By Anonymous on Thursday, August 26, 2004 - 08:34 pm:

We conduct a lot of LC-MS-MS small molecule drug analysis of bioanalytical samples. I have observed that sometimes when protein precipitate extracts from whole blood are diluted (with blank matrix) that the calculated concentration of the drug in the diluted samples can be 3-fold greater than the same sample analyzed with no dilution. Both the non-diluted and diluted samples (e.g. 30-fold dilution) fall within the range of the calibration curve. Has anyone else observed this and do you know the cause?

Thanks!

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By HW Mueller on Thursday, August 26, 2004 - 11:26 pm:

The only unique thing here is that you will still have protein in your extract which may "hold on" to part of the analyte and prevent a normal chromatography. The equilibrium between analyte in the solvent part and that attached to the protein will be shifted toward the solvent part, hence the increase. I would think this to be the more unlikely cause, though. Maybe you inject different volumes for the two types of samples? How do you dilute? Also, sometimes a strong dilution like this results in a very small peak (if it can not be compensated by injection volume) whith accompanying high determination uncertainty. I have often seen this uncertainty to "prefer" a positive deviation, especially if the integration is done by hand.

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By anonymous on Friday, August 27, 2004 - 02:45 pm:

Thanks for your response! I agree that it might be the result of the dilution resulting in a small peak because the resulting peak is near the lower limit of quantitation. Even though this small diluted peak is within the calibration range, I think it might be biased upwards by carryover that gets magnified by the dilution multiple. The injection volumes are the same.