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Analysis of amino acids in body fluids
Amino acid analysis in human plasma, urine and cerebrospinal fluid plays an important role in clinical disease diagnosis and research. Sample preparation is the key to amino acid analysis in body fluid, including collection, centrifugation, storage and protein removal. The blood and urine samples should be collected to ensure that the subjects do not take any drugs that affect the determination of amino acids. At the same time, the blood should be collected on an empty stomach in the morning to avoid the influence of dietary factors on the concentration of amino acids in the blood. The collected samples should be treated as soon as possible to prevent the change of amino acid concentration due to metabolism. The centrifuged sample should not be immediately deproteinized. The centrifuged sample should be stored at a temperature lower than - 18 ℃ to prevent protein hydrolysis.
The methods of protein removal include acid precipitation, organic solvent precipitation, ultrafiltration, high-speed centrifugation and dialysis. The method of protein precipitation by sulfosalicylic acid (SSA) is widely used in the analysis of amino acids in body fluid samples. Qureshi et al. used 30% SSA combined with 1 mol/L HClO4 to remove protein, and most amino acids obtained high recovery. Campanella et al. used SSA method to remove protein, and used PITC pre column derivatization reverse phase high performance liquid chromatography to simultaneously separate and determine cysteine, cystine and other 18 amino acids in human blood and urine samples.
When protein was precipitated by organic solvent, the recovery of amino acids changed greatly, and the recovery of aspartic acid and glutamic acid were higher; The recovery of asparagine and glutamine was low. Uhe et al. compared the measured values of arginine, lysine, ornithine, glycine, serine and glutamic acid by using acetonitrile, trifluoroacetic acid and SSA, respectively The results show that the acid precipitation method is better than the acetonitrile precipitation method. Ultrafiltration and high-speed centrifugation are not suitable for the treatment of oily blood samples, because they are easy to block the filter membrane, and some amino acids will be degraded during treatment.
Dawson et al. used microdialysis technology and capillary electrophoresis to monitor the changes of glutamate and aspartic acid content in the brain. The analysis time was only 4 minutes, and the detection limit reached the level of nmol/L. Qu et al. analyzed the neurotransmitters glutamate, aspartic acid and γ- Aminobutyric acid. Bergquist et al. used microdialysis technology and CBQCA derivatization capillary electrophoresis laser induced fluorescence detection method fmoc-osu Eight kinds of amino acids in the microdialysis samples of the gray substance of mouse adventitia were analyzed.
Gas chromatography can be used to analyze amino acids in body fluids without protein removal. Pietzsch et al. determined homocysteine in human plasma by GC/MS and one-step derivatization of ethyl chloroformate (ECF). Hu š Ek uses GC and ECF derived reagents to analyze 20 kinds of amino acids and 30 kinds of non amino organic acids in plasma at one time. Kataoka et al. used GC method to directly derive a small amount of amino acids in urine and blood samples into isobutyl carbamoyl methyl ester, and separated 21 kinds of protein amino acids and 25 kinds of non protein amino acids on DB-17HT capillary column within 28 minutes. Deng et al. used GC/MS to directly analyze phenylalanine and tyrosine in a drop of blood sample of the newborn to screen whether the newborn suffered from phenylpyruvuria and other diseases
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