Download 7. MATERIALS AND METHOD

6. BRIEF RESUME OF THE INTENDED WORK:
6.1. Need of the study:
HPLC method is being used because it is the prefered method for development
and validation in the recent trend, and validation of a drug is regulatory requirement and
also it ensures that the products and process are fit for their intended use.
Pemetrexed is a drug that belongs to a class of chemotherapeutic drugs known as
folate anti metabolites. The drug was approved by the United States Food and Drug
administration in February 2004 for the treatment of malignant pleural mesothelioma (MPM)
in combination with cisplatin, a platinum-containing chemotherapeutic drug. Then in July
2004, the drug was approved by the FDA as a second line agent for the treatment of advanced
or metastatic non-small cell lung cancer (NSCLC). Currently, the drug is used as a single
agent or in combination with other chemotherapeutic agents for the treatment cancer such as
breast cancer, bladder cancer, colorectal carcinoma and cervical cancer. Pemetrexed is an
antifolate, a substance that blocks the activity of folic acid. Also called folate antagonist.1
According to WHO, Cancer is defined as “Uncontrolled growth and spread of cells.
It can affect almost any part of the body. The growth often invades surrounding tissue and
can metastasize to distant sites”. Cancer is a leading cause of death worldwide and the total
number of cases globally is increasing.2
As on 1 April 2008 Cancer is a leading cause of death worldwide and the total
number of cases globally is increasing. The number of global cancer deaths is projected to
increase 45% from 2007 to 2030 (from 7.9 million to 11.5 million deaths), influenced in part
by an increasing and aging global population. The estimated rise takes into account expected
slight declines in death rates for some cancers in high resource countries. New cases of
cancer in the same period are estimated to jump from 11.3 million in 2007 to 15.5 million in
2030.3
Cancer is a number of related diseases that are characterized by the uncontrolled
proliferation and disorganized growth of cells. Tumor cells invade and destroy normal tissues
and may spread throughout the body via the circulatory systems.4
The body consists of many different organs, which in turn are composed of several different
types of tissues. There are three major categories of tissue-related tumor types: carcinoma,
sarcoma and leukemia/lymphoma. There are also other specialized tumor categories, such as
those of the central nervous system (e.g., brain tumors).
Here are some common examples of site of origin classification:
Adenocarcinoma–originates in glandular tissue.
Blastoma–originates in embryonic tissue of organs.
Carcinoma–originates in epithelial tissue (i.e., tissue that lines organs and tubes).
Leukemia–originates in tissues that form blood cells.
Lymphoma–originates in lymphatic tissue.
Myeloma–originates in bone marrow.
Sarcoma–originates in connective or supportive tissue (e.g., bone, cartilage, muscle) .5
Related substances: Impurities derived from the drug substances and therefore not
including impurities from excipients.
Related substances include degradation products,
synthetic impurities of drug substance, and manufacturing process impurities from the drug
product.
6.2 Literature review :
Sriguru B, Nandha NP et al., have reported on development and validation of stability
indicating HPLC method for the estimation of 5-Fluorouracil and related substances in
topical formulation.
The developed method is found to be specific, reproducible, and
stability indicating. Synergi Polar RP 250×4.6mm 4µ was used as column. Mobile phase
consisted of 0.1M potassium dihydrogen phosphate (KH2PO4) buffer to achieve good
resolution and retention of the analyte and its impurities. The limit of detection (LOD) and
limit of quantification (LOQ) found to be in a range of 0.004 µg/ml and 0.014 µg/ml for 5Fluorouracil and related substances respectively.6
Prakash Sarasambi S, Kalyan Chakravarthy, Purushotham Rao K. have worked on estimation
of Gemcitabine in bulk drug and its Formulation by using Spectrophotometric. Gemcitabine
