Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca An LC-MS/MS based candidate reference method for the quantification of carbamazepine in human serum Judith Taibon a,⁎ , Rupert Schmid a , Stephanie Lucha a , Stephan Pongratz a , Kirill Tarasov a , Christoph Seger b , Christian Timm b , Roland Thiele a , Joachim Mark Herlan a , Uwe Kobold a a Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany b Labormedizinisches Zentrum Dr Risch Anstalt, Landstraße 157, 9494 Schaan, Liechtenstein ARTICLE INFO Keywords: Reference measurement procedure LC-MS/MS Carbamazepine Metabolites Human serum ABSTRACT A validated LC-MS/MS-based candidate reference measurement procedure for the quantification of carbama- zepine is presented in order to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring. Sample preparation was based on protein precipitation using acetonitrile followed by sample dilution. Since the previously listed certified reference material (CRM) SRM 1599 (anticonvulsant drug level assay standard) is no longer available, an ISO certified calibration material was used in this assay. As internal standards deuterated analyte congeners were applied. The method allows the measurement of carba- mazepine, carbamazepine-10,11-epoxide and 10-hydroxy-10,11-dihydrocarbamazepine in the concentration range of 0.1 to 22.0 μg/ml with LODs and LOQs of < 0.1 μg/ml and 0.1 μg/ml, respectively. Comparative measurement of 105 native patient samples using the here presented method showed a good agreement between two independent laboratories with a mean bias of 0.6%. 1. Introduction Carbamazepine (CBZ) is an iminostilbene derivative which is used as an anticonvulsive drug against trigeminal neuralgia, partial epilepsy, generalized tonic-clonic seizures and simple and complex partial sei- zures [1–4]. It is metabolized in the liver, primarily by CYP3A4, to the pharmacologically active metabolite carbamazepine-10,11-epoxide (CBZ-E), which is further degraded to carbamazepine-10,11-dihydr- oxide (DiOH-CBZ) [5,6]. A structurally similar anticonvulsive drug, oxcarbazepine (OXCBZ), has a comparable anticonvulsant efficacy with an improved patient tolerability. In contrast to CBZ the analyte is mainly metabolized by reduction to the active metabolite 10-hydroxy- 10,11-dihydrocarbamazepine (10-OH-CBZ), which is then also de- graded to DiOH-CBZ, a common metabolite with CBZ [7]. Since the use of this antiepileptic drugs, and probably the formation of active meta- bolites, has been associated with a variety of adverse reactions in- cluding cutaneous, haematological, immunological, renal and hepatic disorders [8], therapeutic drug monitoring helps to adjust dosage and to achieve an optimal therapeutic effect while avoiding subtherapeutic and toxic drug levels. Optimum seizure control in patients taking car- bamazepine can be achieved at plasma levels of 4–12 μg/ml [6]. For many years TDM has been performed using immunoassays. However, these methods can suffer with non-specific interferences coming from related compounds, metabolites or matrix effects [9]. Thus, there is still a lack of standardization between different routine assays. Therefore, reference measurement procedures (RMP) are ur- gently needed, which might help to harmonize and standardize such assays. LC-MS methods offer an improved specificity and sensitivity and have shown to be more accurate and precise. Consequently, they are considered as the “gold standard”. Today several analytical methods for the quantification of carbamazepine, oxcarbazepine and their active metabolites are published in literature [10–18]. However, to the best of our knowledge, there is no previous reference method published in literature. The aim of our study was to develop a reference measurement procedure for the quantification of carbamazepine which is me- trological traceable, allows the measurement of the “true value” and thus the standardization and harmonization of lower order measure- ment procedures [19]. Therefore, the primary calibration material used in this assay was an ISO certified reference material (ISO Guide 34, ISO/ IEC 17025, ISO 13485 and ISO 9001). Additionally, with regard to the standardization of routine assays, the two major pharmacologically active compounds carbamazepine- 10,11-epoxide (CBZ-E) and 10-hydroxy-10,11-dihydrocarbamazepine (10-OH-CBZ) were monitored in order to exclude cross-reactivity. http://dx.doi.org/10.1016/j.cca.2017.07.013 Received 18 May 2017; Received in revised form 12 July 2017; Accepted 12 July 2017 ⁎ Corresponding author. E-mail address: judith.taibon@roche.com (J. Taibon). Clinica Chimica Acta 472 (2017) 35–40 Available online 14 July 2017 0009-8981/ © 2017 Published by Elsevier B.V. MARK