Bioequivalence and Bioavailability Forum

Dear Rajendra,

Another vast, but extremely important, question...

Matrix effect is the difference between the mass spectrometric response for an analyte in standard solution an the response for the same analyte at the same concentration in a biological matrix (plama, urine, etc.). Matrix effect is due to co-eluting matrix components that affect the ionisation of your analyte (mostly ion suppression, sometimes ion enhancement). A major source of matrix effect is phospholipids, but there are other matrix components causing matrix effect. You can also get ion suppression from another analyte, a metabolite, concomitant medications, or even from the internal standard.

The main problem is that due to the specificity of LC/MS/MS you just don't see these co-eluting substances.

There are several possible actions to reduce matrix effect problems. One is to obtain cleaner samples: as HS wrote in a previous post somewhere, contrary to the usual belief, you should have a more thorough sample extraction for LC/MS/MS methods than for HPLC/UV ! Protein precipitation can lead to terrible problems. Another action is to have a chromatographic separation between your analyte and the substances causing matrix effect. This means high efficiency chromatographic columns and longer run times. Which means that what you see in many labs (shoot and go: protein precipitation and 1.5 minutes run times) is the best way to get into trouble.

There are two main techniques used to investigate matrix effect:

• post-column infusion. I would consider this as something to be performed during method development, rather than during validation. Basically, you inject your analyte post-column at a constant rate, with a syringe pump and a tee. This will result in a constant signal from your detector. While doing this infusion, inject into your HPLC system a processed blank plasma. If you have matrix effect, this will result in dramatic changes in the signal you observe. At some retention times you will get negative peaks, with almost no signal remaining. This can happen at late retention times, which would affect samples analysed several injections later. The idea is then to optimise the chromatographic conditions in order to elute your analyte at retention times where you have no matrix effect. It can also be used to compare several extractions procedures. But it is considered to be a qualitative test, rather than quantitative.
  
• post extraction spiking. See the paper from Crystal City III for more details.

Other suggestions have been made, such as monitoring phospholipids after in-source collision-induced dissociation, but I would still consider this as experimental.

I would suggest you to have a look at this paper, which may answer quite a few of your questions. It addresses issues such as MS optimisation, cross-talk, choice of IS, non-linearity, matrix effect, interference of metabolites, carry-over, sample extraction, in-source collision-induced dissociation, etc.:

Bakhtiar R., Majumdar T.K. Tracking problems and possible solutions in the quantitative determination of small molecule drugs and metabolites in biological fluids using liquid chromatography - mass spectrometry. Journal of Pharmacological and Toxicological Methods 55 (2007) 227-243.

You can also have a look at this paper. Please note that the authors are from Waters Corporation, and the paper is rather an advertisement for Waters' SPE columns and UPLC systems but it also explains some of the process:

Chambers E, Wagrowski-Diehl D.M., Lu Z., Mazzeo J.R. Systematic and comprehensive strategy for reducing matrix effects in LC/MS.MS analyses. Journal of Chromatography B, 852 (2007) 22-34.

Regards
Ohlbe