|Separation of isomeric metabolites of carbamazepine by liquid chromatography and high resolution accurate mass.
|Hyphenated Techniques for Comprehensive Analysis
|Dr Joanne Roberts
|Dr Moyra McNaughtan
Dr John MacLachlan
|Glasgow Caledonian University
Abstract Information :
The environmental concerns about the presence of excreted pharmaceuticals in wastewater are well documented and the introduction of high resolution accurate mass (HRAM) spectrometers such as Time of Flight and Orbitrap instruments has aided their detection. Although HRAM is a highly specific technique, interferences can occur especially in a complex matrix such as wastewater. This paper describes how some of the problems encountered were overcome when analysing wastewater samples for carbamazepine and its metabolites using a Thermo Scientific Orbitrap Q Exactive HRAM instrument.
Carbamazepine is a widely prescribed drug used to treat epilepsy and neuropathic pain and it is known to be a persistent environmental pollutant. Many drugs are metabolised prior to excretion, however metabolite presence in wastewater is rarely reported. Carbamazepine is excreted mainly as the di-hydroxy metabolite but also forms an epoxide metabolite which is known as being toxic in the environment. In addition to the epoxide and di-hydroxy metabolites there are five mono-hydroxy metabolites of carbamazepine having the same precursor ion exact mass as the epoxide. As well as having the same precursor ion they are all structurally similar and on fragmentation yield the same product ion in high abundance. This makes it difficult to distinguish between the different metabolites even using HRAM and requires careful interpretation of the data. Thus the presence of mono-hydroxy metabolites could result in higher quantities of the epoxide being reported if they are not adequately separated. Therefore, robust chromatography and careful interpretation of the precursor and product ion data is required to ensure the correct analyte was selected and accurately measured. The analysis is further complicated by the breakdown of other carbamazepine metabolites in the ion source to the same precursor and product ions as the epoxide.
The excellent sensitivity and resolution of the instrument aided the ability to inject the samples directly on to the liquid chromatography mass spectrometry (LCMS/MS) system hence, no analytes were missed due to poor recovery during a sample clean-up or concentration step.
A robust LC method was developed ensuring baseline resolution of any interferences, including those partially broken down in the ion source. (Due to the lack of standards, a sample of waste water was used to determine the retention times for all the metabolites.) The MS transition m/z 253.10 → 210.0921 was the most abundant product ion for the epoxide and interferences. Analysis of wastewater samples using the developed LC method determined eight peaks with this transition. With careful selection a more specific transition (m/z 253.10 → 182.0971) was determined for the epoxide which further distinguished it from the interferences. Using the improved chromatography method and the specific transition afforded a good quantitation method for the epoxide.
HRAM is extremely sensitive and selective however, robust chromatography is essential for complex mixtures and care has still to be taken interpreting the HRAM data to prevent interferences and false positives.