saliva by liquid chromatography-electrospray tandem mass spectrometry

Clinica Chimica Acta 375 (2007) 115 – 118 www.elsevier.com/locate/clinchim

Determination of levetiracetam in human plasma/serum/saliva by liquid chromatography-electrospray tandem mass spectrometry Tiedong Guo a,b , Lisa M. Oswald a , Damodara Rao Mendu a , Steven J. Soldin a,c,d,⁎ a

d

Bioanalytical Core Laboratory, General Clinical Research Center, Georgetown University, Washington, DC, USA b Department of Biology, The Catholic University of America, Washington, DC, USA c Departments of Medicine and Pharmacology, Georgetown University, Washington, DC, USA Department of Laboratory Medicine, Children's National Medical Center and Department of Pediatrics and Pathology, The George Washington University School of Medicine, Washington, DC, USA Received 15 May 2006; received in revised form 21 June 2006; accepted 22 June 2006 Available online 27 June 2006

Abstract Background: Levetiracetam (Keppra) is a novel antiepileptic drug recently approved by the U.S. Food and Drug Administration as an add-on therapy in the treatment of partial onset seizures in patients. We developed and describe a simple and rapid high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) assay for the determination of levetiracetam in human matrix (plasma, serum, or saliva). Methods: An API-3000 or API-4000 triple–quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the IonSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing ritonavir as internal standard (IS) for levetiracetam. One hundred microliters of serum (or plasma, saliva) was deproteinized by adding 150 μl of acetonitrile containing internal standard. After centrifugation, 100 μl of supernatant was diluted with 300 μl of water and 10 μl aliquot was injected onto a C-18 column. After a 2.5 min wash the valve was activated to initiate the isocratic elution program which eluted the levetiracetam and internal standard into the MS/MS system. Quantitation by MRM analysis was performed in the positive ion mode. Within-day and between-day imprecision were evaluated for levetiracetam using three levels of in-house controls. Reliability and accuracy of this method were assessed by comparison of targets with external QC material (ChromSystems), between laboratory comparisons and by recovery studies. Results: Within-day coefficients of variation (CVs) were b 6.1% and between-day CVs were b 8.2%. The average spiked recoveries of levetiracetam added to the drug-free human plasma samples were 108% at low concentration level and 103% at high concentration level. Conclusions: The method was found both specific and sensitive for the rapid and accurate measurement of levetiracetam in human matrices and correlated well with the Quest/Chantilly tandem mass spectrometric method (r = 0.983). © 2006 Elsevier B.V. All rights reserved. Keywords: Levetiracetam; HPLC; Tandem mass spectrometry

1. Introduction Levetiracetam [ucb L059; (S)-α-ethyl-2-oxo-pyrrolidine acetamide; Keppra] is a novel antiepileptic drug (AED) approved by the U.S. Food and Drug Administration (FDA) in November 1999 as an add-on therapy in the treatment of partial onset seizures in adults with epilepsy, and following that in June 2005, levetiracetam was approved by FDA for the same indication in ⁎ Corresponding author. Bioanalytical Core Laboratory, Georgetown Clinical Research Center, GM-12A Preclinical Science Building, 3900 Reservoir Road, NW, Washington, DC 20007-2197, USA. Tel.: +1 202 687 8038. E-mail address: [email protected] (S.J. Soldin). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.06.022

children with epilepsy 4 years of age and older. Structurally and mechanistically unrelated to other traditional AEDs, levetiracetam possesses several properties that make it an almost ideal second-line antiepileptic drug in a multidrug therapy in the treatment of partial onset seizures. It has linear and time-invariant pharmacokinetic profile, minimal protein-binding, insignificant hepatic metabolism that produces few drug–drug interactions, and inactive, renally excretable metabolites [1]. Although the drug has a very wide therapeutic index and efficacy in the treatment of a variety of seizure types, it is still worthwhile and advisable to monitor the plasma concentrations of levetiracetam to achieve the optimum therapeutic effect while reducing adverse effects, especially for patients with renal impairment, for elderly

