Determination of perfluorobutane in rat blood by automatic headspace capillary gas chromatography and selected ion monitoring mass spectrometry

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Abstract

A new contrast agent (SonazoidTM; NC100100) for ultrasound imaging has been developed. It is an aqueous suspension of lipid stabilised perfluorobutane (PFB) gas microbubbles. An automatic headspace capillary gas-chromatographic mass spectrometric method using electron impact ionisation was developed for analysis of SonazoidTM PFB in rat blood. The calibration standards were gaseous PFB dissolved in ethanol in the range of 0.5–5000 ng PFB. Fluorotrichloromethane (CFC 11) was used as an internal standard of the method and the MS detector was set to single ion monitoring of the base fragment ions of PFB (m/z 69 and 119) and CFC 11 (m/z 101). The calibration graph, made by plotting the peak area ratios of PFB (m/z 69) to CFC 11(m/z 101) against the amount of PFB, was fitted to a second-order polynomial equation with weighting 1/y2 and found to be reproducible. The limit of quantification of the method was set to 0.4 ng PFB. The between-day variation of the method was below 9.2% relative standard deviation (RSD) and the within-day variation of the method was below 7.6% RSD. The accuracy of the method, as compared to Coulter counter, was estimated by determination of PFB in samples where SonazoidTM was added to saline and found to range from 91.5% to 105.2%. PFB, added as SonazoidTM, was found to be stable for at least 7 months in rat blood samples when stored at −20°C.

Introduction

SonazoidTM (NC100100) is a new ultrasound contrast agent under development for indications such as left ventricular border enhancement, myocardial perfusion mapping, detection of focal lesions in the liver and several vascular applications such as characterisation of the vascularity of tumours [1]. The contrast agent is a lipid-stabilised suspension of perfluorobutane (PFB) gas microbubbles with a median volume diameter of 2.4–3.5 μm.

The perfluorocarbons have structures analogous to the familiar hydrocarbons but possess very different chemical and physical properties. Perfluorocarbons are normally much less reactive than hydrocarbons towards all chemical reagents except alkali metals. Temperatures approaching 1000°C are required to decompose perfluoroethane and perfluoropropane, but even though perfluorocarbons of higher molecular weights are thermally less stable, most perfluorocarbons are stable below 300°C. Completely fluorinated alkanes are essentially non-toxic [2]. Perfluorocarbon liquids have thus been used in vitreoretinal surgery as an instrument for manipulating intraocular tissues [3], [4] and selected perfluorocarbons have been used as tracers for monitoring the movement of the atmosphere [5], [6], [7] and in reservoir studies [8].

Gas chromatography (GC) has been the preferred analytical technique for the determination of perfluorocarbons. One of the requirements for separating volatile compounds is the use of a stationary phase that provides sufficient retention for the compounds to be measured. With the introduction of chemically bonded phases on capillary columns, selectivity could be combined with a high theoretical plate number. Adsorption materials such as Al2O3 and porous polymer types of materials in porous layer open tubular (PLOT) capillary columns have been found to be effective for analyses of several fluorocarbons [9], [10]. Perfluorocarbon compounds have been analysed with alumina PLOT [11], fused-silica SE-30 [12], fused-silica SE-54 [13] and fused-silica DB-Petro 100 [14] capillary columns. The perfluorocarbons have been detected with high sensitivity by electron-capture detection [12], [14], [15], [16] or electron-capture negative ion chemical ionisation mass spectrometry (MS) [13]. GC–MS with electron impact (EI) ionisation has been extensively used for determination of aerosol propellant fluorocarbons in biological tissues [17], [18], [19], [20], [21].

To determine the pharmacokinetic properties of the new contrast agent, SonazoidTM, in animals, we developed an automatic headspace sampler capillary GC–EI–MS method for specific determination of PFB in rat blood. The method was validated and the validation results are presented in this paper.

Section snippets

Materials

PFB, C4F10, was purchased from 3M Speciality Chemicals Division, USA, or from F2 Chemicals Ltd., England. Fluorotrichloromethane (CFC 11) >99% was from Sigma-Aldrich, England. Perfluoropropane, perfluoropentane and perfluorohexane were from Fluorochem, England. SonazoidTM was from Nycomed Imaging AS, Norway. Ethanol (96%) was spiritus fortis and 2-propanol was analytical grade. Sodium chloride (NaCl), 9 mg/ml, was purchased from Kabi Pharmacia AB, Sweden. Heparinised blood was collected from

Results and discussion

The main purpose of this study was to develop a method for the determination of PFB in rat blood after i.v. injection of the ultrasound contrast agent SonazoidTM. Since PFB is a highly volatile substance, headspace injection using an automatic headspace sampler coupled with GC–MS was the method of choice. Fig. 1 shows that PFB was chromatographically separated from some of the closely related perfluorocarbons. CFC 11 was chosen as the internal standard of the method and Fig. 2 shows the EI+

Acknowledgements

We thank Per Christian Sontum for the work with obtaining the Coulter data.

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      An electron microscopy study showed that it is the Kupffer cells, i.e., the macrophages located in the liver sinusoids, which are responsible for the liver uptake of the microbubbles; no liver uptake of microbubbles was observed by parenchymal, stellate or endothelial cells (Kindberg et al. 2003). Gas chromatography coupled to mass spectrometry (GC-MS) is the preferred analytical technique for determination of perfluorocarbons and we have previously described validated methods for analysis of PFB in rat blood (Hvattum et al. 2001), exhaled air from rats (Uran et al. 2005) and rat tissues (Toft et al. 2006). In the present work, we used modifications of these methods to determine the pharmacokinetics of PFB in human blood and exhaled air.

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    1

    Present address: Department of Chemistry and Biotechnology, Agricultural University of Norway, N-1432 Ås, Norway.

    2

    Present address: National Institute of Forensic Toxicology, PO Box 495, Sentrum, N-0105 Oslo, Norway.

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