Development of a UPLC-IDA-ICP-MS/MS method for peptide quantitation in plasma by Se-labelling, and comparison to S-detection of the native peptide

Abstract

A method for quantitation of pharmaceutical peptides in human plasma based on gradient elution UPLC-ICP-MS/MS was developed. The organic solvent from the UPLC eluent was removed by addition of 20% oxygen (O₂) in argon (Ar) prior to entrance to the ICP plasma, and selenium and sulphur were detected as ⁸⁰Se¹⁶O⁺ and ³²S¹⁶O⁺, respectively after addition of O₂ to the collision-reaction-cell (CRC). Flow rates of carrier gas, option gas and cell gas together with sampling depth were optimized for handling gradients of increasing amounts of organic solvents. Sensitivity was highly dependent on the combined flow of option gas and carrier gas, which in turn was dependent on the sampling depth. CRC parameters were manually optimized and compared with the values obtained by the autotune function. In general, manual optimization did not improve signal to noise ratios compared to autotune optimization. Plasma samples were precipitated with 0.1% TFA in acetonitrile and the procedure was optimized taking adsorption, co-precipitation and disturbance of the chromatographic separation of peptides and degradation products into account. Post-column isotope dilution analysis (IDA) was applied for quantitation. Analytical figures of merit including linearity, precision, LOD, LOQ, recovery and accuracy were considered satisfactory. The LODs were 1.5 μg Se L⁻¹ (0.019 μM) and 16 μg S L⁻¹ (0.49 μM), respectively. The method was applied for a stability study showing the degradation of the peptides in plasma.