A Novel HPLC Method for Identification and Quantification of Ramucirumab in Tablet Dosage Form

 

Prashanthi. Y1, Tentu Nageswara Rao*2, Yellapu Srinivas3

1Department of Biochemistry, Acharya Nagarjuna University, Andhra Pradesh, India.

2Department of Chemistry, Mahatma Gandhi University, Nalgonda, Telangana, India.

3Mother Degree & P.G College, Tuni, A.P, India.

*Corresponding Author E-mail: tnraochemistry@gmail.com

 

ABSTRACT:

A novel approach has used for the develop and validate a rapid, accurate, simple, reproducible and an isocratic RP-HPLC method with PDA detector for the estimation of Ramucirumab drug in pharmaceutical dosage forms. Ramucirumab was separated using YMC C18 4.6 X 150 mm, 5 µm analytical column, a Waters HPLC (USA) and a mobile phase consists of methanol and water in the ratio of 80:20 % v/v, delivered at 0.6 ml / min with 20 µl injection volume. The column was maintained at ambient temperature, and the detector was set at wavelength of 320 nm. The system suitability parameter for Ramucirumab such as theoretical plates and tailing factors were found to be 4159 and 1.5 respectively. Linearity was established for Ramucirumab in the range of 20-100 ppm concentration levels with correlation coefficients (r2) of 0.999.  The intra and inter-day precision % RSD values were found to be 0.24 and 0.39 respectively. The limit of detection (LOD) and limit of quantification (LOQ) value were found to be 0.32 and 0.98 µg/ml respectively. The method was validated for all of the above parameters according to International Conference on Harmonization (ICH) guidelines. This method can be used for the estimation and analysis of Ramucirumab drug in Active Pharmaceutical Ingredients and Pharmaceuticals.

 

KEYWORDS: Ramucirumab, Reversed Phase High Performance Liquid Chromatography, Photo Diode Array detector, Cyramza, Estimation.

 

 


INTRODUCTION:

Gastric cancer is currently fifth most common cancer and the third most common causeof cancer deaths worldwide, accounting for almost 9 % of all deaths from cancer1. There were nearly one million cases of gastric cancer were record worldwide in 2012 with approximately 723,000 deaths were recorded2. Stomach cancer is more prevalent in countries outside the US and Europe3. In the US, it was estimated that approximately 22,000 people were diagnosed with gastric cancer in 20144.

 

Gastric cancer is a disease in which tumors forms in the stomach and as it advances it can travel through blood stream and spreads in the liver, lungs and bones of the body system and leads to decrease of survival of the patient5, 6.

 

Vascular Endothelial Growth Factor (VEGF) and Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) mediated signaling and angiogenesis seems to have an important role in the pathogenesis of gastric cancer. VEGF originally known as Vascular Permeability Factor (VPF)7, is a signal protein, produces by cells that stimulates vasculogenesis and angiogenesis. It is a part of the system that restores the oxygen supply to the tissues when blood circulation is inadequate condition 8. VEGF normal function is to create new blood vesicles after injury, new muscles fallowing exercise. When VEGF is overexpressed, it can lead to the formation of solid tumors. VEGF preferentially binds to the VEGFR-2 receptor site and cause the angiogenesis.

 

In animal models of gastric carcinoma, VEGFR-2 inhibition has reduced tumor growth and vascularity. Ramucirumab is a fully human Ig G1 monoclonal antibody, VEGFR-2 antagonist that prevents the binding and receptor mediated pathway activation in endothelial cells. Ramucirumab (IMC-1121 B) trade name is Cyrazma human monoclonal antibody developed for the treatment of solid tumors. It is directed against vascular endothelial growth factor-2 receptor. By binding to the VEGFR-2 it works as a receptor antagonist, blocks the binding of VEGF to VEGFR-2. Ramucirumab has approximately eight times more efficacy for binding the receptor than its natural ligand. After binding to the receptor, Ramucirumab induces conformational changes as well as stearic hindarence for the ligand to bind.

 

Ramucirumab is a new vascular endothelial growth factor receptor-2 (VEGFR-2), monoclonal antibody that has received a considerable momentum and attention in the management and treatment numerous solid tumors [14]. On april 21, 2014 the FDA approved Ramucirumab, as a single agent or with the combination of Paclitaxel for the treatment of gastro-esophageal junction adeno-carcinoma. Ramucirumab has also shown the positive results in the treatment of other solid tumors including metastatic colorectal cancer (mCRC) and advanced non-small cell lung cancer (NSCLC) and it has approved for both diseases.

 

Being a very novel and recently synthesized drug, there are few references for Ramucirumab. Since, there is no reference, in the present literature, concerning analysis of the Ramucirumab drug in the pharmaceuticals. The aims of this study were firstly to develop and optimize the RP-HPLC-PDA conditions for the estimation and analysis of Ramucirumab drug in pharmaceuticals and validate the method, then to apply this method in Active Pharmaceuticals Ingredients and Pharmaceuticals for routine analysis.

