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Bioconjugate Chem. 2000, 11, 113−117

Radio-LC-MS for the Characterization of Bioconjugates

113

99mTc-Labeled

Shuang Liu,* Marisa C. Ziegler, and D. Scott Edwards DuPont Pharmaceuticals Company, Medical Imaging Division, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received August 23, 1999; Revised Manuscript Received October 26, 1999

This report describes the first example of using radio-LC-MS for determining the composition of 99mTc radiopharmaceuticals at the tracer level. The in-line radiometric detector is a useful addition to a standard LC-MS and provides direct correlation between the MS data and the radioactive species in a radiopharmaceutical kit. Complexes [99mTc(HYNICtide)(tricine)(L)] (RP444, L ) TPPTS; RP445, L ) TPPDS; and RP446, L ) TPPMS) were prepared using a decayed generator eluant. All the ternary ligand 99mTc complexes show the expected monoprotonated molecular ions, (M + 1)+, and diprotonated molecular ions, (M + 2)2+. The LC-MS spectral data support the proposed structure and are consistent with those obtained for their corresponding 99Tc analogues. Ternary ligand complexes [99mTc(HYNICtide)(tricine)(L)] (L ) ISONIC-HE and ISONIC-Sorb) are neutral, and the molecular weights are also lower than that of RP444. Using a fresh generator eluant (24 h prior elution), only 1-2 mCi of 99mTc [(7 × 10-12)-(1.5 × 10-11) mol of technetium complex] are required to obtain a reasonably clean mass spectrum. Radio-LC-MS is a quick and accurate analytical tool for characterization of 99mTc radiopharmaceuticals at the tracer level.

INTRODUCTION

Radiopharmaceuticals are drugs containing a radionuclide and are used routinely in nuclear medicine departments for the diagnosis or therapy of various diseases (1-4). Nearly 80% of all radiopharmaceuticals used in nuclear medicine departments are 99mTc-labeled compounds. The reasons for such a preeminent position of 99mTc in clinical practice include its extremely favorable nuclear characteristics (t1/2 ) 6.02 h, Emax ) 142 keV), easy availability, and low cost. Because of the high specific radioactivity for 99mTc radiopharmaceuticals, their concentrations are often very low (10-8-10-7 M). While the radioactivity can be monitored using radiometric detectors, the complete molecular characterization of a 99mTc radiopharmaceutical, using currently available analytical techniques, is not possible at these low concentrations. Therefore, there is a growing need for a quick and accurate method to determine the composition of the radiopharmaceutical in the radiolabeled kit at the tracer (99mTc) level. Mass spectrometry has been used for a number of years as a powerful tool for the study of drug metabolism and biodeposition (5-9). These types of studies have been traditionally carried out by GC-MS techniques. However, the use of LC-MS has been growing in importance, especially since the introduction of the thermospray interface, which works well for compounds of moderate polarity. More recently, with the advent of modern liquidphase ionization techniques, such as electrospray, it has become possible to use LC-MS methods for the structural characterization of highly polar molecules at very low concentrations. LC-MS has been used for characterization of 11C-labeled small molecules, such as [11C]harmine (10, 11), the molecular masses of which are often less than * To whom correspondence should be addressed. Phone: (978) 671-8696. Fax: (978) 436-7500. E-mail: shuang.liu@ dupontpharma.com.

300 Da. To our knowledge, there is no prior literature describing the use of LC-MS for direct characterization of 99mTc radiopharmaceuticals. In this report, we describe the first example of using LC-MS for determining the composition of several 99mTclabeled peptide radiopharmaceuticals (Figure 1). Since 99m Tc radiopharmaceuticals are prepared at very low concentrations, it is very difficult to use the UV profile alone to identify the signals due to the radiopharmaceutical. Therefore, we added an in-line radiometric detector between the LC and MSD. The radiometric detector was used for the identification and the electrospray MS for the composition determination of 99mTc-labeled peptide radiopharmaceuticals. A minimum amount of 1-2 mCi (∼1.0 × 10-11 M for fresh generator eluant with 24 h prior elution time) of low molecular weight and neutral ternary ligand 99mTc complexes is necessary for their characterization by this method. Radio-LC-MS is a quick and accurate analytical tool for characterization of 99mTc radiopharmaceuticals at the tracer level. EXPERIMENTAL SECTION

