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P1‐317: Ultra‐performance 2D‐liquid chromatography–tandem mass spectrometry method for simultaneous analysis of beta‐amyloid peptides: 1‐38, 1‐40 and 1‐42 in human cerebrospinal fluid
Author(s) -
Korecka Magdalena,
Waligorska Teresa,
Trojanowski John,
Shaw Leslie
Publication year - 2012
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2012.05.2003
Subject(s) - chemistry , chromatography , tandem mass spectrometry , high performance liquid chromatography , mass spectrometry , triple quadrupole mass spectrometer , extraction (chemistry) , analyte , selected reaction monitoring , elution , analytical chemistry (journal)
Background: A new ultra performance 2D-LC method with tandem mass spectrometric detection was developed for accurate, reliable and efficient quantification of three amyloid beta peptides, 1-38, 1-40 and 1-42 in human CSF following an initial extraction step using 5M guanidine HCL (GUCL). Methods: An API 5000 triple quadrupole instrument (ABSciex) with an ESI ion source, interfaced with an ACQUITY UPLC (Waters), was used. Artificial CSF containing 4mg/mL bovine serum albumin was used as a matrix for standards and quality control samples. The analytes were isolated from CSF using Oasis MCX m Elution 96-well plates (Waters) after an initial extraction/denaturation step using 5M GUCL. During the first step of chromatography 50 m L of sample extracts undergo on-line dilution/cleaning, using an Xbridge BEH C18 column (Waters). After desalting with a highly aqueous solvent the valve was switched and analytes were transferred to the analytical column (BEH-300, C18, 2.1x150mm, 1.7 mm; Waters). An isocratic gradient of 0.1% ammonia in water and a mixture of organic solvents was used for efficient separation of the three peptides and their internal standards (isotopically labeled). The mass spectrometer was operated in the positive ion mode and MRM scanning. The quadrupoles were set to detect the 4+ molecular ions and their fragments: m/z 1033.9/1001.0 (A b 1-38), m/z 1083.5/1054.3 (A b 1-40) and m/z 1129.5/1079.1 (A b 1-42), m/z 1046.0/1012.5 (15 N-A b 1-38), m/z 1096.0/1066.5 (15 N-A b 1-40) and m/z 1142.5/1091.5 (15 N-A b 1-42). Results: The lower limit of quantification was 100pg/mL for all three peptides (Table 1). The upper limit of quantification was 7500pg/mL for A b 1-38 and 1-40, and 3000pg/mL for A b 1-42. For 4 quality control samples between-day precision varied from 11.1-12.8% (A b 1-38), 7.3-13.2% (A b 1-40) and 10-14.9% (A b 1-42) (Table 2). Mean analytical recovery of the abeta peptides for QC samples was 101.8% (A b 1-38), 100.2% (A b 1-40) and 97.3% (A b 142). Between-day precision for all calibrators not including LLOQ ranged from 4.6% to 16.3% and accuracy ranged from 92 to 109%. Under these conditions no ion suppression of the detection of any ion was found and no carry over effects were observed. Conclusions:We believe this newly developed and validated assay confirms its potential utility for the direct measurement of Abeta peptides in CSF samples and further analyses are underway to demonstrate this. Acknowledgments: We thank Rand Jenkins 1 from PPD Inc. for sharing his extensive experience in development and optimization of the 2-D liquid chromatography tandem mass spectrometry method and Erin Chambers from Waters for Table 2 Precision and accuracy of Ab38, Ab40, Ab42, pTau181 and Total Tau measurements