Development and Validation of an HPLC Method for Analysis of Levetiracetam in Human Plasma
G.V.H. Raju*, S. Ganapathy, D.G. Sankar and P.Y. Naidu
Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530003
*Corresponding Author E-mail: gadepalli_raj@yahoo.com
ABSTRACT:
A simple Reverse Phase liquid chromatographic(HPLC) method for the determination of Levetiracetam in human plasma has been developed. Separation was achieved on a Prontosil C8 (150 x 4.6 mm, 5 µm) column with mobile phase composition of [pH 3.8 buffer: acetonitrile] (Solution A): HPLC grade water (Solution B) in the ratio 60:40 v/v with ultra violet detection at 210nm. Lower limit of quantitation was 200ng/ml.Maximum between-run precision was 5.91%. Mean extraction recovery was found to be 100.02 to 103.06%. Stability study showed that after three freeze-thaw cycles the loss of three quality control samples were less than 10%. Samples were stable at room temperature for 48h and at -20° for 2months. Before injecting onto HPLC system, the processed samples were stable for at least 6h.The method was used to perform bioequivalence study in human.
Levetiracetam is a anticonvulsant drug used in the treatment of epilepsy. It is S-enantiomer of etiracetam, structurally similar to the prototypical nootropic drug piracetam. Along with other anticonvulsants like gabapentin it is also sometimes used in the treatment of neuropathic pain Levetiracetam is a white to off-white crystalline powder with a faint odor and a bitter taste. It is very soluble in water. It is freely soluble in chloroform and in methanol, soluble in ethanol, sparingly soluble in acetonitrile and practically insoluble in n-hexane.
It is official in US pharmacopoeia and few liquid chromatography procedures have been reported for the determination of Levetriacetam and their metabolites in biological fluids. Three of them employed HPLC with UV-detection, and two methods were using GC with NPD-detection. Microemulsion electrokinetic chromatography with UV-detection was utilized in one method, but it lacks suitable sensitivity. Two methods facilitating chiral separation of the S- and Renantiomer of Levetiracetam, one utilizing GC–MS and the other HPLC–UV, were published recently. These methods were designed to investigate in dogs the pharmacokinetic and pharmacodynamic properties of the two enantiomers separately14-16. For routine therapeutic drug monitoring in men, these methods were not appropriate. We here present very simple as well as sensitive new HPLC method for the determination of Levetiracetam in human serum using UV detector which utilizes SPE technique as sample preparation. Separation of analyte from endogenous substances was achieved through Prontosil C8 (150 x 4.6 mm, 5 µm) column. This type of column leads to better retention, very sharp and symmetrical peak shapes and exhibits a very good selectivity for Levetiracetam. The method was validated according to procedures and acceptance criteria based on FDA guidelines and recommendations of ICH.
2.0 EXPERIMENTAL:
2.1 Instrumentation : The waters LC system equipped with 2489 pump and 2487 UV detector was used .The output signal was monitored and integrated using waters Empower 2 software
2.2 Solutions:
2.2.1 Preparation of pH 3.8 buffer solution:
Dissolve 1.36g of Potassium Dihydrogen phosphate (KH2PO4) in 1000mL of milli-Q water and mix well, pH of the solution was adjusted to 3.8±0.05 with ortho Phosphoric acid.
2.2.2 Preparation of Solution A:
A mixture of pH 3.8 buffer, Acetonitrile in the ratio 90:10(v/v) was prepared and filtered through 0.45µm nylon membrane filter prior to use.
2.2.3 Preparation of Solution B:
Milli Q water
2.2.4 Preparation of Mobile phase:
A mixture of Solution A and Solution B in the ratio 60:40(v/v) was prepared and filtered through 0.45µm nylon membrane filter prior to use.
2.2.5 Preparation of 0.1% Ammonia in water : 0.1ml of Ammonia is dissolved in 100ml of water and mixed well
2.2.6 Preparation of Standard Stock solution(5mg/ml): About 50mg of Levetiracetam(LEVI) working standard was accurately weighed and transferred in to a 10mL volumetric flask and dissolved in 5ml of methanol sonicated to dissolve the material and made up to volume with methanol and mixed well.