Materials. TPPTS and N-(2-hydroxyethyl)isonicotinamide (ISONIC-HE)1 was purchased from Aldrich Chemical Co. TPPDS was purchased from STREM Chemicals Co., Newburyport, MA, and was used as received. TPPMS was purchased from TCI America, Portland, OR, and was used without further purification. Na99mTcO4 was obtained from a Technelite 99Mo/99mTc generator, DuPont Pharmaceuticals Company, North Billerica, MA. Syn1 Abbreviations: HYNICtide, cyclo(D-Val-NMeArg-Gly-AspMamb(5-(6-(6-hydrazinonicotinamido)hexanamide))); ISONICHE, N-(2-hydroxyethyl)-isonicotinamide; ISONIC-Sorb, 1-(Nisonicotinamide)-1-deoxy-D-sorbitol; TPPTS, trisodium triphenylphosphine-3,3′,3′′-trisulfonate; TPPDS, disodium triphenylphosphine-3,3′-disulfonate; TPPMS, sodium triphenylphosphine3-monosulfonate.

10.1021/bc990111r CCC: $19.00 © 2000 American Chemical Society Published on Web 12/22/1999

114 Bioconjugate Chem., Vol. 11, No. 1, 2000

Figure 1. Structures for

99mTc

Liu et al.

complexes used in this study.

thesis of 1-(N-isonicotinamide)-1-deoxy-D-sorbitol has been described previously (12). Complexes [99Tc(HYNICtide)(tricine)(L)] (L ) TPPTS, TPPDS, and TPPMS) were prepared according to the published procedure (13, 14). Instruments and Methods. LC-MS spectra were collected using an HP1100 LC/MSD system with APIelectrospray interface. The LC-MS method used a Zorbax C18 column (4.6 mm × 150 mm, 3.5 µm particle size) and a gradient mobile phase starting from 92% solvent A (10 mM ammonium acetate buffer, pH 7.0) and 8% solvent B (methanol) to 100% B at 23 min at a flow rate of 1 mL/min. The MSD parameters are as follows: detection mode gain gas temperature nebullizer pressure radio-detector

positive 1.0 350 °C 60 psig (max) NaI

mass range fragmentor drying gas flow V capillary UV detector

600-2000 30 V 13 L/min 4000 V 230 nm

Synthesisof[99mTc(HYNICtide)(tricine)(L)](TPPTS, TPPDS, and TPPDS). A sealed 10 mL vial containing 0.4 mL of tricine solution (100 mg/mL in 0.25 M succinate buffer, pH 5.0), 0.2 mL of HYNICtide solution (100 µg/ mL in H2O), 0.2 mL of the phosphine coligand solution (25 mg/mL in H2O), and 0.8 mL of decayed 99mTcO4solution (25 mCi/mL in saline) was heated in a boiling water bath for 10 min. After cooling at room temperature, the reaction mixture was analyzed by radio-LC-MS. Synthesis of [99mTc(HYNICtide)(tricine)(L)] (ISONIC-HE and ISONIC-Sorb). A sealed 10 mL vial containing 0.4 mL of tricine solution (100 mg/mL in 0.25 M succinate buffer, pH 5.0), 0.2 mL of HYNICtide solution (100 µg/mL in H2O), 0.2 mL of ISONIC-HE or ISONICSorb solution (50 mg/mL in 0.25 M succinate buffer, pH 5.0), 0.25 mL of the fresh 99mTcO4- eluant (400 mCi/mL in saline), and 25 µL of SnCl2‚2H2O solution (1.0 mg/mL in 0.1 N HCl) was heated at 100 °C for 15 min. After cooling at room temperature, the reaction mixture was analyzed by radio-LC-MS. RESULTS AND DISCUSSION

