Clinical And Biomedical Applications

The most useful application for CE in amino-acid analysis is elucidating the structure of both natural and laboratory-synthesized proteins and polypeptides. In 1991, Bergman and associates (98) demonstrated the utility of CE for preparative purposes because of its reproducibility. These workers also demonstrated that amino-acid composition and sequence analysis of proteins could be achieved using phenylthiocarbamyl derivatized amino. Rapid, reliable results on extremely small sample accelerated industrial adaptations. For example, the aromatic amino acids in citrus juice were measured by Cancalon and Byran (99) and in cheddar cheese by Strickland et al. (100) using CE.

Although the utility of amino-acid identification and quantitation in biological samples is more limited than for industrial uses, their measurement in specific cases can give crucial information about the disease state. Separation of amino acids in biological fluids is also much more difficult than when using pure substances. There is the potential for matrix interference, such as the reaction of the derivatizing agent with other components, and in addition, difficulty in comparing solvent-based standards to amino acids in the samples. Thus amino-acid separation and quantitation in biological samples is more problematic than analysis of model laboratory mixtures. Methods developed using model mixtures frequently do not work when applied to biological samples, making assay modifications necessary. Dankava and Kaniansky (101) assessed limitations of various modes of CZE

in the separation of enantiomers in model and urine matrices. Tryptophan was used as the model analyte and a 90-component mixture of UV light-absorbing organic anions served as the model matrix. Using on-line coupled isotacophoretic sample pretreatment for high sample-load capacity, they were able to separate samples corresponding to 3-6 ^L of undiluted urine.

Cerebrospinal fluid analysis to monitor amino-acid levels using MEKC was reported by Berquist et al. (102) using an on-line derivatization and microdialysis sampling technique. Continuous in vivo monitoring of glutamate and aspartate in neurotransmitters of rats using CZE has been reported by Zhou and coworkers (103). An automated procedure for in vivo monitoring cysteine and glutathione in rat caudate nucleus was also reported by Lada and Kennedy (104). In this study, microdialysis was coupled on-line with CZE. The dialysates were derivatized on-line with monobromo-bimane and transferred to the capillary by a flow-gated interface. Separated thiols were measured on-line by a helium-cadmium LIF. Glutamate in microdialysates of striated muscle were measured with CE in an off-line procedure (105). Additionally, phenylthiohydantoin derivatives of 3- and 4-hydroxyproline were separated using MEKC in bovine skeletal muscle collagen (106).

Diagnostic applications of CE for amino-acid analysis have also expanded. Glutamine measured by CZE and LIF detection in cerebrospinal fluid of children with meningitis was reported by Tucci and coworkers (107). They found glutamine concentration was lower in children with viral and bacterial meningitis and proposed that the lower concentrations might be caused by glutamine use by the bacteria. They confirmed the diagnostic utility of this analytical method in the critical differentiation of meningitis. Shihabi and Friedberg (108) and Lehmann et al. (109) reviewed amino-acid analysis in serum, urine, other body fluids and tissues.

Jellum and coworkers (110) described a multi-component analytical system to determined diagnostic metabolites, such as cysteine and homocys-teine, in urine of patients with various aminoacidopathies and other metabolic disorders. They used a diode-ray detector for fluorescence detection of metabolites derivatized with 9-fluorenylmethyl chloroformate and separated by CZE. They modified and expanded this study using gas chro-matography-mass spectroscopy (GC-MS) in a study on sera of patients collected prior to disease symptoms and held in the Janus-bank (111). Kang et al. (112) determined 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole-derivatized homocysteine, glutathione, and cysteine in plasma. Causse et al. (3) separated and determined homocysteine in plasma using FITC as the derivatizing agent and an argon ion laser for detection. The detection of this intermediary metabolite in the methionine pathway is useful to detect not only genetic metabolic disorders, but also the potential risk for atherosclerosis and some thromboembolic diseases.

Tagliaro et al. (4) used CZE in the determination of serum phenylalanine in a rapid, inexpensive method for diagnosis of phenylketonuria. Also, cysteine in human blood, plasma and urine has been separated and quantitated at the attomole level using an end-column amperometric detection with a gold/mercury amalgam electrode (113). In addition, enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics were analyzed by a metal chelate MEKC method using a cyclodextrin (114). The amino acids were hydrolyzed and derivatized with either dansyl chloride for UV-absorbance detection or with FITC for LIF detection. Enantiomeric identities of serine, beta-hydroxyl-N-methy-valine were confirmed and a nonchiral aminoacid, sarcosine, was found.

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