Fully automated high-performance liquid chromatographic method for the determination of carzelesin (U-80,244) and metabolites (U-76,073 and U-76,074) in human plasma.
Carzelesin (U80,244, I) is a cyclopropylpyrroloindole prodrug analog. The compound exerts its cytotoxic activity after conversion, via U-76,073 (II) to U-76,074 (III) by binding to the DNA minor groove in a sequence selective fashion. In pre-clinical investigations the drug displayed a broad spectrum activity against human xenografts in mice. To enable pharmacokinetic monitoring during the phase I clinical trials, we have developed a selective and sensitive assay for the parent compound and its two metabolites. Sample pre-treatment has been automated by using the ASPEC system and involves solid-phase extraction of the diluted plasma sample (1:3 in 20% v/v acetic acid) on an SPE-C18 precolumn followed by two consecutive washings with water and acetonitrile. The compounds are eluted with 600 microliters of dimethylacetamide and 500 microliters is injected on a Spherisorb-CN column. The sample is chromatographed using a linear gradient from 24% to 60% (v/v) acetonitrile in 20 mM phosphate buffer (pH 6.5). The eluate fraction containing the three compounds is heart-cutted in a 2-ml sample loop, switched onto a Spherisorb-ODS column and separation is accomplished using a mobile phase of acetonitrile-20 mM phosphate buffer pH 6.5 (64:36, v/v). UV detection is used with absorbance monitored at 360 nm. This highly selective method offers a lower limit of detection of less than 1 ng/ml for each of the compounds using 1000 microliters of plasma and enables the quantification of the analytes with an acceptable precision and accuracy over a range of 1 to 50 ng/ml. The assay has been implemented in a phase I clinical trial.
van Tellingen
O
O
Department of Clinical Chemistry, Netherlands Cancer Institute, Amsterdam.
Pels
E M
EM
Henrar
R E
RE
Schaaf
L J
LJ
Padbury
G E
GE
Beijnen
J H
JH
Nooijen
W J
WJ
D000970:Antineoplastic Agents / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D001322:Autoanalysis; D001572:Benzofurans / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D000080890:Duocarmycins; D006801:Humans; D007202:Indicators and Reagents; D007211:Indoles / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D011355:Prodrugs / Q000737:chemistry / Q000493:pharmacokinetics*; D013056:Spectrophotometry, Ultraviolet
D000970:Antineoplastic Agents; D001572:Benzofurans; D000080890:Duocarmycins; D007202:Indicators and Reagents; D007211:Indoles; D011355:Prodrugs; C087957:U 76074; C087955:U 76073; C076570:carzelesin