Radiolabeled compounds have an important role in the discovery and development of new chemical entities in the pharmaceutical industry. They are often used for early studies of metabolism and pharmacokinetics to determine the fate of newly synthesized chemical leads. The use of a radiotag permits the easy identification and quantification of the material in complex matrixes and systems. Certain radiolabeled compounds, such as those

Table 1. LC-MS Spectral Data for [99Tc]RP444, [99Tc]RP445, and [99Tc]RP446 compd

formula

[99Tc]RP444 peak A peak B [99Tc]RP445 [99Tc]RP446

C62H78N14O23PS3Tc C62H78N14O23PS3Tc C62H78N14O23PS3Tc C62H79N14O20PS2Tc C62H80N14O17PSTc

found formula found weight (M + 1)+ (M + 2)2+ 1613.4 1613.4 1613.4 1533.4 1453.3

1614.6 1614.4 1614.7 1534.5 1453.2

807.3 807.2 807.3 767.2 727.2

which incorporate 99mTc are useful as diagnostic agents. Often these compounds are first synthesized in very low concentrations (10-6-10-8 M) and their biological distribution and/or activity must be assessed before further development. While the radioactivity of these compounds permits them to be monitored using radiodetectors, complete molecular characterization of these new structures is not possible at these low levels. Traditionally, characterization of a new 99mTc radiopharmaceutical involves synthesis of the corresponding 99Tc complex at the macroscopic level. The composition and the structure of 99Tc complexes are determined by IR, NMR, FAB-MS, and X-ray crystallography. An HPLC concordance experiment is performed to demonstrate that the same Tc complex is prepared at both the macroscopic and the tracer levels. For peptide-based technetium radiopharmaceuticals, isolation of the 99Tc-peptide complex usually involves multiple step synthesis and tedious HPLC purification. It is also very difficult to grow single crystals for highly water-soluble 99Tc-peptide complexes. The chemistry of some chelating systems may differ at the macroscopic and tracer levels. Thus, it necessary to determine the composition of the radiopharmaceutical at the tracer level. LC-MS of [99Tc]RP444, [99Tc]RP445, and [99Tc]RP446. RP444, RP445, and RP446 are ternary ligand 99m Tc complexes of a HYNIC-conjugated cyclic platelet GPIIb/IIIa receptor antagonist (Figure 1). Both animal and clinical studies have demonstrated the utility of RP444 as a new thrombosis imaging agent. To determine the composition and structure of these ternary ligand complexes, the corresponding 99Tc complexes, [99Tc]RP444, [99Tc]RP445, and [99Tc]RP446 were prepared. Unlike those of the 99mTc analogues, the yields for the ternary ligand 99Tc complexes were generally very low (e10%). These complexes were isolated from the reaction mixture by HPLC. After purification, the 99Tc complexes

Radio-LC-MS for

99mTc-Labeled

Table 2. LC-MS Data for

Bioconjugates

99mTc

Bioconjugate Chem., Vol. 11, No. 1, 2000 115

Complexes

complex

formula

exact mass

found (M + 1)+

found (M + 2)2+

[99mTc(HYNICtide)(tricine)(TPPTS)] (RP444) peak A peak B [99mTc(HYNICtide)(tricine)(TPPDS)] (RP445) [99mTc(HYNICtide)(tricine)(TPPMS)] (RP446) [99mTc(HYNICtide)(tricine)(ISONIC-HE)] [99mTc(HYNICtide)(tricine)(ISONIC-Sorb)]

