The use of highperformance liquid chromatography for separation and detection of amino acidsin plasma                           ADVANCED BIOANALYTICAL TECHNIQUES  BMS4977   ‘The use of high performance liquid chromatographyfor separation and detection of amino acids in plasma’   M00649054         ABSTRACT:The highlysensitive and fast high performance liquid chromatography (HPLC) technique is acapable substitute to the ion exchange chromatography for the separation anddetection of amino acids in plasma.

Diagnosing of individuals from variousdiseases through the analysis free amino acid in the plasma is an importantprocess. The amino acid analysis that produce valid, reliable and reasonableresults is an unavoidable necessity in clinical applications (Yoshida et al.,2018).

The whole process is based on automated precolumn derivatization ofamino acids with o-phthalaldehyde, and separation of the derivatives byreversed-phase chromatography, and the results are quantification byfluorescence method. The low complexity in the sample preparing method andshort sample analysing time makes this one the most suitable for routineanalysis of large number of samples (Terrlink, Leeuwen and Houdijk, 2018). Theresults obtained from this method is comparatively better than any otherconventional method used for the detection of amino acids. HPLC also enables usto detect trace amounts of amino acids which are not detected through otherconventional sources (Fekkes et al.

, 2018). INTRODUCTION:Aminoacids are the largest group of mutually similar nutrients present in the humanbody. It is defined as the organic substance containing both amino and acidgroups. All the amino acids have different biochemical properties and functionbecause of their variation their side chains.

They are generally stable inphysiological pH except glutamine which undergoes rapid oxidation when exposedto water. 300 or more amino acids are known to man and from that only 20 arefound to be the building blocks of protein even though certain non-proteinamino acids plays important role in the cell metabolism (Wu, 2018). Thesequence in which amino acids combines to form a particular protein determinesthe three dimensional structure and function, which differs for each proteins.The role of proteins as antibodies, enzymes, messengers and as source ofstorage and transport are the ones which are commonly studied. But recentstudies also suggest that that they take part in gene transcription and translationand various other complex processes (Jonas Wilson, 2018).Plasma is measuredfor the detection of various diseases because of its relatively simple assayand less physical burden on patients as well as their robustness for otherclinical purposes. Amino acids are the most suitable components for determiningall clinical diagnosing factors because they are ingested or synthesizedendogenously and plays an essential physiological role as a basic metabolitesand a metabolic regulator.

Amino acids are abundantly available in thecirculation and are favourable targets because they gets influenced by metabolicvariations in various organs which induces specific diseases (Shingyoji et al.,2018). The amino acid analysis will also provide knowledge about premarital andprenatal stages for diseases resulting due to inborn errors.

The amino acidsequence is also identified for prolonged diagnosis of various diseases (Liu,2018).Alterationsin any single amino acid can give rise to various disorders. Branches chainamino aciduria, Branched chain organic acidurias, Glutaric acidurias,Phenylketonuria, Homocystinuria and various urea cycle disorders (eg;Hyperammonemia, Neonatal encephalopathy,Alzheimers) are the most commonlystudies amino acid disorders. There can lead to serious life threatening healthissues to the affected individuals (Agamanolis, 2018).

High Performanceliquid chromatography (HPLC) is one of the most accurate, sensitive andreliable technique for the analysis of amino acids in plasma due to its easily achievableand analysing methods which no other separation technique can match. Theability of HPLC to analyse polar and unstable compounds are one of itsoutstanding feature. HPLC also emphasis on the separation of unconjugated andconjugated bile acids without hydrolysis (Nambara and Goto, 2018). Minimal samplevolume and automated precolumn derivitization of amino acids and fluroscentdetection technique are the highlights of this technique. Quantitation of aminoacids becomes usesful due to the minimilastic sample volume in HPLC (Frank andPowers, 2007).

Ion exchange chromatography, which is said tobe the most perfect method for the determination of amino acids, is also widelyused in routine analysis of plasma (Csap, Tth-Psfai and Csap-Kiss, 1991). Gaschromatography, electrophoresis (paper), thin layer chromatography are the variousother highly accurate methods for the detection of amino acids in plasma. Thepurpose of this review was to evaluate the use High Performance Liquidchromatography (HPLC) in the separation and detection of amino acids in plasma. Preparationof physiological samples:Collectionand Storage:It is oneof the most important pre-procedure which needs to be followed in order to getaccurate results. The patient from which the blood is collected is made surethat he is medication free, it’s because the antibodies present will developadditional peaks in the chromatogram which may end up interfering with theamino acid resolution. EDTA containing tubes are used for collecting samplesdue to its anti-coagulating property (Fekkes, 1996). Samples are then stored at-700 C until assayed.SamplePreparation:In thistechnique first the plasma sample is heated, which denatures the proteins inthem.

