| Item type: | Article | ||||||||||||||||||||||||
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| Journal or Publication Title: | Journal of chromatography B | ||||||||||||||||||||||||
| Publisher: | Elsevier | ||||||||||||||||||||||||
| Volume: | 878 | ||||||||||||||||||||||||
| Number of Issue or Book Chapter: | 15-16 | ||||||||||||||||||||||||
| Page Range: | pp. 1103-1112 | ||||||||||||||||||||||||
| Date: | May 2010 | ||||||||||||||||||||||||
| Institutions: | Medicine > Lehrstuhl für Anästhesiologie Medicine > Institut für Funktionelle Genomik > Lehrstuhl für Funktionelle Genomik (Prof. Oefner) | ||||||||||||||||||||||||
| Identification Number: |
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| Classification: |
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| Dewey Decimal Classification: | 500 Science > 500 Natural sciences & mathematics 500 Science > 570 Life sciences 600 Technology > 610 Medical sciences Medicine | ||||||||||||||||||||||||
| Status: | Published | ||||||||||||||||||||||||
| Refereed: | Yes, this version has been refereed | ||||||||||||||||||||||||
| Created at the University of Regensburg: | Yes | ||||||||||||||||||||||||
| Item ID: | 30639 |
Abstract
GC-MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free L- and D-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-L-Val ...
Abstract
GC-MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free L- and D-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-L-Val column. However, the occurrence of racemization during derivatization precluded accurate quantification of AA enantiomers. Derivatization with methyl chloroformate/methanol and separation on an Rt-gammaDEXsa column did not exhibit racemization and yielded ten baseline separated racemates of proteinogenic AAs with resolution values greater than 2.4. However, protein and peptide hydrolysis occurred in serum and urine during the highly exothermal derivatization reaction under alkaline conditions. Removing serum proteins by precipitation before derivatization and performing the reaction at neutral pH enabled the determination of accurate free AA enantiomer concentrations. Accuracy of quantification was validated by an established nonchiral GC-MS method for AA analysis. Reliable quantification was achieved using stable-isotope labeled L-AAs as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) for the D-AAs were in the range of 3.2-446 nM and 0.031-1.95 microM, respectively. Relative standard deviations (N=6) for the measurement of AAs in urine and serum ranged from 0.49-11.10% to 0.70-3.87%, respectively. The method was applied to the analysis of urine from 19 patients with renal insufficiency. In comparison to healthy probands, D-ratios of Ala, Val, Pro, Thr, Asp, and Asn were significantly increased.
Metadata last modified: 25 May 2018 13:15
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