An UHPLC-MS/MS method for simultaneous quantification of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid using micro-elution solid phase extraction

Amyloid beta (A beta) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer's disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from A beta propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of human amyloid beta peptides A beta 1-38, A beta 1-40 and A beta 1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled A beta peptides N-15(51)- A beta 1-38, N-15(53)- A beta 1-40 and N-15(55)- A beta 1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15 ng/mL were prepared. A linear regression (1/x(2) weighting): y = ax + b was used to fit the calibration curves over the concentration range of 0.1-20 ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for A beta 1-38, 6.75% for A beta 1-40 and 10.74% for A beta 1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of A beta 1-38, A beta 1-40 and A beta 1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24 h at 4 degrees C, 4 h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC/MS/MS method was successfully applied to the quantitation of A beta peptides in real human CSF samples. Our work may provide a reference method for simultaneous quantitation of human A beta 1-38, A beta 1-40 and A beta 1-42 from CSF.