On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients

ABSTRACT We developed a high‐throughput method based on on‐line solid‐phase extraction liquid chromatography tandem mass spectrometry (SPE‐LC‐MS/MS) to determine N‐terminal thymosin‐β fragment peptide (N‐acetyl‐seryl‐aspartyl‐lysyl‐proline, Ac‐SDKP) in human plasma samples. Quantification of Ac‐SDKP was performed using direct injection for on‐line SPE based on C18, reversed‐phase LC separation and stable isotope dilution electrospray ionization‐MS/MS in multiple reaction‐monitoring (MRM) mode. The Ac‐SDKP‐13C6, 15N2 (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac‐SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3–100.4% with inter‐day (relative standard deviation, RSD, 0.4–14.1%) and intra‐day (RSD, 0.8–19.7%) assays. This method was applied to the measurement of Ac‐SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre‐hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post‐hemodialyzed subjects, n = 9). All plasma Ac‐SDKP levels were decreased by dialysis. Thus, plasma Ac‐SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis. Copyright © 2011 John Wiley & Sons, Ltd.