Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection
Plasma amino acids are usually analyzed by ion-exchange chromatography (IEC), a reproducible but time consuming method. Here, we test whether plasma amino acids can be analyzed using reverse-phase high performance liquid chromatography (HPLC). Filtered plasma, with S-carboxymethyl- l-cysteine as the...
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| Veröffentlicht in: | Clinica chimica acta 2005-04, Vol.354 (1), p.83-90 |
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| creator | Schwarz, Elisabeth L. Roberts, William L. Pasquali, Marzia |
| description | Plasma amino acids are usually analyzed by ion-exchange chromatography (IEC), a reproducible but time consuming method. Here, we test whether plasma amino acids can be analyzed using reverse-phase high performance liquid chromatography (HPLC).
Filtered plasma, with
S-carboxymethyl-
l-cysteine as the internal standard, was derivatized and analyzed by an Agilent 1100 HPLC system. Primary amino acids were derivatized with
o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C
18 column AAA (4.6×150 mm).
The HPLC method evaluated correlated well with IEC (0.89≤
r≤1.00) with linearity up to 2500 μmol/l. The between- and within-run CVs were |
| doi_str_mv | 10.1016/j.cccn.2004.11.016 |
| format | Article |
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Filtered plasma, with
S-carboxymethyl-
l-cysteine as the internal standard, was derivatized and analyzed by an Agilent 1100 HPLC system. Primary amino acids were derivatized with
o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C
18 column AAA (4.6×150 mm).
The HPLC method evaluated correlated well with IEC (0.89≤
r≤1.00) with linearity up to 2500 μmol/l. The between- and within-run CVs were <6.0%. In addition, this method is able to separate argininosuccinic acid, homocystine and allo-isoleucine, rare but clinically significant amino acids.
This HPLC method was comparable to IEC and could represent an alternative for amino acid analysis.</description><identifier>ISSN: 0009-8981</identifier><identifier>EISSN: 1873-3492</identifier><identifier>DOI: 10.1016/j.cccn.2004.11.016</identifier><identifier>PMID: 15748603</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Amino acid analysis ; Amino Acids - analysis ; Amino Acids - blood ; Chromatography, High Pressure Liquid - methods ; Chromatography, Ion Exchange - methods ; Fluoresceins - chemistry ; HPLC ; Hydrogen-Ion Concentration ; Sensitivity and Specificity ; Spectrometry, Fluorescence - methods ; Spectrophotometry, Ultraviolet - methods ; Time Factors</subject><ispartof>Clinica chimica acta, 2005-04, Vol.354 (1), p.83-90</ispartof><rights>2004 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c354t-ce7c09dce1060b3b1a3b817e3bb29357ce8a1361e3e364361e509bef12206f033</citedby><cites>FETCH-LOGICAL-c354t-ce7c09dce1060b3b1a3b817e3bb29357ce8a1361e3e364361e509bef12206f033</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.cccn.2004.11.016$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27922,27923,45993</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15748603$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Schwarz, Elisabeth L.</creatorcontrib><creatorcontrib>Roberts, William L.</creatorcontrib><creatorcontrib>Pasquali, Marzia</creatorcontrib><title>Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection</title><title>Clinica chimica acta</title><addtitle>Clin Chim Acta</addtitle><description>Plasma amino acids are usually analyzed by ion-exchange chromatography (IEC), a reproducible but time consuming method. Here, we test whether plasma amino acids can be analyzed using reverse-phase high performance liquid chromatography (HPLC).
Filtered plasma, with
S-carboxymethyl-
l-cysteine as the internal standard, was derivatized and analyzed by an Agilent 1100 HPLC system. Primary amino acids were derivatized with
o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C
18 column AAA (4.6×150 mm).
The HPLC method evaluated correlated well with IEC (0.89≤
r≤1.00) with linearity up to 2500 μmol/l. The between- and within-run CVs were <6.0%. In addition, this method is able to separate argininosuccinic acid, homocystine and allo-isoleucine, rare but clinically significant amino acids.
