Evaluation of peptide adsorption-controlled liquid chromatography–tandem mass spectrometric (PAC-LC–MS/MS) method for simple and simultaneous quantitation of amyloid β 1–38, 1–40, 1–42 and 1–43 peptides in dog cerebrospinal fluid

To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography–tandem mass spectrometry (PAC-LC–MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid β 1–38, 1–40, 1–42 and 1–43 peptides (Aβ38, Aβ40, Aβ42 and Aβ43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aβ was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aβ quantitation. To reduce a loss of Aβ during the pretreatment procedures, the dog CSF diluted by water–acetic acid–methanol (2:6:1, v/v/v) was loaded on PAC-LC–MS/MS directly. Quantification of the Aβ in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H5+] and b5+ ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aβ standard calibration solutions using PAC-LC–MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aβ38, Aβ40, Aβ42 and Aβ43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aβ concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aβ standard solution, the freeze–thaw stability and the room temperature stability of Aβ standard solution suggest that the PAC-LC–MS/MS method enables reproducible Aβ quantitation.