plasma by liquid chromatography tandem mass spectrometry

plasma by liquid chromatography tandem mass spectrometry

Clinical Biochemistry 43 (2010) 485–489 Contents lists available at ScienceDirect Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s...

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Clinical Biochemistry 43 (2010) 485–489

Contents lists available at ScienceDirect

Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m

Simultaneous determination of Levetiracetam and its acid metabolite (ucb L057) in serum/plasma by liquid chromatography tandem mass spectrometry Damodara Rao Mendu a, Steven J. Soldin a,b,⁎ a b

Department of Medicine, Bioanalytical Core Laboratory, Georgetown University, Washington, DC, USA Department of Pharmacology, Bioanalytical Core Laboratory, Georgetown University, Washington, DC, USA

a r t i c l e

i n f o

Article history: Received 3 August 2009 Received in revised form 9 November 2009 Accepted 13 November 2009 Available online 23 November 2009 Keywords: Levetiracetam Levetiracetam metabolite antiepileptic drug Tandem mass spectrometry

a b s t r a c t Objective: Levetiracetam and its acid metabolite have almost identical MRMs. They therefore need to be separated chromatographically prior to quantitation. Research design and methods: The sample is deproteinized with acetonitrile containing Ritonavir as internal standard, centrifuged and the supernatant diluted with water (1:2 v/v). Sixty microliters of the supernatant is injected into the LC–MS/MS and Levetiracetam (LEV) and LEV metabolite separated chromatographically at room temperature employing a Supelco C18 column and a 0.1% formic acid methanol gradient at pH of 2.5. Results: The retention times for LEV metabolite, LEV and Ritonavir were 4.50, 5.38 and 9.18 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 0–50 μg/mL for LEV and 0.0–5.0 µg/mL for LEV metabolite. Intra- and inter-run imprecision (n = 10) gave CVs of 2.3– 4.7%, 3.4–8.9% for LEV and 2.9–3.9%, 3.3–7.4% for LEV metabolite. Recoveries of both LEV and LEV metabolite were close to 100%. Results for LEV were compared with those obtained by a commercial reference laboratory (r = 0.974). Conclusion: The procedure is reliable, quick, and inexpensive. LEV and LEV metabolite co-elute using C-18 columns at pHs N 3.0 and previously published methods employing these conditions could therefore be subject to metabolite interference. In this method LEV and LEV metabolite are separated at pH 2.5. The total run time including the washing step is 10 min/sample, making this method suitable when moderate throughput is needed such as in clinical or commercial reference laboratories. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction Levetiracetam (Keppra, (S)-α-ethyl-2-oxo-1-pyrrolidine acetamide) is an antiepileptic medication with efficacy, tolerability, and pharmacokinetic characteristics that make it potentially useful for infants, young children and adults with epilepsy [1–5]. After oral dosing of Levetiracetam in humans, 2 major components are identified in urine: unchanged Levetiracetam (66% of the dose) and its acid metabolite ucb L057 (24% of the dose) [6–8]. The major metabolite ucb L057 (LEV metabolite) is an acidic substance produced by hydrolysis of the acetamide group of Levetiracetam (LEV). Several chromatographic assays have been reported for the measurement of LEV in biological fluids. These involve gas

Abbreviations: MRM, multiple reaction monitoring; LEV, Levetiracetam; MS/MS, tandem mass spectrometry. ⁎ Corresponding author. Bioanalytical Core Laboratory, Room GM12A, Preclinical Science Building, Georgetown University, 3900 Reservoir Road NW, Washington, DC 20007, USA. E-mail address: [email protected] (S.J. Soldin).

