Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.
A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.
Stefan
T L
TL
Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
Ingalls
S T
ST
Gerson
S L
SL
Willson
J K
JK
Hoppel
C L
CL
D000097:Acetonitriles; D000970:Antineoplastic Agents / Q000097:blood* / Q000493:pharmacokinetics; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D006147:Guanine / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D010710:Phosphates; D011786:Quality Control
D000097:Acetonitriles; D000970:Antineoplastic Agents; D002021:Buffers; D010710:Phosphates; C064976:O(6)-benzylguanine; D006147:Guanine; D000432:Methanol; C032159:acetonitrile