Abstract
An analytical method is described whereby the major classes of prostaglandins are fully resolved by microcolumn liquid chromatography and detected at the subfemtomole level by laser-induced fluorescence. The prostaglandins are labeled with the fluorescent reagent 4-bromo-methyl-7-methoxycoumarin and are subsequently separated on a high-efficiency fused-silica microcolumn (0.2 mm i.d., 1.06 m length, 150,000 theoretical plates). The optimal chromatographic conditions consist of a 3-micron octadecylsilica packing material and an isocratic mobile phase of 47.6% methanol, 23.8% acetonitrile, and 28.6% water. The prostaglandin derivatives are detected directly on the microcolumn by laser fluorimetry, using a helium/cadmium laser (325 nm, 15 mW) as the excitation source together with a simple filter/photo-multiplier optical detection system. In real sample matrices, the prostaglandin PGF2 alpha is readily quantifiable from the detection limit (0.3 fmol) to the formulation strength of the therapeutic agent Lutalyse (Upjohn), spanning more than six orders of magnitude in concentration. The simplicity and general applicability of the present analytical methodology and instrumentation suggest that this technique can be used to attack a wide variety of biomedically important problems with exceptional sensitivity and selectivity.
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- BERGSTROEM S., DANIELSSON H., SAMUELSSON B. THE ENZYMATIC FORMATION OF PROSTAGLANDIN E2 FROM ARACHIDONIC ACID PROSTAGLANDINS AND RELATED FACTORS 32. Biochim Biophys Acta. 1964 Jul 15;90:207–210. doi: 10.1016/0304-4165(64)90145-x. [DOI] [PubMed] [Google Scholar]
- Dünges W. 4-Bromomethyl-7-methoxycoumarin as a new fluorescence label for fatty acids. Anal Chem. 1977 Mar;49(3):442–445. doi: 10.1021/ac50011a028. [DOI] [PubMed] [Google Scholar]
- Dünges W., Seiler N. High-performance liquid chromatographic separation of esters of 4-hydroxymethyl-7-methoxy-coumarin. A method for the determination of acidic compounds in the picomole range. J Chromatogr. 1978 May 1;145(3):483–488. doi: 10.1016/s0378-4347(00)81381-x. [DOI] [PubMed] [Google Scholar]
- Karlsson K. E., Wiesler D., Alasandro M., Novotny M. 7-[(Chlorocarbonyl)methoxy]-4-methylcoumarin: a novel fluorescent reagent for the precolumn derivatization of hydroxy compounds in liquid chromatography. Anal Chem. 1985 Jan;57(1):229–234. doi: 10.1021/ac00279a053. [DOI] [PubMed] [Google Scholar]
- Tsuchiya H., Hayashi T., Naruse H., Takagi N. Sensitive high-performance liquid chromatographic method for prostaglandins using a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin. J Chromatogr. 1982 Sep 10;231(2):247–254. doi: 10.1016/s0378-4347(00)81849-6. [DOI] [PubMed] [Google Scholar]
- Turk J., Weiss S. J., Davis J. E., Needleman P. Fluorescent derivatives of prostaglandins and thromboxanes for liquid chromatography. Prostaglandins. 1978 Aug;16(2):291–309. doi: 10.1016/0090-6980(78)90031-x. [DOI] [PubMed] [Google Scholar]
- VAN DORPD, BEERTHUIS R. K., NUGTEREN D. H., VONKEMAN H. THE BIOSYNTHESIS OF PROSTAGLANDINS. Biochim Biophys Acta. 1964 Jul 15;90:204–207. doi: 10.1016/0304-4165(64)90144-8. [DOI] [PubMed] [Google Scholar]
- Watkins W. D., Peterson M. B. Fluorescent/ultraviolet absorbing ester derivative formation and analysis of eicosanoids by high-pressure liquid chromatography. Anal Biochem. 1982 Sep 1;125(1):30–40. doi: 10.1016/0003-2697(82)90379-7. [DOI] [PubMed] [Google Scholar]
- Wintersteiger R., Juan H. Prostaglandin determination with fluorescent reagents. Prostaglandins Leukot Med. 1984 Apr;14(1):25–40. doi: 10.1016/0262-1746(84)90021-0. [DOI] [PubMed] [Google Scholar]
- Yamada K., Onodera M., Aizawa Y. Determination of prostaglandin E2 and the main prostaglandin E metabolite by micro high-performance liquid chromatography using fluorescence derivatization with dansyl hydrazine. J Pharmacol Methods. 1983 Apr;9(2):93–100. doi: 10.1016/0160-5402(83)90001-3. [DOI] [PubMed] [Google Scholar]