chromatography (GC) with nitrogen–phosphorus detection, high performance liquid chromatography (HPLC) and GC–MS [9–15]. Most of these reported methods lack selectivity, sensitivity, and reliability. They are frequently tedious, time-consuming and require a large sample volume. Three methods are available for analysis of LEV using tandem mass spectrometry [16–18]. Two of these use solid phase extraction, which is cumbersome and tedious. However, those procedures do not afford the simultaneous measurement of LEV and its metabolite. A few methods facilitating chiral separation of the S- and R-enantiomers of Levetiracetam utilizing GC–MS were published [19–21] and there are 2 published methods for the determination of ucb L057 by LC/ESI/MS using single ion monitoring (SIM) in urine and plasma samples [22–23]. Employing a single quadrupole and SIM lacks the sophistication and specificity afforded by tandem mass spectrometry. LEV and LEV metabolite cannot be quantified separately if they co-elute due to similar Q1 and Q3 ions and molecular weights that differ by only 1 amu. We found that co-elution occurred unless we used a mobile phase with a pH close to 2.5. The presence of metabolite would therefore lead to inaccurate measurement of the parent drug due to their co-elution at higher pHs. Periodic plasma

0009-9120/$ – see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.11.008

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Table 1 (A) Gradient parameters for LEV and LEV metabolite (Solvent A: 0.1% formic acid in 2% methanol, Solvent B: 0.1% formic acid in 100% methanol) Time (min)

Solvent A (%)

0.0 2.5

100 100

Assay procedure

Elution

2.6 9.0 10.5

concentration of 50 ng/mL in acetonitrile was used as internal standard (IS).

Solvent A (%)

Solvent B (%)

97 0 0

3 100 100

(B) MRM transition ions and compound-dependent parameters Analyte

MRM transition

Declustering potential (V)

Focusing potential (V)

Collusion Collision cell exit energy potential (V) (V)

LEV LEV Metabolite Ritonavir

171→126 172.5→126 721→296.2

61 60 96

100 200 200

20 37 28

8 10 22

level monitoring is very important both for successful therapy and for evaluating potential drug interactions and adverse effects of LEV. The objective of the present report was to develop and validate a rapid, reliable, and accurate LC–electrospray MS/MS method for the simultaneous determination of LEV and its metabolite ucb L057 in human plasma/serum. The present method has been successfully utilized in therapeutic drug monitoring of LEV and ucb L057 by analysis of plasma samples of patients treated with LEV for seizures. This is the first report describing the simultaneous measurement of both the analytes in plasma/serum.

The chromatographic system, Shimadzu (Shimadzu Scientific Instruments, Columbia, MD, USA), consisted of LC-10ADvp pumps (three), autoinjector (SIL-HTA), and a degasser (DGU-14A). Separation of the analytes was performed on a Supelco C18 column (3 μm, 3.3 cm × 3 mm). The mobile phase consisted of a methanol– 0.1% formic acid gradient (Table 1A) used at a flow rate of 1 mL/ min and an electrospray ionization interface (ESI) used in the positive mode coupled to an API 3000 tandem triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). Ammonium acetate buffers were avoided as they have pHs N3.0 resulting in co-elution of LEV and LEV metabolite. Tuning parameters of MS/ MS were optimized by direct infusion of solutions of LEV, LEV metabolite (ucb L057) and internal standard (IS) Ritonavir in 100% methanol containing 0.1% formic acid at a flow rate of 10 μL/min using a syringe pump. The MRM transitions m/z 171→126, 172.5→126 and 721→296.2 were selected for quantification of LEV, LEV metabolite and IS, respectively. Ultra-pure nitrogen gas was used as curtain and collision gas. The main working parameters of the mass spectrometer were: collision gas 6, curtain gas 10, nebulizer gas 8, ion spray voltage 5500 V, probe temperature 550 °C, and dwell time 150 ms. The compound dependent parameters were summarized in Table 1B. The data were processed by employing Analyst 1.4.2 version.

