Simultaneous Quantification of Amino Metabolites in Multiple Metabolic Pathways Using Ultra-High Performance Liquid Chromatography with Tandem-mass Spectrometry

Abstract

Metabolites containing amino groups cover multiple pathways and play important roles in redox homeostasis and biosyntheses of proteins, nucleotides and neurotransmitters. Here, we report a new method for simultaneous quantification of 124 such metabolites. This is achieved by derivatization-assisted sensitivity enhancement with 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate (5-AIQC) followed with comprehensive analysis using ultra-high performance liquid chromatography and electrospray ionization tandem mass spectrometry (UHPLC-MS/MS). In an one-pot manner, this quantification method enables simultaneous coverage of 20 important metabolic pathways including protein biosynthesis/degradation, biosyntheses of catecholamines, arginine and glutathione, metabolisms of homocysteine, taurine-hypotaurine etc. Compared with the reported ones, this method is capable of simultaneously quantifying thiols, disulfides and other oxidation-prone analytes in a single run and suitable for quantifying aromatic amino metabolites. This method is also much more sensitive for all tested metabolites with LODs well below 50 fmol (at sub-fmol for most tested analytes) and shows good precision for retention time and quantitation with inter-day and intra-day relative standard deviations (RSDs) below 15% and good recovery from renal cancer tissue, rat urine and plasma. The method was further applied to quantify the amino metabolites in silkworm hemolymph from multiple developmental stages showing its applicability in metabolomics and perhaps some clinical chemistry studies.

Figure 1

Synthesis of 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate (5-AIQC).

Figure 2

Schemes for 5-AIQC derivatization of amino analytes with thiol and disulfide groups in one pot.

Figure 3

UHPLC-MS/MS chromatograms for some sets of the 5-AIQC-tagged amino analytes having the same pseudomolecular ions (m/z at unit resolution). (A) ion m/z 260 (A1: sarcosine; A2: β-alanine; A3: L-alanine; A4: 2-amino-2-methyl-1-propanol); ion m/z 274 (A21: γ-aminobutyric acid; A22: DL-3-aminoisobutyric acid; A23: 2-aminoisobutyric acid; A24: L-2-aminobutyric acid); ion m/z 302 (A31: trans-4-hydroxy-L-proline; A32: 6-aminocaproic acid; A33: L-isoleucine; A34: L-leucine; A35: L-norleucine); ion m/z 324 (A41: (±)-octopamine; A42: dopamine; A43: 3-hydroxyanthranilic acid; A44: 3-aminosalicylic acid); ion m/z 340 (A51: L-cysteic acid; A52: 3-methyl-L-histidine; A53: 1-methyl-L-histidine; A54: (−)-norepinephrine; A55: 5-hydroxydopamine). (B) ion m/z 288 (B1: 5-aminovaleric acid; B2: L-valine; B3: L-norvaline); ion m/z 308 (B21: tyramine; B22: 3-aminobenzoic acid; B23: 4-aminobenzoic acid); ion m/z 318 (B31: L-glutamic acid; B32: o-acetyl-L-serine; B33: 4-hydroxy-L-isoleucine); ion m/z 350 (B41: D-mannosamine; B42: D-(+)-glucosamine; B43: D-(+)-galactosamine); ion m/z 373 (B51: asymmetric dimethylarginine; B52: cysteamine; B53: Ala-Leu). (C) ion m/z 280 (C1: hypotaurine; C2: 4-aminophenol); ion m/z 282 (C21: histamine; C22: cystathionine); ion m/z 290 (C31: D-homoserine; C32: L-threonine); ion m/z 334 (C41: 1-deoxynojirimycin; C42: DL-ethionine); ion m/z 336 (C51: DL-methionine sulfoxide; C52: DL-phenylalanine); ion m/z 352 (C61: DL-methionine sulfone; C62: L-tyrosine); ion m/z 359 (C71: L-homoarginine; C72: Nα-acetyl-L-lysine); ion m/z 208 (C81: 1,3-diaminopropane; C82: 1,2-diaminopropane).

Figure 4

UHPLC-MS/MS chromatograms for the 5-AIQC-tagged oxidation-prone amino analytes including (a) these containing thiol and disulfide groups and (b) the aromatic metabolites from three aromatic amino acids (phenylalanine, tyrosine and tryptophan).

Figure 5

UHPLC-MS/MS chromatograms for the 5-AIQC-tagged amino metabolites in multiple metabolic pathways including (a) protein biosynthesis/degradation, (b) urea cycle, (c) folate-associated homocysteine metabolism, (d) biosynthesis of monoamine neurotransmitters and (e) tryptophan-mediated kynurenine pathway.