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Table 1 Parameters

2.2. Chemicals and solutions Value

(a) Tandem mass spectrometer main working parameters for API-3000 Nebulizer gas 8 7000 Turbo ionspray gas (cm3/min) Curtain gas 10 Collision gas 3 Ionspray voltage (V) 5000 Probe temperature (°C) 500 Dwell time per transition (ms) 200 (b) Tandem mass spectrometer main working parameters for API-4000 Ion source gas 1 50 Ion source gas 2 20 Curtain gas 20 Collision gas 6 Ionspray voltage (V) 5500 Probe temperature (°C) 500 Dwell time per transition (ms) 200

patients with decreasing renal function, for pediatric patients with higher apparent clearance of levetiracetam than in adults and to assess patient adherence. A number of excellent reviews of levetiracetam have been recently published [1–6] regarding its pharmacokinetics, pharmacodynamics, efficacy, and tolerability. There have also been several papers describing the measurement of levetiracetam in human plasma via gas chromatography-mass spectrometry [7], capillary electrophoresis [8], or HPLC with UV detector [9–15]. Tandem mass spectrometry (MS/MS) is being increasingly used to detect and quantify drugs and endogenous compounds due to its high specificity, sensitivity, and simultaneous multianalyte analyzing ability. The objective of this study was to develop a simple and rapid assay for the accurate determination of levetiracetam in human fluids employing multiple reaction monitoring (MRM) in tandem mass spectrometry. 2. Materials and methods 2.1. Instrumentation An API-3000 or API-4000 tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, California/ Concord, Ontario, Canada) equipped with electrospray ionization (ESI, Applied Biosystems/MDS SCIEX) source was used to perform the analysis. All MS data were collected in the positive ion mode. The high-performance liquid chromatography system consisted of 3 Shimadzu SCL-10vp pumps, Shimadzu SIL-HTa autosampler, and Shimadzu DGU-14A degasser (Shimadzu Scientific Instruments, Columbia, MD). The chromatographic separation of the analyte was performed on a Supelco LC-18-DB (3.3 cm × 3.0 mm, 3 μm particle size) chromatographic column equipped with Supelco Discovery C- 18 (1.0 cm × 3.0, 3 μm particle size) guard column with identical packing material (Supelco, St. Louis, MO) at room temperature. Data were collected and processed by Analyst 1.4.1 software package (SCIEX). The main working parameters are listed in Table 1a–b.

Levetiracetam was obtained from UCB (Smyrna, GA). Twoconcentration-level quality control samples (lyophilized human serum) were from ChromSystems (Munich, Germany). Deionized water was produced using Barnstead B-PURE Water System (Dubuque, IA). HPLC-grade methanol and optimagrade acetonitrile were from Fisher Scientific (Fair Lawn, NJ). Ammonium acetate and acetic acid were from Sigma-Aldrich (St. Louis, MO). A levetiracetam stock solution was prepared by dissolving 10.0 mg of levetiracetam in 10.0 ml methanol and was stored at −20 °C until use. A 0.1 mg/ml working solution was obtained by diluting 1.0 ml of stock solution with water to give a final volume of 10 ml. By spiking drug free human plasma/serum with the working solution, standard solutions were obtained in the concentrations of 1.0, 5.0, 10.0, 25.0, and 50.0 μg/ml, which cover the therapeutic concentrations of levetiracetam in patient samples. Three levels of in-house quality controls of levetiracetam in human plasma were prepared in the same way to evaluate the within-day and between-day precision, as well as the accuracy of the method. A solution of ritonavir at concentration of 20 ng/ml in acetonitrile was used as internal standard. 2.3. Sample preparation To a 1.5 ml conical plastic Eppendorf centrifuge tube, 100 μl of standard, quality control or patient sample containing levetiracetam was mixed with 150 μl of 20 ng/ml ritonavir solution in acetonitrile to precipitate proteins. The tube was capped, vortexed vigorously for at least 30 s, and the precipitated proteins were separated by centrifugation at 13,000 rpm for 10 min. One hundred microliters of supernatant in the centrifuge tube was transferred into and mixed with 300 μl water in an autosampler vial. Addition of water decreases the acetonitrile concentration in the supernatant in order to reduce the elutropic effect caused by the acetonitrile-rich supernatant. It also allows us to avoid the use of the insert for the autosampler vial. A 10-μl aliquot of the mixture was injected into the LC-MS-MS system. Sample preparation was performed at room temperature. 2.4. LC-MS-MS procedure The procedure is based on an online extraction/cleaning of the injected sample with subsequent introduction into the mass spectrometer by using a switching valve. Aliquots of 10 μl were Table 2 HPLC timetable

Cleaning (1.0 ml/min) Elution (1.0 ml/min)

Time (min)

Solvent A (%)

Solvent B (%)

0.00 2.50 2.51 5.00

100 100 0 0

0 0 100 100

Solvent A, a mixture of 15 mmol/l ammonium acetate and methanol (98:2, v/v), 0.1% acetic acid. Solvent B: a mixture of 15 mmol/l ammonium acetate and methanol (3:97, v/v), 0.1% acetic acid.

