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Anal. Chem. 1988, 60,514-516
Fluorescence Detection of Gadolinium Chelates Separated by Reversed-Phase High-Performance Liquid Chromatography J a m e s J. Hagan, Susan Cicero Taylor, and Michael F. Tweedle* Contrast Media Research Department, The Squibb Institute for Medical Research, P.O. Box 191, New Brunswick, New Jersey 08903
A fluorescence detection method to determine the quantlty of free Gd( III ) In a strong Gd-llgand preparation Is descrlbed. The free or uncomplexed Gd( I I I ) in the preparation is first cheleted with ethylenediamlnetetraacetic acid (EDTA) to form Gd(EDTA)-, which Is separated from the Gd-ligand by reversed-phase hlgh-performance ilquld chromatography. The fluorescence emission of Gd( 111) in the Gd(EDTA)- and the Gd-ilgand peaks Is monitored at 316 nm after excltation at 280 nm. Peak areas are a linear function of Gd( III ) concentration. Values of several concentrations of Gd( III ) determined by a reference method, inductively coupled plasma atomic emission spectrometry (ICP-AES), and by the experimental method agree to within 16.7% at 0.06 mM Gd(111) and 0.5% at 5.52 mM Gd(II1). The limit of detection defined as S / N = 2 is 27 ng of Gd.
Paramagnetic contrast agents are becoming important diagnostic drugs for nuclear magnetic resonance imaging (MRI) ( I ) . MRI signal intensity in the tissues depends on the relaxation times of water protons. The longitudinal relaxation time, TI, and the transverse relaxation time, T,, of the water protons in tissues vary according to their immediate environment. Injected paramagnetic compounds shorten the TI and T2of the water protons, which affects the signal intensity resulting in enhanced tissue contrast that depends on the in vivo distribution of the paramagnetic compound. The most effective paramagnetic ion is Gd(II1). The relatively high toxicity of the Gd(II1) aqua ion is drastically reduced by complexing with a strong ligand. Shown in Figure 1 are two chelating ligands, the Gd(II1) chelates of which are being developed as MRI contrast agents (2, 3 ) . The di-N-methylglucamine salt of gadolinium diethylenetriaminepentaacetic acid, Gd(DTPA)2-,has been administered clinically to enhance contrast in MRI images (2). Its intravenous LD,, in rats is 10 mmol/kg compared to 0.50 mmol/kg for uncomplexed Gd(II1) (2). Because of the importance of complexing Gd(II1) ion prior to injection, a method was sought that would detect
