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 C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (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.

Mass spectrometric quantification of AcSDKP‐NH2 in human plasma and urine and comparison with an immunoassay

“…Recent studies have demonstrated the ability of C18 analytical columns to retain the endogenous AcSDKP peptide. [27,28] Thus, the chromatographic behavior of AcSDKP-NH 2 was first evaluated on classic reversed-phase columns such as the Zorbax Stable Bond C18 (Agilent Technology, Les Ulis, France) or the Polaris C18-A (Varian, Palo Alto, USA). In contrast with the behavior of endogenous AcSDKP, [27,28] poor retention of the amidated peptide was observed (data not shown).…”

“…[27,28] Thus, the chromatographic behavior of AcSDKP-NH 2 was first evaluated on classic reversed-phase columns such as the Zorbax Stable Bond C18 (Agilent Technology, Les Ulis, France) or the Polaris C18-A (Varian, Palo Alto, USA). In contrast with the behavior of endogenous AcSDKP, [27,28] poor retention of the amidated peptide was observed (data not shown). This discrepancy could be caused by the increased hydrophilicity of AcSDKP-NH 2 which can be explained by the positive charge afforded by the C-terminal amidation under acidic conditions.…”

“…[21] Indeed, the LC/MS/MS analysis of endogenous AcSDKP has a limit of quantification of 0.1 ng/mL in human plasma with variability below 10%. [28] Thus, the LC/MS/MS method associated with sample extraction and chromatographic separation allows analyte concentration and purification leading to more sensitive quantification compared with EIA. It is worth noting that endogenous AcSDKP could not be detected by the EIA [23] or by the LC/ MS/MS assay owing to the chromatographic separation and the mass signal discrimination (see blank samples in Fig.…”

New techniques of on-line biological sample processing and their application in the field of biopharmaceutical analysis

“…Thus, it should be a powerful method for preclinical and clinical PK study of puerarin and other isoflavonoids drugs. Currently, the on-line SPE–LC–MS technique has been widely used in analysis of complex biological matrices, such as plasma 11 , 12 , serum 13 and tissue homogenates 14 , etc.…”

An analytical quality by design approach towards a simple and novel HPLC-UV method for quantification of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline

“…Given the potential clinical applications of the peptide, a reliable analytical method is required to facilitate the formulation of the peptide, as well as assess the associated in vivo profile. Although a limited number of quantitative assays for the peptide or its analogue have been reported, these techniques either involved enzyme immunoassays (EIA) [11,12] or liquid chromatography-tandem mass spectrometry (LC-MS/MS) [13][14][15][16]. Pradelles et al first described an EIA for Ac-SDKP in 1990, using acetylcholinesterase-Ac-SDKP conjugate as the tracer, rabbit antiserum, and mouse anti-rabbit IgG antibody-coated 96-well plate.…”