GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine
In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5<i>H</i>-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measu...
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MDPI AG
2021-05-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/11/3206 |
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| author | Olga Begou Kathrin Weber Bibiana Beckmann Dimitrios Tsikas |
| author_facet | Olga Begou Kathrin Weber Bibiana Beckmann Dimitrios Tsikas |
| author_sort | Olga Begou |
| collection | DOAJ |
| description | In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5<i>H</i>-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d<sub>0</sub>-creatinine), [<i>methylo</i>-<sup>2</sup>H<sub>3</sub>]creatinine (d<sub>3</sub>-creatinine, internal standard) and creatine (d<sub>0</sub>-creatine) with <i>N</i>,<i>O</i>-<i>bis</i>(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting <i>N</i>,<i>N</i>,<i>O</i>-<i>tris</i>(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of <i>m</i>/<i>z</i> 271 (d<sub>0</sub>-creatinine/d<sub>0</sub>-creatine) and <i>m</i>/<i>z</i> 274 (d<sub>3</sub>-creatinine) in the NICI mode. In the PICI mode, SIM of <i>m</i>/<i>z</i> 256, <i>m</i>/<i>z</i> 259, <i>m</i>/<i>z</i> 272 and <i>m</i>/<i>z</i> 275 was performed. BSTFA derivatization of d<sub>0</sub>-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone. |
| first_indexed | 2024-03-10T10:59:46Z |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-10T10:59:46Z |
| publishDate | 2021-05-01 |
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| series | Molecules |
| spelling | doaj.art-c1de54e5f86749759107261b3923e30b2023-11-21T21:37:54ZengMDPI AGMolecules1420-30492021-05-012611320610.3390/molecules26113206GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human UrineOlga Begou0Kathrin Weber1Bibiana Beckmann2Dimitrios Tsikas3Institute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, GermanyIn consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5<i>H</i>-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d<sub>0</sub>-creatinine), [<i>methylo</i>-<sup>2</sup>H<sub>3</sub>]creatinine (d<sub>3</sub>-creatinine, internal standard) and creatine (d<sub>0</sub>-creatine) with <i>N</i>,<i>O</i>-<i>bis</i>(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting <i>N</i>,<i>N</i>,<i>O</i>-<i>tris</i>(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of <i>m</i>/<i>z</i> 271 (d<sub>0</sub>-creatinine/d<sub>0</sub>-creatine) and <i>m</i>/<i>z</i> 274 (d<sub>3</sub>-creatinine) in the NICI mode. In the PICI mode, SIM of <i>m</i>/<i>z</i> 256, <i>m</i>/<i>z</i> 259, <i>m</i>/<i>z</i> 272 and <i>m</i>/<i>z</i> 275 was performed. BSTFA derivatization of d<sub>0</sub>-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.https://www.mdpi.com/1420-3049/26/11/3206BSTFAcreatinecreatininederivatizationquantificationsilylation |
| spellingShingle | Olga Begou Kathrin Weber Bibiana Beckmann Dimitrios Tsikas GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine Molecules BSTFA creatine creatinine derivatization quantification silylation |
| title | GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine |
| title_full | GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine |
| title_fullStr | GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine |
| title_full_unstemmed | GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine |
| title_short | GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine |
| title_sort | gc ms studies on derivatization of creatinine and creatine by bstfa and their measurement in human urine |
| topic | BSTFA creatine creatinine derivatization quantification silylation |
| url | https://www.mdpi.com/1420-3049/26/11/3206 |
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