Rachel Wall Ph.D.

rachel.wall@dcu.ie

Background

Cancer is the second largest cause of mortality in Western society. Typical treatments are surgery, chemotherapy and radiotherapy.

Chemotherapy involves the administration of a cytotoxic agent at a concentration sufficient to kill a maximum number of cancer cells while minimising the toxicity to normal tissue.

Studies suggest that the improvement of chemotherapeutic treatment (efficacy, toxicity, morbidity) is observed where chemotherapy administration is coupled with accurate measurement of the component drugs.

Currently in Ireland, there are no facilities or expertise to integrate accurate chemotherapy drug and metabolite determination into the clinical oncology setting.

Mass spectral (MS) analysis as a detection system (coupled to LC) is a relatively new tool in the quantitation and analysis of drug samples. It can be used to identify and quantitate known and unknown compounds and to elucidate specific, structural information about drugs and their metabolites, thus MS can be used to simultaneously measure a number of drugs and their metabolites.

These features make LC-MS particularly suited to analyse complex mixtures of drugs and their metabolites from biological sources including patient serum. Cancer chemotherapy generally employs “cocktails” of drugs to treat patients. Accurate, sensitive and rapid estimation of all drugs present and their metabolites may potentially allow much-improved individualisation of patient treatment. Also, these methods will be used to examine and answer fundamental questions regarding cellular movement of chemotherapy drugs and optimise strategies to increase drug uptake

Techniques

LC-MS, HPLC-UV, fluorescence and cell culture are some of the techniques used in this project

Results

A MS method was developed for the analysis of the anthracycline drugs. Detailed optimisation strategies increased the ionisation intensities by four orders of magnitude to 10 6 -10 7 , thus allowing the analysis of low concentrations of analyte.

Figure 1 outlines an LC-MS chromatograph (UV trace, extracted ion current (EIC) for the appropriate molecular weight of the analyte and mass spectra) of a mixture of three anthracycline drugs, doxo-, epi- & daunorubicin, eluting at

2.7, 3.1 & 5.0min respectively. It is immediately clear that the MS affords more sensitivity than the UV.

Figure 1 LC-MS Chromatogram of a mixture of Anthracyclines Drugs

An Ion-Trap and a Triple-Quadrupole Mass Spectrometer have been used for the development of an LC-MS assay for the determination of epirubicin.

UV sensitivity disappears at sub µg/ml levels with a detection limit (LOD) of 0.92µM (500ng/ml).

The MS calibration curve was linear over the range 9-18500nM using the Ion Trap. The LOD was 9nM while the lower limit of quantitation (LLOQ) was 18nM (10ng/ml).

The calibration curve of epirubicin analysed by the Triple Quadrupole Instrument is shown in figure 2 . The calibration curve for epirubicin was linear over the range of 2-1000nM (1-543ng/ml). The LOD was between 0.5-1nM (0.27-0.55ng/ml) while the LLOQ was 2nM (1ng/ml).

Figure 2 Calibration Curve of Epirubicin (2-1000nM)

Partners

National Institute for Cellular Biotechnology

Funding

Cancer Research Ireland

Outcomes and Activities

A research visit to the University at Buffalo, State University of New York to R.M.Straubinger et al. in the School of Pharmacy and Pharmaceutical Sciences in March 2004.