Predicted influence of sample hematocrit on injected mass of internal standard in mass spectrometry assays utilizing simple protein precipitation for sample preparation.
BACKGROUND
The injected mass of internal standard is often monitored in mass spectrometry assays to flag aberrant specimen processing. When simple protein precipitation is used for sample preparation, the protein volume (solids fraction) of the specimen is in principle a variable affecting the final concentration of the internal standard in the liquid phase. We examined the predicted extent of this variation for an example of protein precipitation for sample preparation used in a mass spectrometry assay for immunosuppressants in whole blood.


METHODS
Liquid and solid mass fractions of samples with hematocrit ranging from 15% to 60% were measured after protein precipitation of samples using a whole blood/precipitating reagent ratio of 1:4. Relative supernatant volumes as a function of hematocrit were determined.


RESULTS
Liquid volume variation was consistent with a predicted variation in the internal standard concentration of only approximately +/-1% across this hematocrit range. Data were in close agreement with calculations based simply on protein density and concentration.


CONCLUSIONS
Hematocrit affects the injected mass of internal standard in assays that utilize protein precipitation for sample preparation, due to predictable variation in solids and liquid fractions. The effect is small, however, compared to the typical variation observed for injected mass of internal standard.
Cushman-Vokoun
Allison M
AM
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-3135, USA.
Plath
Kim B
KB
Landmark
James D
JD
Stickle
Douglas F
DF
D000818:Animals; D011232:Chemical Precipitation; D006400:Hematocrit; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D011506:Proteins / Q000737:chemistry*; D012015:Reference Standards; D015203:Reproducibility of Results
D011506:Proteins