iTrEnDi: In Situ Trimethylation Enhancement Using Diazomethane: Improved and Expanded Glycerophospholipid and Sphingolipid Analyses via a Microscale Autonomous Derivatization Platform

Abstract

Trimethylation enhancement using diazomethane (TrEnDi) is a derivatization technique that significantly enhances the signal intensity of glycerophospholipid species in mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses. Here, we describe a novel apparatus that is able to conduct in situ TrEnDi (iTrEnDi) by generating and immediately reacting small amounts of gaseous diazoalkane with analyte molecules. iTrEnDi allows complete and rapid methylation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and sphingomyelin (SM) in a safe manner by removing any need for direct handling of dangerous diazoalkane solutions. iTrEnDi-modified PC ([PCTr]+) and PE ([PETr]+) showed similar sensitivity enhancements and fragmentation patterns compared to our previously reported methodology. iTrEnDi yielded dimethylated PA ([PATr]), which exhibited dramatically improved chromatographic behavior and a 14-fold increase in liquid chromatography MS (LCMS) sensitivity compared to unmodified PA. In comparison to in-solution-based TrEnDi, iTrEnDi demonstrated a modest decrease in sensitivity, likely due to analyte losses during handling. However, the enhanced safety benefits of iTrEnDi coupled with its ease of use and capacity for automation, as well as its accommodation of more-reactive diazoalkane species, vastly improve the accessibility and utility of this derivatization technique. Finally, as a proof of concept, iTrEnDi was used to produce diazoethane (DZE), a more-reactive diazoalkane than diazomethane. Reaction between DZE and PC yielded ethylated [PCTr]+, which fragmented via MS/MS to produce a high-intensity characteristic fragment ion, enabling a novel and highly sensitive precursor ion scan.

Enhanced Characterization of Cardiolipins via Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry

“…Considering that signal dilution and spectral complexity are partially due to the acidity of the phosphate groups, the chemical derivatization of CL to modify the acid groups and disfavor zwitterions was enabled. Notably, the methylation of GPL and CL phosphate and amine functional groups using diazomethane-based chemistry has been demonstrated to improve positive mode ionization efficiency. ,− The methylation of CL phosphate groups using TMSD is reportedly efficient and promotes the detection of ammonium-adducted CL [M + NH 4 ] + in the positive ion mode over the protonated [M + H] + ion. − Here, TMSD was performed to methylate CL phosphate groups, as illustrated in Figure D, to limit sodium adduction and focus the signal to the singly sodium-cationized [M + Na] + ion, ultimately curtailing signal dilution and spectral complexity. To illustrate this outcome, CL (16:0/18:1­(9Z))/(16:0/18:1­(9Z)) was derivatized using TMSD and subsequently ionized via nESI, resulting in an abundant ion of m/z 1456.03 corresponding to [M + Na] + (Figure B) of the methylated structure.…”

Trimethylation Enhancement using Diazomethane (TrEnDi) Enhances Mass Spectrometry-Based Analysis of Glufosinate and 3-(Methylphosphinico)propionic Acid in Complex Matrices

“…54 Due to the dangers associated with using an ethereal solution of DZM, a new method of TrEnDi was developed in which DZM was created in situ (iTrEnDi). 59 Once the DZM was created, it was used immediately in its gaseous form resulting in full methylation of the glycerophospholipids tested. 59 An aqueous solution of potassium hydroxide was added dropwise to an aqueous solution of DZM precursor -N-methyl-Nnitrosourea (NMU) -and diethyl ether which resulted in the formation of DZM.…”

“…59 Once the DZM was created, it was used immediately in its gaseous form resulting in full methylation of the glycerophospholipids tested. 59 An aqueous solution of potassium hydroxide was added dropwise to an aqueous solution of DZM precursor -N-methyl-Nnitrosourea (NMU) -and diethyl ether which resulted in the formation of DZM. 59 Once the diazomethane was formed, it flowed directly into the analyte vial containing the dissolved analytes where derivatization occurred.…”

“…59 An aqueous solution of potassium hydroxide was added dropwise to an aqueous solution of DZM precursor -N-methyl-Nnitrosourea (NMU) -and diethyl ether which resulted in the formation of DZM. 59 Once the diazomethane was formed, it flowed directly into the analyte vial containing the dissolved analytes where derivatization occurred. 59 The nitrogen saturated ether vial was present to ensure positive flow throughout the system to make sure the DZM was flowing into the analyte vial as shown in Figure 9.…”

“…59 Once the diazomethane was formed, it flowed directly into the analyte vial containing the dissolved analytes where derivatization occurred. 59 The nitrogen saturated ether vial was present to ensure positive flow throughout the system to make sure the DZM was flowing into the analyte vial as shown in Figure 9. PC; however, in solution TrEnDi shows better sensitivity than iTrEnDi.…”