BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF LENVATINIB BY RP-HPLC METHOD

BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF LENVATINIB BY RP-HPLC METHOD

G. Krishna Veni ¹, A. Ajitha ^{* 1} and K. Abbulu ²

Department of Pharmaceutical Analysis ¹, Department of Pharmaceutics ², CMR College of Pharmacy, Medchal, Kandlakoya, Hyderabad - 500055, Telangana India.

ABSTRACT: A rapid, fast, accurate, and precise method was developed for the estimation of lenvatinib in human plasma using methotrexate as an internal standard by reverse-phase high-performance liquid chromate-graphy (RP-HPLC). The separation was carried out by using zodiasil C18 (150 × 4.6 mm, 5 m) as a stationary phase and 0.01 N sodium di-hydrogen phosphate (pH: 4.8): acetonitrile in the ratio of 45:55 % v/v. The analysis was performed at a flow rate of 1.0 ml/min using PDA detector at 240 nm. The column temperature was maintained at 30 °C for better separation and resolution. The retention time of lenvatinib was found to be 4.508 min. The % coefficient of variation of lenvatinib was found to be 2.66%. The % recovery was found to be 94.758%. The method was found to be linear between the concentration range of 28-1120 ng/mL (r² = 0.999). The lower limits of quantification were 28 ng/mL, which reach the level drug possibly found in human plasma. Further, the reported method was validated as per the ICH guidelines and found to be well within a suitable range. In the future, this method can be used for clinical and pharmacokinetic studies.

Lenvatinib, Methotrexate, RP-HPLC, Method development, ICH Guidelines, Validation

INTRODUCTION: Thyroid cancer is the 6^th most common cancer in women. It is the most common cancer in women aged 20 to 34. About 2% of cases occur in children and teens. Lenvatnib is an oral anti-cancer drug that is mainly used to treat radioiodine-refractory differentiated thyroid cancer ¹. Lenvatinib belongs to the class of quinolines which is the carboxamide of 4 - {3 -chloro - 4 (cyclopropylcarbamoyl) amino phenoxy} - 7 - methoxyquinoline - 6 - carboxamide ². Lenvatinb drug is approved in the year 2018 ^{5, 6}.

A literature review reveals that very few analytical methods have been reported for the determination of lenvatinib by UPLC ³, RP-HPLC method ^{4, 7}. However, a literature review reveals that no method is reported for the determination of lenvatinib in human plasma ⁸ by RP-HPLC. Hence, a precise, sensitive, accurate, selective, reproducible, and rapid analytical technique for the estimation of lenvatinib in human plasma is developed and validated as per ICH guidelines ⁹. The chemical structure of lenvatinib is shown in Fig. 1.

FIG. 1: CHEMICAL STRUCTURE OF LENVATINIB

MATERIALS AND METHODS:

Reagents and Chemicals: The drug sample of lenvatinib and methotrexate was obtained from BMR pharma and chemicals (Hyderabad, India).

All reagents and solvents were of analytical and HPLC grade including acetonitrile (HPLC Grade), water (HPLC Grade), potassium dihydrogen phosphate, methanol (HPLC grade), were from Rankem, Lab Chemicals, Haryana.

Instrumentation and Chromatographic Conditions: The HPLC system used was watered HPLC 2695 system equipped with quaternary pumps, photodiode array detector, and autosampler integrated with empower 2 software.  Zodiacal (150 mm × 4.6mm, 5mm) column was used for separation.

The mobile phase was 0.01 N sodium dihydrogen phosphate pH (4.8) acetonitrile 45:55 (v/v). The flow rate was maintained at 1.0 ml/min, and the effluent was monitored at 240 nm.

Preparation of Solutions:

Preparation of Lenvatinib Stock Solution (140 µg/ml): Take 14 mg of lenvatinib in 100 ml volumetric flask and make the volume with diluent to produce 140 µg/ml.

Preparation of Lenvatinib Spiking Solutions  (0.028 µg/ml to 11.2 µg/ml): From the above  lenvatinib stock solution 0.05 ml, 0.1 ml, 0.15 ml,  0.6 ml, 1.0 ml, 1.2 ml, 1.6 ml and 2.0 ml was  pipette and transferred to 8 individual 25 ml  volumetric flask and make up the volume up to the  mark with diluents to produce 0.28 µg/ml, 0.56  µg/ml, 0.84 µg/ml, 2.24 µg/ml, 5.60 µg/ml, 6.72  µg/ml,  8.96  µg/ml  and  11.2 µg/ml.

Preparation of Calibration and Quality Control Samples: Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 28 ng/ml, 56 ng/ml, 84 ng/ml, 336 ng/ml, 560 ng/ml, 672 ng/ml, 896 ng/ml and 1120 ng/ml.

Preparation of Internal Standard Solution (Methotrexate):

Stock-1: Take 5 mg of methotrexate in 100 ml volumetric flask and make up the volume with diluents to produce 50 µg/ml.

Stock-2: From the above solution, take 1 ml of solution into 10 ml volumetric flask and make up the volume with diluents to produce 5 µg/ml solutions.

Note: From the above solution, take 0.5 ml of solution and spiking blank plasma with working stock dilutions of analytes to produce 1 µg/ml internal standard concentration.

Extraction Procedure: Take 750 µl of plasma and 500 µl of internal standard, 250 µl of lenvatinib from the spiking solutions of both into a centrifuging tube and add 1 ml of acetonitrile go for cyclomixer for 15 sec. Then vertex for 2 min and finally centrifuge for 5 min at 3200 rpm speed. After the centrifugation collect the sample and filter it by using polyvinylidene fluoride 0.45 µ filter and directly inject 10 µL into HPLC.

