A pre-validation precision study was performed to evaluate the performance of the Hoofnagle Lab ApoE LC-MS/MS assay.  To evaluate intra- and inter-day imprecision, two separate cerebral spinal fluid (CSF) pools were created from leftover clinical lumbar tap CSF samples: a ‘Normal’ pool and a ‘High’ pool which was spiked with 10ug/mL each of recombinant ApoE2, ApoE3, and ApoE4 (PeproTech).  A 5x5 study was performed by analyzing five replicates of each sample pool prepared on five different days.  Additionally, a 3x5 study was performed using five individual CSF samples (Patients 1-5) were evaluated by analyzing three replicates on five different days. 

Results:  Inspection of the individual Patient chromatograms for the proteotypic peptides monitored enabled determination of their ApoE genotype as indicated in the tables below.  No ApoE2 was evident in the Normal Pool, which can be expected given ApoE allele frequency.  Table 1 shows intra-day precision for normal and high pools was less than target

Table 1.  Intra-day precision (%CV).

Table 2.  Inter-day precision (Total Sum of Squares).

ApoE2, E3, and E4 isoforms are analyzed from cerebral spinal fluid samples using the following sample preparation procedure.  

LC-MS/MS analysis of tryptic peptides was performed using a Waters Aquity UPLC coupled to a Waters TQ-s mass pectrometer.