is a pyramidine analog which is used in the treatment of cancer. The method is based on
three simple, sensitive, accurate and economical spectrophotometric methods has been
developed for the estimation of gemcitabine in bulk drug and pharmaceutical formulation.
Method A is based on UV spectrophotometric measurements in which gemcitabine was
dissolved in double distilled water and exhibited absorption maximum at 266 nm and obeyed
Beer’s law in the concentration range of 5-30 g/ml. Method B is based on measurements of
first order derivative spectroscopy adopted to eliminate spectral interference, in which
derivative amplitude was measured at 260.5 nm. Method C is based on calculation of area
under curve for analysis of Gemcitabine in wavelength range of 260-271 nm. The analysis
were validated for accuracy, precision, Limit of Detection (LOD), Limit of Quantification
(LOQ).7
Renaud Respaud, Jean-Francois Tournamile et al., validation on liquid chromatography
coupled to ultraviolet and evaporative light scattering detection (HPLC-UV-ELSD) method
for determination of L-glutamic acid, a potential degradation product of pemetrexed and for
the quantification of pemetrexed. This is an ion-pairing, reversed-phase method. Synergi
MAX-RP C12 4 μm was used as column, trideca fluoroheptanoic acid as mobile phase it is
an aqueous solution and acetonitrile under gradient elution mode.
L-Glutamic acid was
detected by ELSD, and pemetrexed detected by UV at 254 nm. This method validated
according to SFSTP and ICH guidelines.8
Monique W.J, Bastiaan Nuijen et al., have worked on development and validation for the inv
estigational anticancer agent imexon and identification of its degradation products. Imexon is
a derivative of 4-imino-1, 3-diazabicyclo [3, 1, 0]-hexan-2-one and it is a class of 2cyanoaziridine
derivatives.
Pharmaceutical
development
of imexon necessitated
the
availability of an assay for the quantification and purity determination of imexon active
pharmaceutical ingredient (API) and the drug is a pharmaceutical dosage form. The method
was developed and detection by liquid chromatographic (LC) method with ultraviolet (UV)
using a reverse phase column with phosphate buffer (pH 6.5 mM) as mobile phase and uv
detection at 230 nm. The method was found to be linear over the concentration range of
interest of 1.0–25μg/ml, precise, accurate, and stability-indicating. LC–mass spectrometry
(MS) and on-line photodiode array (PDA) use as detection enabled us to propose structures
for four degradation products.9
Olivia Roth, Odilie Spreux-Varoquaux et al., have performed on Imatinib assay by HPLC
with photodiode-array UV detection in plasma from patients with chronic myeloid leukemia
Comparison with LC-MS/MS. Imatinib is a competitive inhibitor of BCR–ABL tyrosine
kinase, is now the first-line treatment for chronic myelogenous leukemia (CML).
The
method was developed on a high-performance liquid chromatography (HPLC) method with
UV/Diode array detection for (DAD) for trough
imatinib concentration determination
in human plasma. The calibration curve was prepared in blank human plasma. Results with
these two methods were compared using Deming regression, chi-square test, and sign test.10
Ramachandra R Y, Srinivasan R et al., have reported on LC and LC–MS/MS study of forced
decomposition behavior of anastrozole and establishment of validated stability indicating anal
ytical method for impurities estimation in low dose anastrozole tablets. In this method had
the ability to separate anastrazole from there degradation products, impurities and relative
compounds such as excipients found in the tablet dosage form. This method column using
reversed phase high performance liquid chromatography (RP-HPLC). Water was used as
mobile phase-A, and acetonitrile as mobile phase-B and uv detection at 215 nm.
The
same method was also extended to LC–MS/MS studies which were carried out to identify the
degradation product. The method is rapid, direct, specific, accurate, precise, stabilityindicating and validated for the routine analysis in the finished dosage form. The method also
