Determination of Methylglyoxal-bis(Guanylhydrazone) in Body Fluids by Ion-Pair Chromatography

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Abstract
Methylglyoxal-bis(guanylhydrazone), Methyl-G, is a potent antineoplastic agent currently undergoing Phase I clinical trials. Serum, ascitic and pleural fluids, and urine are deproteinized with methanol, supernatant is evaporated, residue is redissolved in the eluent, lipids are removed with carbon tetrachloride, and an aliquot of the aqueous layer injected into the Chromatograph. Ethylglyoxal-bis(guanylhydrazone) (Ethyl-G) is the internal standard. The mobile phase is a mixture of an aqueous buffer (containing 0.004 M heptane and pentane sulfonic acid, 90%:10%, buffered to pH 3.5) and methanol (68%:32%). The ion-pair complex is retained on a µBondapak C18 column, eluted with a flow of 2.0 mL/min. Absorbance is measured at 280 nm. Detectability: 30 ng/mL (0.11 µM) in serum, ascitic and pleural fluids, 300 ng/mL (1.1 µM) in urine. Calibration curves (peak height ratios of Methyl-G/Ethyl-G plotted against known drug concentrations) were linear in the 0.1–30 µg/mL range. Correlation coefficients were 0.999; coefficients of variation for reproducibility were <5%. Residual blood levels of Methyl-G persist for several days. Methyl-G was found to pass into ascitic fluid.