Chromatography Forum

Dear colleagues,

Please, help me with finding a solution for the following problem.
I have to fing a % of binding of a set of drug candidates with proteins in blood plasma at concentration levels of 5 and 0.5 ug/ml.
For this purpose, I should develop a method for LC/MS/MS analysis of samples from equilibrium dialysis using the following protocol: www.piercenet.com/files/2034as4.pdf
In brief, according to this protocol, a phosphate buffer is used for sample preparation and the sample after protein precipitation with AcN is subject to LC/MS/MS.

The problem is that, as follows from my preliminary experiments with such model compounds as warfarine and ranitidine, the presence of a phosphate buffer in sample results in a 30-fold sensitivity loss and a chromatographic peak shape degradation (severe tailing and broadening).

I know that phosphate buffer is not recommended for MS/MS, but I can't avoid using it. I have also tried other sample preparation techniques such as LLE and SPE, but it didn't help.

I also observe the peak shape degradation not only using MS/MS, but also using UV detection (UVD is between HLPC and MS/MS systems) - so, this means that phosphate influences also chromatographic efficiency?? It's quite surprising for me.

LC/MS/MS conditions are as follows:
1100 Agilent HPLC + ABI QTrap MS/MS (MRM mode), column Chromolith 4.6x100mm with RP-18e phase, flow rate 2.7 ml/min, split approx. 1:12 prior to injection into MS/MS, injection volume 50 ul, mobile phase 80%ACN with 0.1% FA. For ranitidine determination the run time is about 1 minute, RT of ranitidine is about 0.5 min, the flow is diverted to waste for 0.4 minutes.

Please, advise, what else could we try to solve the problem with peak shape degradation and sensitivity loss in presence of phosphate?

Thank you in advance.
Elena

Assuming that you are not able to send 2.7L/min through your column it seems this is another none-chromatography that the poor MS is supposed to compensate. If you did a chromatography you would not have to worry about phosphates getting into the MS. Also, since you seem to have no retention the peak integration in your case is meaningless.

Assuming that you are not able to send 2.7L/min through your column it seems this is another none-chromatography that the poor MS is supposed to compensate. If you did a chromatography you would not have to worry about phosphates getting into the MS. Also, since you seem to have no retention the peak integration in your case is meaningless.

I am sorry, it was a misprint. Of course, the flow rate is 2700 ul/min (or 2.7 ml/min).

I figured that, this is why I said you seem to be doing a none-chromatography. Well, just guessing "over the thumb" you may have a bit of exclusion.

I figured that, this is why I said you seem to be doing a none-chromatography. Well, just guessing "over the thumb" you may have a bit of exclusion.

Sorry again, I am not a native English speaker and I'm afraid I don't understand what you mean under "none-chromatography" and what you suggest. Is it some kind of slang? Google didn't help me.

The void volume of your column is about 1 mL. Ranitidine is exactly unretained, and with the mobile phase composition that you are using, I expect that most analytes of practical interest will be unretained. With other words, you will get the exact same results (=overlap with phosphate and other interferences) if you inject the ranitidin sample without the column.
If you want to set up a generic method for multiple analytes, you need to run a gradient. This is best done with very short columns. If you contact me, I can send you an article on how to do this.

It should read non-chrom.... It means, as Uwe also pointed out, you are not doing a chromatography.

If you want to set up a generic method for multiple analytes, you need to run a gradient. This is best done with very short columns. If you contact me, I can send you an article on how to do this.

Dear Uwe, thank you for your advise and for the useful article.

Elena