Detection and Quantification of Lanthanide Complexes in Cell Lysates by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Davide Corpillo,* Claudia Cabella, ² Simonetta Geninatti Crich, ‡ Alessandro Barge, ‡ and Silvio Aime ‡ Laboratorio Integrato Metodologie Avanzate, Bioindustry Park Canavese, 10010 Colleretto Giacosa, Via Ribes 5, Italy Gadolinium (III) complexes are under intense scrutiny as contrast agents for magnetic resonance imaging. Al- though currently used mainly as extracellular agents, there is a growing interest to exploit their contrast enhancing ability in the intracellular environment. To ascertain the preservation of their chemical integrity upon the intracellular entrapment, it is necessary to have a method for their dosage in the cell lysates. Herein, a mass spectrometric method for detection and quantification of gadolinium complexes in cell lysates is reported. The detection by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was carried out by using a non-acidic matrix (2 ,4 ,6 -trihy- droxyacetophenone), which does not allow any leakage of gadolinium from the complex. Quantification has been possible by using as an internal standard an ytterbium complex with the same ligand of the analyte. Ytterbium was chosen because, among the lanthanides, it is the one with the isotopic distribution pattern the most similar to that of gadolinium. Sensitivity was enough to detect low micromolar quantities of a cationic complex and high micromolar quantities of a neutral complex in cell lysates of rat hepatoma cells. In the case of anionic complexes, sensitivity was too low for quantitative analysis. To the best of our knowledge, this is the first report concerning the quantification of metal complexes by MALDI-TOF-MS. The advent of magnetic resonance imaging (MRI) 1 for clinical diagnosis has brought an increasing interest in design and synthesis of paramagnetic complexes for their potential use as contrast enhancers. 2 Among the paramagnetic ions, some lan- thanides, and particularly gadolinium (III), 3 have been shown to be the most useful for this application because they possess a large magnetic moment and a long electronic relaxation time. 4 Highly stable Gd chelates are currently used in clinical settings as reporters of blood flow and organ perfusion. The next generation will deal with systems endowed with targeting capabili- ties, and some applications will involve imaging probes that are able to be specifically entrapped into cells. Recently, Gd complexes have also been proposed for neutron capture therapy (NCT). 5-7 In the latter case, entrapment into the cell is mandatory for the success of the therapy because neutron- activated Gd nuclei emit Auger electrons whose cell damaging effects are confined to few micrometers. Both for therapeutic and diagnostic applications, the determi- nation of the amount of internalized intact Gd complex is of fundamental importance for monitoring the efficacy of the pro- posed method. Actually, the determination of Gd ions would have been much easier, but it may be misleading because the biological environment may lead to partial metal decomplexation. Therefore, one has to discard very sensitive techniques, such as inductively coupled plasma mass spectrometry (ICP-MS), X-ray spectromi- croscopy, and time-of-flight secondary ion mass spectrometry (TOF-SIMS), because they are not able to discriminate between Gd complexes and other gadolinium-containing compounds aris- ing from the speciation of Gd ions released from the complex. The specific uptake of Gd complexes has already been shown in several cell cultures. 3,8,9 Among the techniques which can be used for quantification of complexes in cell lysates, nuclear magnetic resonance (NMR) has an inherently low sensitivity; high- pressure liquid chromatography (HPLC) needs further work for optimization of separation procedures; and electrospray ionization mass spectrometry (ESI-MS) has a low tolerance toward sample contaminants, especially salts. * To whom correspondence should be addressed. Fax: +39 0125 53028. E-mail: davide.corpillo@ unito.it. † Current address: Bracco Imaging SpA, Bioindustry Park Canavese, Coller- etto Giacosa, Italy. ‡ Current address: Dipartimento di Chimica IFM, Universita ` di Torino, Torino, Italy. (1) Beckmann, N.; Laurent, D.; Tigani, B.; Panizzutti, R.; Rudin, M. Drug Discovery Today 2004 , 9, 35-42. (2) Wisner, E. R.; Aho-Sharon, K. L.; Bennett, M. J.; Penn, S. G.; Lebrilla, C. B.; Nants, M. H. J. Med. Chem. 1997 , 40, 3992-3996. (3) Aime, S.; Cabella, C.; Colombatto, S.; Geninatti Crich, S.; Gianolio, E.; Maggioni, F. J. Magn. Reson. Imaging 2002 , 16, 394-406. (4) Runge, V. M.; Clanton, J. A.; Price, A. C.; Wehr, C. J.; Herzer, W. A.; Partain, C. L.; James, A. E. Magn. Reson. Imaging 1985 , 3, 43-55. (5) Shih, J. L.; Brugger, R. M. Med. Phys. 1992 , 19, 733-744. (6) De Stasio, G.; Casalbore, P.; Pallini, R.; Gilbert, B.; Sanita `, F.; Ciotti, M. T.; Rosi, G.; Festinesi, A.; Larocca, L. M.; Rinelli, A.; Perret, D.; Mogk, D. W.; Perfetti, P.; Mehta, M. 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