C62H78N14O23PS3Tc C62H78N14O23PS3Tc C62H78N14O23PS3Tc C62H79N14O20PS2Tc C62H80N14O17PSTc C52H73N16O16Tc C56H81N16O20Tc

1613.4 1613.4 1613.4 1533.4 1454.5 1277.1 1397.2

1613.8 1613.8 1613.8 1534.5 1454.3 1277.3 1398.3

807.5 807.2 807.4 767.7 727.8 639.3 699.2

were obtained only in microgram quantities, which is enough for LC-MS, but not enough for extensive NMR studies. All three 99Tc complexes were analyzed by LC-MS using both direct flow injection (FIA) MS and LC/MS. For the initial sample run, a number of 1.5-2.0 min isocratic flow injections were made without the use of an HPLC column. The fragmentor voltage, drying gas temperature and drying gas flow were varied until maximum signal strength was obtained for the first sample. Once the MS parameter optimization was completed, a Zorbax C18 reversed-phase column (4.6 mm × 150 mm, 3.5 µm) was installed. The concentration of these complexes was about 10 µM. The injection volume was 5-20 µL. Under these conditions, all three 99Tc complexes yielded sufficiently intense MS peaks. LC-MS results, expressed as massto-charge ratios, were compared to the expected molecular weights of these complexes. The results are summarized in Table 1. In general, all three 99Tc complexes show the expected monoprotonated molecular ions, (M + 1)+, and diprotonated molecular ions, (M + 2)2+. The molecular ions were detected in their acid forms because the sulfonato groups of the phosphine coligands became protonated when the mobile phase and electrolytes were evaporated in the desolvation chamber. The LC-MS spectral data support the proposed structure (Figure 1) and is completely consistent with the 1:1:1:1 composition for Tc:HYNICtide:tricine:TPPTS, as determined via a series of mixed-ligand experiment on the tracer (99mTc) level (13). [99Tc]RP444 shows two peaks (peak 1 and peak 2) in both the HPLC chromatogram and MSD profile. Both peaks show almost identical fragmentation patterns and the same molecular ions. The observation of the predominant diprotonated molecular ions, (M + 2)2+, is characteristic of both labeled and unlabeled HYNICtide. The fact that these three complexes all show the diprotonated molecular ions suggests that they share the same coordination sphere in the technetium chelate. The two proton acceptors are probably from the carboxylateoxygen and the uncoordinated pyridine-nitrogen atoms. LC-MS of Complexes [99mTc(HYNICtide)(tricine)(L)] (L ) TPPTS, TPPDS, TPPMS, ISONIC-HE, and ISONIC-Sorb). RP444, RP445, and RP446 were prepared using a decayed generator eluant. The concentration of the total technetium (99mTc and 99Tc) was calculated to be approximately 1.5 × 10-6 M. A sample of the reaction solution (200 µL) containing ∼3.0 × 10-10 mol of technetium (99mTc and 99Tc) was injected and a gradient LC method was used for the LC-MS analysis. We used a reversed-phase Zorbax C18 column and a mixture of acetonitrile and 10 mM ammonium acetate buffer as the mobile phase. The flow rate for the LC was 1.0 mL/ min, which is common for high-flow electrospray interfaces. Under these conditions, RP444 shows two radiometric peaks (peak A and peak B) in the radio-HPLC chromatogram and MSD profile (Figure 2). This method was used for the characterization of several 99mTc complexes comprised of the same peptide sequence.

Figure 2. Radio-LC-MS concordance for RP444 (decayed generator eluant, ∼1.5 × 10-10 mol injected).

The LC-MS results are summarized in Table 2. All the ternary ligand 99mTc complexes show the expected monoprotonated molecular ions, (M + 1)+, and diprotonated molecular ions, (M + 2)2+. The LC-MS spectral data support the proposed structure (Figure 1) and are consistent with those obtained by FAB-MS for their corresponding 99Tc analogues (15). Previously (14), we have demonstrated by a chirality experiment that the presence of two radiometric peaks in the HPLC chromatograms of RP444 is due to the resolution of diastereomers, which result from the presence of chiral cyclic peptide and the formation of two enantiomers of the technetium chelate. In this study, both peak A and peak B show identical fragmentation patterns and the same molecular ions, providing direct evidence that the two species are really due to diastereomeric forms of RP444. RP444, RP445, and RP446 were also prepared using a fresh generator eluant. Since they were prepared at much lower concentrations (∼2 × 10-7 M), it is very difficult to determine their retention times based on either the UV (λ ) 220 nm) trace or the MS profiles (Figure 2). There are two approaches to solve the problem. In the first approach, we used the corresponding 99Tc analogue to identify the radiopharmaceutical peak in the MSD profile. In the second approach, we installed an in-line radio-detector before the MSD. The retention time of the 99mTc radiopharmaceutical recorded by the radio-detector is clearly correlated with the location of the tracer level electrospray MS peak (Figure 2). This approach is

116 Bioconjugate Chem., Vol. 11, No. 1, 2000

Figure 3. LC-MS spectrum of RP444 (decayed generator eluant, ∼3.0 × 10-10 mol injected).

