Critical sample pretreatment in monitoring dried blood spot citrulline.
BACKGROUND
Plasma Citrulline concentration has been correlated to functional enterocyte mass. Dried blood spot (DBS) analysis using LC-MSMS reduces sample amount needed. We optimized DBS elution to increase precision and accuracy of DBS LC-MSMS analysis.


METHODS
DBS control samples were eluted in varying pH (2.2-7.0) and for varying times (15-75 min) and Cit, Arg and Orn were analyzed using LC-MSMS, with and without derivatization. In 20 volunteers, the DBS LC-MSMS assay was correlated with a plasma ion exchange HPLC method.


RESULTS
For Citrulline an extraction optimum was obtained at pH 2.6, whereas lower Arginine concentrations were found using low extraction pH. Increasing elution times lead to increased concentrations. Within-run CV was higher with, compared to without derivatization. No close association could be found between plasma HPLC and DBS LC-MSMS concentrations.


CONCLUSIONS
Analysis of amino acids on DBS using LC-MSMS should be optimized regarding the purpose of the assay. In our study, most optimal results were obtained without derivatization and elution in pH 2.6 for 45 min. Cellular amino acids in DBS might influence the correlation of Cit with severity of enteral disorders. Therefore, further evaluation of DBS Cit as a marker for enteral disorders is warranted.
Wuyts
Birgitte
B
Clinical Chemistry, University Hospital Ghent, Gent, Belgium. brigitte.wuyts@UGent.be
Stove
Veronique
V
Goossens
Linde
L
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002956:Citrulline / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D008991:Monitoring, Physiologic / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D000596:Amino Acids; D002956:Citrulline; D001120:Arginine