Journal of Analytical Toxicology, Vol. 30, January/February 2006
Drug Monitoringand Toxicology:A Procedure for the Monitoringof Levetiracetamand Zonisamide by HPLC-UV JoEtta M. Juenke1,*, Paul I. Brown 1, Francis M. Urry 1,2, and Gwendolyn A. McMillin 1,2 7ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Inc., Salt Lake City, Utah 84108 and 2Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132
I Abstract { article describes a rapid isocratic high-performance liquid chromatographic (HPLC) method for the simultaneous measurement of the anticonvulsants levetiracetam and zonisamide. Monitoring these drugs is important for detecting potentially toxic concentrations, particularly in patients with renal impairment, but no commercial assaysare currently available. Following a liquid-liquid extraction, levetiracetam (5-150 pg/mL) and zonisamide ( 5 - 8 0 pg/mL) are quantitated by HPLC-UV. The assay's limit of quantitation, linearity, imprecision, and accuracy adequately cover the therapeutic range of these drugs. The assay should be attractive to clinical laboratories because the run time for quantification of both drugs is approximately 5 rain per sample, and no interferences are currently known. This
Introduction Levetiracetam (Keppra | and zonisamide (Zonegran| are anticonvulsant drugs that were approved in 1999 for use in the United States. Levetiracetam is chemically unrelated to the existing anticonvulsant drugs, and its exact mechanism of action is yet unknown. Current studies suggest that it does not act via the traditional targets for anticonvulsant drugs (1). Levetiracetam was most effective in newer animal models, including pilocarpine- and kainic acid-induced seizures (2). It is rapidly absorbed after oral administration, with peak plasma levels at 1-1.5 h. It is not extensively metabolized, producing only one nonreactive carboxylic metabolite. Because it is not metabolized by the liver, it is free of nonlinear elimination kinetics and autoinduction. Because it is not dependent on the cytochrome P450 isoenzymes and is not protein bound, no drug-drug interactions have been described (2,3). It does, however, have a relatively short half-life of 6-8 h and is primarily eliminated in * Author to whomcorrespondenceshouldbe addressed:JoEttaM. Juenke,ARUPInstitutefor Clinical andExperimentalPatho]ogy,ARUPLaboratoriesInc., 500 ChipetaWay, SaltLake City, Utah 84108.E-mail:
[email protected].
urine, so dosing may be adjusted based on renal function. Levetiracetam has been approved as adjunctive therapy for partialonset seizures in adults but may have a broader spectrum of efficacy. Tentative target therapeutic range is 6.0 to 20 I~g/mL (4). It is currently under study for refactory epilepsy (5), benzodiazepine withdrawl (6), monoclonus (7,8), mood stablizing properties (9), as well as a study utilizing it as a single drug for the treatment of partial onset seizures (10). Zonisamide is also a structurally unique anticonvulsant drug. With an anticonvusant profile similar to phenytoin, carbamazepine, and valproic acid combined, zonisamide is an extremely effective broad-spectrum drug. Its favorable pharmacokinetic profile includes rapid absorption, peak concentrations within approximately 4 h post-dose, a long-half life of approximately 63 h, and protein binding less than 40%. Because zonisamide is a substrate of cytochrome P450 2C19 and 3A4, it is subject to many drug-drug interactions, including other anticonvulsants (11,12). Zonisamide has an unusual pattern of distribution with concentrations in erythrocytes, liver, kidney, and adrenal glands twice that of plasma and other tissues (13). In rat studies, zonisamide has eight metabolites, and approximately 33% of a dose is eliminated in urine unchanged. The major metabolites are 2-(sulfamoylacely)-phenol glucuronide at 12.6%, N-acetyl-3-(sulfamoylmethyl)-l,2-benzisoxazole at 7.7%, and glucuronided zonisamide at 7.6%, and the remaining five account for less than 5% of a dose (14) and are therapeutically inactive. Zonisamide is currently approved as adjunctive therapy for partial-onset seizures in adults (15). The tentative target therapeutic range is 10 to 38 I~g/mL (4). Other applications under investigation include weight loss (16,17), hypsarrhythmia in infants (18,19), and bipolar depression (20). Unlike levetiracetam, several significant adverse effects have been seen with zonisamide therapy. These include smoldering myeloma (21), psychotic episodes (22), and urinary stone formations (23). Potentially fatal sulfonamide reactions have also occurred following zonisamide therapy. Monitoring of these drugs is recommended for compliance and for monitoring the effects of polypharmacy.
