4 CHAPTER 4 80 transitions were individually optimized for targeted analytes and respective isotopically labelled internal standards. Data acquisition and pre-processing was performed using LabSolutions LCMS Version 5.97 SP1 (Shimadzu Corporation). Data quality was monitored using regular injection of quality control (QC) samples prepared from pooled CSF. Samples were injected in a randomized order. QC samples were used to correct for inter-batch variations using the in-house developed mzQuality workflow (available at http://www.mzQuality.nl). Relative standard deviations (RSDs) of peak area ratios were calculated for each targeted analyte detected in the QC samples. The absolute concentration of the eCBs in CSF samples was calculated using calibration samples. Quality control was set on RSDqc < 15% and blank effect < 15%. The data as relative response ratios (target area/ISTD area; unit free) of these metabolites after QC correction were calculated and converted to concentrations. Statistical analysis Principal component analysis (PCA) was performed on the concentration measurements of all measured eCBs together. Based on the first two principal components outliers were excluded by visually inspecting the plots. Log10-transformed eCB concentrations (pM) were used for further statistical analyses. Multivariate linear regression was used to detect metabolite differences between migraineurs and healthy controls for each eCB separately. Based on literature, BMI, sex, age, weekly alcohol consumption, cigarettes per day, and lifetime depression were identified as potential covariates and therefore included as independent variables. Prior to data analysis missing data for relevant covariates was imputed with Multiple Imputation by Chained Equations (MICE).32 For MICE, we used 10 imputations and 30 iterations as settings and included the following variables: Log10 eCB concentrations, age, sex, BMI, migraine without aura/migraine with aura/control-stratus, lifetime depression score, and the amount of alcohol and smoking per week. Regression analyses were performed on the imputed data and results were pooled. All data analyses were performed using R version 4.0.1. and RStudio version 1.4.1717 (RStudio: Integrated Development for R. RStudio, PBC).33 P-values of ≤ 0.05 were considered statistically significant. Data Availability The data that support the findings of this study are available from the corresponding author, upon reasonable request. Results Clinical characteristics We reliably detected three endocannabinoids, AEA, 2-AG, and DHEA in the CSF of 195 individuals with migraine (n = 98 migraine with aura; n = 97 migraine without aura) and 94 healthy controls. Other endocannabinoids did not comply to the quality control criteria and for 5
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