Addendum to anthracycline assay by high-pressure liquid chromatography: Modification of mobile phase for aminocyanosilica colums used for anthracycline assay
✍ Scribed by Reich, Steven D. ;Martinez, Lorraine ;Krehel, Walter W. ;Bickler, J. Robert
- Publisher
- John Wiley and Sons
- Year
- 1981
- Tongue
- English
- Weight
- 288 KB
- Volume
- 70
- Category
- Article
- ISSN
- 0022-3549
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✦ Synopsis
plasma since aglycones of the drug and metabolites are not well resolved and elute near the solvent front, where there are high levels of nonspecific fluorescence due to plasma constituents.
Aglycone separation is not a problem in the in uitro assays since there are few interfering substances. Separation of aglycones from deoxyaglycones can be accomplished for most anthracyclines using the aminocyanosilica column if a less polar mobile phase is used. The parent compound, however, will have a longer retention time under these conditions.
Marcellomycin is an anthracycline with a three-sugar chain linked by a glycosidic bond to pyrromycione (22). Mussettamycin and pyrromycin are the di-and monosaccharides in this series, respectively. As can be seen from Table 11, these four anthracyclines can be separated easily by this system.
The relative peak heights of test compounds compared to doxorubicin in this HPLC system depend on three major factors: ( a ) the molar fluorescence of the compounds at the excitation-emission bandwidths used in the fluorescence detector, ( b ) the extraction efficiency of the chloroform-isopropanol solvent, and ( c ) the retention time of the compound. Since retention times of the compounds are short, there is not much spreading of peak widths so the third factor does not influence peak heights substantially. However, the other two factors are important since the relative fluorescence of carubicin is -2.5-3 times that of doxorubicin in this system. For example, 50 ng of carubicidml produces a peak height that is -33% of that due to 400 ng of doxorubicin/ml. This result means that the fluorescence due to 50 ng of carubicin is equivalent to that due to 132 ng of doxorubicin/ml. Since the standard curve for carubicin is linear, it is not necessary to determine if this greater relative fluorescence is due to differences in molar fluorescence or to extraction efficiency. The relative fluorescence of doxorubicin, daunorubicin, and marcellomycin appears to be comparable when these drugs are extracted from pooled plasma.
The examples reported here demonstrate the usefulness and versatility of HPLC to anthracycline research. There is good evidence that doxorubicin toxicity can be predicted on the basis of elevated drug exposure or the area under the plasma concentration curve (23). If this observation holds for anthracycline analogs, then drug monitoring could warn against toxicity during early clinical trials. This HPLC system would then be useful in obtaining concentrations within a reasonable time with accuracy and reproducibility.