 

MATERIALS AND METHODS:

Chemicals and reagents:

The pure drug of Ramucirumab was kindly provided as a gift by MSN Pharmaceuticals Private Limited (Hyderabad, India).  HPLC grade of Acetonitrile, Methanol and Water were purchased from Merck (Germany). Analytical reagents of ortho-phosphoric acid, potassium dihydrogen phosphate and di potassium mono hydrogen phosphate were purchased from Merck (Germany).

 

Equipment:

The analysis was performed by using a chromatographic system from a Waters (USA) Separation module of 2695 with 996 PDA detector. For the data acquisition and analysis Empower software version-2 was employed. An ultra-sonic water bath sonicator model SE60US, Enertech company (USA), digital PH meter (AD 102U, ADWA) digital weighing balance ER 200A from Ascocet (USA) and UV double beam spectrometer (UV 3000+ UV win software) (India) were used in the study.

 

Chromatographic conditions:

Ramucirumab was analyzed in YMC C18 (4.6 X 150 mm), 5 µm particles sized analytical column. For the elution of Ramucirumab drug we used the mobile phase of methanol and water in the ratio of 80:20 % v/v delivered at 0.6 ml/min flow rate. The mobile phase was filtered through 0.22 µ nylon membrane filter under vacuum filtration. The injection volume was 20 µl and the detector was set at wavelength of 320 nm. The auto-sampler and column temperatures were maintained at ambient condition. The chromatographic program run time was 6 min.

 

Preparation of mobile phase:

The mobile phase used under the chromatographic conditions was prepared by the mixing of HPLC grades of methanol and water in the ratio of 80:20 % v/v. Then this mobile phase mixture was degassed by using an ultrasonic water bath for 5 minutes, and filtered through 0.22 µ nylon membrane filters under vacuum filtration system.

 

Preparation of diluent:

Mobile phase was used as diluent.

 

Preparation of sample solution of Ramucirumab:

10 mg of Ramucirumab tablet powder was accurately weighed and transferred into a 10 ml clean and dry volumetric flask, added about 2 ml of diluent and sonicate to dissolve it completely and making volume up to the mark with same diluent. Further pipetted out 10 ml of above stock solution in to a 100 ml clean volumetric flask and diluted up to the mark with diluent.

 

Preparation of standard solution of Ramucirumab:

10 mg of Ramucirumab working standard was accurately weighed and transferred into a 10 ml clean dry volumetric flask and add about 2 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution).Further pipette out 1.0 ml from the above stock solution into a 10 ml volumetric flask and was diluted up to the mark with diluent.

 

 

 

Validation parameters:

Selection of wavelength:

Prepared 10 ppm concentration solution of Ramucirumab in the mobile phase and scanned using the UV double beam spectrometer with in the wavelength region of 200-400nm. The maximum of absorption wavelength was selected for the detection of Ramucirumab wavelength.

Percentage of Assay:

10 µl from standard and sample solution of Ramucirumab drug were injected in triplicate into the chromatographic system and the peak areas were measured for Ramucirumab. The % Assay was calculated by comparing the peak area of standard and sample chromatogram by using formula.


 

Assay %

=

Sample area

X

Dilution sample

X

P

X

Avg.wt.

X

100

Standard Area

Dilution of standard

100

Lc

Where,

P    =    Percentage purity of working standard

Lc  =    Label claim of drug in mg/ml.

 

 

 


System suitability test:

At the first HPLC system was optimized as per the chromatographic conditions. 10 µl of the standard and sample solution of Ramucirumab drug were injected in triplicate in to the chromatographic system. To ascertain the system suitability for the proposed method, the parameters such as retention time, theoretical plate and tailing factors were calculated.

 

Specificity:

The specificity of the method was carried out to determine whether there is any interference of any impurities in the retention time of analytical peak. The specificity was performed by the injecting of blank, standard and sample solution of Ramucirumab solution.

 

Linearity:

Prepared 20, 40, 60, 80, and 100 ppm concentration levels of calibration standard solutions of Ramucirumab and injected into the chromatographic system. A linear regression was used to plot the calibration graph of peak area (on y-axis) versus concentration (on x-axis) of Ramucirumab. Each peak area was used to calculate the correlation coefficient (r2) 9.

 

Accuracy:

The accuracy of the method was determined by calculating the percentage of recovery values of Ramucirumab by the method of standard addition. Known amount of standard solution of Ramucirumab at 50, 100 and 150 % were added to pre-quantified the sample solution of Ramucirumab and injected into the chromatographic system. Each standard solution was prepared in triplicate and analyzed.  The peak area of each level was used to calculate the percentage of recovery at each level10, 11.