Figure 4. LC-MS spectrum of [99mTc(HYNICtide)(tricine)(ISONIC-HE)] (fresh generator eluant, ∼5.0 × 10-11 mol injected).

particularly useful for 99mTc complexes with low UV absorption. It should be noted that the minimum amount of injected activity necessary to obtain interpretable results is very much dependent on the charge and molecular weight of the 99mTc complex. For example, RP444 is highly charged and usually requires a larger amount of activity to obtain a mass spectrum with signal-to-noise ratio of >3. Thus, the decayed generator eluant is recommended to increase the technetium concentration. The alternative is to use multiple injections to concentrate the radiopharmaceutical on the LC column under isocratic conditions. Figure 3 shows the LC-MS spectrum of RP444 using the decayed generator eluant containing ∼3.0 × 10-10 mol of technetium complex. Using a fresh generator eluant (24 h prior elution), at least 10 mCi of 99mTc (∼7 × 10-11 mol of technetium complex) is required to obtain an interpretable mass spectrum. Complexes [99mTc(HYNICtide)(tricine)(L)] (L ) ISONICHE and ISONIC-Sorb) are neutral, and the molecular weights are also lower than that of RP444. Using a fresh generator eluant (24 h prior elution), only 1-2 mCi of 99mTc [(7 × 10-12)-(1.5 × 10-11) mol of technetium complex] are required to obtain a reasonably clean mass spectrum. Figure 4 shows the LC-MS spectrum of [99mTc(HYNICtide)(tricine)(ISONIC-HE)] using a fresh generator eluant containing ∼5.0 × 10-11 mol of technetium complex. The radio-LC-MS method used in this study is particularly useful for 99mTc-radiolabeled bioconjugates, the

Liu et al.

corresponding 99Tc analogues of which cannot be readily prepared. For example, complexes [99Tc(HYNICtide)(tricine)(ISONIC-HE)] and [99Tc(HYNICtide)(tricine)(ISONIC-Sorb)] could not be prepared according to the procedure used for the preparation [99Tc]RP444, [99Tc]RP445, and [99Tc]RP446 (15). This is mainly due to the competition between HYNICtide, which contains a pyridine-N donor, and the corresponding functionalized pyridine coligand. At the tracer level (99mTc), the molar ratio of ISONIC-HE to HYNICtide is ∼2000:1. It is possible for ISONIC-HE to compete with HYNICtide and form the ternary ligand technetium complex. At the macroscopic level (99Tc), this ratio is very difficult to achieve. HYNICtide can compete with ISONIC-HE in bonding to the technetium. Syntheses of [99Tc(HYNICtide)(tricine)(ISONIC-HE)] and [99Tc(HYNICtide)(tricine)(ISONIC-HE)] become extremely difficult. Using the LC-MS method, we are able to determine the composition of ternary ligand complexes [99mTc(HYNICtide)(tricine)(ISONIC-HE)] and [99mTc(HYNICtide)(tricine)(ISONICHE)] at the tracer level. In conclusion, this study reported the first example of using LC-MS for the direct characterization of 99mTc radiopharmaceuticals. Radio-LC-MS is a quick and accurate analytical tool for determining the composition of 99m Tc radiopharmaceuticals at the tracer level. Mass spectrometry by itself cannot determine the structure of compounds. It supplements structural information obtained from other analytical tools such as NMR. Therefore, radio-LC-MS may also be a complimentary technique for structure determination of radiolabeled compounds by analyzing the fragmentation patterns of the mass spectra. Radio-LC-MS has the potential to become a routine analytical tool for new radiopharmaceuticals labeled with 99mTc or other radioisotopes such as 186Re and 177Lu. ACKNOWLEDGMENT