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117

Fig. 1. LC-MS/MS MRM chromatograms of a plasma sample. Levetiracetam m/z 171→126; ritonavir (IS) m/z 721→296. The concentration for levetiracetam in the patient sample is 1.0 μg/ml, and the ritonavir (IS) 20 ng/ml).

injected by the autosampler onto a Supelco LC-18-DB column coupled with Supelco Discovery C-18 guard column. The drug and internal standard adhered to the column, which was washed with loading buffer A (2% methanol in 15 mmol/l ammonium acetate (v/v), 0.1% acetic acid) at a flow rate of 1.0 ml/min. After 2.5 min of washing, the switch valve (VICI, Valco Instruments Co Inc, Houston, TX) was activated, and the column was eluted with solvent B (97% methanol in 15 mmol/ l ammonium acetate (v/v), 0.1% acetic acid) at a flow rate of 1.0 ml/min (Table 2). The eluted sample was introduced into the ion source without splitting. The chromatographic separation is shown in Fig. 1. Detection and quantitation were carried out by ESI-MS/MS in MRM mode monitoring the transitions m/z 171.0→126.0 for levetiracetam and m/z 721.3→296.1 for ritonavir (IS). The compound-dependent parameters are listed in Table 3. Calibration, using internal standardization, was done by linear regression analysis. Peak area ratio between target analyte and its internal standard was used for quantitation. 2.5. Accuracy, imprecision and specificity The accuracy of the method was evaluated by: (a) comparison with the results from external quality control samples obtained from ChromSystems and (b) performing recovery studies. Imprecision was assessed by assaying in-house controls at 3 Table 3 MRM transitions and compound-dependent parameters Analyte

MRM transition

Instrument

DP

EP

CE

CXP

Levetiracetam

171.2/126.1

Ritonavir (IS)

721.3/296.1

API-3000 API-4000 API-3000 API-4000

10 30 50 30

6 10 10 10

20 21 28 28

6 6 15 15

concentration levels in replicates. In-house controls were prepared in the same way as the standards. Specificity was assessed by injecting a sample of CAP PT control material containing therapeutic concentrations of acetaminophen, salicylate, carbamazepine, phenobarbital, phenytoin, valproic acid, digoxin, lithium, theophylline, caffeine, gentamicin, tobramycin, amikacin, vancomycin and methotrexate. The method was compared to results from the Quest Diagnostics/Chantilly tandem mass spectrometric procedure. 3. Results and discussion Due to the high specificity of MRM mode offered by the tandem mass spectrometry, no matrix interference was observed at the retention time (2.90 min) of the analyte under the isocratic conditions employed in this method. The sensitivity of the method is more than satisfactory since concentrations of levetiracetam in plasma usually range from 2 to 25 μg/ml [2]. With the simple sample preparation and 10 μl injection of the diluted supernatant of the sample, the MS/MS-based method can still easily reach lower limit of detection of 0.1 μg/ml (extrapolated from the Table 4 Tandem MS within-day and between-day imprecision Sample Between-day Level 1 Level 2 Level 3 Within-day Level 1 Level 2 Level 3

Replicates (n)

Measured mean (μg/ml)

CV (%)

20 20 10

2.16 12.9 25.2

8.2 6.2 5.3

10 10 10

2.1 12.7 25.3

6.1 5.2 4.9

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Table 5 Recovery study

Table 7 Comparison with ChromSystems quality control samples

Sample

Replicates (n)

Target value (μg/ml)

Measured mean (μg/ml)

Recovery (%)

Sample

Replicates (n)

Target value (μg/ml)

Measured mean (μg/ml)

CV (%)

Level 1 Level 2

10 10

2.0 12.5

2.16 12.88

108 103

Level 1 Level 2

12 12

8.54 51.1

8.79 52.1

6.5 5.1

reading of 3 S.D. over the baseline noise). The within-day and between-day imprecision at three levels are shown in Table 4. Between-day results (replicate n = 20) gave a CVof 8.2% at a low level, 6.2% at the mid level, and 5.3% at the high level, respectively. The mean recovery for levetiracetam was found to be 108% at the low concentration level, and 103% at high concentration level (Table 5). The method was employed to measure the concentration of levetiracetam in patient's serum or saliva samples, and results from a typical patient are listed in Table 6. In this case the levetiracetam concentrations in saliva had a slightly lower value compared with that of serum when the samples were collected at almost the same time point. Bigger discrepancies were sometimes noted for other samples. Levetiracetam is not strongly bound to serum proteins. This varies between individuals with saliva concentrations sometimes greater than simultaneously drawn serum samples. The procedure has been employed for over 1 year during which time we measured N 600 patient samples. Our method was found to be reliable, reproducible and accurate as attested by the comparison of our results with the target values provided by ChromSystems (see Table 7). None of the drugs present in the CAP PT sample interfered in the method. Finally, our method compared well with the tandem mass spectrometric procedure used by Quest Diagnostics/Chantilly. Correlation coefficient = 0.983, linear regression equation, Quest = 0.91 Georgetown + 2.3, n = 20. In summary, a simple and rapid HPLC-ESI-MS/MS based method for the determination of levetiracetam in human plasma/ serum or saliva samples was developed and validated. This method requires as little as 50 μl of patient sample and minimal sample preparation — protein removal by acetonitrile. The high specificity and sensitivity offered by MS/MS running in MRM mode eliminate the possible interferences from other drugs and normal plasma constituents. The total run time including the washing step is 5 min/sample, making this method suitable for high-throughput environment such as clinical or reference laboratories. Acknowledgements This study has been supported in part by NIH/NINDS RO1NS045656. It was also supported in part by a grant M01RR13297 from the General Clinical Research Center Program of

