Optimized 13C-TrEnDi Enhances the Sensitivity of Plasmenyl Ether Glycerophospholipids and Demonstrates Compatibility with Other Derivatization Strategies.

The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses.

Trimethylation Enhancement Using Diazomethane (TrEnDi) Enables Enhanced Detection of Glufosinate and 3-(Methylphosphinico)propionic Acid from Complex Canola Samples.

Over the past century, agriculture practices have transitioned from manual cultivation to the use of an array of chemical herbicides for weed control including phosphinothricin, or glufosinate (GLUF). Consequently, the potential for long-term residual GLUF exposure in the food chain has increased, highlighting the need for improved analytical strategies for its detection, as well as the detection of its main breakdown product 3-(methylphosphinico)propionic acid (MPPA). Chemical derivatization strategies have been developed to improve the detection of GLUF and MPPA via liquid chromatography tandem mass spectrometry analyses. Herein, we employ trimethylation enhancement using diazomethane (TrEnDi) for the first time as a means to confer analytical advantages via quantitatively derivatizing these analytes into permethylated GLUF ([GLUFTr]+) and MPPA ([MPPATr+H]+). Comparing [GLUFTr]+ and [MPPATr+H]+ to underivatized counterparts, TrEnDi yields 2.8-fold and 1.7-fold improvements in reversed-phase chromatographic retention, respectively, while MS-based sensitivity is enhanced 4.1-fold and 11.0-fold, respectively. Successful analyte derivatization (with >99% yields) was further demonstrated on a commercial herbicide solution imparting consistent analytical enhancements. To investigate the benefits of TrEnDi in a bona fide agricultural scenario, simple aqueous extractions from distinct parts of field-grown canola plants were performed to quantify GLUF and MPPA before and after TrEnDi derivatization. In their underivatized forms, GLUF and MPPA were undetectable in all field samples, whereas [GLUFTr]+ and [MPPATr+H]+ were readily quantifiable using the same analysis conditions. Our results demonstrate that TrEnDi continues to be a useful tool to enhance the analytical characteristics of organic molecules that are traditionally difficult to detect.

An In Silico Database for Automated Feature Identification of High-Resolution Tandem Mass Spectrometry 13C-Trimethylation Enhancement Using Diazomethane (13C-TrEnDi)-Modified Lipid Data.

13C-Trimethylation enhancement using diazomethane (13C-TrEnDi) is a chemical derivatization technique that uses 13C-labeled diazomethane to increase mass spectrometry (MS) signal intensities for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid classes, both of which are of major interest in biochemistry. In silico mass spectrometry databases have become mainstays in lipidomics experiments; however, 13C-TrEnDi-modified PC and PE species have altered m/z and fragmentation patterns from their native counterparts. To build a database of 13C-TrEnDi-modified PC and PE species, a lipid extract from nutritional yeast was derivatized and fragmentation spectra of modified PC and PE species were mined using diagnostic fragmentation filtering by searching 13C-TrEnDi-modified headgroups with m/z 199 (PC) and 202 (PE). Identities of 25 PC and 10 PE species were assigned after comparing to predicted masses from the Lipid Maps Structure Database with no false positive identifications observed; neutral lipids could still be annotated after derivatization. Collision energies from 16 to 52 eV were examined, resulting in three additional class-specific fragment ions emerging, as well as a combined sn-1/sn-2 fragment ion, allowing sum-composition level annotations to be assigned. Using the Lipid Blast templates, a NIST-compatible 13C-TrEnDi database was produced based on fragmentation spectra observed at 36 eV and tested on HEK 293T cell lipid extracts, identifying 47 PC and 24 PE species, representing a 1.8-fold and 2.2-fold increase in annotations, respectively. The 13C-TrEnDi database is freely available, MS vendor-independent, and widely compatible with MS data processing pipelines, increasing the throughput and accessibility of TrEnDi for lipidomics applications.

Improved Chromatography and MS-Based Detection of Glyphosate and Aminomethylphosphonic Acid Using iTrEnDi.

Glyphosate (GLY), a synthetic, nonselective systemic herbicide that is particularly effective against perennial weeds, is the most used weedkiller in the world. There are growing concerns over GLY accumulation in the environment and the attendant human health-associated risks, and despite increased attention in the media, GLY and its breakdown product aminomethylphosphonic acid (AMPA) remain elusive to many analytical strategies. Chemical derivatization coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) addresses the challenge of quantifying low levels of GLY and AMPA in complex samples. Here we demonstrate the use of in situ trimethylation enhancement using diazomethane (iTrEnDi) to derivatize GLY and AMPA into permethylated products ([GLYTr]+ and [AMPATr]+, respectively) prior to analysis via HPLC-MS. iTrEnDi produced quantitative yields and resulted in a 12-340-fold increases in HPLC-MS-based sensitivity for [GLYTr]+ and [AMPATr]+, respectively, compared with underivatized counterparts. The limits of detection of derivatized compounds were found to be 0.99 ng/L for [GLYTr]+ and 1.30 ng/L for [AMPATr]+, demonstrating significant sensitivity improvements compared to previously established derivatization techniques. iTrEnDi is compatible with the direct derivatization of Roundup formulations. Finally, as proof of principle, a simple aqueous extraction followed by iTrEnDi enabled the detection of [GLYTr]+ and [AMPATr]+ on the exterior of field-grown soybeans that were sprayed with Roundup. Overall, iTrEnDi ameliorates issues relating to low proton affinity and chromatographic retention, boosting HPLC-MS-based sensitivity and enabling the elucidation of elusive analytes such as GLY and AMPA within agricultural systems.

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

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.