THE USE

OF
HIGH-PERFORMANCE LIPID
CHROMATOGRAPHY
FOR THE
SEPARATION AND DETECTION
OF
AMINO ACID
IN
PLASMA
High-performance liquid chromatography is an advanced technology applied in biomedical
pharmaceutical and clinical research used to separate, identify samples (compounds) in a
mixture. The principle of this technique is based on the movement of analytes from the mobile
phase to the stationary phase which makes use of a high-pressure pump that elutes liquid
solvents that interact with absorbent allowing flow rate through the column and are detected.
The schematic of an HPLC instrument typically includes a solvent reservoir, a degasser, pumps
(these can be high/low-pressure pumps), an autosampler, a column containing the
adsorbent/stationary phase, and a detector. The analysis of amino acid is important being the
bodybuilding block therefore increase or decrease the level of amino acid is as a result of the
inability of the body to metabolize amino acid or certain medical condition, inherited medical
disorders are diagnosed using physiological fluids (blood, CSF, saliva, urine, etc.). Henderson
JW et al 2000; Hamnillon P.B et al 1959)

There are different types of HPLC they include Normal–phase chromatography, Partition
chromatography, Size-exclusion chromatography, Ion-exchange chromatography, Displacement
chromatography, Reversed-phase chromatography but this review is focused on the separation
of amino acid in plasma using Ion Exchange Chromatography (IEC) and Reverse Phase high-
performance liquid chromatography, the former is based on the use of cation exchange resin
with post-column derivatization with ninhydrin or OPA while the latter is based on reverse phase
pre-column derivatization with OPA or PITC, FMOC, however, a chromatographic system must
be specific, sensitive, linear, reproducible and specific. ( Krok KA et al 1991; Henderson JW et
al 2000; Hamnillon P.B et al 1959; Benson J.R 1975).

A blood sample is usually required is usually, samples are collected between 8-9 am and patient
are free of any form of medication e.g. Antibiotics, contraceptives, and other metabolites as they
may affect the resolution of amino acid in protein, Heparin or EDTA or silicon (SST) bottle is
preferable as it prevents platelet activation which can increase taurine and
phosphoethanolamine, hemolysis must be prevented as can lead to a false increase in aspartic
acid, glutamic acid, PEA and taurine, sample are stored at 4 C to reduce the elevated level of
glutamic acid, serine, cysteine and arginine, the blood sample is spun and separated at 2500g
for 15-20 min, if the sample is not used immediately they are stored at -15c to prevent
hydrolysis of protein. (Schael A et al 1987 Perry T. L et al 1969; Bremer H. J et al 1981). After
taking note of all the sample preparation they undergo deproteinization. This is a process of
removing the protein, it is very important to deproteinize as a precipitate on either of the HPLC
systems can lead to blockage and damage to the instrument and this is done by a various
method which includes;

 Ultrafiltration,
 Ultracentrifugation
 Equilibrium Dialysis
 Precipitation

Precipitation Ultrafiltration ultracentrifugation Equilibrium Dialysis

An amino acid is Complete removal of Complete removal of Loss of amino acid
recovered and protein protein
consistent
DEPROTEINZATION

Deproteinization is done using 30% 5-sulfosalicylic acid (SSA)with HCI, acetonitrile (ACN),
trichloroacetic acid (TCA)

5-sulphosalicylic acid ↑ aspartic and glutamic ↓ asparagine and tryptophan, cysteine,

(SSA) and 1 M HCI acid glutamine and proline (not
determined)
acetonitrile (ACN), ↓ aspartic acid glycine, Tryptophan (total
serine, and glutamic recovery)
trichloroacetic acid (TCA ↓amino acid ↓ aspartic acid and
glutamic acid

 SSA is either added to plasma or serum as a 3% solution, (4 parts of 3% SSA to l part of plasma or
as a solid substance, 30-40 mg/ml of fluid).
 This mixture is vortexed immediately.
 After centrifugation, an aliquot of the somewhat turbid supernatant can be applied directly to
the column.
 Immediate vortex mixing followed by standing for 15 min at 4°C centrifugation
 Supernatant which after adjustment of pH, can be used directly for amino-acid analysis. (Bremer
H.J et al 1981; Fekkes D et al 1995).

DERIVATIZATION
The determinations of amino acids in physiological fluids mostly involve the use of ion-exchange
chromatography (IEC) or reversed-phase high-performance liquid chromatography (HPLC),
which are post-column derivatization and post-column derivatization respectively.

RP HPLC IEC CHROMATOGRAPHY

shorter analysis time Cation-exchange resins
lower cost of instrumentation and post-column derivatization with ninhydrin or
maintenance, higher sensitivity and flexibility -α phthaldialdehyde (OPA)
Pre column derivatized with OPA, Commercial amino-acid analyzer.
phenylisothiocyanate (PITC),
5-dimethylamino-lnaphthalenesulphonyl
chloride (dansyl) or
9- fluorenylmethyl chloroformate (FMOC).
 Reagent Used for Derivatization

Reagents ADVANTAGES DISADVANTAGES

FDNB (l-Fluoro-2,4-
dinitrobenzene), FDNPAA (I-
Fluoro-2,4-dinitrophenyl-5-L-
alanine)
amide), FDNDEA (N, N-
Diethyl-2,4-dinitro-5-
fluoroaniline)
FMOC (9-Fluorenylmethyl
chloroformate)
PITC (Phenylisothiocyanate)
OPA (α-Phthaldialdehyde)
Dansyl
It takes 5 30,50 minutes
respectively
Determination of secondary
amino acid.
It takes 5minutes
Widely used for derivatization
derivatives are very stable,
No side product formed
It takes 20min
Primary amino acid
Usually <1 minute.
Excellent linearity
It takes 35 minutes
Formation of side-product
after derivatization
The excess reagent used to
stop derivatization and
hydrolysis
Sample preparation is lengthy
and requires vacuum
evaporation during the
derivatization
Inability to detect cysteine and
secondary amino acids and the
instability of formed
derivatives
Interfering side-products

For the ion-exchange chromatography technique, amino acids eluted were mixed at a high-
temperature reaction coil with ninhydrin to form colored compounds after passing through
different PH, Temperature, and ionic strength, the colored compound formed are then measured
photometrically at 570 nm (amino acids) and 440 nm (amino acids). The run time is usually 1 hr. 30 min.
While the reverse phase HPLC, amino acids are separated in plasma in a very short time using a
photodiode array detector, however two injections per sample are required, and the wide
analytic measurement of amino acid.