2.2.7 Preparation of Internal Standard solution(2mg/ml): 10mg of Levetiracetam Impurity B(4-{[1-(aminocarbonyl)propyl]amino}butanoyl chloride) was accurately weighed and transferred into 5ml volumetric flask and dissolved in 2ml of methanol and made up to volume with water and mixed well.
2.2.8 Preparation of Sample: sample was prepared by using Solid phase extraction technique.25µl of internal standard solution was admixed with 0.5ml of sample in ria vial to it 0.5ml of 0.1% Ammonia water is added. 1ml of this premixed sample was loaded onto each strata extraction cartridge which was kept for equilibration .After loading sample on to cartridge, cartridge was washed with water, OPA in 5% methanol and 10% methanol and drained out by applying vacuum. Finally the sample was collected in to a suitable vessel by selecting methanol as eluent.
2.2.9 Chromatographic Conditions:
A Prontosil C8( 150 x 4.6 mm, 5 µm packing) column was used for analysis at column temperature 25ºC .The mobile phase was pumped through the column at a flow rate of 1.0mL/min.The Sample injection Volume was 20µL.The UV detector was set to a wavelength of 210 nm for the detection.
2.3 Method development:10-11
To develop a suitable and robust LC method for the determination of Levetiracetam in plasma different mobile phases and columns were employed to achieve the best separation and resolution. The method development was started with a C18 column using a mobile phase of pH 2.8 buffer: Acetonitrile: Methanol in the ratio 75:10:15.In the above condition elution was very broad for Levetiracetam. Early elution with little separation was observed with mobile phase consisting of pH 3.0 buffer: Acetonitrile: Methanol in the ratio 60:25:15. Finally the mobile phase consisting 60% of pH 3.8 buffer: Acetonitrile and 40% of water was found to be appropriate, allowing good separation and symmetrical peak at a flow rate of 1.0mL/min using Prontosil, C8 (150 x 4.6 mm, 5 µm packing) column. The Chromatogram of Levetiracetam sample spiked in plasma using the proposed method is shown in Fig. 2.In the proposed method the resolution is more than 2 between the Levetiracetam and internal standard. Levetiracetam and its related compounds show significant UV absorbance at Wavelength 210 nm .Hence this wavelength has been chosen for detection in the analysis of Levetiracetam.
2.3.1 Column Selection:
Based on the retention time and separation Prontosil, C8 (150 x 4.6 mm, 5 µm packing) column was selected as suitable column for the analysis of Levetiracetam in Human plasma.
3.0 Method Validation:12-13
The developed bioanalytical method for determination of Levetiracetam in Human plasma was Extensively validated as per FDA guidelines and recommendations of ICH. Stock solutions of Levetiracetam and internal standard were prepared in methanol and kept at -20°. Working solutions were prepared by diluting the stock solutions of standard with methanol. For calibration curve eleven different concentrations (0.2 µg/ml, 0.5 µg/ml, , 0.6 µg/ml ,1 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 30 µg/ml , 40 µg/ml, 50 µg/ml )in plasma were prepared by adding required volume of working solution of analyte to blank plasma. For internal standard the final concentration in plasma was 25µg/ml. The plasma samples were subjected to the sample preparation procedure and injected onto HPLC. Plasma calibration curve was prepared by taking area ratio of analyte to internal standard as Y-axis and concentration of analyte (µg/ml) as X-axis. Six replicates of calibration curve were prepared taking each concentration for six times.
3.1 Extraction recovery:
The extraction recovery of analyte was determined by measuring the peak areas of the drug from the prepared plasma quality control samples. 0.6 µg/ml, 10 µg/ml and 30 µg/ml plasma samples were taken as LQC (low quality control), MQC (medium quality control) and HQC (high quality control) samples respectively. The peak areas of extracted LQC, MQC and HQC were compared to the absolute peak area of the unextracted samples containing the same concentration of the drug as 100%. To obtain good extraction efficiency the extraction recovery of Levetiracetam was determined using five replicates of each QC samples.
