Anal. Chem. 2008, 80, 8163–8170
Speciation Analysis of Gadolinium-Based MRI Contrast Agents in Blood Plasma by Hydrophilic Interaction Chromatography/Electrospray Mass Spectrometry Jens Ku¨nnemeyer,† Lydia Terborg,† Sascha Nowak,† Andy Scheffer,† Lena Telgmann,† Faruk Tokmak,‡ Andreas Gu¨nsel,§ Gerhard Wiesmu ¨ ller,§ Stephan Reichelt,| and Uwe Karst*,† University of Mu¨nster, Institute of Inorganic and Analytical Chemistry, Corrensstrasse 30, 48149 Mu¨nster, Germany, Ruhr-University of Bochum, Marienhospital Herne, Department of Medicine I, Ho¨lkeskampring 40, 44625 Herne, Germany, Environmental Specimen Bank for Human Tissues, Domagkstrasse 11, 48149 Mu¨nster, Germany, and Center for Radiology and Nuclear Medicine Mu¨nster, Von-Steuben-Strasse 10a, 48143 Mu¨nster, Germany The first analytical method for simultaneous speciation analysis of five of the most important gadolinium-based magnetic resonance imaging (MRI) contrast agents in blood plasma samples was developed. Gd-DTPA (Magnevist), Gd-BT-DO3A (Gadovist), Gd-DOTA (Dotarem), Gd-DTPA-BMA (Omniscan), and Gd-BOPTA (Multihance) were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospray mass spectrometry (ESI-MS). Spiking experiments of blank plasma with Magnevist and Gadovist were performed to determine the analytical figures of merit and the recovery rates. The limits of detection ranged from 1 × 10-7 to 1 × 10-6 mol/L depending on the ionization properties of the individual compounds, and limits of quantification ranged from 5 × 10-7 to 5 × 10-6 mol/L. The linear concentration range comprised 2 orders of magnitude. With application of this method, blood plasma samples of 10 healthy volunteers, with Magnevist or Gadovist medication, were analyzed for Gd-DTPA and Gd-BTDO3A, respectively. The obtained results were successfully validated with inductively coupled plasma-optical emission spectroscopy (ICP-OES). Gadolinium-based contrast agents are widely used to enhance the contrast of images in magnetic resonance imaging (MRI) for more than 20 years. Most of the contrast agents are based on strongly paramagnetic metal ions, such as iron, manganese, or gadolinium. These metal ions exhibit a large magnetic momentum, altering the relaxation times of protons in their vicinity and thereby changing the signal intensity of protons of either normal or * To whom the correspondence should be addressed. Prof. Dr. Uwe Karst, University of Mu ¨ nster, Institute of Inorganic and Analytical Chemistry, Corrensstrasse 30, 48149 Mu ¨ nster, Germany. Phone: +49-251-8333141. Fax: +49-2518336013. E-mail:
[email protected]. † University of Mu ¨ nster. ‡ Ruhr-University of Bochum. § Environmental Specimen Bank for Human Tissues. | Center for Radiology and Nuclear Medicine Mu ¨ nster. 10.1021/ac801264j CCC: $40.75 2008 American Chemical Society Published on Web 09/27/2008
pathological tissues.1 It is known that free Gd3+ is toxic to the human body and may cause spleen, liver, and bone damage.2 Moreover, free Gd3+ binds to serum proteins and leads to macrophage depletion.3 To circumvent the toxic effects of free Gd3+, but maintaining the excellent magnetic properties for MRI, the free ions are commonly chelated by polyaminocarboxylic acid compounds. Among these complexes, the most well-known examples are Gd-DTPA, Gd-DTPA-BMA, Gd-BOPTA, Gd-DOTA, and Gd-BT-DO3A. Figure 1 shows the chemical structures of the five predominantly applied MRI contrast agents and the trademarks of the respective drugs. In general, contrast agents are classified into linear (Gd-DTPA, Gd-DTPA-BMA, and Gd-BOPTA) and macrocyclic compounds (Gd-DOTA and Gd-BT-DO3A). For ionic contrast agents (Gd-DTPA, Gd-BOPTA, and Gd-DOTA), meglumine is commonly used as a counterion. It is estimated that 25-30% of all MRI procedures are performed with the help of contrast agents, resulting in about 20 million applications per year.4 Generally, complexation of Gd3+ leads