Separation of isomeric metabolites of carbamazepine by liquid chromatography and high resolution mass spectrometry (HRMS)

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Abstract

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 TOF 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 some of the problems encountered and how they 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. It is known to be a persistent environmental pollutant and in some cases the concentration even increases during wastewater treatment. Many drugs are metabolised prior to excretion and the metabolite presence in wastewater is rarely reported. Carbamazepine is excreted mainly as the di-hydroxy metabolite but also forms an epoxide metabolite which is reported 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 highest abundance. This makes it difficult to distinguish between the different metabolites even using HRAM and requires careful interpretation of the data. The mono-hydroxy metabolites are a potential analytical interference and could result in higher quantities of the epoxide being reported if they are not adequately separated. Therefore, good chromatography and careful interpretation of the precursor and product ion data was required to ensure the correct analyte was selected and accurately measured. The analysis was further complicated by the breakdown of other carbamazepine metabolites in the ion source to the same precursor and product ions as the epoxide. Ion source breakdown was confirmed by infusion of solutions of the individual analyte directly into the ion source.
A good LC method was developed to ensure baseline resolution of the interferences, including those partially broken down in the ion source. This was particularly challenging and used a wastewater sample since standards for all the metabolites were not available. The MS transition of 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 of 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. The excellent sensitivity 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.

HRAM is extremely sensitive and selective however, good chromatography is essential for complex mixtures and care has still to be taken interpreting the HRAM data to prevent interferences and false positives.
Original languageEnglish
Publication statusPublished - 24 Jan 2018

Keywords

  • carbamazepine
  • environmental chemistry
  • LCMS
  • waste water

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