Materials and methods Chemicals and reagents Levetiracetam and ucb L057 were supplied by UCB Pharma (Brussels, Belgium). The internal standard (IS), Ritonavir, was purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Water was purified using a Millipore Synergy water device (Millipore, Bedford, MA, USA). All other chemicals and reagents were of analytical grade and solvents were of HPLC grade. Stock standards, calibration standards and quality control samples Stock solutions of LEV and LEV metabolite (99.5% purity) were prepared separately by dissolving 10 mg of each powder in 10 mL methanol. This stock solution (1.0 mg/mL) was further diluted in methanol to yield a working standard solution of 100 μg/mL for LEV and LEV metabolite and then stored at -80 °C. Calibration standards containing both LEV and LEV metabolite were prepared by spiking drug-free human plasma with LEV working solution at concentrations of 0.0, 1.0, 5.0, 10.0, 25.0, 50.0 μg/mL and also with LEV metabolite at concentrations of 0, 1.0, 2.0, 3.0, 4.0, 5.0 μg/mL. The quality control (QC) samples were prepared in drug-free human plasma at concentrations of 1.0 and 4.0 μg/mL for LEV metabolite. The quality controls for LEV were obtained from Chromsystems (Munich, Germany) at a concentration of 12.5, 25.0 and 50.0 μg/mL. The standards and quality control samples were aliquoted into Eppendorf polypropylene tubes and kept frozen at -80 °C. The within-day and between-day imprecisions of the method were evaluated by using these quality controls (n = 10). A solution of Ritonavir at a

Fig. 1. Chromatograms shown are from a patient plasma sample. The concentrations found were: LEV, 19.8 μg/mL and LEV metabolite, 3.9 μg/mL.

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Table 2 (A) Within-run and between-run imprecisions for LEV and LEV metabolite controls Level 1

LEV LEV metabolite

Within-run Between-run Within-run Between-run

Level 2

Level 3

n

Mean (μg/mL)

Percent CV

Mean (μg/mL)

Percent CV

Mean (μg/mL)

Percent CV

12.5 12.5 2.0 2.0

4.7 8.9 3.9 7.4

25 25 4.0 4.0

2.8 5.9 2.9 3.3

50 50

2.3 3.4

10 10 10 10

(B) Recovery study Compound name

Control

Replicates (n)

Target value (μg/mL)

Measured mean (μg/mL)

Recovery (%)

LEV

1 2 3 1 2

10 10 10 10 10

10 25 50 2 4

10.08 25.11 49.87 2.03 4.08

100.8 100.4 99.8 101.5 102.1

LEV metabolite

Prior to assay, frozen samples including calibrators, QC samples or patient samples were thawed at ambient temperature and then vortexmixed for 30 s. Protein precipitation was accomplished by mixing 50 μL of the sample (serum/plasma, calibrators or QCs) with 100 μL of acetonitrile containing the IS. The samples were vortexed for 30 s and the precipitated proteins were separated by centrifugation at 10,000 rpm for 10 min. One hundred microliters of the clear supernatant from the centrifuged sample was mixed with 200 μL of water, transferred into autosampler vials and 60 μL injected into the LC–MS/MS. Gradient chromatographic conditions are shown in Table 1A. Chromatography was performed at room temperature with a total run time of 10 min. Imprecision studies QC samples at concentrations of 12.5, 25.0 and 50.0 μg/mL for LEV and different samples with concentrations of 1.0 and 4.0 μg/mL for LEV metabolite were assayed (n = 10) to assess intra- and inter-run imprecision.

Patient samples Patient samples (serum/plasma) were obtained from the commercial reference laboratory and were stored at -80 °C until analysis. All patient identifiers had been removed. Method comparison Results from the newly developed LC–MS/MS assay were compared with those obtained employing the LC–MS/MS method in routine use at the commercial reference laboratory. Fifty patient plasma samples were analyzed by both methods. No method comparison studies were possible for LEV metabolite. LEV results by both methods were compared using linear regression analysis (Graphpad Prism) and Bland–Altman plots to assess bias [24]. Results and discussion Liquid chromatography–tandem mass spectrometry

Linearity The linearity for both analytes was investigated by diluting the high calibrator with calibrator “drug-free plasma” (S0) to give the following concentrations: 0, 1.0, 5.0, 10.0, 25.0, 50.0 μg/mL for LEV and 0, 1.0, 2.0, 3.0, 4.0, 5.0 μg/mL for LEV metabolite.

