The use of high
performance liquid chromatography for separation and detection of amino acids
‘The use of high performance liquid chromatography
for separation and detection of amino acids in plasma’
sensitive and fast high performance liquid chromatography (HPLC) technique is a
capable substitute to the ion exchange chromatography for the separation and
detection of amino acids in plasma. Diagnosing of individuals from various
diseases through the analysis free amino acid in the plasma is an important
process. The amino acid analysis that produce valid, reliable and reasonable
results is an unavoidable necessity in clinical applications (Yoshida et al.,
2018). The whole process is based on automated precolumn derivatization of
amino acids with o-phthalaldehyde, and separation of the derivatives by
reversed-phase chromatography, and the results are quantification by
fluorescence method. The low complexity in the sample preparing method and
short sample analysing time makes this one the most suitable for routine
analysis of large number of samples (Terrlink, Leeuwen and Houdijk, 2018). The
results obtained from this method is comparatively better than any other
conventional method used for the detection of amino acids. HPLC also enables us
to detect trace amounts of amino acids which are not detected through other
conventional sources (Fekkes et al., 2018).
acids are the largest group of mutually similar nutrients present in the human
body. It is defined as the organic substance containing both amino and acid
groups. All the amino acids have different biochemical properties and function
because of their variation their side chains. They are generally stable in
physiological pH except glutamine which undergoes rapid oxidation when exposed
to water. 300 or more amino acids are known to man and from that only 20 are
found to be the building blocks of protein even though certain non-protein
amino acids plays important role in the cell metabolism (Wu, 2018). The
sequence in which amino acids combines to form a particular protein determines
the three dimensional structure and function, which differs for each proteins.
The role of proteins as antibodies, enzymes, messengers and as source of
storage and transport are the ones which are commonly studied. But recent
studies also suggest that that they take part in gene transcription and translation
and various other complex processes (Jonas Wilson, 2018).
Plasma is measured
for the detection of various diseases because of its relatively simple assay
and less physical burden on patients as well as their robustness for other
clinical purposes. Amino acids are the most suitable components for determining
all clinical diagnosing factors because they are ingested or synthesized
endogenously and plays an essential physiological role as a basic metabolites
and a metabolic regulator. Amino acids are abundantly available in the
circulation and are favourable targets because they gets influenced by metabolic
variations in various organs which induces specific diseases (Shingyoji et al.,
2018). The amino acid analysis will also provide knowledge about premarital and
prenatal stages for diseases resulting due to inborn errors. The amino acid
sequence is also identified for prolonged diagnosis of various diseases (Liu,
in any single amino acid can give rise to various disorders. Branches chain
amino aciduria, Branched chain organic acidurias, Glutaric acidurias,
Phenylketonuria, Homocystinuria and various urea cycle disorders (eg;
Hyperammonemia, Neonatal encephalopathy,Alzheimers) are the most commonly
studies amino acid disorders. There can lead to serious life threatening health
issues to the affected individuals (Agamanolis, 2018).
liquid chromatography (HPLC) is one of the most accurate, sensitive and
reliable technique for the analysis of amino acids in plasma due to its easily achievable
and analysing methods which no other separation technique can match. The
ability of HPLC to analyse polar and unstable compounds are one of its
outstanding feature. HPLC also emphasis on the separation of unconjugated and
conjugated bile acids without hydrolysis (Nambara and Goto, 2018). Minimal sample
volume and automated precolumn derivitization of amino acids and fluroscent
detection technique are the highlights of this technique. Quantitation of amino
acids becomes usesful due to the minimilastic sample volume in HPLC (Frank and
Ion exchange chromatography, which is said to
be the most perfect method for the determination of amino acids, is also widely
used in routine analysis of plasma (Csap, Tth-Psfai and Csap-Kiss, 1991). Gas
chromatography, electrophoresis (paper), thin layer chromatography are the various
other highly accurate methods for the detection of amino acids in plasma. The
purpose of this review was to evaluate the use High Performance Liquid
chromatography (HPLC) in the separation and detection of amino acids in plasma.
