In determining the recovery of thiamine diphosphate extracted from lysed whole blood, 40 nmol/L was spiked into a six aliquots of the same pool of lysed whole blood and extracted, and six additional aliquots of this same pool were extracted as "blanks" to which 40 nmol/L was added after post-extraction.
The extraction procedure is basically a dilution with water, derivatization & incubation using an alkaline potassium ferricyanide solution, a protein crash using 70% perchloric acid, centrifugation, and finally collection of the supernatant.
Despite good chromatography, we are unable to reproduce a 40 nmol/L nominal spike in the extracted aliquots. The determined concentration is always the baseline endogenous level, while the post-spiked samples always return a definitive ~40 nmol/L spike in concentration despite using the same spiking solution in both sets of samples. Any ideas on what could be happening?
Hi, I have run into a similar problem with a different analyte in plasma. Have you tried a range of pre-extraction spiking concentrations, including some that are considerably higher than 40 nmol/L? In my case, it turned out there were some problems with the stability of the analyte under the extraction conditions, and using a different protein crash method helped somewhat.
As Anita said, that's the problem. Chemically ferrocyanide and perclorate react and react very well (as dry solid are used as igniter, indeed). So that's the problem use a more bland protein precipitation medium.
Thank you both for the direction. I'm currently investigating alternative crash methods. I'll be sure to repeat the spike and recovery method in each crash protocol. Thanks again!
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