Table 6 Exemplar levetiracetam concentrations of patient serum/saliva samples taken at different time points Sample_time point

Measured value (μg/ml)

Sample_time point

Measured value (μg/ml)

Serum_7:37 am Serum_8:07 am

5.7 30.7

Saliva_7:40 am Saliva_8:10 am

5.6 27.9

the National Center for Research Resources, National Institutes of Health, Department of Health and Human Services, Bethesda, MD. It was supported in part by grant 1 U10HD45993-02 of the National Institute of Child Health and Development, Bethesda, MD. References [1] Patsalos PN. Pharmacokinetic profile of levetiracetam: toward ideal characteristics. Pharmacol Ther 2000;85:77–85. [2] Perucca E, Bialer M. The clinical pharmacokinetics of the newer antiepileptic drugs: focus on topiromate, levetiracetam. Relative bioavailability and bioequivalence of a 10% oral solution (750 mg). Clin Pharmacokinet 1996;31:29–46. [3] Coupez R, Straetemans R, Sehgal G, Stockis A, Lu Z(S). Levetiracetam: relative bioavailability and bioequivalence of a 10% oral solution (750 mg) and 750-mg tablets. J Clin Pharm 2003;43:1370–6. [4] Cramer JA, De Rue K, Devinsky O, Edrich P, Trimble MR. A systematic review of the behavioral effects of levetiracetam in adults with epilepsy, cognitive disorders, or an anxiety disorder during clinical trials. Epilepsy Behav 2003;4:124–32. [5] Devinsky O, Elger C. Efficacy of levetiracetam in partial seizures. Epileptic Disord 2003;5(Suppl 1):S27–31. [6] Yerby MS. New antiepileptic drugs for the treatment of epilepsy. http:// www.seizures.net/articles_epilepsy/New_AED3_03.html. [7] Isoherranen N, Roeder M, Soback S, Yagen B, Schurig V, Bialer M. Enantioselective analysis of levetiracetam and its enantiomer R-alphaethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection. J Chromatogr B 2000;745:325–32. [8] Shihabi ZK, Oles K, Hinsdale M. Analysis of the antiepileptic drug Keppra by capillary electrophoresis. J Chromatogr A 2003;1004:9–12. [9] Vermeij TA, Edelbroek PM. High-performance liquid chromatographic and megabore gas–liquid chromatographic determination of levetiracetam (ucb L059) in human serum after solid-phase extraction. J Chromatogr B 1994;662:134–9. [10] Ratnaraj N, Doheny HC, Patsalos PN. A micromethod for the determination of the new antiepileptic drug levetiracetam (ucb L059) in serum or plasma by high performance liquid chromatography. Ther Drug Monit 1996;18:154–7. [11] Grim SA, Ryan M, Miles MV, et al. Correlation of levetiracetam concentrations between serum and saliva. Ther Drug Monit 2003;25:61–6. [12] Pucci V, Bugamelli F, Mandrioli R, Ferranti A, Kenndler E, Raggi MA. High-performance liquid chromatographic determination of levetiracetam in human plasma: comparison of different sample clean-up procedures. Biomed Chromatogr 2004;18:37–44. [13] Rao BM, Ravi R, Shyam Sundar Reddy B, et al. A validated chiral LC method for the enantioselective analysis of levetiracetam and its enantiomer R-alpha-ethyl-2-oxo-pyrrolidine acetamide on amylose-based stationary phase. J Pharm Biomed Anal 2004;35:1017–26. [14] Martens-Lobenhoffer J, Bode-Boger SM. Determination of levetiracetam in human plasma with minimal sample pretreatment. J Chromatogr B 2005;819:197–200. [15] Juenke J, Brown PI, Urry FM, McMillin GA. Drug monitoring and toxicology: a procedure for the monitoring of levetiracetam and zonisamide by HPLC–UV. J Anal Toxicol 2006;30:27–30.