH's, which give very little tailing but will not be applicable for direct injection of plasma samples. I tried the suggested method at pH 6 with Na2HPO4: ACN with heptane sulfonate, which also resulted in tailing. Mobile phase with 50mM KH2PO4+1%TEA:ACN at pH 3.0 has also been tried with a very low retention time of 2.3 min.

can you please suggest me some other options to try?

Thanks in advance

[Bryan Evans]

Hi Sonal -

For direct injection of plasma, I'd recommend trying Cadenza HS-C18.

Here is some application data - feel free to contact me if you would like to learn more - thank you.

http://www.imtakt.com/TecInfo/TI302E.pdf
http://www.imtakt.com/TecInfo/TI296E.pdf

[Kelly Johnson]

What is the detection method?

[AdrianF]

Why do you need to use plasma? I have found serum much better to handle as plasma gives precipitates on freezing. Solid phase extraction is another option.

The use of 1% TEA is excessive usually 0.1% is enough to stop tailing -retention should then be later.

[sonal]

The detection method is fluoroscence.

What concentration of octane sulfonate should I use as an ion pairing agent? The suggested is 3.5mM in one of the papers. But, it is not being recommended in the book by Synder at the concentartion of ACN which I am using i.e 25%. Would it make a difference to use C-10/12 sulfonates instead? The Rt that I am getting is 24 min with 50mM KH2PO4+3.5mM Octane sulfonate:ACN (75:25) at 1ml/min

[AdrianF]

C10-C12 sulphonates will increase retention times, it would be better to use pentane sulphonic acid if you want to go along this route. You need to be aiming for a much shorter retention time to give adequate quantitation

[tom jupille]

What concentration of octane sulfonate should I use as an ion pairing agent?

Concentration and chain length are more or less equivalent variables for the alkyl sulfonates. What matters is the concentration of charge on the stationary phase surface, so more of a long-chain or less of a short-chain give about the same results. While rough guidelines (as in the Snyder, Kirkland, & Glajch book) can be given, they are just that: rough guidelines. You have to optimize the concentration and chain length for your particular sample.

3.5 mM is already fairly low in concentration; if this were my problem and I wanted a shorter elution time, I would go down in chain length from octane to heptane, keeping the same concentration for the first try, and then tweaking concentration as appropriate.

[zokitano]

When you're using ion-pair reagent I suppose it is dissolved in to the mobile phase. Maybe my question is little bit off topic, but did anyone try to pair the analyte molecules (or ions, exactly) with ion-pair reagent prior to injection into the HPLC system?
I mean did anyone try to add an appropriate amount of the ion-pair reagent only in the standard and sample solutions, and with this, to avoid putting the reagent into the mobile phase?

If anyone tried this kind of analyses, do you get any good and reproducible results as when you've used the ion-pair reagent into the mobile phase?

Best regards

[Uwe Neue]

From this standpoint, "ion-pair" is a misnomer. You need to look at "ion-pair chromatography" as the chromatography of ions on a C18 column coated with a hydrophobic counterion. It is effectively ion-exchange in disguise.

Consequence: you need to coat the packing with the ion-pair reagent first. Once it is completely coated, you will get reproducible chromatography of the ions that you want to separate.

[HW Mueller]

All this tweaking, etc., etc., with 6-7% (weight) blood protein in the sample, at pH 3 or near that?
Now, since that last chain on restricted access columns I found out (thanks, Bryan, for the information) that Imtakt Co. (manufacturer of this type of column in Kyoto) recommends to micro-filter plasma/serum samples prior to injection. Furthermore, they admonish to be very careful in handling the proteins....... In other words, one should do only indirect injection.

Incidentally, AdrianF, our experience with thousands of frozen plasma or serum samples would indicate that there are other proteins, than those involved in fibrin formation, which can precipitate due to freezing and thawing.