Fig. 2. Correlation between LEV concentrations in patient samples measured by a commercial reference laboratory LC–MS/MS and the new LC–MS/MS method. The regression equation found is: new LC–MS/MS = 0.905 commercial reference laboratory - 0.08, with Syx = 3.15, r = 0.974, n = 50.

Optimization of the analytical conditions was carried out in a three-step process. First, infusion of the standards was performed in positive ion scan mode to investigate the [M+H]+ ion of LEV (m/z 171 amu) and LEV metabolite (m/z 172.5 amu), and for IS (m/z 721 amu), respectively. The product ion scan mode was used to determine the most abundant product ion for each analyte. Among the product ions the most abundant was the ion at m/z 126 amu for LEV, the ion at

Fig. 3. Bland–Altman plot of the new LC–MS/MS vs. the commercial reference laboratory LC–MS/MS (percent difference between methods versus the mean of both methods).

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m/z 126 amu for LEV metabolite and 296.2 amu for IS. Consequently, the transitions m/z 171→126 (LEV), 172.5→126 (LEV metabolite) and 721→296.2 (IS) amu were chosen for the MRM analytical mode. LC–MS/MS analysis by the MRM mode combined with liquid chromatography separation was performed with a C18 column using a methanol–formic acid linear gradient. Fig. 1 shows representative chromatograms on a plasma sample for LEV, LEV metabolite and IS. Note that the formic acid concentration remains constant throughout the gradient. Different mobile phase compositions were evaluated for chromatographic separation of LEV, LEV metabolite and Ritonavir. The buffer pH was varied to assess the pH effect on elution profile. The pH of the mobile phase was found to markedly affect the retention times of LEV, LEV metabolite and Ritonavir. At pHs between 3.0 and 5.0 (using acetic acid and ammonium acetate isocratic buffer) LEV and LEV metabolite co-eluted. At pH 2.5 (using 0.1% formic acid), LEV metabolite is presumably not ionized (pK value is not available) and consequently could be present as R-COOH, which causes an increase in retention time allowing for the chromatographic separation of LEV and LEV metabolite. The calibration data for LEV and LEV metabolite were obtained and the linearity curves showed a linear response over a wide range of concentrations (for LEV: 1–50 μg/mL, LEV metabolite: 0.1–5.0 μg/mL). The r2 values of the calibration curves for LEV and LEV metabolite in plasma were 0.999 and 0.997, respectively. Due to the high specificity of the MRM mode available when using tandem mass spectrometry, no matrix interference was observed in 20 blank serum and plasma samples. Likewise, recovery studies using plasma or serum showed that either matrix could be used and provided the same result. The retention times for LEV, LEV metabolite and Ritonavir were 4.50 min, 5.38, and 9.17, respectively under the gradient conditions employed in this method. The sensitivity of the method is more than satisfactory since concentrations of LEV in plasma usually range from 2 to 25 μg/mL [25] and the LEV metabolite concentration found was usually between 1 and 5 μg/mL in patients. The LLOD was taken as 3 times the baseline noise. The LLOQ (approximately twice the LLOD) for LEV and LEV metabolite were found to be 0.01 μg/mL and 0.03 μg/mL, respectively. At these concentrations measurement of both LEV and LEV metabolite have CVs of approximately 12%. LEV and LEV metabolite are stable in both serum and plasma for many months when frozen at -80 °C or -20 °C. Stability at 4 °C was at least 48 h. Samples were stored in plastic vials and serum or heparinized plasma frozen at -80 °C. Within-day and between-day imprecisions were evaluated employing 3 different controls for LEV and 2 different controls for LEV metabolite (n = 10, see Table 2). The resulting CVs were acceptable at all concentrations studied. The results employing our new LC–MS/MS procedure were compared with those obtained at a commercial reference laboratory using their tandem MS/MS method and are shown in Fig. 2. We could not report comparisons for LEV metabolite since no comparative method existed. The Bland–Altman plots shown in Fig. 3 reveal that the commercial reference laboratory method showed a positive bias of 9.3%, which could in part be due to coelution and quantification of the LEV metabolite in the commercial laboratory method. Higher concentrations of the metabolite may be found when LEV concentrations are N20 μg/mL or when samples are left on the red cells too long allowing hydrolysis of the parent drug to the metabolite as demonstrated by Patsalos et al. [26]. The major metabolic pathway is the enzymatic hydrolysis of the acetamide group, which produces the carboxylic acid metabolite, ucb L057 (24% of dose) and is not dependent on liver cytochrome P-450 isoenzymes. Significantly, LEV metabolite ucb L057 is not pharmacologically active and therefore separation of drug from metabolite is important [7,27–28]. The LEV and LEV metabolite percent ratios