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Journal of Analytical Toxicology, Vol. 30, )anuary/February 2006
A number of methods have been described for measuring zonisamide and levetiracetam independently. Methods include high-performance liquid chromatography (HPLC) for both drugs (24-29), gas chromatography for levetiracetam (30), and enzyme immunoassay (31) for zonisamide. Here we describe a single, rapid isocratic HPLC assay using IN detection for the quantification of both levetiracetam and zonisamide.
recording. The detector wavelength was 220 nrn. Separation was performed on a 50- x 2.0-ram i.d. Luna Phenyl Hexyl column (Phenomenex,Torrance, CA) with 5-1amspherical particles connected to a phenylpropyl 4- x 3.0-mm guard column (Phenomenex) at a flow rate of 0.5 mL/min (isocratic) maintained at 70~ The mobile phase consisted of 1 L deionized water, one vial of Waters (Milford,MA) D4 mobile phase modifier (dibutylammonium phosphate), and 100 rnL of methanol. HPLC-grade methanol, chloroform, isopropranol, and hydrochloric acid were purchased from Fisher Scientific (Hampton NH). The mobile phasewas filtered through a 0.45I~m nylon membrane under reduced pressure and degassed prior to the addition of the D4 mobile phase modifier. The internal standard (IS), UCB 17025, and levetiracetam were gifts from UCB Pharma (Brussels, Belgiurn). Zonisamide was purchased from Sigma-Aldrich (Dallas, TX). The primary stock solutions (100 g/L) of levetiracetam,zonisamide, and the IS were prepared in methanol. Figure 1 shows the chemical structures. Samples were prepared by transferring 0.5 mL of serum or plasma [positive control, negative control (blank), and calibrators] into respective screw-top, round-bottom, glass tubes. Then 100 laL of IS solution was added to each tube followed by 0.25 mL of 4M HCl. The tubes were capped and vortex mixed for approximately 30 s, and 5 mL of chloroform/isopropanol (90:10) was added. The tubes were rocked for 10 rain and then centrifuged for 10 min at approximately 2500 rpm. The supernatant was transferred to a clean, silanized, screw-top, round-bottom, glass tube where it was completely dried under desiccated air at 50-60~ after which the residue was reconstituted with 0.2 mL of mobile phase. The solution was transferred to an autosampler vial, capped tightly, and 15 ]aLwas then injected onto the column. A ratio of the peak height of the analyte divided by the peak height of the internal standard is used to quantify the analytes of interest from the calibration curve.
Methods The assay system included an automated Agilent (Agilent Technologies,Palo Alto, CA) 1100 series LC with autosampler, column heater, and variable IN wavelength detector utilizing Chemstation (Agilent Technologies) software for result
\ NH;t UCB 17025
Zonisamide
0
fCHa
o Levetiracetam
Figure1. The chemicalstructuresof UCB 17025,zonisamide,and levetiracetam.
A
B
~
C
i
~
Results
_
Time (0-4 rain)
T i m e ( 0 - 4 rain)
E
_
T i m e (C~4rain)
T i m e {0-4 rain)
F
o
= o
: lo
: zo
: ~
~ ~
0 $o
9
ltg
* n
t ~
i 49
Reference method (rag/L) L C - M S
Reference method ( m g / L ) L C - M S
Levetiracetam comparison
Zonisamlde comparison
Figure 2. Chromatograms obtained from blank plasma (A), representative patient on levetiracetam therapy (B), representative patient on zonisamide therapy (C), and calibrator 40/50 mg/L (D). The peaksof interest, levetiracetam at 1.33 min, the IS at 1.95 min, and zonisamide at 2.90 min for a total run time of 3.5 min. Also shown is a graph containing method comparison data for levetiracetam (E) and method comparison data for zonisamide (F).