to a good tolerance of contrast agents so that adverse effects are very exceptional.5,6 However, recent publications have shown that serious complications have occurred after administration of Gd-based contrast agents in an increasing number of cases. Patients with renal failure have shown severe intoxication and inflammatory symptoms7 or have suffered from a fatal hardening of the skin. This newly diagnosed syndrome, known as nephrogenic systemic fibrosis (NSF) or nephrogenic fibrosing dermopathy (NFD), was first (1) European Medicines Agency (EMeA). Public Assessment Report (EPAR): London, U.K., June 26, 2007. (2) Sieber, M. A.; Pietsch, H.; Walter, J.; Haider, W.; Frenzel, T.; Weinmann, H. J. Invest. Radiol. 2008, 43, 65–75. (3) Ide, M.; Kuwamura, M.; Kotani, T.; Sawamoto, O.; Yamate, J. J. Comp.Pathol. 2005, 133, 92–102. (4) Idee, J. M.; Berthommier, C.; Goulas, V.; Corot, C.; Santus, R.; Hermine, C.; Schaefer, M.; Bonnemain, B. Biometals 1998, 11, 113–123. (5) Bourrinet, P.; Martel, E.; El Amrani, A. I.; Champeroux, P.; Richard, S.; Fauchou, N.; Le Coz, F.; Drici, M.; Bonnemain, B.; Gaillard, S. Invest. Radiol. 2007, 42, 63–77. (6) Herborn, C. U.; Honold, E.; Wolf, M.; Kemper, J.; Kinner, S.; Adam, G.; Barkhausen, J. Invest. Radiol. 2007, 42, 58–62. (7) Tokmak, F.; Schieren, G.; Lefringhausen, L.; van Bracht, M.; Perings, C.; Willers, R.; Gu ¨ nsel, A.; Kemper, F.; Wiesmu ¨ ller, G. A.; Rump, L. C. Am. J. Kidney Dis. 2008, 51, 976–986.
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008
8163
Figure 1. Chemical structures of the contrast agent complexes, the meglumine counterion, and the trademarks of the respective drugs.
recognized in the USA in 1997, described by Cowper in 20008 and is only observed for terminally renal impaired patients. NSF patients show a thickening and hardening of the skin of the extremities, and NSF may be associated with a systemic involvement of the inner organs, including the lungs, liver, and the heart. Patients may have severe sharp pains and may be unable to fully extend their joints. In critical cases, NSF might contribute to death by scarring of body organs.1 The detailed pathogenesis of NSF is not yet fully understood and is discussed elsewhere.1,2,9 However, one supposed trigger for NSF is the result of a combination of renal impairment and medication with Gd-containing contrast agents.10-12 Because of the renal failure, dwell times of the contrasting agents are much higher than in healthy individuals. Under these conditions, stabilities of the contrast agents are likely to be insufficient and it is reasonable to hypothesize an accumulation of metabolites which then attract free fibrocytes, initiating fibrosis.13 Hence, the selective and sensitive determination of Gdbased MRI contrast agents in blood plasma can be of great importance for further investigations of possible pathomechanisms of this newly observed syndrome. To investigate Gd-based contrast agents with the goal to study toxicity and safety aspects, only the total concentrations of Gd were commonly determined in biological fluids, such as plasma, serum, urine, and feces, by element-selective techniques, such as inductively coupled plasma-optical emission spectroscopy (ICPOES), inductively coupled plasma-mass spectrometry (ICP-MS), (8) Cowper, S. E.; Robin, H. S.; Steinberg, S. M.; Su, L. D.; Gupta, S.; LeBoit, P. E. Lancet 2000, 356, 1000–1001. (9) Idee, J. M.; Port, M.; Raynal, I.; Schaefer, M.; Le Greneur, S.; Corot, C. Fund. Clin. Pharmacol. 2006, 20, 563–576. (10) Grobner, T. Nephrol., Dial., Transplant. 2006, 21, 1104–1108. (11) Marckmann, P.; Skov, L.; Rossen, K.; Dupont, A.; Damholt, M. B.; Heaf, J. G.; Thomsen, H. S. J. Am. Soc. Nephrol. 2006, 17, 2359–2362. (12) Broome, D. R.; Girguis, M. S.; Baron, P. W.; Cottrell, A. C.; Kjellin, I.; Kirk, G. A. Am. J. Roentgenol. 2007, 188, 586–592. (13) Morcos, S. K. Br. J. Radiol. 2007, 80, 73–76.
