Dilution Followed By Injection

Sample dilution is used to reduce the concentration of salts and endogenous materials in a sample matrix and is commonly applied to urine. Drug concentrations in urine are usually fairly high and allow this dilution without an adverse effect on sensitivity. While the amount of protein in a sample is usually a concern, protein concentrations in urine are negligible under normal physiological conditions. An example of a urine dilution procedure is reported for indinavir in which 1 mL urine was diluted with 650 |vL acetonitrile so that the resulting concentration of organic in the sample was equal to or less than that of the mobile phase. An aliquot of 6 yL was injected into an LC-MS/MS system [7]. Although co-eluting endogenous species in urine were not seen in the selected ion-monitoring mode, their presence did adversely affect the ionization of analytes, leading to increased variation in MS/MS responses.

Dilution is not typically used for plasma due to the high amounts of protein present and the greater effect that dilution has on sensitivity. However, dilution can be very attractive for the minimal effort and time involved. In one report [31], dog-plasma samples were centrifuged, pipetted into wells of a microplate, and then placed on a pipetting workstation. A volume of 15 yL plasma was diluted with 485 yL of a solution of water/methanol/formic acid (70:30:0.1, v/v/v) containing internal standard. The samples were sealed and mixed; the dilution resulted in a slightly viscous solution with no observed precipitation. An aliquot of 5 yL was injected into an LC-MS/MS system. The limit of quantitation for the dilution assay (2 ng/mL) was 400 times higher than that of a more selective procedure that also concentrates the an-alyte (liquid-liquid extraction; 5-pg/mL limit of quantitation). However, the advantage offered by the dilution procedure was fifty times greater throughput. In this case, throughput was a more important consideration than analyte sensitivity.

Was this article helpful?

0 0

Post a comment