Purpose

Free amino acids in cell culture media, blood plasma, and other matrices can be separated, identified, and quantified via Hitachi amino acid analyzers. This method is for accurate determination and quantification of amino acids

Summary

Samples are prepared for analysis by diluting a portion of the sample with a deproteinizing solution (5-sulfosalicylic acid hydrate), internal standard (L-2-Amino-3-Guanidinopropionic Acid Hydrochloride), and if necessary a lithium buffer to achieve an appropriate dilution. The mixture is then vortexed, micro-centrifuged, and filtered into an HPLC vial.

Amino acid analysis is performed on Hitachi HPLC amino acid analyzers. Amino acids are separated by cation exchange chromatography using various lithium eluents. Absorbance is measured at 440 and 570 nm following post-column color development by Ninhydrin reagent. Appropriate standards are run to ensure each sequence is running properly and are used in the quantification of samples using peak areas and applying an external standard technique.

Limits of detection are 0.3 to 1.0 micromoles per liter. There is no high-end limit as the sample can always be diluted down to an appropriate detection range. 

Amino Acids Quantified

L-Alanine

L-Arginine

L-Asparagine**

L-Aspartic Acid

L-Cysteine*

L-Cystine

L-Ethanolamine

Gamma-Aminobutyric Acid

L-Glutamic Acid

L-Glutamine

L-Glycine

L-Histidine

Hydroxy-L-Proline

L-Isoleucine

L-Leucine

L-Lysine

L-Methionine

L-Ornithine

L-Phenylalanine

L-Proline

L-Citrulline

L-Serine

*L-Cysteine is not included in the standard panel for Free Amino Acid Analysis and readily decomposes into L-Cystine. Thus, Cysteine analysis is often to check for presence rather than quantity since its rate of decomposition is unpredictable. It can be quantified but needs freshly made specialty standards.

**L-Asparagine eludes as part of a triad with L-Glutamic Acid and L-Glutamine. If the Glutamic Acid concentration is less than 5 micromoles per liter, the Asparagine is baseline resolved. To calculate the most accurate results for the baseline resolved Asparagine, a specialty standard containing Asparagine is run with a sample to obtain more accurate quantification. Without the specialty standard with low Glutamic Acid, results may be up to 20% off for reported Asparagine.

***L-Alanyl-Glutamine is not included in the standard panel for Free Amino Acid Analysis but can be quantified by request using freshly made specialty standards.