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 Directed Isotopic Positional Derivatization (DIPD) “barcode pattern” signals from background noise, in a biological sample comprising

• A) i) derivatizing the one or more compounds with one or more of 13C methylchloroformate, 13C ethylchloroformate, 13C methanol, 13C ethanol, 13C acetyl anhydride, 13C labeled N,O-bistrifluoroacetamide (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 one or more 13C labeled or 15N DIPD standards for quantitating the one or more compounds by derivatizing the one or more compounds with DIPD reagents including trimethylsilyl-13C-diazomethane (TMS-13CHN2), and introducing the 13C labeled or 15N DIPD standards into a compound sample already derivatized with DIPD 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 DIPD 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 DIPD reagents, such as 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.