Benzoyl chloride derivatization with liquid chromatography-mass spectrometry for targeted metabolomics of neurochemicals in biological samples

Abstract

Widely targeted metabolomic assays are useful because they provide quantitative data on large groups of related compounds. We report a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method that utilizes benzoyl chloride labeling for 70 neurologically relevant compounds, including catecholamines, indoleamines, amino acids, polyamines, trace amines, antioxidants, energy compounds, and their metabolites. The method includes neurotransmitters and metabolites found in both vertebrates and insects. This method was applied to analyze microdialysate from rats, human cerebrospinal fluid, human serum, fly tissue homogenate, and fly hemolymph, demonstrating its broad versatility for multiple physiological contexts and model systems. Limits of detection for most assayed compounds were below 10nM, relative standard deviations were below 10%, and carryover was less than 5% for 70 compounds separated in 20min, with a total analysis time of 33min. This broadly applicable method provides robust monitoring of multiple analytes, utilizes small sample sizes, and can be applied to diverse matrices. The assay will be of value for evaluating normal physiological changes in metabolism in neurochemical systems. The results demonstrate the utility of benzoyl chloride labeling with HPLC-MS/MS for widely targeted metabolomics assays.

Keywords: Brain; Derivatization; Drosophila; HPLC; Mass spectrometry; Neurochemistry.

Figure 1

Normalized effect of sodium borate versus sodium carbonate buffer on calibration slope for select analytes. Standards made using sodium borate buffer and sodium carbonate buffer were analyzed with LC-MS in triplicate. A 6-point calibration curve for all analytes of interest was made to determine the average calibration slope for each analyte (n = 3 for each concentration tested). For the calibrations, the high concentrations were 20 nM for ACh, 5HT, NE, NM, DA and 3MT; 200 nM for Hist, GABA, 5HIAA, HVA, and DOPAC; and 2 µM for Tau, Ser, Asp, Ado, Gly, and Glu; followed by serial dilution. Analyte to internal standard ratios were plotted against known concentrations and a linear trend line was applied to determine slope (A). Sodium carbonate slopes were normalized to sodium borate slopes. Significant improvements to Ado, Gly, Hist, NE, DA, and DOPAC occurred when using 100 mM sodium carbonate as the buffer. Slopes were decreased for 5HT, NM, 3MT, and HVA. Unpaired two-tailed Students t test statistics were performed (B). Data expressed as percent borate ± SD. *p < 0.05, n = 3.

Figure 2

Chemical structures of neurochemicals enhanced by sodium carbonate buffer (A). Structures of neurochemical reduced with carbonate buffer (B).

Figure 3

Fragmentation patterns for select benzoyl labeled compounds. Analytes were detected by MS/MS under collision activated dissociation (CAD) conditions. While the benzoyl fragment of 105 m/z was the most abundant product ion for most analytes detected, unique fragments were chosen for detection to increase the selectivity of the assay for these compounds.

Figure 4

Reconstructed ion chromatogram of 70 compounds detected in 20 min. Extracted ion chromatograms for each compound at the highest concentration calibration standard run, were normalized to highest intensity and overlaid.

Figure 5

Metabolites showing significant differences between sated and starved states in fly hemolymph. Metabolite concentrations were normalized to total protein content, and then normalized to the sated sample. Each sample was run in triplicate. Unpaired two-tailed Students t tests were performed, and the Holm-Bonferroni correction was used. Data expressed as average ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

Recovery of four isotopically labeled metabolites spiked into plasma prior to solvent precipitation and derivatization. Percent recovery calculated as measured concentration after precipitation, relative to concentration spiked into serum. The average of three extraction replicates is shown. Error bars represent the standard error of the mean.