The deproteinised plasma is then centrifuged at 2000rpm for 10 minutes (at0-4OC). Deproteinization process is done through various othertechniques which include acid precipitation, ultra-filtration, ultracentrifugationand addition of organic solvents (Fekkes, 1996). The then produced supernatant iscollected for HPLC analysis.  Florescentintensity is not lost during this technique. Certain studies suggest thatdeprotenization and addition of other acids leads to reduced yield of manyphenylthiocarbamyl  amino acids (M CAristoy et al, 1792)Chromatographicequipment:HPLCmethod quantifies primary amino acids in plasma by automated precolumnderivatization with ortho-phthalaldehyde. The HPLC required for the analysisdepends upon the detection limit we require. The system consists of dual pistonpump, photodiode array detector (DAD), florescence detector (FD) and auto samplerfor chromatographic analysis (Furst P et al,1990). Mobile phase was made ofbuffer solutions( 0.

4% tetrahydrofuran and 4mol/L KOH mixed HPLC grade water).The buffers were filtered through degassed helium (pure). The chromatographic separationwas done at 220C. The flowrate in the mobile phase should beadjusted in accordance with the process. The fluorometrical values ranges from340-460nm.

The whole runtime of a single sample and standard is up to a maximumof 30-35 minutes. After every single run the column is equilibrated completelyin the mobile phase. DataReduction:Theobtained chromatogram was compared to a serum standard.

Peaks which exceededthe heights of templates and which has retention times corresponding to theamino 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 thepeaks with the height of the standards (Fekkes, 1996).Recoveryand variability:Therecovery is calculated to be the difference between the peaked and non-peakedsamples. The recovery percentage stands between 90 -110%.

              Fig: High-Performance LiquidChromatography HPLC System (Waters.com. (2018))     DISCUSSION:The HPLCtechnique has found its place in all the clinical laboratories due to itsability to analysis a wide range of compounds. The accurate estimation of aminoacids from plasma requires specificity, sensitivity and high efficiency.

Thismethod has proven all of the above criteria’s but the precipitation of thesample remains a drawback. Furthermore, this technique should be able toresolve and quantify all the amino acids. For the quantification of certainamino acids like cysteine and homocysteine, their reduced form can only beestimated, so this also remains a drawback. HPLC also lacks specificity inquantifying certain –SH group containing amino acids due to their low fluorescenceproperty (Babu et al., 2002). The ortho-phthalaldehydemethod showcased good intra-laboratory variability for a wide range of aminoacids and the preliminary data showed variability of HPLC similar to that ofIon exchange chromatography.

The use of ortho-phthalaldehyde enabled todetermine 24 major amino acids within the time frame of 12 minutes.  Precision, reproducibility, simpler procedureand 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 gradientand the processing without the column heater are also the critical pros ofHPLC. CONCLUSION:HPLC hasproduced valid information for preliminary diagnosis in the initiation oftreatment in various inborn errors of metabolism (Bidlingmuyer et al, 1984).

HPLCseems to be an evolving technique for amino acid qualitative results and quantitativeamino acid analysis. The efficiency of this method can be increased in theinitial stages itself by reducing the delay between the collection andpreparation of the sample. The possible delay causes the amino acids tometabolise which may later arise as errors while quantitating. The 30-35 minuteturnaround time is of great advantage for emergency diagnosis and monitoring ofsamples. HPLC also enables a low retention time which makes the amino acididentification process more objective. The faster and more sensitive HPLCmethod which utilizes pre-column derivatization can be a good alternative tothe most efficient Ion exchange chromatography. HPLC method inherited with ortho-phthalaldehydemethod was the most accurate method for the determination of amino acids fromplasma due to their sensitivity and reliability.

Therefore we can say that theHPLC method used here for the monitoring of amino acids from plasma givessatisfactory separation, sensitivity and reliable results.       REFERENCE:1.      Agamanolis, D. (2018).

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Validation of the determination of amino acids inplasma by high-performance liquid chromatography using automated pre-columnderivatization with o-phthaldialdehyde.7.      Frank, M.

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Validation of an analytical method for human plasma freeamino acids by high-performance liquid chromatography ionization massspectrometry using automated precolumn derivatization.       

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