This HPLC method was comparable to IEC and could represent an alternative for amino acid analysis.</description><subject>Amino acid analysis</subject><subject>Amino Acids - analysis</subject><subject>Amino Acids - blood</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Chromatography, Ion Exchange - methods</subject><subject>Fluoresceins - chemistry</subject><subject>HPLC</subject><subject>Hydrogen-Ion Concentration</subject><subject>Sensitivity and Specificity</subject><subject>Spectrometry, Fluorescence - methods</subject><subject>Spectrophotometry, Ultraviolet - methods</subject><subject>Time Factors</subject><issn>0009-8981</issn><issn>1873-3492</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kEtLxDAUhYMozjj6B1xIVu5ac5s-wY0M6ggDulBchjS5ZTK0TU06yvx7U2bAnav74NzDPR8h18BiYJDfbWOlVB8njKUxQBxWJ2QOZcEjnlbJKZkzxqqorEqYkQvvt2FMWQ7nZAZZkZY543Py-dDLdu-Np7ahQyt9J6nsTG-pVEZ7Wu_p6m29pD9m3NBhY0erjdVIpXNyT2WvadPurEOvsFdINY6oRmP7S3LWyNbj1bEuyMfT4_tyFa1fn1-WD-tI8SwdI4WFYpVWCCxnNa9B8rqEAnldJxXPCoWlBJ4DcuR5OjUZq2psIElY3jDOF-T24Ds4-7VDP4rOhF_aVvZod17kU1Aeoi5IchAqZ7132IjBmU66vQAmJpxiKyacYsIpAERYhaObo_uu7lD_nRz5BcH9QYAh47dBJ7wyEwltXAAhtDX_-f8C5nOF7Q</recordid><startdate>20050401</startdate><enddate>20050401</enddate><creator>Schwarz, Elisabeth L.</creator><creator>Roberts, William L.</creator><creator>Pasquali, Marzia</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20050401</creationdate><title>Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection</title><author>Schwarz, Elisabeth L. ; Roberts, William L. ; Pasquali, Marzia</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c354t-ce7c09dce1060b3b1a3b817e3bb29357ce8a1361e3e364361e509bef12206f033</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Amino acid analysis</topic><topic>Amino Acids - analysis</topic><topic>Amino Acids - blood</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Chromatography, Ion Exchange - methods</topic><topic>Fluoresceins - chemistry</topic><topic>HPLC</topic><topic>Hydrogen-Ion Concentration</topic><topic>Sensitivity and Specificity</topic><topic>Spectrometry, Fluorescence - methods</topic><topic>Spectrophotometry, Ultraviolet - methods</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Schwarz, Elisabeth L.</creatorcontrib><creatorcontrib>Roberts, William L.</creatorcontrib><creatorcontrib>Pasquali, Marzia</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Clinica chimica acta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Schwarz, Elisabeth L.</au><au>Roberts, William L.</au><au>Pasquali, Marzia</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection</atitle><jtitle>Clinica chimica acta</jtitle><addtitle>Clin Chim Acta</addtitle><date>2005-04-01</date><risdate>2005</risdate><volume>354</volume><issue>1</issue><spage>83</spage><epage>90</epage><pages>83-90</pages><issn>0009-8981</issn><eissn>1873-3492</eissn><abstract>Plasma amino acids are usually analyzed by ion-exchange chromatography (IEC), a reproducible but time consuming method. Here, we test whether plasma amino acids can be analyzed using reverse-phase high performance liquid chromatography (HPLC).
Filtered plasma, with
S-carboxymethyl-
l-cysteine as the internal standard, was derivatized and analyzed by an Agilent 1100 HPLC system. Primary amino acids were derivatized with
o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C
18 column AAA (4.6×150 mm).
The HPLC method evaluated correlated well with IEC (0.89≤
r≤1.00) with linearity up to 2500 μmol/l. The between- and within-run CVs were <6.0%. In addition, this method is able to separate argininosuccinic acid, homocystine and allo-isoleucine, rare but clinically significant amino acids.
This HPLC method was comparable to IEC and could represent an alternative for amino acid analysis.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>15748603</pmid><doi>10.1016/j.cccn.2004.11.016</doi><tpages>8</tpages></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Amino acid analysis Amino Acids - analysis Amino Acids - blood Chromatography, High Pressure Liquid - methods Chromatography, Ion Exchange - methods Fluoresceins - chemistry HPLC Hydrogen-Ion Concentration Sensitivity and Specificity Spectrometry, Fluorescence - methods Spectrophotometry, Ultraviolet - methods Time Factors |
| title | Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection |
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