 

Limit of detection and limit of quantification:

Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 X SD/S and   10 X SD/S respectively as per ICH guidelines12. Where SD is the standard deviation response (Y-intercept) and S is the slope of the calibration curve. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio 3). The LOQ is the smallest concentration of analyte which gives the response that can be accurately quantified (signal to noise ratio 10).

 

Precision studies:

Intra-day precision (Repeatability):

Method precision for Ramucirumab drug solution was checked by repeatability. We prepared five injection samples of same concentration of 100 µg/ml of Ramucirumab drug solution and injected into the chromatographic system. The peak area of each injection was used to calculate the % RSD.

 

Inter-day precision (Reproducibility):

We performed the intermediate precision test. This study was performed with same injections of repeatability test. For evaluating the intermediate precision we analyzed six injections of 100 µg/ml concentration of Ramucirumab drug on different days by different columns of same dimensions by different analysts. Each injection area was used to calculate the % RSD.

 

RESULTS AND DISCUSSIONS:

Chromatographic conditions:

Ramucirumab is a newly developed human monoclonal antibody, showing positive results in the treatments and also approved for the gastric carcinoma, advanced non-small cell lung cancer (NSCLC) and metastatic colorectal cancer (mCRC) treatments. Being a newly developed drug, Literarure review reveals that there was no study was conducted for the estimation and analysis of Ramucirumab drug by HPLC method. Our study addresses that Ramucirumab needs a rapid and sensitive analytical method for the routine analysis in the pharmaceuticals. We developed and validated the RP-HPLC method with PDA for the estimation of Ramucirumab. While developing the method, we trialed with different types of column, and mobile phases and different flow rates.

 

 

In the first approach, INERSIL RP 18 4.6 X 150 mm, 5 µm analytical column was used for the separation of drug. Methanol and phosphate buffer in the ratio of 80:20 % v/v was used to elution of of the drug with the 1 ml/min flow rate. The injection volume was 10 µl, and the detector wavelength was set at 320 nm. The program run time was 10 minutes. This trial shows no good peak separation, so more trials were requiredto get good peak.

 

In the second trial INERSIL RP 18 4.6 X 150 mm, 5 µm analytical column, acetonitrile and water in the 70:30 % v/v ratio mobile phase was employed. We injected the 10 µl injection volumes, and the flow rate was 1 ml / min. the detector wavelength was set at 320 nm and the program run time was 10 minutes. In this trial no proper peak separation was obtained. So still more trials were required for good peak.

 

In the next trial Agilent C8 4.5 X 150 mm, 5 µm analytical column was employed for the analysis. Mobile phase consists of acetonitrile and phosphate buffer solution of pH at 6.8 was used at 1 ml / min. 10 µl was the injection volume, detector wavelength was set at 320 nm and the program run time was 10 minutes. In this trial Ramucirumab was eluted but there was no proper baseline and retention time. So we performed further trial.

 

In the last trial, YMC C18 4.6 X 150 mm, 5 µm analytical column, mobile phase consists of methanol and water in the 80:20 % v/v ratio was employed for the analysis. 20 µl was the injection volume, 0.6 ml / min was the flow rate, and the detector wavelength was set at 320 nm. The program run time was 6.0 minutes. In this trial, the peak separation and shape was good. We found the retention time of Ramucirumab at 2.497 min. This chromatographic condition was taken as a final method for the method validation studies. The spectra were extracted from the PDA detector in the chromatographic conditions.

 

Assay validation:

Selection of wavelength:

Ramucirumab was shown a maximum of UV absorbance at 320 nm. So we have taken 320 nm as a detection wavelength of Ramucirumab. The chromatogram was shown in Fig. No. 1.

 

Fig. No.1 Spectrum showing overlapping spectrum of Ramucirumab

 

 

Percentage of Assay:

We performed the % Assay study for Ramucirumab drug and we found the % purity of Ramucirumab drug in pharmaceutical dosage form was 98.94 %. The obtained results were represented in the Table No. 1.

 

Table:1 Assay of marketed formulation of Ramucirumab

Drug

Ramucirumab capsule label claim (mg)

Amount found (mg)

% label claim ± % RSD (n=3)

Ramucirumab

10

9.89

98.94 ±0.29

n = number of determination, % RSD = Relative Standard Deviation

 

System suitability:

By performing system suitability test for Ramucirumab drug, we found the retention time for Ramucirumab was 2.425 min. Theoretical plates and tailing factors were found to be 4159.0 and 1.5 respectively. The obtained results were shown in the Table No. 2.

 

Table No.2 system suitability parameter for Ramucirumab

Parameter (n=6)

Ramucirumab

Retention time

2.425

Theoretical plates

4159.0

Tailing factor

1.5

 

Specificity:

Figure No.2 shows that there was no interference of impurity or any endogenous peak in retention time of Ramucirumab drug analytical peak. The obtained chromatograms for blank, standard and sample solution of Ramucirumab were shown in Fig. No. 3, 4 and 5 respectively.