Authors would like to thank Dr. Joseph L. Glajch and Neal Williams for their technical assistance. LITERATURE CITED (1) Jurisson, S ., and Lydon, J. D. (1999) Potential technetium small molecule radiopharmaceuticals. Chem. Rev. 99, 22052218. (2) Anderson, C. J., and Welch, M. J. (1999) Radiometal labeled agents (non-technetium) for diagnostic imaging. Chem. Rev. 99, 2219-2234. (3) Liu, S., and Edwards, D. S. (1999) 99mTc-labeled small peptides as diagnostic radiopharmaceuticals. Chem. Rev. 99, 2235-2268. (4) Volkert, W. A., and Hoffman, T. J. (1999) Therapeutic radiopharmaceuticals. Chem. Rev. 99, 2269-2292. (5) Lee, M. S., Kerns, E. H., Hail, M. E., Liu, J.-P., and Volk, K. J. (1997) Recent applications of LC-MS techniques for the structure identification of drug metabolites and related compounds. Liq. Chromatogr. Mass Spectrom. 15, 542-558. (6) Niessen, W. M. A., and Tinke, A. P. (1995) Liquid chromatography-mass spectrometry: general principles and instrumentation. J. Chromatogr. A 703, 37-57. (7) Gelpe´, E. (1995) Biomedical and biochemical applications of liquid chromatography-mass spectrometry. J. Chromatogr. 703, 59-80. (8) Regnier, F., and Huang, H. (1996) Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry. J. Chromatogr. 750, 3-10. (9) Bruins, A. P. (1991) Liquid chromatography-mass spectrometry with ionspray and electrospray interfaces in pharmaceutical and biomedical research. J. Chromatogr. 554, 3946.

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Bioconjugates

(10) Hyllbrant, B., Tyrefors, N., Markides, K. E., and La¨ngstro¨m. (1997) LC-radio-UV coupled to MS for improved sensitivity and selectivity in analysis of PET radiopharmaceuticals. J. Labeled Compd. Radiopharm. 40, 197-198. (11) Hyllbrant, B., Tyrefors, N., Markides, K. E., and La¨ngstro¨m. (1995) Improved sensitivity and selectivity in the determination of specific radioactivity by means of radioHPLC-MS. J. Labeled Compd. Radiopharm. 37, 704-705. (12) Liu, S., Edwards, D. S., Harris, A. R. (1998) Functionalized pyridine analogues as coligands for 99mTc labeling of HYNICconjugated biomolecules. In Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine (M. Nicolini, G. Banoli, and U. Mazzi, Eds.) Vol. 5, pp 661-668, SGEditoriali, Padova. (13) Edwards, D. S., Liu, S., Barrett, J. A., Harris, A. R., Looby, R. J., Ziegler, M. C., Heminway, S. J., and Carroll, T. R. (1997)

Bioconjugate Chem., Vol. 11, No. 1, 2000 117 New and versatile ternary ligand system for technetium radiopharmaceuticals: water soluble phosphines and tricine as coligands in labeling a hydrazinonicotinamide-modified cyclic glycoprotein Iib/IIIa receptor antagonist with 99mTc. Bioconjugate Chem. 8, 146-154. (14) Liu, S., Edwards, D. S., Harris, A. R. (1998) A novel ternary ligand system for 99mTc-labeling of hydrazino nicotinamidemodified biologically active molecules using imine-N-containing heterocycles as coligands. Bioconjugate Chem. 9, 583595. (15) Liu, S., Edwards, D. S., Harris, A. R., Hemingway, S. J., and Barrett, J. A. (1999) Technetium complexes of a hydrazinonicotinamide-conjugated cyclic peptide and 2-hydrazinopyridine: synthesis and characterization. Inorg. Chem. 38, 1326-1335.

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