3.2 Accuracy and precision:
Precision and accuracy were also determined from LQC (0.6 µg/ml), MQC (10 µg/ml) and HQC (30 µg/ml). Five replicates of each concentration were analyzed on the same day to determine the within-run accuracy and precision of the method. To confirm the between-run accuracy and precision five replicates of each concentration on the first day and four replicates of each concentration on second day and third day were analyzed.
3.3 Stability study:
The stability of Levetiracetam in plasma was evaluated with four studies; a short-term stability study, a long-term stability study, a freeze thaw study and stability in processed sample. Plasma blank samples were spiked with Levetiracetam at concentration of 0.6 µg/ml (LQC), 10 µg/ml (MQC) and 30 µg/ml (HQC) and each concentration was carried out for five times. Plasma samples were extracted and subsequent HPLC analysis was carried out as described previously.
Short-term stability test was performed at room temperature. Plasma samples spiked with Levetiracetam were kept at room temperature for 6 h, extracted and then analyzed. The long term stability study was carried out with plasma blank samples spiked with Levetiracetam, which were stored -20° and they were analyzed periodically 2months against a standard curve prepared on the analysis day. For freeze thaw stability spiked samples were analyzed immediately after preparation and on a daily basis after repeated freeze thaw cycles at -20° on three consecutive days. Finally the stability in the processed sample ready for injection was determined at two level of concentration, 0.6 µg/ml (LQC) and 30 µg/ml (HQC). The processed QC samples ready for injection were kept for 6h before HPLC analysis.
3.4 The Lower limit of quantitation (LLOQ) and limit of detection (LOD):
The Lower limit of quantitation (LLOQ) and Limit of detection (LOD) were determined from the peak signal and noise level (S/N) as ten and three times the baseline noise, respectively.
3.5 Pharmacokinetic parameters:
The above mentioned bioanalytical method was used in bioequivalence study of Levetiracetam. It was an open, randomized crossover study to assess relative bioavailability of Levetiracetam in twelve healthy male volunteers following single dose administration of Levetiracetam 500 mg tablet. Test preparation was Levetiracetam 500 mg tablet manufactured by Orchid Healthcare. Tablet Keppra containing 500mg of Levetiracetam, was used as Reference preparation. The pharmacokinetic parameters like area under the plasma-concentration–time curve from time zero to the last measurable Levetiracetam sample time and to infinity (AUC0-t and AUC0-inf), maximum concentration (Cmax), time to maximum concentration (Tmax), elimination rate constant (Kel) and elimination half-life (t1/2) were determined for the period of 0 to 36h.
RESULTS AND DISCUSSION:
Representative chromatogram of blank plasma and plasma spiked with Levetiracetam and internal standard are shown in figs. 1 and 2, respectively. Retention time for the Levetiracetam and internal standard were 8.189 min and 12.099 min, respectively. No interfering peaks at these times were found in the chromatogram obtained from blank plasma. Good separation and baselines with low background noise were observed. The chromatographic run time was 18minutes for plasma sample analysis. The linearity of the calibration curve was evaluated by calculating the r2 (regression coefficient) value. The standard curves of Levetiracetam in human plasma were linear over the ranges of 0.2 to 50 µg/ml and the regression coefficients (r2) were over 0.998 from each standard curve of six separate runs. The limit of detection defined as three times the base noise was 0.1 µg/ml for this analytical method and the lower limit of quantitation defined as ten times the base noise was 0.2 µg/ml.
Fig. 1: Representative chromatogram of blank plasma
Fig. 2: Representative chromatogram of plasma spiked with Levetiracetam and internal standard.
TABLE 1: WITHIN-RUN ACCURACY AND PRECISION OF THE ANALYTICAL METHOD FOR LEVETIRACETAM
|
Q. C. Samples |
LQC |
MQC |
HQC |
|
Concentration spiked (µg/ml) |
0.6 |
10 |
30 |
|
Concentration found |
0.609 |
10.824 |
29.952 |
|
S.D. |
0.024 |
0.037 |
0.048 |
|
C.V.% |
3.94 |
0.34 |
0.16 |
|
% Nominal |
101.5 |
108.24 |
99.84 |
LQC, MQC, HQC are low quality control, medium quality control, high quality control samples of 150, 900 and 2100 ng/ml. Data obtained are average of six observations (n= 6) where SD means Standard Deviation, CV% (precision) means coefficient of variation calculated as (SD/Mean concentration found)×100 and% nominal means accuracy which is calculated as (Concentration found/ Concentration spiked)×00.