of physiological samples:
It is one
of the most important pre-procedure which needs to be followed in order to get
accurate results. The patient from which the blood is collected is made sure
that he is medication free, it’s because the antibodies present will develop
additional peaks in the chromatogram which may end up interfering with the
amino acid resolution. EDTA containing tubes are used for collecting samples
due to its anti-coagulating property (Fekkes, 1996). Samples are then stored at
-700 C until assayed.
technique first the plasma sample is heated, which denatures the proteins in
them. The deproteinised plasma is then centrifuged at 2000rpm for 10 minutes (at
0-4OC). Deproteinization process is done through various other
techniques which include acid precipitation, ultra-filtration, ultracentrifugation
and addition of organic solvents (Fekkes, 1996). The then produced supernatant is
collected for HPLC analysis. Florescent
intensity is not lost during this technique. Certain studies suggest that
deprotenization and addition of other acids leads to reduced yield of many
phenylthiocarbamyl amino acids (M C
Aristoy et al, 1792)
method quantifies primary amino acids in plasma by automated precolumn
derivatization with ortho-phthalaldehyde. The HPLC required for the analysis
depends upon the detection limit we require. The system consists of dual piston
pump, photodiode array detector (DAD), florescence detector (FD) and auto sampler
for chromatographic analysis (Furst P et al,1990). Mobile phase was made of
buffer solutions( 0.4% tetrahydrofuran and 4mol/L KOH mixed HPLC grade water).
The buffers were filtered through degassed helium (pure). The chromatographic separation
was done at 220C. The flowrate in the mobile phase should be
adjusted in accordance with the process. The fluorometrical values ranges from
340-460nm. The whole runtime of a single sample and standard is up to a maximum
of 30-35 minutes. After every single run the column is equilibrated completely
in the mobile phase.
obtained chromatogram was compared to a serum standard. Peaks which exceeded
the heights of templates and which has retention times corresponding to the
amino acids was documented as the peaks/elevations of a particular amino acids.
Through this process most of the amino acids can be detected by comparing the
peaks with the height of the standards (Fekkes, 1996).
recovery is calculated to be the difference between the peaked and non-peaked
samples. The recovery percentage stands between 90 -110%.
Fig: High-Performance Liquid
Chromatography HPLC System (Waters.com. (2018))
technique has found its place in all the clinical laboratories due to its
ability to analysis a wide range of compounds. The accurate estimation of amino
acids from plasma requires specificity, sensitivity and high efficiency. This
method has proven all of the above criteria’s but the precipitation of the
sample remains a drawback. Furthermore, this technique should be able to
resolve and quantify all the amino acids. For the quantification of certain
amino acids like cysteine and homocysteine, their reduced form can only be
estimated, so this also remains a drawback. HPLC also lacks specificity in
quantifying certain –SH group containing amino acids due to their low fluorescence
property (Babu et al., 2002).
method showcased good intra-laboratory variability for a wide range of amino
acids and the preliminary data showed variability of HPLC similar to that of
Ion exchange chromatography. The use of ortho-phthalaldehyde enabled to
determine 24 major amino acids within the time frame of 12 minutes. Precision, reproducibility, simpler procedure
and low time consuming are all the pros which can be seen for the HPLC technique.
The low sample intake and no separate processing and the use of simplified gradient
and the processing without the column heater are also the critical pros of
produced valid information for preliminary diagnosis in the initiation of
treatment in various inborn errors of metabolism (Bidlingmuyer et al, 1984). HPLC
seems to be an evolving technique for amino acid qualitative results and quantitative
amino acid analysis. The efficiency of this method can be increased in the
initial stages itself by reducing the delay between the collection and
preparation of the sample. The possible delay causes the amino acids to
metabolise which may later arise as errors while quantitating. The 30-35 minute
turnaround time is of great advantage for emergency diagnosis and monitoring of
samples. HPLC also enables a low retention time which makes the amino acid
identification process more objective. The faster and more sensitive HPLC
method which utilizes pre-column derivatization can be a good alternative to
the most efficient Ion exchange chromatography. HPLC method inherited with ortho-phthalaldehyde
method was the most accurate method for the determination of amino acids from
plasma due to their sensitivity and reliability. Therefore we can say that the
HPLC method used here for the monitoring of amino acids from plasma gives
satisfactory separation, sensitivity and reliable results.
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