Typical applications of HPLC in biomedicine

Many of the points described above are illustrated by the following brief descriptions of a number of typical practical examples. 

1 Creatinine in urine. The assay of creatinine in body fluids is one of the core assays in clinical chemistry since its level in blood and urine reflects the functional status of the kidney. There are many methods for its assay ranging from the simple colorimetric Jafle reaction to dedicated creatinine analysers using discrete sampling technologies. For many metabolic assays, the so-called creatinine correction can be applied since creatinine excretion is considered to be constant throughout the day. The clinic therefore only needs to collect a random specimen of urine rather than a full 24 h specimen. 

3 Indoleamines in urine. 5-Hydroxyindolacetic acid (5-HIAA) is the principle metabolite of the neurotransmitter 5-HT and is excreted in urine. It is naturally fluorescent (excitation 280 nm and emission 360 nm, using a xenon lamp). The selectivity of fluorescence detection means that a very simple and rapid assay is possible. Provided the pH of the eluent is controlled in the region 5.1-5.4 (the exact value depends upon the make of Cl8 columns being used) 5-HIAA can be resolved from the front and 

In contrast nucleosides are bases of moderate hydrophobicity. They are easily resolved by reversed phase HPLC but again their complexity in biofluids means gradient elution is usually required. 

5 Amino acid analysis. There are some 20 amino acids found in proteins and these are released by overnight hydrolysis in 6M HCl. Plasma and urine contain an even larger number of amino acids or related compounds. At low pH, amino acids are cations and for 40 years have been separated by cation exchange column, chromatography. The problem with amino adds is that in general they possess no chromophores by which they can all be detected. In the traditional amino add analyser, their detection was accomplished by a post-column reaction with nin-hydrin which forms a purple colour on heating with an amino acid at pH 5.5. This colour, Ruhemann s purple, is formed with all primary amino acids and can be detected at 570 nm. Secondary amino acids such as proline form a yellow chromophore measurable at 440 nm. 

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