HPLC-ICP-MS compared with radiochemical detection for metabolite profiling of 3 H-bromohexine in rat urine and faeces Berit Packert Jensen,* a Bente Gammelgaard, a Steen Honore´ Hansen a and Jan Vanggaard Andersen b a Department of Analytical Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark. E-mail: bpj@dfuni.dk b Drug Metabolism, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Ma ˚løv, Denmark Received 28th September 2004, Accepted 20th January 2005 First published as an Advance Article on the web 7th February 2005 3 H-Bromohexine was dosed to rats as a model compound to allow comparison of HPLC-ICP-MS detection on bromine to radiochemical detection in an in vivo drug metabolism study. Metabolite profiles were obtained in urine and faeces extracts. No influence of the methanol gradient on the bromine response was observed in the range of 18–75% methanol. The sensitivity obtained by HPLC-ICP-MS was almost two orders of magnitude better than on-line 3 H radiochemical detection. For ICP-MS, the limit of detection was calculated to be 69 nM Br (injection volume 100 ml), corresponding to an absolute limit of detection of 1.3 ng of bromohexine on-column. This allowed ICP-MS detection of several minor metabolites that were not detected using radiochemical detection. Furthermore, metabolites that had lost the radioactive label were detected due to the bromine in the metabolites. As ICP-MS is also more selective than UV and molecular MS detection, it could thus be applied as an alternative detector in drug metabolism studies. Introduction An important part of drug discovery and development is investigation of the metabolism of candidate compounds. In in vivo studies in rats for example, the rate and route of excretion are determined in an ‘‘excretion balance’’ study, in which the amount of compound-related material is determined in the excreta. Furthermore, HPLC profiling of the samples provides information on the metabolic fate of compounds and, depending on the type of detector, quantification or identifica- tion of the metabolites can be determined. As potential meta- bolites are not necessarily known at early stages of the development process, radiolabelling is the usual approach to detecting and quantifying metabolites in biomatrices. How- ever, this requires synthesis of radiochemically labelled drug candidates, a time-consuming and expensive process. Radiola- belled drug compounds are thus often not available until later in the drug development process. An alternative method of detection would thus be advantageous to overcome the issues of radiolabelling. For drug candidates containing elements detectable by ICP- MS, these types of metabolism studies can be performed using ICP-MS detection instead of radiochemical detection. For both radiochemical and ICP-MS detection, structure-indepen- dent response is obtained and only metabolites containing the radioactive label/specific element will be detected. Although ICP-MS detection is not as free from background interferences as radiochemical detection, it is still a very selective method of detection compared with, for example, electrospray mass spec- trometry (ESI-MS), and sample preparation can thus be mini- mised. The ICP-MS detectable elements most often present in drug compounds are the halogens chlorine, bromine and iodine, as well as sulfur and phosphorus. Furthermore, the metals platinum and gold are found in a few drug compounds. HPLC-ICP-MS has indeed been applied to drug metabolism studies, as recently reviewed by Wind and Lehmann. 1 For bromine detection in particular, qualitative studies have been made on bromine-containing compounds 2–4 and bromobrady- kinin metabolites have been assessed quantitatively. 5 The use of a radiolabel along with ICP-MS detection has only been investigated on a platinum-containing compound. 6 This study therefore compares ICP-MS detection on bromine with radio- chemical detection applied in an in vivo drug metabolism study in rats, determining the excretion balance and metabolite profiling of urine and faeces extracts. 3 H-Bromohexine was chosen as a model compound for a likely drug candidate as it is known to form a range of metabolites. 7,8 In addition, as organic solvents are known to influence the bromine sensitivity in the ICP-MS; 9,10 this study evaluates the stability of the bromine response throughout the methanol gradient used for the chromatographic profiling of samples. Experimental ICP-MS The ICP-MS instrument was a PE Sciex Elan 6000 (Perkin- Elmer, Norwalk, CT, USA) equipped with a MicroMist AR30- 1-F-02 glass concentric nebuliser and a cooled (4 1C) cyclonic spraychamber (Glass Expansion, Romainmotier, Switzerland). Sampler and skimmer cones were made of platinum. The plasma and auxiliary argon gas flow rates were 14 and 1.2 l min 1 , respectively. The nebulisation argon gas flow rate, rf power and lens voltage were optimised using a solution of 25 mM NaBr in 50% methanol and 0.1% formic acid. Data acquisition parameters were: isotopes monitored, 79 Br and 81 Br; dwell time, 300 ms for flow injection and 500 ms for chromatography; sweeps per reading, 1; readings per replicate, variable. Peak areas were determined by TurboChrom soft- ware (PerkinElmer). Radiochemical detection A Tri-carb 2900TR Liquid Scintillation Analyzer (Packard, Groningen, The Netherlands) was used to measure total 3 H at 0–18.2 keV. For on-line flow scintillation, the radiochemical detector was a Flo-one 150 TR detector (Packard) equipped with a 500 ml flow cell. ARTICLE www.rsc.org/jaas DOI: 10.1039/b415003a 204 J. Anal. At. Spectrom., 2005, 20 , 204–209 This journal is & The Royal Society of Chemistry 2005