8164
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008
or atomic absorption spectroscopy (AAS).14-18 To gather more detailed information about the Gd species, chromatographic or electrophoretic techniques are required to separate the particular gadolinium compounds and to detect them individually. Several approaches are described in the literature, employing a variety of separation techniques, coupled to optical, element-, and massselective detectors. The use of capillary electrophoresis (CE) for the investigation of single contrast agents has been described by Vetterlein19,20 and Campa.21 Bettmer determined Gd-DTPA and Gd3+ by coupling size exclusion chromatography (SEC) to ICPMS.22 Vora and Chellquist applied ion exchange chromatography (IEC)23 and ion pair chromatography (IPC)24 with UV-vis detection. HPLC methods based on reversed phase (RP) chromatography were introduced by Tweedle, Hvattum, Behra-Miellet, and Arbughi.25-28 Despite the great variety of analytical tech(14) Normann, P. T.; Joffe, P.; Martinsen, I.; Thomsen, H. S. J. Pharm. Biomed. Anal. 2000, 22, 939–947. (15) Puttagunta, N. R.; Gibby, W. A.; Smith, G. T. Invest. Radiol. 1996, 31, 739– 742. (16) Okada, S.; Katagiri, K.; Kumazaki, T.; Yokoyama, H. Acta Radiol. 2001, 42, 339–341. (17) Frame, E. M. S.; Uzgiris, E. E. Analyst 1998, 123, 675–679. (18) Saussereau, E.; Lacroix, C.; Cattaneo, A.; Mahieu, L.; Goulle, J. P. Forensic Sci. Int. 2008, 176, 54–57. (19) Vetterlein, K.; Buche, K.; Hildebrand, M.; Scriba, G. K. E.; Lehmann, J. Electrophoresis 2006, 27, 2400–2412. (20) Vetterlein, K.; Bergmann, U.; Buche, K.; Walker, M.; Lehmann, J.; Linscheid, M. W.; Scriba, G. K. E.; Hildebrand, M. Electrophoresis 2007, 28, 3088– 3099. (21) Campa, C.; Rossi, M.; Flamigni, A.; Baiutti, E.; Coslovi, A.; Calabi, L. Electrophoresis 2005, 26, 1533–1540. (22) Loreti, V.; Bettmer, J. Anal. Bioanal. Chem. 2004, 379, 1050–1054. (23) Chellquist, E. M.; Dicken, C. M. J. Pharm. Biomed. Anal. 1993, 11, 139– 143. (24) Vora, M. M.; Wukovnig, S.; Finn, R. D.; Emran, A. M.; Boothe, T. E.; Kothari, P. J. J. Chromatogr. 1986, 369, 187–192. (25) Hagan, J. J.; Taylor, S. C.; Tweedle, M. F. Anal. Chem. 1988, 60, 514–516. (26) Hvattum, E.; Normann, P. T.; Jamieson, G. C.; Lai, J. J.; Skotland, T. J. Pharm. Biomed. Anal. 1995, 13, 927–932.
niques, the majority of the presented methods exhibit certain limitations or drawbacks. Methods applying optical detection techniques frequently do not offer the required sensitivity to determine very low concentrations and generally exhibit an insufficient selectivity for the analysis in complex matrixes such as blood plasma, serum, and urine. Moreover, time-consuming derivatization steps are required in some cases. Some methods, such as capillary electrophoresis or ion chromatography, are limited to ionic analytes, and most of the described methods were used for qualitative analysis only. In 1994, Tweedle found out that the separation efficiency of the employed RP18 column is unsatisfactory for the simultaneous separation of Gd-CDTA, Gd-EDTA, Gd-DTPA, and Gd-DOTA, obviously due to the insufficient retention of the ionic compounds to the stationary C18 phase. However, hydrophilic interaction chromatography (HILIC) offers a high separation efficiency for polar and ionic compounds29,30 and can easily be coupled to ESI-MS, facilitating a sensitive and selective detection.31,32 Hence, hydrophilic interaction chromatography combined with a selective and sensitive detector should be a powerful tool for the determination of highly polar and ionic Gd-chelates used as contrast agents in MRI. This manuscript describes the development and application of a respective HILIC/ ESI-MS speciation analysis method for the determination of the most important Gd-based MRI contrast agents in blood plasma samples. EXPERIMENTAL SECTION Chemicals. Diethylenetriaminepentaacetic acid (DTPA), diethylenetriaminepentaacetic acid gadolinium(III) dihydrogen salt hydrate (Gd-DTPA), and diethylenetriaminepentaacetic acid iron(III) dihydrogen salt hydrate (Fe-DTPA) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Formic acid, ammonium formate, and ammonium hydroxide solution (25%) were purchased from Fluka Chemie GmbH (Buchs, Switzerland). Europium standard (1,000 mg/L Eu), gadolinium standard (1,000 mg/L Gd) for ICP-OES (traceable to NIST SRM 3118a, lot 992004), methanol, and acetonitrile for HPLC were obtained from Merck KGaA (Darmstadt, Germany). Heparin sodium solution (5 000 units/mL) was obtained under the name of Liquemin from Hoffmann-La Roche (Wyhlen, Germany). All contrast agent infusion solutions were obtained from the respective pharmaceutical companies: Gadovist (Gd-BT-DO3A, 1.0 mol/L) and Magnevist (Gd-DTPA, 0.5 mol/L) from Bayer Schering Pharma AG (Berlin, Germany); Omniscan (Gd-DTPA-BMA, 0.5 mol/L) from GE Healthcare Buchler (Braunschweig, Germany); Dotarem (GdDOTA, 0.5 mol/L) from Guerbet (Sulzbach, Germany); and Multihance (Gd-BOPTA, 0.5 mol/L) from Bracco-Altana Pharma (Konstanz, Germany). All chemicals were used in the highest quality available. Water used for HPLC was purified using a Milli-Q Gradient A 10 system and filtered through a 0.22 µm Millipak 40 filter (Millipore, Billerica, MA). (27) Behra-Miellet, J.; Briand, G.; Kouach, M.; Gressier, B.; Cazin, M.; Cazin, J. C. Biomed. Chromatogr. 1998, 12, 21–26. (28) Arbughi, T.; Bertani, F.; Celeste, R.; Grotti, A.; Sillari, S.; Tirone, P. J. Chromatogr., B 1998, 713, 415–426. (29) Alpert, A. J. J. Chromatogr. 1990, 499, 177–196. (30) Alpert, A. J. J. Chromatogr. 1988, 444, 269–274. (31) Guo, Y.; Gaiki, S. J. Chromatogr., A 2005, 1074, 71–80. (32) Schlichtherle-Cerny, H.; Affolter, M.; Cerny, C. Anal. Chem. 2003, 75, 2349– 2354.
