rp-hplc method for the estimation of pemetrexed ... - Ijsidonline.info

Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries
An International peer
Review Journal for Science
Research Article
Available online through www.ijsidonline.info
RP-HPLC METHOD FOR THE ESTIMATION OF PEMETREXED ASSAY IN
FORMULATIONS.
L. Maheswara Reddy 1* , P. Raveendra Reddy 1 , L. Bhaskar Reddy and K. Janardhan Reddy 2
1Department of Chemistry, Sri Krishnadevaraya University, Anantapur-515 055, Andhra Pradesh, India.
2Department of Nanomaterial Chemistry, Donguguk University, 707 Seokjang-Dong, Gyeongju-780 714, Republic of Korea
Received: 20.07.2011
Modified: 29.07.2011
Published: 12.08.2011
Keywords: Pemetrexed,
RP-HPLC
and validation
*Corresponding Author
Address:
Name: L.Maheswara Redddy
Place: Hyderabad
E-mail: mahi_reddy67@yahoo.com
ABSTRACT
Formulations
A simple RP-HPLC method was developed and validatied for
method development
the determination of Pemetrexed in pharmaceutical dosage
forms. Mobile phase composed of buffer (1.0ml of
orthophosporic acid into 1000 ml of Milli-Q water) and
acetonitrile in the ratio of 85: 15 v/v, Inertsil C18 (150×4.6
mm, 5µm) HPLC column and absorbance was measured at
230nm. Column oven temperature maintained at 25 o C,
injection volume was 5µL and 12min runtime was seletected
for analysis. Method validation was performed as per ICH
guidelines, with respect to linearity, accuracy, precision,
ruggedness and robustness. Linearity was evaluated and
found to be linear in the range of 117ppm to 780ppm,
correlation coefficient is 0.999. Recovery studies also
checked and found to be within the limit. This method has
applicable for regular analysis.
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INTRODUCTION
Pemetrexed (1-10) is a chemotherapeutic drug, used for the treatment of malignant pleural
mesothelioma (MPM) in combination with cisplatin, a platinum-containing chemotherapeutic use. It is
used as a single agent or in combination with other chemotherapeutic agents for the treatment of other
types of cancer such as breast cancer, bladder cancer, colorectal cacinoma and cervical cancer. The
dosage of pemetrexed is depending on a number of factors, including body height, weight and response to
this medication. Pemetrexed available in injection dosage form. It is injected slowly (infused) into a vein.
Each infusion takes about 10 minutes. In most cases, you will receive one infusion every 21 days. The
serious side effects are pale skin, easy bruising or bleeding, unusual weakness, fever, chills, body aches,
chest pain, trouble breathing and swelling. Pemetrexed is a suitable third-line treatment option with
good efficacy and tolerable toxicity profile for Non-small Cell Lung Cancer (NSCLC). Figure-1 represents
the chemical structure of Pemetrexed.
Figure-1: Chemical structure of Pemetrexed
Pemetrexed and its dosage froms have reported methods. In the present study developed the
simple, less runtime and accurate RP-HPLC method for the quantification of pemetrexed in bulk and all
type of dosage forms.
MATERIALS AND METHODS
Chemicals and Reagents
HPLC grade acetonitrile and water were purchased from Merck, AR grade orthophospharic acid,
Sodium chloride, Hydrochloric acid, Sodium hydroxide, Hydrogen peroxide and other reagents were
procured from S.D. Fine chemicals Ltd. High pure standards and all market samples were used for this
study. GL Science makes Inertsil column was used for this product.
Instrumentation: Waters make 2489 model LC system was used for chromatographic analysis and
equipment operated with Empower software.
Chromatographic conditions:
Buffer
: Transferred 1.0mL of orthophosporic acid into 1000 ml of Milli-Q water,
mixed and degassed.
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Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
Mobile Phase : Mixed the buffer and Acetonitrile in the ratio 85:15v/v and degassed.
Diluent
Column
Flow rate
Wavelength
: 0.9% Sodium chloride solution
: Inertsil 150x4.6mm 5µm.