TABLE 2: BETWEEN-RUN ACCURACY AND PRECISION OF THE ANALYTICAL METHOD FOR LEVETIRACETAM
|
Q. C. Samples |
LQC |
MQC |
HQC |
|
Concentration spiked (ng/ml) |
0.6 |
10 |
30 |
|
Concentration found |
0.592 |
10.451 |
29.560 |
|
S.D. |
0.035 |
0.555 |
0.643 |
|
C.V.% |
5.91 |
5.31 |
2.18 |
|
% Nominal |
98.64 |
104.51 |
98.53 |
LQC, MQC, HQC are low quality control, medium quality control, high quality control samples of 150, 900 and 2100 ng/ml. Data obtained are average of 6 observations (n= 6) where SD means Standard Deviation, CV% (precision) means Coefficient of Variation calculated as (SD/Mean concentration found) ×100 and % nominal means accuracy which is calculated as (Concentration found/ Concentration spiked) ×100
Within- and between-run precision and accuracy of the method was assessed by analyzing the QC samples spiked with known amount of Levetiracetam according to the procedure described in the previous section. Results are shown in Table 1 and 2. The accuracy of this bioanalytical method for within- and between-run was from 99.84 to 108.24% and from 98.53 to 104.51%, respectively. The %RSD within- and between-run precision ranged from 0.16 to 3.94% and from 2.18 to 5.91%, respectively.
The recovery of Levetiracetam from plasma was determined in accordance with the method described in the previous section. The recoveries (mean) of Levetiracetam from plasma were found to be 103.06% at 0.6 µg/ml, 101.16% at 10 µg/ ml and 100.02% at 30 µg/ml.
The stability of Levetiracetam in plasma was determined under various conditions according to the procedure described in the earlier section. Short-term stability test performed at room temperature showed that QC samples were stable for 48h (mean recoveries were 99.147%, and 98.977% at LQC and HQC, respectively). The long- term stability results indicated that Levetiracetam samples were stable for a period of 2 months, with an average recovery of 98.883%. No significant decrease of Levetiracetam concentration in plasma was detected after exposing samples to three freeze/thaw cycles and mean recovery was found to be 103.570 and 95.898% at LQC and HQC, respectively. Finally, the stability in the processed sample ready for injection was also determined. Result showed that three QC samples were stable at least for 6h with loss not higher than 10%.
The above mentioned bioanalytical method was used in the plasma analysis of a bioequivalence study of Levetiracetam as described in the earlier section. Fig.3 shows the mean plasma level of Levetiracetam for test and reference preparation after the oral administration of a single dose 500 mg of Levetiracetam in 8 healthy human volunteers. Maximum plasma concentration (Cmax) ranged from 12.463 to 18.354 µg/ml at 0. 5 to 1.25 h (tmax). The half life (t1/2) ranged from 5.633 to 7.317 h. Also the mean value of area under the concentration time curve (AUC0-t) obtained was 146.712 ± 11.655 µg x h/ml and AUC0-inf was found to be 150.029 ± 11.229 µg x h/ml. Relative bioavailability of test preparation was 95.62% to that of reference preparation and both the products were bioequivalent.
Fig. 3: Mean Plasma Concentration Time Curve for Test and Reference Product.
From the above discussion it is found that the analytical method for analysis of Levetiracetam in plasma is simple, rapid and sensitive. The main advantage of this method is the use of soild extraction procedure for sample preparation, which shows less interference from plasma. It also uses UV detection, which is less costly than LC-MS (Liquid chromatography coupled with mass spectrometry). It can be used as a reliable assay method in the study of Pharmacokinetics of Levetiracetam as well as bioavailability / bioequivalence study.
ACKNOWLEDGEMENTS:
The authors would like to acknowledge the Orchid Healthcare in providing the samples and to conduct the study.
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Received on 31.03.2010 Modified on 11.04.2010
Accepted on 20.04.2010 © AJRC All right reserved
Asian J. Research Chem. 3(3): July- Sept. 2010; Page 776-780