Reagent and method for detection of carboxylic acids by mass spectrometry
Inventors
Gelb, Michael H. • Turecek, Frantisek • Bollinger, James
Assignees
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Publication Number
US-9045421-B2
Publication Date
2015-06-02
Expiration Date
Abstract
Method and reagent for converting a carboxylic acid to a positively charge amide are described. The method and reagent facilitate positive ion mass spectral analysis of carboxylic acids. The method includes reacting a carboxylic acid with a compound having formula I: wherein A and B are aromatic rings, ring A includes a quaternized nitrogen and has n additional ring atoms, each additional ring atom optionally substituted with an RA group, and n is an integer from 4 to 10, and ring B includes a carbon atom and has m additional ring atoms, each additional ring atom optionally substituted with an RB group, and m is an integer from 4 to 10. The compound includes at least one RA or RB group, and the at least one RA and RB group is -L-N(Z)H; and X− is a counterion.
Core Innovation
The invention relates to converting carboxylic acids to permanently positively charged amide derivatives to enable positive-ion LC/ESI-MS/MS detection. The derivatization uses a quaternary ammonium reagent of a defined formula that contains aromatic rings with a quaternized nitrogen and a counterion X−. The reagent includes at least one -L-N(Z)H substituent, where L is an alkylene and Z is hydrogen or an alkyl group.
A core aspect described in the document is the behavior of the tag versus the analyte in LC/ESI-MS/MS, where fragmentation occurs in the analyte portion rather than the tag. This rationale is presented to support effective fragmentation patterns for the positively charged derivatives during detection. The described approach targets analysis of eicosanoids and related carboxylic acid analytes under positive-ion mass spectrometric conditions.
The document further provides a derivatization kit concept that includes the quaternary ammonium reagent, including AMPP salts as an example, such as N-(4-aminomethylphenyl)pyridinium. The overall formulation and characterization focus on achieving permanently positive charge on the resulting amide derivatives and obtaining measurable LC/ESI-MS/MS fragmentation and performance metrics for the analyzed analytes.
Claims Coverage
The partial information includes two independent claim families directed to a compound formula and a compound formula with a counterion requirement. Across these, the core coverage centers on inventive structural elements containing -L-N(Z)H, quaternized aromatic nitrogen, and a counterion X−, with additional refinement in dependent claims and a kit claim for derivatizing carboxylic acids.
Quaternary-ammonium amide-derivatization compound scaffold with -L-N(Z)H and counterion X−
A compound having a formula wherein R1–R10 are independently hydrogen, an alkyl group, and -L-N(Z)H, wherein L is an alkylene, Z is hydrogen or an alkyl group including from one to twenty carbon atoms, with at least one of R1–R10 being -L-N(Z)H, and X− is a counterion.
Counterion-defined compound of specified formula
A compound having a formula wherein X− is a counterion.
The provided independent claims primarily cover compounds defined by the stated general structural formula incorporating at least one -L-N(Z)H substituent and the presence of a counterion X−, with additional dependent refinements and a kit claim that includes indicia/instructions for derivatizing a carboxylic acid.
Stated Advantages
Improves positive-ion LC/ESI-MS/MS detection of carboxylic acids by forming permanently positively charged amide derivatives.
Supports fragmentation in the analyte portion rather than the tag in LC/ESI-MS/MS.
Provides performance improvements versus underivatized negative-ion detection, including sensitivity improvements and described LOQ/CV/recovery performance.
Provides derivatization yield and side-product suppression as described, including high AMPP amide coupling yield and low N-acyl urea side product.
Demonstrates reproducibility and evaluation in described real-sample contexts, including serum and stimulated bronchial epithelial cells.
Documented Applications
Derivatizing carboxylic acids in a sample for positive-ion LC/ESI-MS/MS detection using the quaternary ammonium reagent.
Measuring eicosanoids and related carboxylic acid analytes, including PGD2, PGE2, PGF2α, arachidonic acid, HETEs, LTB4, TxB2, and 6-keto-PGF1α, using LC/ESI-MS/MS fragmentation targets described in the document.
Use in a derivatization kit that includes a container containing the compound and printed indicia/instructions for using the compound to derivatize a carboxylic acid.
Application to real biological matrices evaluated as described, including serum and stimulated bronchial epithelial cells, including bronchoalveolar lavage fluid and primary bronchial epithelial cells as referenced in the provided summary.
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