: 1.5 mL per minute
: 230nm
Inj. Volume : 5µL
Run time
: 12minutes
Standard Preparation (500ppm): Weigh about 50 mg of Pemetrexed di-sodium WS in to 100ml
volumetric flask, add 75ml diluent , dissolve and dilute to volume with same and mix well.
Sample Preparation: Prepared the all dosage forms equivalent to 500ppm of pemetrexed with diluent.
System suitability: The USP tailing factor for standard peak should be not more than 2.0; the % relative
standard deviation should be not more than 2.0% and USP theoretical plates for standard solution should
be not less than 2000.
Calculation:
% of Assay: Area of test sample x Concentration of Std x Potency of Std.
Area of Std. sample x Concentration of sample
Optimization of Chromatographic Conditions:
RESULTS AND DISCUSSION
Chromatographic conditions were optimised by using various mobile phases containing
phosphate, sulphate, acetate buffers at pH 2-6 and various organic solvents such as acetonitile, methanol
and ethanol and different make HPLC columns. Finally, optimised chromatographic conditions with
orthophosphate buffer, acetonitrile as solvent and Inertsil C18 column. Figure-2 and 3 represent the
diluent and standard solution chromatograms.
Figure-2: Blank chromatogram
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Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
System Suitability:
Figure-3: Standard solution
System suitability parameters were evaluated from five replicate standard injections and found to
be within the limit. Calculated the % RSD (0.1%) from five replicate injections for pemetrexed peak area
and was found to be within the limit (The acceptable % of relative standard deviation of peak areas of
pemetrexed should be not more than 2%), tailing factor of the active peak in standard solution is 1.1.
(Acceptance limit is not more than 2.0) and the area ratio between two peaks is within the limit. Table-1
represents the system suitability results.
Specificity:
Table-1: System suitability results
System suitability Results
Std. No
Pemetrexed
01 5857358
02 5880172
03 5868972
04 5869287
05 5870451
Average 5869248
%RSD 0.1%
Specificity of the method was checked by conducting placebo interference and stress study with
acid (0.1N HCl), base (0.1N NaOH), hydrogen peroxide (1% hydrogen peroxide for 15 min), and thermal
water for 15 min, humidity, UV light and sun light degradation at 60 o C. Placebo and degradation products
have no interference with the pemetrexed peak. Peak purity results indicate that the method was more
specific and accurate. Peak purity results were tabulated in table-2. Figure-4 to 12 represents the peak
purity flat in all stress study conditions.
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Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
0 .3 6
0 .3 4
0 .3 2
0 .3 0
0 .2 8
0 .2 6
0 .2 4
0 .2 2
P u rit y
N o is e + S o lve n t ( 1 . 0 0 )
Pemtrexed - 5.367
9 0 .0 0
8 0 .0 0
7 0 .0 0
6 0 .0 0
AU
0 .2 0
0 .1 8
0 .1 6
0 .1 4
5 0 .0 0
4 0 .0 0
Degrees
0 .1 2
0 .1 0
0 .0 8
0 .0 6
0 .0 4
0 .0 2
0 .0 0
- 0 .0 2
3 0 .0 0
2 0 .0 0
1 0 .0 0
0 . 0 0
5 . 0 0 5 .1 0 5 .2 0 5 . 3 0 5 . 4 0 5 .5 0 5 . 6 0 5 . 7 0 5 .8 0 5 .9 0 6 . 0 0 6 .1 0 6 .2 0
M in u te s
Figure-4: Purity plot of peroxide stressed sample
Table-2: Stress study results
Sr No Name of Sample Purity Angle Purity Threshold Peak purity
01 Peroxide Stress (1%) 0.179 1.216 Pass
02 Acid Stress (0.1N/1hrs) 0.143 1.178 Pass
03 UV Stress 0.189 1.247 Pass
04 Humidity stress 0.180 1.228 Pass
05 Sunlight Stress 0.193 1.235 Pass
06 Thermal Stress 0.186 1.235 Pass
07 Base Stress (0.1N/ 2hrs) 2.005 3.528 Pass
08 Water Stress 0.151 1.180 Pass
09 Unstressed 0.160 1.209 Pass
0.024
0.022
0.020
0.018
0.016
0.014
P urity
Nois e+ S olvent (1.00)
Pemtrexed - 5.376
90.00
80.00
70.00
60.00
AU
0.012
0.010
50.00
40.00
Degrees
0.008
0.006
0.004
0.002
30.00
20.00
10.00
0.000
0.00
5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65 5.70 5.75 5.80 5.85
Minutes
Figure-5: Purity plot of Acid stressed sample.
