Liquid chromatography of the potential memory-enhancing agent CL 275,838 and its main metabolites, using a post-column photochemical reactor and fluorimetric detection.
On irradiation with short-wavelength ultraviolet light, the potential memory-enhancing compound CL 275,838 (I) and its desbenzyl derivative CL 286,527 (metabolite II) are cleaved into the highly fluorescent derivative CL 228,346 (metabolite IV). This reaction was exploited for the sensitive and selective detection of these compounds in human and animal plasma, after reversed-phase high-performance liquid chromatography on a Supelco LC18 DB column (15 cm x 4.6 mm I.D.) at room temperature. The parent compound and its metabolites were isolated from plasma constituents using the Sep-Pak C18 Plus cartridge, with satisfactory recovery (76-90%) and selectivity. The detection limits were ca. 1.25, 5 and 0.3 ng/ml for I, II and IV, respectively, using 1 ml of plasma. The validation procedure, which includes analysis of multiple ascending calibration curves based on between-day values and replicate analysis of quality control samples analysed with each standard curve, indicated acceptable precision and accuracy of the method within the concentration ranges investigated, the overall coefficient of variation and relative error being less than 10%. The method was successfully applied to plasma samples from healthy volunteers and animals after single of multiple doses of compound I. Metabolites II and IV were detectable in plasma of all species, the former at higher concentrations than the parent compound and metabolite IV. Together with the fact that metabolite II retains much of the parent compound's biological activity in vivo and in vitro, this suggests that it may contribute to the pharmacological effects of compound I.
Caccia
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Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Confalonieri
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Guiso
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Celeste
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Marini
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D000328:Adult; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D006801:Humans; D008297:Male; D008568:Memory / Q000187:drug effects*; D010777:Photochemistry; D010879:Piperazines / Q000097:blood* / Q000378:metabolism / Q000494:pharmacology; D011720:Pyrazoles / Q000097:blood* / Q000378:metabolism / Q000494:pharmacology; D011743:Pyrimidines / Q000097:blood* / Q000378:metabolism / Q000494:pharmacology; D011786:Quality Control; D051381:Rats; D012016:Reference Values; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D010879:Piperazines; D011720:Pyrazoles; D011743:Pyrimidines; C074871:CL 275838