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Quantification of Underivatised Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC-ESI-MS/MS.
MedLine Citation:
PMID:  22125148     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting.
Authors:
Giuseppe Giordano; Iole Maria Di Gangi; Antonina Gucciardi; Mauro Naturale
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  828     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2012  
Date Detail:
Created Date:  2011-11-29     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  219-42     Citation Subset:  IM    
Affiliation:
Department of Pediatrics, University of Padova, Padova, Italy, giordano@pediatria.unipd.it.
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