28
The linearity of the method was evaluatedby analyzing in-house C-18-filtered blood bank plasma to which zonisamide/levetiracetamwas added at the followingconcentrations (rng/L): 5/5, 10/10, 20/30, 40/50, 80/100, and 100/150 (n = 36). This experimentwas performed three times over a period of two months. Calibration materials were also analyzed by a nationally recognized reference laboratory to validate accuracy. Each calibrator was then analyzed in duplicate and concentrations were calculated from a four-point calibration curve containing calibrators of 5/5, 20/30, 40/50, and 80/150 mg/L. Using criteria of accuracy within 85-115% of the target value, the assay was found to be linear to 80/150 mg/L for zonisamide and levetiracetam, respectively. The
Journal of AnalyticalToxicology,Vol. 30, January/February2006
coefficient of variation (CV) averaged 3.5% for zonisamide and 7.1% for levetiracetam, respectively. Analytical performance at 5 mfi/L for each drug includes recovery of drug between 88 and 109% and a CV of 5.5% for zonisamideand recovery of drug between 84 and115% and a CV of 5.5% for ]evetiracetam. This concentration was the lowest concentration tested that produced a peak twice as great as the baseline noise of a plasma blank. As a result, 5/5 mg/L was selected as the lower limit of quantitation and as the low calibrator for the assay. Figure 2 illustrates chromatograms obtained from blank plasma (A), representative patient on levetiracetam therapy (B), representative patient on zonisamide therapy (C), and calibrator 40/50 mg/L (D). The peaks of interest, levetiracetam at 1.33 min, the IS at 1.95 min, and zonisamide at 2.90 rain for a total run time of 4.0 rain. To study potential assay interference, 48 de-identified patient samples submitted for the testing of various therapeutic drugs were processed. An apparent interference was observed with one sample, which resolved after re-injection. The sample just prior was then re-examined and found to contain clonazepam, which had eluted in the next injection and providedthe apparent interference. Extending the analytical runtime to 4 rain will prevent this interfering carryover. Other drugs assayed at therapeutic concentrations that did not interfer with this assay include: carbamazepine, 10-11-carbamazepineepoxide, and hydroxy metabolites; oxcarbazepine and its monohydroxy metabolite; gabapentin; vigabitrin; phenytoin and metabolites; felbamate; lamotrigine; phenobarbital; primidone; acetaminophen; salicylate; ibuprofen; amitriptyline; nortriptyline; desipramine; doxepin and nordoxepin; imipramine; clomipramine and metabolite; quetiapine; valproic acid; topiramate; mephenytoin and metabolite; amiodarone and desethylamiodarone; methsuximide and normethsuximide; ethotoin; clozapine; and sertraline. Carryover was studied by injecting a high calibrator (80/150 mg/L zonisamide/levetiracetam) followedby blank plasma over three seperate runs, over three separate days. No carryover was detected at these concentrations. The concentration is significantly greater than those seen in therapeutic monitoring of these drugs. Thirty de-identified patient samples testing positive for levetiracetam were compared with another reference laboratory LC-mass spectrometry (MS) assay. The linear regression equation for correlation, where y is the HPLC method, was y = 0.99x + 0.55, r = 0.98, with a standard error value of 1.59. Thirty de-identified patient samples testing positive for zonisamide were compared with another reference laboratory LC-MS assay. The linear regression equation for correlation, where y is the HPLC method, was y = 1.01x - 0.04, r = 0.97 with a standard error value of 2.52 (Figures 2E and F).
Conclusions This paper describes a robust assay for the measurement of levetiracetam and zonisamide concurrently by HPLC-UV.The
phenyl-hexyl column employs hexyl alkyl groups instead of traditional propyl chains, giving it added stability and remarkable selectivity for aromatic, amine, and polar compounds. Column lifetime generally supercedes 2000 injections (data not shown), with the pre-column being replaced every other day or after approximately 300 injections. The extraction process avoids the use of solid-phase extraction, which substantially decreases set-up time.
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