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 10 nM, relative standard deviations were below 10%, and carryover was less than 5% for 70 compounds separated in 20 min, with a total analysis time of 33 min. 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.

A Systematic Search and Mapping Review of Studies on Intracerebral Microdialysis of Amino Acids, and Systematized Review of Studies on Circadian Rhythms

“…Only one study described the baseline microdialysate levels for all our compounds of interest [26], and 4 more described 8 out of 9 [4272829]. Most of the studies reported only one (1246 studies) or two (548 studies) of these compounds.…”

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

“…The supernatant was then extracted with acetonitrile. Aliquots of 20 l were subsequently derivatized with benzoyl chloride as described previously (30). The samples were analyzed in triplicate for the presence of 5Ј-deoxyadenosine by HPLC-tandem mass spectrometry, using an Acquity HSS T3 C18 chromatography column (1 mm ϫ 100 mm, 1.8 m, 100 Å pore size) on a Waters nanoAcquity ultra performance liquid chromatograph interfaced to an Agilent 6410 triple quadrupole mass spectrometer.…”

“…The extracts were incubated anaerobically at room temperature with 5 mM DTT and 5 mM dithionite for 30 min prior to the addition of 200 M SAM. After 1 h the nucleotide pool was extracted and analyzed by LC-MS using established protocols (30). No 5Ј-dA was detected in cell extracts lacking viperin, whereas 5Ј-dA was readily detected extracts prepared from viperin-expressing HEK293T cells (Fig.…”

Repeatability of gradient ultrahigh pressure liquid chromatography–tandem mass spectrometry methods in instrument-controlled thermal environments

“…For the quantitative UHPLC-MS/MS analyses used to monitor neurotransmitters regularly employed in our lab [48,49], the most important parameters for identifying and quantifying peaks are the elution time and peak area ratio between the analyte and its 13 C-labeled internal standard. Because the axial thermal gradient is higher in quasi-adiabatic environments like the still-air oven mode, larger shifts in the elution time between consecutive runs could be observed.…”

“…Benzoyl chloride (BzCl) derivatization of neurotransmitter samples has been described previously [48,49] and is only summarized here. Prior to analysis, the thawed 10 µL concentrated mixture described above was diluted 10-fold in HPLC grade water to a final volume of 100 µL.…”