Instrumentation. For LC/MS measurements, an HPLC system from Shimadzu (Duisburg, Germany), consisting of two LC-10ADVP pumps, a DGC-14A degasser, a SIL-HTA autosampler, a CTO-10AVP column oven, and a SPD-10AVVP UV detector that was coupled to either an API 2000 or QTRAP mass spectrometer from Applied Biosystems (Darmstadt, Germany), both equipped with an electrospray ionization (ESI) TurboIonSpray source. The software used for controlling the HPLC, API 2000, and QTRAP and for quantitative data processing was Analyst 1.4.1 (Applied Biosystems). For the calculation of isotopic patterns of the respective gadolinium compounds, the IsotopePattern software (Bruker Daltonics GmbH, Bremen, Germany) was used. For ICPOES measurements, a Spectro CIROSCCD ICP optical emission spectrometer (Spectro Analytical Instruments, Kleve, Germany) with axial plasma viewing was used. All ICP operating parameters, such as gas flows and positioning of the discharge container in front of the optical interface, were adjusted by the Smart Analyzer CIROSCCD software (version 3.2, Spectro). Stock and Standard Solutions. Stock solutions with a concentration of 10 and 50 mmol/L of each contrast agent were prepared by dilution of the respective infusion solution with purified water. A 10 mM DTPA stock solution was prepared by dissolving the free acid in purified water. To improve the solubility of DTPA, 1% of ammonium hydroxide solution (25%) was added. A europium-DTPA stock solution was prepared by adding a 5% excess volume of an equimolar DTPA solution to an appropriate amount of the certified Eu standard solution for ICP (1 000 mg/L Eu), to obtain a final Eu-DTPA concentration of 3.29 × 10-3 mol/L (500 mg/L Eu). Eu-DTPA was used as an internal standard. HILIC calibration standard solutions were prepared by dilution of appropriate amounts of the respective 10 mM contrast agent and internal standard stock solutions with eluent B. For HILIC measurements, seven calibration standard solutions were prepared in the range from 1 × 10-6 to 1 × 10-4 mol/L (0.786-15.72 mg/L Gd), containing Gd-DTPA and Gd-BT-DO3A, while Eu-DTPA was kept at a constant concentration of 3.29 × 10-5 mol/L (5 mg/L Eu). Calibration solutions for ICP-OES were prepared by dilution of distinct amounts of certified Eu and Gd stock solutions for ICP with a 2% solution of nitric acid in purified water. Volumetric flasks for ICP-OES measurements were pretreated with 2% suprapure HNO3 and purified water to minimize adsorption effects. For ICP measurements, five calibration solutions were prepared in the range from 0.1 to 2.0 mg/L, containing both elements, Gd and Eu. Sampling of Blood Plasma. Blood plasma samples were obtained from 10 healthy volunteers, six male and four female subjects, ages 29-69 years, with an average age of 45. Five patients were treated with Magnevist, the other five with Gadovist. Samples were collected via an intravenous cannula into a syringe and transferred immediately into a centrifuge tube, containing 100 µL (500 units) of Liquemin to suppress blood coagulation. Prior to the MRI procedure, 10 mL of whole blood was taken as a sample blank. Subsequently, a solution of either 0.5 M Magnevist or 1.0 M Gadovist at a dose of 0.1 mmol contrast agent per kilogram of body weight was applied to the subject. At 30 min after the MRI procedure, another 10 mL of whole blood was taken and stored at room temperature for the short period between sampling and blood plasma preparation (typically