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Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
0 .4 0
0 .3 5
0 .3 0
0 .2 5
P u rit y
N o is e + S o lve n t (1 . 0 0 )
Pemtrexed - 5.381
9 0 .0 0
8 0 .0 0
7 0 .0 0
6 0 .0 0
AU
0 .2 0
0 .1 5
5 0 .0 0
4 0 .0 0
Degrees
0 .1 0
3 0 .0 0
0 .0 5
2 0 .0 0
1 0 .0 0
0 .0 0
0 .0 0
5 .0 0 5 .1 0 5 .2 0 5 .3 0 5 .4 0 5 .5 0 5 .6 0 5 .7 0 5 .8 0 5 .9 0 6 .0 0 6 .1 0 6 .2 0
M in u te s
Figure-6: Purity plot of UV stressed sample.
0 .40
0 .35
0 .30
0 .25
P u rity
N o is e+ S o lve n t (1 .0 0 )
Pemtrexed - 5.371
9 0.0 0
8 0.0 0
7 0.0 0
6 0.0 0
AU
0 .20
0 .15
5 0.0 0
4 0.0 0
Degrees
0 .10
0 .05
0 .00
3 0.0 0
2 0.0 0
1 0.0 0
0 .0 0
5.0 0 5.1 0 5 .2 0 5 .3 0 5 .4 0 5 .5 0 5 .6 0 5 .7 0 5.8 0 5 .9 0 6 .0 0 6 .10 6 .20
Min u te s
Figure-7: Purity plot of Humidity stressed sample.
0 . 4 0
0 . 3 5
0 . 3 0
0 . 2 5
P u rit y
N o is e + S o lve n t (1 . 0 0 )
Pemtrexed - 5.382
9 0 . 0 0
8 0 . 0 0
7 0 . 0 0
6 0 . 0 0
AU
0 . 2 0
0 . 1 5
5 0 . 0 0
4 0 . 0 0
Degrees
0 . 1 0
0 . 0 5
3 0 . 0 0
2 0 . 0 0
1 0 . 0 0
0 . 0 0
0 .0 0
5 .0 0 5 .1 0 5 .2 0 5 . 3 0 5 . 4 0 5 . 5 0 5 . 6 0 5 . 7 0 5 .8 0 5 .9 0 6 .0 0 6 .1 0
M in u te s
Figure-8: Purity plot of Sunlight stressed sample.
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Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
0 .4 0
0 .3 5
0 .3 0
0 .2 5
P u rit y
N o is e + S o lve n t (1 . 0 0 )
Pemtrexed - 5.379
9 0 .0 0
8 0 .0 0
7 0 .0 0
6 0 .0 0
AU
0 .2 0
0 .1 5
5 0 .0 0
4 0 .0 0
Degrees
0 .1 0
3 0 .0 0
0 .0 5
2 0 .0 0
1 0 .0 0
0 .0 0
0 .0 0
4 .9 0 5 .0 0 5 .1 0 5 .2 0 5 .3 0 5 .4 0 5 .5 0 5 .6 0 5 .7 0 5 .8 0 5 .9 0 6 .0 0 6 .1 0
M in u te s
Figure-9: Purity plot of Thermal stressed sample.
0.024
0.022
0.020
0.018
0.016
0.014
P urity
Noise+Solvent (1.00)
Pemtrexed - 5.376
90.00
80.00
70.00
60.00
AU
0.012
0.010
50.00
40.00
Degrees
0.008
0.006
0.004
0.002
30.00
20.00
10.00
0.000
0.00
5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65 5.70 5.75 5.80 5.85
Minutes
Figure-10: Purity plot of Base stressed sample.
