by Chi Man Ng and Wilhad M. Reuter AL
The Benefits of HPLC Coupled With Mass Spectrometry for the Direct Quantitation of Underivatized Amino Acids
Amino acids are critical biological components having multiple metabolic functions, particu- larly when linked together to form proteins, and have any number of important roles. Thus, amino acid analysis plays a key role in the accurate quantitation of proteins and peptides, used for determining both the total protein in biomedical samples and the quantity of puri- fied proteins and peptides for biochemistry research. While traditional methods have proved useful in analyzing amino acids, time- consuming and costly derivatization steps are usually needed. The addition of these steps can also impact the quality of the analysis. The following discussion covers existing methods, and introduces a new process that eliminates the derivatization step while providing compa- rable results.
Traditional methods for
quantifying amino acids Analysis of amino acids and their hydrolysates is typically carried out using HPLC with pre- or post-column derivatization for improved ana- lyte retention or sensitivity, respectively. If only primary amino acids are required, o-phthal- aldehyde (OPA) pre-column derivatization is effective. However, if the analysis requires separation of both primary and secondary amino acids, other approaches must be used. These include pre-column derivatization using reagents such as AccQ-Tag (Waters, Milford, Mass.) or fluorenylmethyloxycarbonyl (FMOC),
Table 1 – HPLC method parameters Column
Intrada amino acid, 5 µm, 100 × 3 mm column (Imtakt, Portland, Ore.)
Solvent A: acetonitrile/tetrahydrofuran (THF)/25-mM ammonium formate/formic acid; 9/75/16/0.3 (v/v/v/v) Solvent B: acetonitrile/100-mM ammonium formate; 20/80 (v/v) Solvent program
Mobile phase
1 2 3 4 5 6
Analysis time Flow rate Pressure
Oven temp Detection
Injection volume 25 min
0.400 mL/min 2100 psi 35 °C
Altus SQ MS 4 µL
Sampling (data) rate 2 pts/sec
Time (min) Flow rate (mL/min) %A %B Curve Initial 5.00
12.00 19.00 25.00 25.10
0.400 0.400 0.400 0.400 0.400 0.400
100.0 0.0 Initial
100.0 0.0 6 83.0 17.0 6 0.0 100.0 6 0.0 100.0 11 100.0 0.0 6
and post-column derivatization using Thioflour (Pickering Laboratories, Mountain View, Calif.).
AMERICAN LABORATORY 22 APRIL 2016
Although these methods are well-understood and have been well-received, the ability to
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