Analysis of Amino Acids

Pharmacopoeial Discussion Group (PDG) is the Pharmacopoeial Conference that performs harmonization activities on the test methods and monographs of the European Pharmacopoeia (EP), India Pharmacopoeia (IPC), Japanese Pharmacopoeia (JP) and the United States Pharmacopoeia (USP).
https://www.pmda.go.jp/rs-std-jp/standards-development/jp/0005.html

Analytical methods for amino acids in the pharmacopoeia of each country were agreed on October 26, 2016 in the three regions of Japan, the United States, and Europe, and have been implemented since December 1, 2017.
https://www.pmda.go.jp/files/000215408.pdf

An overview of analytical methods is provided below. For further details, please refer to the pharmacopoeia of each country.
Japan; https://jpdb.nihs.go.jp/kyokuhou/files/000788362.pdf
USA; https://www.usp.org/harmonization-standards/pdg/biotechnology/amino-acid-determination
Europe; http://www.uspbpep.com/ep60/2.2.56.%20amino%20acid%20analysis%2020256e.pdf

Amino Acid Analysis

Methods	Postcolumn detection techniques	Precolumn detection techniques
Principle	The free amino acids are separated by ion-exchange chromatography followed by derivatization and detection.	After derivatization of the free amino acids, they are separated by reverse-phase HPLC.
Sample requirement	5-10 μg of protein per assay	0.5-1.0 μg of protein per assay
Characteristics	It can be utilized for samples containing small amounts of buffer components such as salts and urea.	The sensitivity is high. It may be influenced by buffer salts in the samples. It may also result in multiple derivatives of a given amino acid, which complicates the result interpretation.

Each of the amino acid analysis methods

Method	Analysis method	Separation method	Wavelength for detection	Detection limit	Linearity range	Sample requirement
Method 1	Postcolumn Ninhydrin Detection General Principle	Ion-exchange chromatography	570 nm and 440 nm (proline)	10 pmol 50 pmol for proline	20 to 500 pmol	1 μg or more
Method 2	Postcolumn OPA Fluorometric Detection General Principle	Ion-exchange chromatography	Excitation; 348 nm Emission; 450 nm	A few 10 pmol The oxidation with sodium hypochlorite allows secondary amines (imino acids, such as proline) to react with OPA.	A few pmol to a few tens nmol	500 ng or more
Method 3	Precolumn PITC Derivatization General Principle	Reverse-phase HPLC	245 nm	1 pmol	20 to 500 pmol	500 ng or more
Method 4	Precolumn AQC Derivatization General Principle	Reverse-phase HPLC	Excitation; 250 nm Emission; 395 nm	About 40 to 320 fmol About 800 fmol for cysteine	2.5 to 200 μmol/L	30 ng
Method 5	Precolumn OPA Derivatization General Principle	Reverse-phase HPLC	Excitation; 348 nm Emission; 450 nm	50 fmol (practical limit; 1 pmol) This technique does not detect amino acids that exist as secondary amines (e.g., proline).	-	-
Method 6	Precolumn DABS-CI Derivatization General Principle	Reverse-phase HPLC	436 nm	About 1 pmol	2 to 5 pmol	10 to 30 ng
Method 7	Precolumn FMOC-Cl Derivatization General Principle	Reverse-phase HPLC	Excitation; 260 nm Emission; 313 nm	The low fmol range Only the histidine derivative shows any breakdown.	0.1 to 50 μmol/L	-
Method 8	Precolumn NBD-F Derivatization General Principle	Reverse-phase HPLC	Excitation; 480 nm Emission; 530 nm	About 10 fmol	-	About 1.5 mg*

* JP states 1.5μg.
OPA; o-Phthalaldehyde
PITC; Phenylisothiocyanate
AQC; 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
DABS-Cl; (dimethylamino)azobenzenesulfonyl chloride
FMOC-Cl; 9-fluorenylmethyl chloroformate
NBD-F; 7-fluoro-4-nitrobenzene-2-oxa-1,3-diazole