Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 964, Issue -, Pages 116-127Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2014.03.025
Keywords
Amino acids; HPLC; Hydrolysates; OPA (o-phthaldialdehyde); Proteins
Funding
- National Basic Research Program of China [2013CB127302]
- National Natural Science Foundation of China [31172217, 31272450]
- China Postdoctoral Science Foundation [2012T50163]
- Chinese Universities Scientific Fund [2013RC002]
- Program for New Century Excellent Talents in University [NCET-12-0522]
- National Research Initiative Competitive Grants from the Animal Reproduction Program [2008-35203-19120, 2011-67015-20028]
- Animal Growth & Nutrient Utilization Program of the USDA National Institute of Food and Agriculture [2008-35206-18764, 2014-67015-21770]
- Texas A&M AgriLife Research [H-8200]
- Program for Beijing Municipal Excellent Talents
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Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCI (110 degrees C for 24h), 4.2M NaOH (105 degrees C for 20h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60 degrees C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25 degrees C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25 degrees C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal). (C) 2014 Elsevier B.V. All rights reserved.
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