RESULTS AND DISCUSSION:

Method Development: Chromatographic conditions used was stationary phase zodiasil (150 mm × 4.6 mm, 5 mm), mobile phase 0.01N sodium dihydrogen phosphate pH (4.8): acetonitrile 45:55 (v/v) and flow rate was maintained at 1.0 ml/min and detection wavelength was 240 nm, injection volume of 10 µl and column temperature was set to 30 °C. The retention time of lenvatinib and metho-trexate was found to be 4.508 min and 2.278 min respectively. The chromatogram of lenvatinib obtained by optimized conditions is shown in Fig. 2.

FIG. 2: OPTIMIZED CHROMATOGRAM

Bio-analytical Method Validation: The analytical method developed for the estimation of lenvatinib is validated as per ICH guidelines.

System Suitability: All the system suitability variables were within the range as per ICH rules. The % coefficient of variation for system suitability analysis was in the range of 0.04-0.46 for retention time (RT) and 0.72% for the area ratio (analyte area/IS area). The results were reported in Table 1.

Selectivity/Specificity: To establish the selectivity of the method, possible interference at the retention time of lenvatinib and internal standard due to endogenous plasma components were checked during the validation. Selectivity was performed by testing six batches of K2 EDTA blank plasma and the mass detection of extracted blank plasma gave good selectivity of both drug and internal standard. No interferences were found at the retention times of analytes and internal standards.

Representative chromatograms of standard blank and blank with an internal standard sample using pooled plasma are represented in Fig. 3, 4, 5, 6.

TABLE 1:  SYSTEM SUITABILITY OF LENVATINIB

Linearity: Calibration was found to be linear over the concentration range of 28 to 1120 ng /ml for lenvatinib. The coefficient of correlation (r²) value was found consistently greater than 0.999 in all the cases. This indicates a high linearity of results and an excellent correlation between peak area ratios for each concentration of analytes. A representative calibration curve is shown in Fig. 7, which is obtained during the third precision and accuracy batch. Back calculated concentrations obtained for 3 calibration curves are summarized in Table 2.

FIG. 7: CALIBRATION CURVE OF LENVATINIB

TABLE 2: LINEARITY OF LENVATINIB

Precision and Accuracy: The intraday and inter-day accuracy and precision were assessed by analyzing six replicates at five different QC levels like LLOQ, LQC, MQC and HQC. Accuracy and precision method performance was evaluated by determined by six replicate analyses for lenvatinib at four concentration levels, i.e., 84 ng/ml (LQC), 560 ng/ml (MQC) and 896 ng/ml (HQC).

TABLE 3: ACCURACY AND PRECISION DATA FOR INTRA-DAY RUNS OF LENVATINIB

The intra-day and inter-day accuracy of plasma samples were assessed and excellent mean % accuracy was obtained with range varied from 99.22%-101.66% for intraday and 99.10%-101.90 for inter-day respectively. The precision (% CV) of the analyte and plasma samples were calculated and found to be 0.45%-11.99% for intraday and 0.64%-11.17% for inter-day, respectively. The results are summarized in Table 3, 4.

TABLE 4: ACCURACY AND PRECISION DATA FOR INTER-DAY RUNS OF LENVATINIB

Recovery: Recovery was determined by measuring the peak areas obtained from prepared plasma samples with those extracted blank plasma spiked with standards containing the same area with a known amount of lenvatinib. The overall % mean recovery for lenvatinib was found to be 94.75%.

The recoveries obtained for lenvatinib at 3 QC concentration levels are summarized in Table 5 respectively. The overall % mean recovery for methotrexate was found to be 94.12%. The recovery results of methotrexate are summarized in Table 6.

TABLE 5:  RECOVERY-LENVATINIB

TABLE 6: RECOVERY OF METHOTREXATE (IS)

Stabilities:

Long Term Stock Solution Stability or Lenvatinib: In bench-top stability, six replicates of LQC and HQC samples (84 and 896 ng/ml) were analyzed for 9 h at room temperature on the laboratory bench. The % means stability was calculated and found to 100.33% for LQC and 101.87% for HQC respectively Table 7.

Matrix Samples Stability at -28 ± 5 °C and -80 ± 5 °C for 37 Days: Long term stock solution stability for the lenvatinib was determined at a concentration of LQC-HQC level after a storage period of 37 days at -28 °C and -80 °C in the refrigerator. 101.90%, 101.29% and 100.24%, 100.31 at 28 ± 5 °C and 101.31%, 99.70% and 100.39%, 100.32 at 28 ± 5 °C separately. The long term stability of lenvatinib is presented in Table 8 and 9.

TABLE 7: LONG TERM STOCK SOLUTION STABILITY OF LENVATINIB (ZERO DAY)

TABLE 8: MATRIX SAMPLES STABILITY AT -28 ± 5 °C LENVATINIB (37 DAYS)

TABLE 9: MATRIX SAMPLES STABILITY AT – 80 ± 5 °C-LENVATINIB (37 DAYS)

CONCLUSION: The proposed method was validated as per the ICH guidelines and found to be well within the acceptable range. The method is simple, rapid, accurate, precise, and appropriate for pharmacokinetic and therapeutic drug monitoring in the clinical laboratories.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: The authors declare that he/she has no conflicts of interest.

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   How to cite this article:

   Veni GK, Ajitha A and Abbulu K: Bioanalytical method development and validation of lenvatinib by RP-HPLC method. Int J Pharm Sci & Res 2020; 11(7): 3313-19. doi: 10.13040/IJPSR.0975-8232.11(7).3313-19.

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