37 Environmental Analysis & Electrochemistry
Experiments were conducted to confi rm the additional peaks were due to in-source fragmentation. A solution of EsliCBZ was infused directly into the ion source at a fl ow of 10 µL/min.
Analysis of wastewater samples using the developed LC method determined multiple peaks with the transition m/z 253.10 → 210.0921 for some samples. The 3 main product ions from 253.10 were m/z 180.0814, 182.0971 and 210.0921. From the CBZEP standard another product ion at m/z 254.0812 was observed. Figure 3B demonstrates the transition m/z 253.10 → 210.0921 is less specifi c, especially for CBZEP but would be the ion of choice for the monohydroxy metabolites. The peaks eluting before trans- CBZdiOH (7.6 minutes) are thought to be the glucuronide metabolites. With careful selection a more specifi c 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 specifi c transition afforded a good quantitation method for CBZEP. Unknown ‘A’ had a precursor ion m/z 255.1133 and was not 10,11-dihydro-10-hydroxycarbamazepine as both had different retention times. The isolation window for the tMS2 experiment was changed to 2 m/z which removed this interference.
From the CBZEP standard, the transitions 253.10 → 182.0971 and 253.10 → 254.0812, were specifi c to CBZEP and using these product ions CBZEP was separated from the other analytes (Figure 3 C and E). The product ion with m/z 254.0812 is unusual in that it has a higher molecular weight than the precursor ion. The accurate mass fi tted for a
molecular formula of C15H12NO3+. This can be explained by the epoxide ring opening to a di-hydroxy and the loss of NH2 (Figure 4B).
Figure 2. In-source fragmentation of EsliCBZ (A) and trans-CBZdiOH (B) introduced into the ion source by infusion at a concentration of 1000ng/mL.
Infusion of a solution of EsliCBZ into the ion source (Figure 2A) produced the expected precursor ion for EsliCBZ at m/z 297.1227, a sodium adduct m/z 319.1045 and an in-source breakdown product of m/z 237.1017 consistent with CBZ. During chromatography EsliCBZ elutes from the column and on entering the ion source partially breaks down to CBZ. This means at the retention time for EsliCBZ there are two precursor ions present, EsliCBZ at m/z 297.1227 and the break down ion at m/z 237.1017. Both of these will travel through the mass spectrometer and produce their respective product ions in the collision cell.
A similar breakdown was observed when trans-CBZdiOH was infused into the ion-source.
Infusion of a solution of trans-CBZdiOH into the ion source (Figure 2B) produced the expected precursor ion for trans-CBZdiOH m/z 271.1075 and a breakdown ion m/z 253.0969 consistent with CBZEP and CBZmonohydroxy. The infusion experiments were carried out under normal ion-source conditions and no fragmentation energy was applied in the ion source.
These two infusion experiments prove some conversion can take place in the ion source. Where conversion has occurred the analytes are well separated by retention time. However, the monohydroxy metabolites have yet to be tested and this adds to the complexity of analysing CBZ and its metabolites. 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.
Due to the lack of mono-hydroxy standards, a sample of wastewater was used to determine the retention times for all the metabolites. The excellent sensitivity and resolution of the instrument aided the ability to inject the samples directly on to the liquid chromatography mass spectrometry system with only fi ltration of the samples prior to analysis, this was limited to a few samples. This would ensure no analytes were missed due to poor recovery during a sample clean-up or concentration step.
Figure 4. Comparison of experimental and theoretical pattern of CBZEP product ion (253.10 → 254.0809) and the extracted ion chromatogram of the 13C isotope of CBZEP = 254.1002.
It was necessary to confi rm the product ion (m/z 254.0812) was not the 13C isotope of CBZEP. The theoretical 13C isotope for CBZEP is in the inset (Figure 4C). Comparing the experimental ion and the theoretical isotope pattern for CBZEP confi rms this is a product ion of CBZEP (m/z 254.0810) and not the 13C isotope (m/z 254.1005). Using an instrument with lesser resolution the product ion for CBZEP (m/z 254.0821) could not be used as it would not be possible to distinguish it from the 13C isotope (m/z 254.1015)
HRMS is extremely sensitive and selective however, robust chromatography is still essential for complex mixtures and care has still to be taken interpreting the HRMS data to prevent interferences and false positives.
Acknowledgements
The LCMS/MS system used in this study was funded by the noPILLS project, as part of EU funding from the Transnational Territorial Cooperation programme INTERREG IVB NWE; this support is greatly acknowledged. This work was also partly funded by the Phos4You project, through INTERREG VB North-West Europe programme (2014- 2020) under the grant NWE292. I would also like to thank my PhD supervisors Dr Moyra McNaughtan and Dr John MacLachlan.
References
1. SNIFFER, 2010. Methodology for the analysis of selected pharmaceuticals and drugs of abuse in sediments and sludge, Available at:
http://www.sniffer.org.uk/ fi les/9113/4183/7992/ER09_Final_e-version_FINAL_3May101.pdf.
2. Roberts, J.B., 2017. Determination and identifi cation of drug and chemical metabolites in waste water by LCMS/MS. PhD thesis, Glasgow Caledonian University.
https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.726806
3. Miao, X.-S. and Metcalfe, C.D., 2003. Determination of carbamazepine and its metabolites in aqueous samples using liquid chromatography-electrospray tandem mass spectrometry. Analytical Chemistry, 75(15), pp.3731–3738. Available at:
http://www.ncbi.
nlm.nih.gov/pubmed/14572037.
Figure 3. Ions at m/z 253.0977 in a wastewater sample.
4. Bahlmann, A., Brack, W., Schneider, R.J. and Krauss, M., 2014. Carbamazepine and its metabolites in wastewater: Analytical pitfalls and occurrence in Germany and Portugal. Water Research, 57, pp.101–114.#
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