An official website of the United States government
The analysis of organic acids in complex mixtures by LC‐MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)‐MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N ‐(4‐aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC‐MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N ‐(4‐aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25‐to‐2100‐fold. The effect of the derivatization group on sensitivity, which increased by at least 200‐fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC‐MS, especially for those that are undetectable in their native form.
more »
« less
Piperidine Derivatization to Improve Organic Acid Detection with SFC-MS
The analysis of organic acids in complex mixtures by LC-MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native form. These compounds have also been difficult to measure using SFC-MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N-(4-aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC-MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N-(4-aminophenyl)piperidine to achieve detection limits down to 0.5 ppb. The effect of the derivatization group on sensitivity and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC-MS, especially for those that are undetectable in their native form.
more »
« less
- Award ID(s):
- 1904919
- PAR ID:
- 10484994
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Journal of separation science
- ISSN:
- 1615-9314
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Derivatization reactions are commonly used in mass spectrometry to improve analyte signals, specifically by enhancing the ionization efficiency of those compounds. Vicinal diols are one group of biologically important compounds that have been commonly derivatized using boronic acid. In this study, a boronic acid with a tertiary amine was adapted for the derivatization of vicinal diol metabolites in B73 maize tissue cross‐sections for mass spectrometry imaging analysis. Using this method, dozens of vicinal diol metabolites were derivatized, effectively improving the signal of those metabolites. Many of these metabolites were tentatively assigned using high‐resolution accurate mass measurements. In addition, reaction interference and cross‐reactivity with various other functional groups were systematically studied to verify data interpretation.more » « less
-
Spatial lipidomics is a powerful technique for understanding the complexity of the lipidome in biological systems through mass spectrometry imaging (MSI). Recent advancements have enabled isomer-selected MSI (iMSI) of lipids in biological samples using both online and off-line derivatization strategies. Despite these impressive developments, most iMSI techniques are limited to either positive or negative ion mode analysis, restricting the molecular coverage achievable in a single experiment. Additionally, derivatization efficiency often varies across lipid classes, presenting challenges for comprehensive lipid analysis. In this study, we introduce tetrakis(4-carboxyphenyl)porphyrin (TCPP) as a universal photosensitizer that facilitates online lipid derivatization in both positive and negative ionization modes via singlet oxygen (1O2) reaction. This method enables the identification and localization of both acyl chain compositions and lipid carbon-carbon (C=C) isomers in liquid extraction-based ambient ionization techniques. We have also employed sodium fluoride (NaF) as a solvent dopant to enhance the analysis of low-abundance and poorly ionizable biomolecules. By integrating these online derivatization and signal enhancement strategies with nanospray desorption electrospray ionization (nano-DESI), we achieved dual polarity iMSI within the same sample. We demonstrate imaging of low-abundance isomeric lipids, which were otherwise below the noise level. Notably, TCPP significantly enhances the efficiency of the online derivatization of unsaturated fatty acids, for which other photosensitizers are inefficient. This novel approach allows for the imaging of isomeric fatty acids and phospholipids from multiple classes in the same experiment, revealing their distinct spatial localization within biological tissues.more » « less
-
null (Ed.)We present the analysis of formaldehyde (HCHO) in anhydrous methanol (CH 3 OH) as a case study to quantify HCHO in non-aqueous samples. At higher concentrations (C > 0.07 M), we detect a product of HCHO, methoxy methanol (MM, CH 3 OCH 2 OH), by Fourier transform infrared spectroscopy, FTIR. Formaldehyde reacts with CH 3 OH, CD 3 OH, and CD 3 OD as shown by FTIR with a characteristic spectral feature around 1,195 cm −1 for CH 3 OH used for the qualitative detection of MM, a formaldehyde derivative in neat methanol. Ab initio calculations support this assignment. The extinction coefficient for 1,195 cm −1 is in the order of 1.4 × 10 2 M −1 cm −1 , which makes the detection limit by FTIR in the order of 0.07 M. For lower concentrations, we performed the quantitative analysis of non-aqueous samples by derivatization with dinitrophenylhydrazine (DNPH). The derivatization uses an aqueous H 2 SO 4 solution to yield the formaldehyde derivatized hydrazone. Ba(OH) 2 removes sulfate ions from the derivatized samples and a final extraction with isobutyl acetate to yield a 1:1 methanol: isobutyl acetate solvent for injection for electrospray ionization (ESI). The ESI analysis gave a linear calibration curve for concentrations from 10 to 200 µM with a time-of-flight analyzer (TOF). The detection and quantification limits are 7.8 and 26 μM, respectively, for a linear correlation with R 2 > 0.99. We propose that the formaldehyde in CH 3 OH is in equilibrium with the MM species, without evidence of HCHO in solution. In the presence of water, the peaks for MM become less resolved, as expected from the well-known equilibria of HCHO that favors the formation of methylene glycol and polymeric species. Our results show that HCHO, in methanol does not exist in the aldehyde form as the main chemical species. Still, HCHO is in equilibrium between the production of MM and the formation of hydrated species in the presence of water. We demonstrate the ESI-MS analysis of HCHO from a non-aqueous TiO 2 suspension in methanol. Detection of HCHO after illumination of the colloid indicates that methanol photooxidation yields formaldehyde in equilibrium with the solvent.more » « less
-
The quantification of amino acids as freely dissolved compounds or from hydrolyzed peptides and proteins is a common endeavor in biomedical and environmental sciences. In order to avoid the drawbacks of derivatization and application challenges of tandem mass spectrometry, we present here a robust 13-min liquid chromatography coupled with a full-scan mass spectrometry method to achieve rapid detection and quantification of 30 amino acids without derivatization. We combined hydrophilic interaction liquid chromatography with heated electrospray ionization and high-resolution mass spectrometry operated with polarity switching to analyze the 20 proteinogenic amino acids, ornithine, citrulline, homoserine, cystine, and six acetylated amino acids. We obtained high mass accuracy and good precision of the targeted amino acids. Limits of detection ranged from 0.017 to 1.3 μM. Noteworthy for environmental samples, we found comparable ionization efficiency and quantitative detection for the majority of the analytes prepared with pure water versus a high-salt solution. We applied the method to profile carbon source-dependent secretions of amino acids by Pseudomonas protegens Pf-5, a well-known plant-beneficial bacterium.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- The DOI is not currently available.
