A multi-matrix HILIC-MS/MS method for the quantitation of endogenous small molecule neurological biomarkerN-acetyl aspartic acid (NAA)

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Abstract

A multi-matrix hydrophilic interaction liquid chromatography tandem mass spectrometric method (HILIC-MS/MS) was developed for the quantitation of N-Acetyl Aspartic acid (NAA) using stable isotope labeled internal standard, D3-NAA in various biological matrices such as human plasma, human CSF, mouse plasma, brain and spinal cord. A high throughput 96-well plate format supported liquid extraction (SLE) procedure was developed and used for sample preparation. Mass spectrometric analysis of NAA was performed using selected reaction monitoring transitions in positive electrospray ionization mode. As NAA is endogenously present, a surrogate matrix approach was used for quantitation of NAA and the method was qualified over linear calibration curve range of 0.01–10 μg/mL. Intra and inter assay precision indicated by percent relative standard deviation (%RSD) was less than 7.1% for low, medium, medium high and high QCs. The accuracy of the method ranged from 92.6-107.0% of nominal concentration for within-run and between-run for the same QCs. Extraction recovery of NAA and D3-NAA was greater than 76%. Stability of NAA was established in the above biological matrices under bench top (RT, 5 h), freeze thaw (–20 ± 10 °C, 3 cycles) and moues/human plasma sample collection (Wet ice, RT) conditions. HILIC-MS/MS method was then used to quantify and compare the NAA levels in human plasma and CSF of ALS patients versus control human subjects. NAA CSF levels in control human subjects (73.3 ± 31.0 ng/mL, N = 10) were found to be slightly higher than ALS patients (46.1 ± 22.6 ng/mL, N = 10) (P = 0.04). No differences were observed in NAA plasma levels in human control subjects (49.7 ± 13.8 ng/mL, N = 9) as compared to ALS patients (49.6 ± 8.1 ng/mL, N = 10) (P = 0.983). NAA endogenous concentrations in mouse plasma, brain and spinal cord were found to be 243.8 ± 56.8 ng/mL (N = 6), 1029.8 ± 115.2 μg/g tissue weight (N = 5) and 487.6 ± 178.4 μg/g tissue weight (N = 5) respectively.

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