Fluorescence detection reversed phase liquid chromatography

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

FF Shih, AD Kalmer. Determination of glutamine and asparagine by isocratic elution reverse phase liquid chromatography with fluorescent detection. J Liq Chromatogr 7 1169-1183, 1984. 

The liquid chromatographic procedure [622] uses reversed phase liquid chromatography with fluorescence detection to separate all 16 PAHs completely. The method is sensitive and so selective as often to allow the method to be applied without clean-up procedure. For gas chromatographic methods, detection limits are about lpg L 1 whereas for the liquid chromatographic methods, limits are between 1 and lOOpg L 1 for two- and three-ring aromatics and below lpg L 1 for the four-, five- and six-ring compounds. 

The OPA reagent was first reported in 1971 by Roth as a postcolimm fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 

In 2000, Nohta et al. described a method for the determination of biologically active polyamines by intramolecular excimer-forming derivatization with 4-(l-Pjo ene)butyric acid N-hydroxysuccinimide ester (PSE) [34], By this method, dipyrene-labeled putrescine, cadaverine, spermidine, and spermine could be separated by reversed-phase liquid chromatography and specifically detected by the excimer fluorescence at 475 nm with excitation at 345 nm. The excimer fluorescence-emission wavelength is far different from that of the monomer fluorescence-emission wavelength (375 nm) derived from the excess PSE reagent, the hydrolysate product (4-(l-pyrene)butyric acid), and other monopyrene-labeled derivatives. In real biological samples, various monopyrene-labeled derivatives are formed by reaction with PSE and severely interfere with the determination of polyamines. 

AQC is one of the best precolumn fluorescence derivatization reagents for amino compounds [29]. Currently, MS/MS detection is frequently used for the selective determination of biological substances in complex mixtures. The reagent AQC reacts with primary and secondary amines to form aminoquinoline-labeled compounds via a carbamide linkage. These derivatives are separated by reversed-phase liquid chromatography and can be monitored by electrospray ionization—mass spectrometry. The loss of the aminoquinoline tag occurs readily and can be monitored by MS/MS detection, thus, metabolite analysis of amino compounds can be carried out [35]. 

Holtzapple, C.K. Buckley, S.A. Stanker, L.H. Determination of fluoroquinolones in serum using an on-line clean-up column coupled to high-performance immunoafflnity-reversed-phase liquid chromatography, J.Chromatogr.B, 2001, 754, 1-9. [LOD 1.7 ng/mL fluorescence detection]... 

Bai, F. Kirstein, M.N. Hanna, S.K. lacono, L.C. Johnston, B. Stewart, C.F. Determination of plasma topotecan and its metabolite Al-desmethyl topotecan as both lactone and total form by reversed-phase liquid chromatography with fluorescence detection, J.Chromatogr.B, 2003, 784, 225-232. 

Hara, S., Yamaguchi, M., Takemore, Y. and Sakamura, M. Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection. /. Chromatogr. 377 111-119, 1986. 

An enzyme reactor with immobilized 3 -hydroxysteroid dehydrogenase has been successfully used for the analysis of residues of 17 -methyltestosterone in trout by high-performance liquid chromatography (HPLC) (269). Following their separation by reversed-phase chromatography, the major tissue metabolites of 17 -methyltestosterone, namely 5 -androstane-17 -methyl-3, 17 -diol, and 5 -androstane-17 -methyl-3, 17 -diol, were enzymatically modified in the presence of a coreactant, nicotinamide-adenine dinucleotide (NAD), to the corresponding ketone. The position at 3 was enzymatically oxidized, and NADH, the reduced form of NAD, was produced as a coproduct and subjected to fluorescence detection. Reoxidation of NADH to NAD provides the possibility for electrochemical detection. 

SL Abidi, TL Mounts, KA Rennick. Reversed-phase high performance liquid chromatography of phospholipids with fluorescence detection. J Chromatogr 639 175-184,1993. 

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