[Bryan Evans]

I think I read the post a little bit too quickly (my mistake). Anyways, if you are using ion pairing chromatography - it would be best to do some sample clean up prior to injection (SPE or protein precipitation). I don't know what column you are using right now - but if it's a typical C18 phase - it is being used as a reverse phase / protein trap column all in one - and the column lifetime will be poor.

Hans - thanks for reading our instruction manual, it probably needs to be edited a little bit. For direct injection - the "filter" in the manual should be replaced with "guard column+cartridge" - otherwise its (as you pointed out) indirect injection!

Anyways - i'll follow up with you in an email. I don't want to turn this discussion into a discussion about our products.

[HW Mueller]

Really, I mentioned the Imtakt column as a representative for restricted access columns, and because I was very positively surprised that Imtakt was apparently? of the same opinion: In my experience it is strongly preferable (over directly attached guards, filters) to use a separate filter, or even a precipitation, before injecting. It seems fair that people who consider this method get some independent opinions.

[Bryan Evans]

I think there may be some confusion here. For 99.9999% of the columns in the world - we recommend sample clean up prior to injection of biological samples (centrifuging, SPE, protein precipitation, filtering, ect.). We recommend doing this for all of our stationary phases as well.

If anyone wants to inject biological samples directly, then they should try Cadenza HS-C18 - it's a novel stationary phase that was designed for this (the chromatograms show the large abundant proteins quickly eluting from the column).

For Cadenza HS-C18, we always recommend centrifugion - whether or not samples need additional filtration prior to injection is dependent upon plasma concentration. If the samples are diluted with an internal standard - then it is possible to skip filtration (just use a guard column). If there is no dilution, then filtration should be evaluated.

[Uwe Neue]

How many problems you have with a method depends on the quality of the method. Restricted access packings will get rid of large molecular weight interferences, but they can't do anything about low molecular weight interferences. I put their ability to prevent interference and column contamination at about the same level as a protein precipitation procedure with acetonitrile. Selective detection with either MS or fluorescence is a big step forward to make a method work, and fluorescence is generally superior to MS. But even with fluorescence you can suffer from signal suppression, but probably not as badly as in MS.

[HW Mueller]

Bryan, there is no confusion here. I again checked the Imtakt "Instruction Sheet Cadenza HS-C!8" which you e-mailed me. On the first page there is the heading "Mobile Phase and Sample Filtration", under which is stated: "Please filter the mobile phase and and sample solution through either a 0,45µm or 0.2µm membrane filter prior to injection." On the second page, under the heading "Sample Pretreatment" it says: "Plasma (blood serum) samples should be pre-treated in advance with a 0.45µm filter and centrifuge so that there is no turbidity."
Again I must say that I admire Imtakt for such real world advice.
In another chain it was mentioned that freezing and thawing plasma/serum samples can precipitate some proteins, here I may add that some patients yield turbid plasma/serum freshly at preparation. In our hands some patient´s plasma/serum even plugged 0.45 µm filters immediately. That is why we opted for a simple Na2SO4 partial precipitation of proteins ahead of the injection. Furthermore we finally used restricted access columns only as a first step in 3-step HPLC methods.
The quoted statements of Imtakt (and some not quoted here) also indicate that there has been no breath-taking breakthrough in this methodology since the Pinkerton, Supelco, Merck, and Chrompack (now Varian?) columns tried here. That is, the advice of Imtakt and that which I gave still seems to have relevance.
In fairness I must say that we had no problem analysing spiked cortisol in plasma/serum of ourselves (indicating that we were fairly normal) via direct injection into a Pinkerton. This would indicate that in a pharmaceutical setting, where normal people were used in tests, the direct approach might be worthwhile when more or less frequent guard column wastage can be tolerated. But this leads to the question: Is there somebody out there who can give a documented example of a routine analysis (in a clinical setting) using only direct injection of plasma/serum on a restricted access column? No indication of protins and or peptides getting through to a MS or fluorescence (many proteins/peptides fluoresce) detector?