Environmental Analysis & Electrochemistry
Separation of isomeric metabolites of carbamazepine by liquid chromatography and high resolution accurate mass
Joanne Roberts, Glasgow Caledonian University,
Joanne.Roberts@
gcu.ac.uk
The environmental concerns about the presence of excreted pharmaceuticals in wastewater are well documented [1] and the introduction of high resolution mass spectrometry (HRMS) such as Time of Flight and Orbitrap instruments has aided their detection. Although HRMS is a highly specifi c technique, interferences can occur especially in complex matrixes. This paper describes how some of the problems encountered were overcome when analysing wastewater samples for carbamazepine (CBZ) and its metabolites using a Thermo Scientifi c Orbitrap Q Exactive HRMS instrument [2].
CBZ is a widely prescribed drug used to treat epilepsy and neuropathic pain and it is known to be a persistent environmental pollutant which is not broken down during wastewater treatment.
Most drugs are metabolised prior to excretion, however detection of these metabolites in wastewater is only beginning to be routinely tested. CBZ is excreted mainly as the trans-10,11-dihydro-10,11-dihydroxycarbamazepine (trans-CBZdiOH) metabolite but also forms an epoxide metabolite, carbamazepine epoxide (CBZEP), which gives negative side effects in humans [3] and is toxic in the environment. There are also fi ve mono-hydroxy metabolites of CBZ having the same precursor ion exact mass as CBZEP and because they are all structurally similar yield the same product ion in highest abundance on fragmentation. Oxcarbazepine (OxCBZ) is a replacement for CBZ which was developed as it does not metabolise to the toxic CBZEP but, it also has the same molecular formula as CBZEP and the monohydroxy metabolites. This makes it diffi cult to distinguish between the different metabolites even using HRMS and requires careful interpretation of the data and good chromatography to ensure the mono-hydroxy metabolites are separated from CBZEP and a false high concentration is not reported. Another new drug substitute for CBZ is eslicarbazepine (EsliCBZ) which was also included in this study.
A summary of the chemical structures, precursor ion and molecular formula of the analytes included in this study are shown in Table 1.
Table 1. Chemical structures and precursor ions in this study.
Ionisation was by electrospray in positive ionisation mode with a spray voltage of 3.5 kV. The sheath and auxiliary gas were 45 and 10 arbitrary units respectively and the capillary and auxillary temperature were both 300ºC. The range of the full MS-SIM experiment was 50 – 750 m/z, the target ions were specifi ed in the targeted MS2 method with an isolation window of 4 m/z. For both experiments the mass resolution was 35000.
The chromatography column was a Waters Atlantis® dC18 chromatography column 150 × 2.1 mm, particle size 3 µm.
Mobile phase A was methanol and B was 0.1% formic acid in Ultra-pure water. The LC gradient started at 99% B for 2 minutes then to 70% B over 3 minutes and maintained at that for 11 minutes. The composition of B was dropped to 1% over 1 minute and maintained at that for 3 minutes. Finally returning to 99% B over 1 minute and re-equilibrated for 9 minutes. The flow rate was 0.2 mL/minute and injection volume of 10 μL.
Solutions containing CBZ, cis-CBZdiOH, trans-CBZdiOH, CBZEP, OxCBZ, EsliCBZ and CBZ-10hydoxydihydro at a concentration of 1000 ng/mL were first injected separately to determine the retention time of each analyte. These were then injected as a mixture.
On processing the mixture three peaks were observed at the specifi c transition of 237.11 → 194.0963 for CBZ (Figure 1A). This was a synthetic mixture and the solution should not contain any other drugs with this specifi c transition or precursor ion and the loss of specifi city was initially concerning. The interference at 7.9 minutes corresponded to EsliCBZ and at 12.5 minutes 10,11-dihydro-10- hydroxycarbamazepine with transitions 297.12 → 194.0963 and 255.11 → 194.0963 respectively. Although these have the same product ion the precursor ion is very different for each.
Similar contamination was observed for CBZEP at 9.5 minutes, this time the interferences had retention time of 6.4 and 7.2 minutes which were consistent for cis- and trans-CBZdiOH.
The mass spectrometer used for the analysis was a Thermo Scientifi c Q-Exactive Orbitrap mass spectrometer, fi tted with a Dionex Ultimate 3000 RS Pump, Dionex Ultimate 3000 RS Autosampler (Temperature controlled at 10ºC) and Dionex Ultimate 3000 RS Column Compartment (Temperature controlled at 30ºC).
The software was Chromeleon®, Xcalibur™ and Tracefi nder™.
Figure 1. Chromatograms of carbamazepine, eslicarbazepine and 10,11-dihydo-10- hydroxycarbamazepine.
Closer inspection suggested this may be due to fragmentation in the ion-source. Although electrospray is a soft ionisation technique some analytes can break down in the ion source [4].
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