Drug monitoring: simultaneous analysis of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, and zonisamide by HPLC-UV and a rapid GC method using a nitrogen-phosphorus detector for levetiracetam

Abstract

A high-performance liquid chromatography (HPLC) assay using UV detection is described for the simultaneous measurement of the newer generation anti-epileptic medications lamotrigine, oxcarbazepine (parent drug and active metabolite 10- hydroxycarbazepine), and zonisamide. Detection of all four compounds can be done at 230 nm; however, there is a potential interference with zonisamide in patients on clonazepam therapy. Therefore, the method uses dual wavelength detection: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for lamotrigine and zonisamide. In addition, a simple gas chromatography method using a nitrogen-phosphorus detector is described for the measurement of levetiracetam, another of the recently approved anti-epileptic medications. For both methods, limits of quantitation, linearities, accuracies, and imprecisions cover the therapeutic range for drug monitoring of patients. A wide variety of clinical drugs, including other anti-epileptic drugs, do not interfere with these assays. These procedures would be of special interest to clinical laboratories, particularly due to the limited availability of immunoassays for newer generation anti-epileptic medications and that therapeutic uses of these drugs are expanding beyond epilepsy to other neurologic and psychiatric disorders.

Figure 1

Chemical structures of lamotrigine, levetiracetam, zonisamide, oxcarbazepine, 10-hydroxycarbazepine (pharmacologically active metabolite of oxcarbazepine), and 10,11-dihydroxy-trans-10,11-dihydrocarbamazepine (therapeutically inactive metabolite of oxcarbazepine).

Figure 2

(Upper panel) Chromatogram from HPLC analysis of a blank plasma sample spiked with lamotrigine (5 μg/mL), zonisamide (15 μg/mL), oxcarbazepine (2 μg/mL), 10-hydroxycarbazepine (15 μg/mL) and the internal standard chloramphenicol using 230 nm wavelength detection. Note that all compounds are readily detected at this wavelength but potential interference with zonisamide occurs in patients taking clonazepam (not shown). (Lower panel) Chromatogram from HPLC analysis of a blank plasma sample spiked with lamotrigine (5 μg/mL), zonisamide (15 μg/mL), oxcarbazepine (2 μg/mL), 10-hydroxycarbazepine (15 μg/mL) and the internal standard chloramphenicol using 270 nm wavelength detection. At this wavelength, 10-hydroxycarbazepine (*) and oxcarbazepine (**) are poorly detected compared to 230 nm.

Figure 3

(Upper panel) Chromatogram of a plasma sample from a patient on chronic oxcarbazepine therapy using 230 nm wavelength detection. Determined concentrations: 10-hydroxycarbazepine (11.2 μg/mL) and oxcarbazepine (< 0.75 μg/mL). (Lower panel) Chromatogram of a plasma sample from a patient on chronic lamotrigine and zonisamide therapy using 270 nm wavelength detection. Determined concentrations: lamotrigine (4.3 μg/mL) and zonisamide (16.5 μg/mL).

Figure 4

Chromatogram from GC analysis of a plasma sample from a patient on chronic levetiracetam therapy. Determined levetiracetam concentration: 18.8 μg/mL.