 

Fig. No.2 Chromatogram showing blank (Mobile phase)

 

 Fig. No.3 chromatogram showing standard solution of Ramucirumab

 

 

Fig. No.4 chromatogram showing sample solution of

 


Ramucirumab:

Linearity:

The linearity of the method was evaluated over a concentration range of 20 – 100 µg / ml. We found correlation coefficient to be 0.999. A perfect linearity was obtained in the concentration versus area curve for standard. The mean values for the regression equation were (y = 9601.5x - 97107) where y – axis was the peak area and X – axis was the concentration analyte in µg / ml. The obtained results were summarized in the Table No. 3.

 

Table No.3 linearity data for Ramucirumab

Parameters

Values (µg/ml)

Regression equation

Y = 9601.5x - 97107

r2

0.999

LOQ

0.98

LOD

0.32

Analytical parameters of linearity and limit of detection and limit of quantification obtained for Ramucirumab drug

 

Accuracy:

Accuracy or recovery study was performed for 50, 100, and 150 % concentrations of Ramucirumab drug solution. The results were in accordance with the ICH guide lines. According to ICH guidelines the percentage of recovery should be with in the 98 – 102 % range. The obtained results were presented in the Table No. 4.

 

Table No.4: Accuracy (Recovery) study data for Ramucirumab

Sample name

Amount added (mg) (n=3)

Amount found (mg) (n=3)

% recovery

Statistical analysis

S:50%

S:50%

S:50%

5

4.94

98.8

 

% RSD-0.23

4.96

99.2

4.96

99.2

S:100%

S:100%

S:100%

 

10

 

9.96

99.60

 

% RSD-0.15

9.97

99.70

9.99

99.90

S:150%

S:150%

S:150%

 

15

 

15.01

100.06

 

% RSD-0.08

15.01

100.06

15.03

100.2

n = number of determination, % RSD = Relative Standard Deviation

 

Limit of detection and limit of quantification:

LOD and LOQ were determined by analysis of standard solution of Ramucirumab drug. We found the LOD and LOQ values were to be 0.32 µg / ml and 0.98 µg / ml. The obtained values for LOD were taken on the basis of signal-to-noise ratio 3 and for LOQ signal-to-noise ratio 10. These results were in agreement with ICH guidelines. The obtained results were presented in the Table No.3.

Precision:

Repeatability study was evaluated by analyzing six samples of 100 ppm concentration of Ramucirumab drug. We found the system precision % RSD value was 0.24%. The results were shown in Table No. 5.

 

Table No. 5: method precision (Repeatability) data for Ramucirumab

S.No

Concentration (µg/ml)

Retention Time

Peak Area

1

100

2.423

693877

2

100

2.424

696531

3

100

2.424

693977

4

100

2.424

695278

5

100

2.423

697676

Mean

 

2.423

695468

SD

 

0.000548

1642.7

%RSD

 

0.02

0.24

SD = Standard Deviation, % RSD = Relative Standard Deviation

 

Table No.6 Reproducibility data for Ramucirumab

Reproducibity

% RSD

Instrument 1 (n =6)

 

Pa ratio (n =6)

0.37

Rt ratio (n =6)

0.17

Instrument 2 (n =6)

 

Pa ratio (n =6)

0.39

Rt ratio (n =6)

0.15

n = number of determination, % RSD = Relative Standard Deviation, Pa = peak area, Rt = retention time

 

The reproducibility % RSD values were shown in Table No.6. The outcome results were in accordance with ICH guidelines. According to the ICH guidelines the % RSD for standard injection it should be less than 2%. So our results indicated that the developed method was validated the precision.

 

CONCLUSION:

A fast, accurate and an isocratic RP-HPLC method for the estimation of Ramucirumab drug in the pharmaceutical dosage form was established and validated as per ICH guidelines. Linearity was achieved for Ixazomib in the range of 20 – 100 µg/ml with correlation coefficient (r2) 0.999. The % recovery was achieved in the range of 98 – 102 % which was within the acceptance criteria. The % RSD was less than 2 % which proved the precision of the developed method. The advantage of this method is suitable retention time and high sensitivity. The effect of flow rate and organic composition of mobile phase on system suitability parameters were investigated to obtain the optimum chromatographic conditions. The obtained results suggested that the developed method was validated for precision, accuracy, linearity, LOD and LOQ, and robustness. Hence this method can be used for the routine analysis of Ixazomib drug in pharmaceuticals.

 

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Received on 25.08.2018       Accepted on 26.09.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(4): 209-214.

DOI: 10.5958/2231-5675.2018.00038.8