Chromatography Forum

Dear users,

I would like to know if there is any literature on the effect of diluting plasma samples prior to SPE. Does dilution affect the SPE proces and if so, in what way?

Thanks for your help.

Regards Bert

Dilution can have an impact.

the literature i will point you to is the bioanalytical method validation guidance.

it says "The ability to dilute samples originally above the upper limit of the standard curve should be demonstrated by accuracy and precision parameters in the validation."

So if this is what you are doing, have already done, or are trying to justify a validation extension may be your safest route.


the white paper also discusses dilutions

Quantitative Bioanalytical Methods Validation andI mplementation: Best Practices for Chromatographic and Ligand Binding Assays

Dear users,

I would like to know if there is any literature on the effect of diluting plasma samples prior to SPE. Does dilution affect the SPE proces and if so, in what way?

Thanks for your help.

Regards Bert

Bert, It kind of depends what you dilute with

Personally, I would advise diluting with a (pretested) blank plasma so you are not altering the base marix prior to the extraction process

Bert, are you talking about blood plasma? (Some people have used this word in a different context here, recently). If so, are you talking about advantages due to lowering the viscosity? Or changing the retention characteristics of some components?
Anyway, I would check the extensive literature by manufacturers to get some examples.

Thanks for you replies.

I recently had a discussion with a colleague: for preparation of calibration samples, they spiked 1 ml of plasma with different volumes of standard solutions. The dilution of the plasma was up to 25%. They argued that the volume of plasma applied on the SPE columns was always 1 ml and did not validate the effect of dilution. In my lab, we always try to minimize the dilution and spike all calibration samples with the same volume of (different) dilutions of standard solutions. So I was wondering whether there are any solid references on this subject

Regards Bert

So that is your problem after all. Isn´t it obvious that one would check whether dilution changes recovery?

bert,

I have been out of the bio game for a while but I agree with your procedure for standard preparation. That is best practice. keep the plasma volume and standard spike volume the same for your calibrators and QC's.

I was refering to sample dilutions (with matrix) in my earlier post. If i recall it is frowned upon dilute/spike plasma with standard (in organic solvent) more than 10%.

There are advantages to the dilution of plasma prior to SPE. The first one is that dilution reduces the viscosity of the sample, and the SPE runs more smoothly. Second, we always add the internal standard into the diluent, which creates an automatic standardization. Third, you need to adjust/manipulate the pH of the plasma sample for optimizing or standardizing the chromatographic conditions of your SPE method. Fourth, the addition of a strong acid is a general tool to free any analyte that is bound to protein.

You can get good information on SPE in general by getting the Oasis Application booklet from Waters.

Thanks for your comments! I think that I can conclude that different dilutions of calibration samples is not the way to go.

You should make up the difference in solvent volume with blank solvent. So each of your calibration standards contains the same amount of solvent but different levels of analytes.

e.g.

prepare std 0 by adding 1mL plasma and 0.1mL solvent

prepare std 5 by adding 1mL plasma, 0.05mL solvent and 0.05mL stock at 10ng/mL

prepare std 10 by adding 1mL plasma and 0.1mL of stock at 10ng/mL

You can test dilution effects by preparing QCs at over the calibration curve (OTC) and then diluting before sample prep and analysis. If you get 100% recovery then dilution is fine. But make sure your validation dilution is the same as any sample analysis dilution. Don't do a 4x dilution during validation and then a 1000x dilution post validation on study samples without running more OTC QC's at higher dilution factors.

As said above try and dilute with blank matrix. But if you are dealing with an endogenous component, you can always dilute with saline or DIW. You would then demonstrate this works during validation with the OTC QCs.