title: An efficient HPLC method for the analysis of isomeric purity of technetium-99m-exametazime and identity confirmation using LC-MS.
abstract: 99mTc-exametazime (99mTc-d,l-HMPAO, 99mTc-d,l-hexamethylpropyleneamine oxime) is a neutral rather unstable complex of short-lived 99mTc (t(1/2)=6 h) with the d,l-isomer (mixture of D,D- and L,L-isomers) of a bis-amine bis-oxime tetraligand. It is widely used for measurement of regional cerebral perfusion in nuclear medicine. The meso-isomer (D,L-form) should not be present in a preparation as it is not retained in brain and thus does not provide clinically useful information. Meso-HMPAO is removed from the ligand during the synthesis procedure by repeated recrystallization, but can still be present as impurity in d,l-isomer. Due to the lack of a suitable chromatographic method for analysis of the isomeric purity of 99mTc-exametazime preparations, United States Pharmacopoeia 25 (USP 25) prescribes a biological test in rats for quality control purpose. In this study, we developed a suitable high-performance liquid chromatography (HPLC) method which allows to demonstrate the relative amounts of d,l- and meso-isomer in 99mTc-exametazime and so obviates the need for a biodistribution test in animals as part of the quality control. Due to the low concentrations in which 99mTc-d,l-HMPAO is obtained (typically 2-6 ng/ml), confirmation of the identity of 99mTc-d,l-HMPAO in the monograph of the European Pharmacopoeia is now performed only indirectly by TLC and assessment of its retention time on RP-HPLC. To investigate the potential of radio-LC-MS for assessment of the identity of 99mTc-exametazime, 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO prepared using a Tc-rich eluate were analyzed using a radio-LC-MS system equipped with a time-of-flight mass spectrometer with electrospray ionization. The main peak in the radiometric channel coincided with the molecular ion mass of 99mTc-d,l-HMPAO in the mass spectrometer channel and the measured accurate mass differed only by 0.26 ppm from the theoretical mass. The identity of 99mTc-meso-HMPAO was also confirmed. Thus, radio-LC-MS allowed to obtain strong evidence for the structure of 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO at nanomolar concentration. It is concluded that radio-LC-MS can become a sensitive aid in quality control of "no carrier added" radiopharmaceutical preparations.
authors.affiliation: Laboratory of Radiopharmaceutical Chemistry, UZ Gasthuisberg, University of Leuven, Herestraat 49, B-3000, Leuven, Belgium
authors.forename: D
authors.initials: D
authors.lastname: Vanderghinste
authors.forename: M
authors.initials: M
authors.lastname: Van Eeckhoudt
authors.forename: C
authors.initials: C
authors.lastname: Terwinghe
authors.forename: L
authors.initials: L
authors.lastname: Mortelmans
authors.forename: G M
authors.initials: GM
authors.lastname: Bormans
authors.forename: A M
authors.initials: AM
authors.lastname: Verbruggen
authors.forename: H P
authors.initials: HP
authors.lastname: Vanbilloen
mesh_terms: D002074:Butanones / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004340:Drug Contamination; D013058:Mass Spectrometry / Q000379:methods; D013667:Technetium / Q000032:analysis* / Q000737:chemistry; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D002074:Butanones; D013667:Technetium; C078058:exametazime