0 .4 0
0 .3 5
0 .3 0
0 .2 5
P u rit y
N o is e + S o lve n t (1 . 0 0 )
Pemtrexed - 5.367
9 0 .0 0
8 0 .0 0
7 0 .0 0
6 0 .0 0
AU
0 .2 0
0 .1 5
5 0 .0 0
4 0 .0 0
Degrees
0 .1 0
0 .0 5
3 0 .0 0
2 0 .0 0
1 0 .0 0
0 .0 0
0 .0 0
5 .0 0 5 .1 0 5 .2 0 5 .3 0 5 .4 0 5 .5 0 5 .6 0 5 .7 0 5 .8 0 5 .9 0 6 .0 0 6 .1 0
M in u te s
Figure-11: Purity plot of Water stressed sample.
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Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
0 .3 5
0 .3 0
0 .2 5
P u rit y
N o is e + S o lve n t (1 . 0 0 )
Pemtrexed - 5.359
9 0 .0 0
8 0 .0 0
7 0 .0 0
6 0 .0 0
AU
0 .2 0
0 .1 5
5 0 .0 0
4 0 .0 0
Degrees
0 .1 0
3 0 .0 0
0 .0 5
2 0 .0 0
1 0 .0 0
0 .0 0
0 . 0 0
5 . 0 0 5 . 1 0 5 .2 0 5 .3 0 5 . 4 0 5 . 5 0 5 . 6 0 5 .7 0 5 .8 0 5 . 9 0 6 .0 0 6 .1 0 6 .2 0
M in u te s
Precision:
Figure12: Purity plot of unstressed sample.
Precision of the test method was evaluated by analysing six test samples. Test solutions were
prepared as per the proposed method and intermediate precision also evaluated by using different
preparations, analyst and instruments. Precision and intermediate precision results were tabulated in
table-3.
Accuracy:
Sample No.
Table-3: Precision Results
%ASSAY OF PEMETREXED
ANALYST-1 ANALYST-2
1 101.4 101.6
2 101.2 100.7
3 100.5 101.2
4 100.9 102.2
5 101.4 101.6
6 101.4 100.8
Average 101.1 101.3
% RSD 0.4% 0.54%
Accuracy of the method was determined by analysing with placebo and standard. Standard stock
solution was spiked in to the placebo solution at different concentration levels. Results were found to be
within in the limit (Between 97% and 103%) and tabulated in table-4.
Table-4: Accuracy Results
Sample no. Spike level ‘mg/mL’ added
‘mg/mL’ found Mean %
(recovered) recovery
1 50% 0.250 0.253 101.20
2 80% 0.400 0.397 99.25
3 100% 0.500 0.508 101.60
4 133% 0.666 0.668 100.30
5 200% 1.000 0.989 98.90
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Linearity:
Maheswara Reddy et al., IJSID 2011, 1 (1), 1-10
The linearity of the method was checked from 117ppm to 780ppmand the results shown in table-
5. The correlation coefficient, 0.999 was indicates the linearity of the test method.
Ruggedness:
Table-5: Linearity Results
% Level Standard
concentration(mg/
ml)
Pemetrexed
Area
24% 117 1404747
60% 293 3512651
80% 390 4683354
100% 488 5853487
120% 585 7036505
160% 780 9408045
Coefficient of correlation (r) 0.9999
Slope (m) 12069
Y-Intercept -20225
Coefficient of Regression (r2) 1.0000
System to system /Analyst to Analyst/column to Column variability study was conducted under
similar conditions at different times for ruggedness of the method. A study to establish stability of
standard and test preparation on bench top was conducted over period of two days. Confirms the
standard and test solution were stable during this assay of 2 days period. Comparison of both the results
obtained on two different HPLC systems, different column and different analysts shows that the related
assay method is rugged for system to system /analyst to analyst/column to column variability.
Robustness:
The experimental conditions were purposely altered for the determination of robustness of
method such as variation in organic phase composition in mobile phase, flow rate, column temperature
but no significant change in assay value was observed.
CONCLUSION
The developed RP-HPLC method for the determination of pemetrexed is more precise, accurate
and selective with good system suitability, ruggedness and robustness by satisfactory results. This
method can be successfully used for determination of pemetrexed in bulk and formulation samples
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