Table 3 Mean percent ratio of LEV metabolite/LEV in 50 patient samples. Parameter

LEV (μg/mL)

LEV metabolite (μg/mL)

Percent ratio of LEV metabolite/LEV

Mean Percent CV

18.3 70.0

1.7 100.7

9.1 71.2

are also shown in Table 3 and show a mean concentration of 9.1% of the parent drug. The correlation coefficient (r = 0.974, Fig. 2) is good and there is a mean percent bias of 9.3% (commercial reference laboratory gives results 9.3% higher than our new method, Fig. 3). As the MRMs of LEV and LEV metabolite are similar but not identical, we have also assessed the LEV metabolite effect on a sample containing a low LEV concentration. The sample was assayed before and after addition of 5 μg/mL of LEV metabolite. The concentration measured by the commercial laboratory increased from 1.0 μg/mL prior to addition to 2.7 μg/mL post-addition. Our new method revealed no interference in the LEV level, which was found to be 0.3 μg/mL both before and after addition of 5 μg/mL LEV metabolite. So in this case the commercial reference laboratory provided LEV results, which were clearly affected by LEV metabolite concentrations. Finally Table 3 shows that in the 50 patient samples studied the mean LEV metabolite concentration found is 9.1% of the LEV concentration measured. Conclusions We describe a new LC–MS/MS method for the simultaneous measurement of LEV and LEV metabolite in human plasma. This method requires 50 μL of patient sample and minimal sample preparation. The high specificity and sensitivity offered by MS/MS running in MRM mode eliminate the possible interferences from other drugs and normal plasma constituents. LEV and LEV metabolite are separated chromatographically. This is necessary as they have very similar MRMs. The total run time including the washing step is 10 min/sample, making this method suitable when moderate throughput is needed such as in clinical or commercial reference laboratories. Separation of LEV and LEV metabolite is important as the latter is not pharmacologically active and therefore should not be included in LEV measurement. In summary current LC–MS/MS methods used in many laboratories provide results for LEV, which contain a small and variable contribution from LEV metabolite. Acknowledgments Dr. S.J. Soldin is supported in part by Grant M01-RR13297 from the General Clinical Research Center Program of the National Center for Research Resources, National Institutes of Health, Department of Health and Human Services, Bethesda, MD. References [1] Hovinga CA. Levetiracetam: a novel antiepileptic drug. Pharmacotherapy 2001;21: 1375–88. [2] Cereghino JJ, Biton V, Abou-Khalil B, Dreifuss F, Gauer LJ, Leppik I. Levetiracetam for partial seizures: results of a double-blind, randomized clinical trial. Neurology 2000;55:236–42. [3] Shorvon S, Löwenthal A, Janz D, Bileen E, Loiseau P. Multicenter double-blind, randomized, placebo-controlled trial of Levetiracetam as add-on therapy in patients with refractory partial seizures. Epilepsia 2000;41:1179–86. [4] Ben-Menachem E, Falter U. Efficacy and tolerability of Levetiracetam 3000 mg/day in patients with refractory partial seizures: a multicenter, double-blind, responder-selected study evaluating monotherapy. Epilepsia 2000;41:1276–83. [5] Betts T, Waegemans T, Crawford P. A multicentre, double-blind, randomized, parallel group study to evaluate the tolerability and efficacy of two oral doses of Levetiracetam, 2000 mg daily and 4000 mg daily, without titration in patients with refractory epilepsy. Seizure 2000;9:80–7.

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