Annachiara D'Urso (Presenter)
Fondazione IRCCS Istituto Nazionale Neurologico Ca
Bio: At present Fellowship at Clinical Pharmacology Service at Clinical Pathology and Medical Genetics Unit of Fondazione IRCCS Istituto Nazionale Neurologico Carlo Besta – Milano (Italy). Master’s degree in Biology at University of Milano – Bicocca, Department of Biotechnology and Biosciences.
Authorship: Annachiara D’Urso(1), Valentino Coniglione (2), Sara Brambilla (1), Elisa Bassotti (2), Mara Matteucci (2), Luigi Motti (3), Ugo de Grazia (1).
1) Fondazione IRCCS Istituto Nazionale Neurologico Carlo Besta - Milano 2) Eureka S.r.l. Lab Division 3) Alifax Group - diagnostic instrumentation
| Short Abstract In the past 4 years we tested 56776 samples of patients treated for different kind of epilepsy. 27296 samples were analyzed by chromatography and 29480 by different immunological methods. Approximately one hundred samples of both groups were also retested by LC-MS/MS with a commercial kit (Alifax-Eureka, Italy). The remaining of samples to be tested in chromatography were then tested only by LC-MS/MS with same kit on a Thermo-Fisher TSQ Quantum Access Max triple quadrupole. This kit showed for all the analyzed drugs (carbazepine, oxcarbazepina and their active metabolites, phenytoin, phenobarbital, primidone, topiramate, lamotrigine, levetiracetam, felbamate, zonisamide, rufinamide, lacosamide) consistent and reproducible recovery, sensitivity, selectivity, linearity and high correlation in comparative assays. This kit is now widely used in our TDM routine. |
Long Abstract
Introduction
The treatment of the epileptic patients requires a multidisciplinary medical, pharmacological, psychological and social expertise. In this context, therefore, drug therapy and subsequent monitoring play a decisive role in the immediate seizure control and in the medium and long-term prevention of relapse. Currently, thanks to the monitoring of antiepileptic drug concentrations in plasma is possible in many cases to customize the therapy to each individual patients.
Our lab is performing therapeutic drug monitoring (TDM) of anti-epileptics drugs (AEDs) from at least 30 years. In the past 4 years we performed 56776 TDM of patients treated for different kind of epilepsy. Among them 27296 were analyzed by chromatography (either HPLC-UV or LC-MS/MS) and 29480 by different immunological methods. Approximately one hundred samples of both group were also retested by LC-MS/MS with a commercial kit from Alifax-Eureka. This study was designed to evaluate the performances of this newly released kit using a validated high performance liquid chromatography–ultraviolet (HPLC-UV) method or immunometric assays as reference method.
Methods
Immunometric detection is routinely adopted in our lab for: carbazepine (CBZ), phenytoin (PHT), phenobarbital (PB), and topiramate (TPM) while chromatographic determination is devoted to quantify oxcarbazepine and its metabolite 10-Monohydroxy-dihydro-carbamazepin (OXA/MHD), carbamazepine-epoxide (CBZ-EPOX), primidone (PRM), lamotrigine (LMTG), levetiracetam (LEV), felbamate (FELB), zonisamide (ZON), rufinamide (RUF), lacosamide (LACO).
Immunometric assays were performed on a Abbot Architect Plus apparatus with Abbot kits according to manufacturer instructions and they involve carbazepine (i_Carbazepine), phenytoin (i_Phenytoin), phenobarbital (i_Phenobarbital), and topiramate. The last was adapted on Architect from an Ark kit.
HPLC-UV assays were performed using different kits from Alifax-Eureka as: AE3-FAST (ESM, PRM, LMTG, FELB, OXA, MHD, CBZ-EPOX), AE4-FAST (ZON), AE5-FAST (LACO, RUF, DESM) and LEVETIRACETAM kit (LEV) on a Shimadzu system.
LC-MS/MS assays were carried out on a Thermo-Fisher mass spectrometer TSQ Quantum Access Max triple quadrupole with an UHPLC module Trascend equipped with a C18 Hypersil Gold column (50x2.1 mm, 1,9 µm particle) and the dedicated Alifax-Eureka kit for 18 different AEDs according to manufacturer instruction with some modification for a better reagent-instrument tuning.
For all chromatographic tests preanalyitical procedure is simple and fast being composed by deproteinization followed by vortexing, centrifugation, recovery of the samples and subsequent mixing in a stabilizing solution before injection. We tested both serum and plasma samples.
Valproic acid and metosuximide were not tested in this study.
Results
LC-MS/MS method shows linear reproducible calibration curves for all the considered analytes.
CVs intra and inter day are within accepted values (intra-series 4.32-7.24%; inter-series 4.88-9.96%). All curves are linear at concentrations two or three fold above maximum value of therapeutic range, depending on the substance. Recovery is consistently above 96%. Neither analytical interferences nor matrix effect were observed.
Moreover the determinations carried out on real samples (eg patients undergoing TDM) have shown excellent correlation values with those obtained with the reference methods. Of course, the greatest bias was found towards immunoassays because of the difference between the two techniques.
Samples in stabilizing solution are stable for at least one week at 4°C.
Conclusion
The Alifax-Eureka kit for determination of anti-epileptic drugs in plasma/serum, resulted, in our hands, very easy to use for sample preparation and subsequent analytical step.
Moreover it is sensitive, specific, linear and robust despite the number of different analytes tested. In the first version this kit doesn’t include deuterated internal standards. After the introduction of such standards it’s strength is noticeably enhanced. Although we spent some time in kit-instrument tuning, we established very good chromatographic conditions allowing us to perform rapid analysis with no matrix effect and no analytical interferences. In order to increase the test performances we set two different methods on the instrument: one dedicated to levetiracetam and zonisamide, the other for the remaining drugs. We tested also online purification but we don’t achieve significant improvement so actually we do not include this process in the analysis. A new study is now ongoing to include perampanel in our panel of AEDs.
References & Acknowledgements:
1. Progress report on new antiepileptic drugs: A summary of the Tenth Eilat Conference (EILAT X) Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T and White HS. Epilepsy Research (2010) 92, 89—124
2. Progress report on new antiepileptic drugs: A summary of the Eleventh Eilat Conference (EILAT XI) Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T and White HS. Epilepsy Research (2013) 103, 2—30
3. Drug Interactions with the Newer Antiepileptic Drugs (AEDs)—Part 1: Pharmacokinetic and Pharmacodynamic Interactions Between AEDs. Patsalos PN. Clin Pharmacokinet (2013) 52:927–966
4. Drug Interactions with the Newer Antiepileptic Drugs (AEDs)—Part 2: Pharmacokinetic and Pharmacodynamic Interactions Between AEDs and Drugs Used to Treat Non-Epilepsy Disorders Patsalos PN. Clin Pharmacokinet (2013) 52:1045–1061
5. Interactions between antiepileptic drugs, and between antiepileptic drugs and other drugs. Zaccara G, Perucca E. Epileptic Disord 2014; 16 (4): 409-32
6. Advances in anti-epileptic drug testing. Krasowski MD, McMillin GA. Clinica Chimica Acta 436 (2014) 224–236.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |