Use of 13C and 15N derivatization reagents for gas and liquid chromatography-mass spectroscopy chemical identification and quantification

A method for metabolomic/lipidomic profiling of one or more compounds, by distinguishing signals of derivatives from background noise, in a biological sample comprising

• A) i) derivatizing the one or more compounds with one or more 13C labeled or 15N labeled derivatization agents and one or more matching unlabeled derivatization reagents, wherein the ratio between the labeled derivatization reagents and matching unlabeled derivatization reagents is selected to enhance signal to noise ratio, wherein the 13C labeled or 15N labeled derivatization agents are selected from the group consisting of 13C methylchloroformate, 13C ethylchloroformate, 13C methanol, 13C ethanol, 13C acetyl anhydride, 13C labeled N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), 13C labeled N-(tert.-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA), trimethylsilyl-13C-diazomethane (TMS-13CHN2), 13C and 15N phenylisothiocyanate (PITC), 13C and 15N aniline, and 13C and 15N O-benzylhydroxylamine;
• or
• ii) making two or more 13C labeled or 15N labeled standards for quantitating the one or more compounds by derivatizing the one or more compounds with two or more 13C labeled or 15N labeled derivatization reagents, wherein the ratio between the two or more derivatization reagents is selected to enhance signal to noise ratio, and introducing the 13C labeled or 15N labeled standards into a compound sample already derivatized with matching unlabeled derivatization reagents or 13C labeled or 15N labeled derivatization reagents;
• and, for i) or ii),
• calculating the number of chemical groups on the molecule that can be derivatized, using chemical ionization (CI) and high resolution gas chromatography-mass spectroscopy (GC/MS), or for the molecular ion with LC/MS, from the mass shift from unlabeled reagents, aided globally from the exact registration of peaks between labeled and unlabeled reagents;
• narrowing the chemical formulae possible for compounds not in a database;
• and
• comparing electron impact ionization (EI) spectra from matching 13C and 15N labeled barcode spectral patterns to reveal a composition of EI fragment identity to further aid chemical formulae identification;
• comparing molecular ion and possibly MS/MS fragments generated by LC/MS to reveal a composition of MS/MS fragment identity to further aid chemical formulae identification;
• and
• B) i) for GC/MS, if amino group numbers need to be confirmed for an unknown compound, derivatizing the compounds containing the unknown with 99% 13C methylchloroformate (MCF) and unlabeled methanol vs. unlabeled MCF/methanol, wherein each 2 dalton mass shift seen on CI for each unknown represents one amino group;
• comparing the EI spectra from matching unlabeled and labeled derivatization reagents to reveal composition of EI fragment identity to further aid chemical formulae identification;
• and
• comparing spectra with that from the sample derivatized with unlabeled silylation reagents to further narrow chemical formulae;
• and/or
• ii) if carboxyl group numbers need to be confirmed for an unknown compound, derivatizing the compounds containing the unknown with unlabeled methylchloroformate and 99% 13C methanol vs. unlabeled MCF/methanol, wherein each dalton mass shift seen on CI for each unknown represents one carboxyl group;
• comparing EI spectra from matching unlabeled and labeled derivatization reagents to reveal composition of EI fragment identity to further aid chemical formulae identification;
• and
• comparing spectra with that from compounds derivatized with unlabeled silylation and/or MCF reagents to further narrow chemical formulae;
• and/or
• iii) if hydroxyl group numbers need to be confirmed for an unknown compound, derivatizing the compounds containing the unknown with 99% 13C acetic anhydride vs. unlabeled acetic anhydride, wherein each dalton mass shift on CI for each unknown represents one hydroxyl group;
• iv) comparing EI spectra from matching unlabeled and labeled derivatization reagents to reveal composition of EI fragment identity to further aid chemical formulae identification;
• and
• v) comparing spectra with that from the sample derivatized with unlabeled silylation and/or MCF and methanol reagents to further narrow chemical formulae;
• and/or
• vi) determining the number of carbonyl, carboxyl, phosphoryl or amino/amine groups of a compound by assessing the molecular ion and/or MS/MS profiles by LC/MS using labeled reagents selected from the group consisting of 13C and 15N aniline, 13C and 15N PITC, and 13C and 15N o-BHA;
• thereby metabolomic/lipidomic profiling one or more compounds in a biological sample.