evaluated active drug substance.11
Hui Li et al., have performed on determination of blood methotrexate by high performance
liquid chromatography with online post-column electrochemical oxidation and fluorescence
detection. HPLC method was developed for the determination of plasma methotrexate level,
whole-blood
methotrexate
(MTX)
level,
and
whole-blood
total
methotrexate
(MTX + MTXPG) level. The MTXPG converted into methotrexate (MTX) to enzymatically.
Then add 20 μl NaOH solutions (0.5 M) and 0.8 ml methanol and mixed well and
centrifugation, a 0.5ml aliquot of the supernatant was evaporated to dryness and re-dissolved
in 0.2 ml hydrochloric acid (10 M). Methylene chloride (0.2 ml) was added and mixed well.
After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the
MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by
a fluorescence detector.12
Srinivasu P, Senlin Zhou., et al., have worked on estimation and investigational an anticancer agent SR271425 and its metabolites in mouse plasma by High-performance liquid
chromatographic method. This method is simple and reliable. HPLC method was developed
for the estimation of a new anti-cancer agent that belongs to the thioxanthone class,
SR271425 in mouse plasma. The chromatography technique was performed on a reverse
phase c18 was used as column, methanol 10 mM phosphate buffer, pH 3.5 (45:55) as mobile
phase standard. At a flow-rate of 0.8 ml/min for first 10 min and 1.4 ml/min for the next 15
min with UV–Vis at 264 nm.13
Devi T.S, Gayathri S et al., estimation of paclitaxel drugs by HPLC method. Paclitaxel is an
anti-leukemic, anti-tumor or in general an anti-neoplastic agent. It is first isolated from the
bark of the pacific Yew tree, Tax us breviofolia. Many pharmaceutical drugs analyzed using
high performance liquid chromatography method. In the present work, analysis of Paclitaxel
drugs such as Nanoxel and Oncotaxel are done by HPLC method.
HPLC analysis of
Paclitaxel drugs was carried out on Shimadzu prominence C18 column phenomenex Gemini
(250×4.6mm, 5μm) with KH2PO4 -Acetonitrile (60:40) as the mobile phase and flow rate of
2ml/min at UV detection wavelength of 230nm.14
Leanne, Karen Gelmon et al., have worked on validation of a high-performance liquid
chromatographic assay method for quantification of total vincristine sulfate in human plasma
following administration of vincristine sulfate liposome injection. Validation of a high
performance liquid chromatographic (HPLC) is described assay method for quantitation of
total vincristine sulfate (VINC) in human plasma. The chromatography method using a
waters Symmetry C8 (250 mm×4.6 mm I’d) analytical column, a Waters Delta-Pak ODS
guard column with a mobile phase of 34.9% water–0.1% diethyl amine (pH 7.0)–
40%acetonitrile–25% methanol pumped isocratically at 1.0 ml min−1 detection with
ultraviolet at 297 nm.15
Deeanna L Thomas Burke G, have reported on simultaneous quantitation of the lactone and c
arboxylate forms of camptothecin anticancer
drugs
by
high
performance
liquid
chromatographic method. The well documented hydrolysis of the α-hydroxyl-δ-lactone ring
moiety in camptothecin and related analogues is routinely monitored using high-performance
liquid chromatography (HPLC). Previous HPLC separations of the lactone and carboxyl ate
forms of camptothecins have often required mobile phases containing three to four
components the reagents are, ion-pairing reagent to provide adequate retention of the
carboxylate form of the drug, buffer is control the ionic strength and pH of the mobile phase,
acetonitrile to control the retention of the lactone form and, in some instances,
sodium dodecyl sulfate to reduce peak tailing.16
6.3 Objective of the study:
The objective of validation of the HPLC analytical method for related substances of
Pemetrexed for Injection is to establish documented evidence that provides an assurance that
the specified method will consistently provide accurate test results that evaluate a product
against its specification by laboratory studies.
Method validation:

Carry out the method validation as per related substances procedure.

Calibration of equipment, maintenance of verification related documents as per sop.

Handling of Instruments, data generation and reporting.

Preservation and circulation of verification reports.

Compilation of analytical results and generation of validation report with respect to
each parameter against the specified success criteria, monitoring the chromatograms.
Carry out the analytical method for related substances of Pemetrexed for Injection by
High Performance Liquid Chromatography (HPLC) method, using the following conditions.
Evaluate certain parameters for HPLC method to ensure compliance as per ICH Q3 B
(R) Guidelines.
Analytical method validation for related substances of Pemetrexed for Injection shall
be performed by carrying out the following analytical parameters.
1.
System Suitability
2.
Specificity
3.
Precision
3.1. Method Repeatability
3.2. Method Reprodubility
4.
Linearity and Range
5.
Accuracy
6.
Ruggedness
7.
Roubstness
8.
Solution stability
9. Revalidation criteria
1. SYSTEM SUITABILITY:
Inject 20 µL of standard solution into the chromatograph and record the suitability parameters
as per procedure.
2. SPECIFICITY:
Specificity is the ability to assess unequivocally the analyte in the presence of components
that may be expected to be present, such as impurities and excipients.

Prepare diluents and inject into the chromatographic system in triplicate.

Prepare a solution of excipient mixture (placebo) and inject into the chromatographic
system in triplicate.

Prepare and inject standard preparation into the HPLC system in triplicate.

Prepare a test solution with placebo and drug substance powder containing 0.5 % v/v
of Impurity and analyze as per test procedure and record the retention times of peaks.

Inject all the above solutions in triplicate and record the retention times of the peaks
present.
3. PRECISION:
Precision is the measure of either the degree of reproducibility or of repeatability of the
analytical method under normal operating conditions. Determine the precision of analytical
procedure the degree of agreement among individual test results in the procedure applied
repeatedly to multiple samplings of a homogeneous sample.
The precision of an analytical procedure is usually expressed as the standard deviation (SD)
or relative standard deviation (RSD).
3.1 Method Repeatability
3.2 Method reproducibility
3.1 METHOD REPEATABILITY:
Determine the precision of test method by spiking test preparation with Impurities solution to
get 0.5 % v/v of Impurity in pemetrexed for injection (0.2 mg/ml of pemetrexed disodium)
and will be tested through the complete analytical procedure from sample preparation to final
result. Repeatability should be assessed using a minimum of 6 determinations and calculate
relative standard deviation % of impurities.
3.2 METHOD REPRODUCIBILITY:
Method reproducibility is demonstrated by 6 determinations of the same samples tested by
another analytical group or in another laboratory (collaborative study).
Determine the
precision of test method by spiking test preparation with Impurities solution to get 0.5 % v/v
of Impurity in pemetrexed for injection (0.2 mg/ml of pemetrexed Disodium) and will be
tested through the complete analytical procedure from sample preparation to final result.
Reproducibility should be assessed using a minimum of 6 determinations and calculate
relative standard deviation % of impurities.
4. LINEARITY AND RANGE:
Linearity is to validate the ability to elicit test results that are directly proportional to the
concentration of analyte in sample within a given range. Linearity and range has to be studied
for the Pemetrexed Disodium and impurity in the range of LOQ levels to 120% (LOQ, 80%,
90%, 100%, 110%, & 120%) of the specified limit of each impurity (i.e. 0.5 % of Impurity).
Estimate the degree of linearity by calculating the correlation coefficient, Regression
coefficient (r2), Y-intercept ,residual sum of squares and Y-intercept / response at 100% of
working concentration and slope should be calculated and reported by injecting each of the
above solutions ( LOQ to 120%) in triplicate and 120% solution in six replicates. Establish a
plot of data for analyte response Vs Concentration.
5. ACCURACY:
Acc Accuracy is to validate the closeness of test results obtained by the analytical procedure to
the true value. The accuracy should be established across the specified range of the analytical
method. To validate the test method can accurately quantify impurities within the Pemetrexed
for injection. Inject the samples in triplicate by spiking test preparation with 80%, 100%, and
120% to the target concentration of 0.5 % v/v of Impurity. Calculate the % recovery of
impurities.
6. RUGGEDNESS:
The ruggedness of an analytical method is the degree of reproducibility of test results
obtained by the analysis of the same samples by different analysts. It is a measure of
reproducibility of test results under normal operational conditions from analyst to analyst.
The ruggedness of an analytical method is determined by analysis of aliquotes from
homogeneous lots, by different analysts, by different equipment and different days. This has
to be compared to the precision of the assay by different analysts.
Determine the Ruggedness of test method by spiking test preparation with Impurities solution
to get 0.5 % v/v of Impurity in pemetrexed for injection 500 mg/vial (0.2 mg/mL of
pemetrexed disodium) and will be tested through the complete analytical procedure from
sample preparation to final result. It should be assessed using a minimum of 6 determinations
and calculate relative standard deviation % of impurities.
It is considered necessary to study these effects individually and within the same
laboratory.
7. Robustness:
Robustness is to validate the analytical procedure capacity to remain unaffected by small but
deliberate variation in method parameters and provides indication of its reliability during
normal usage. Evaluate the analytical method robustness for the following typical variation
from set procedure.

Influence of variations of flow rate in mobile phase. (± 0.2 mL of the specified flow
rate, Normal flow rate 1.0 ml / min).

Influence of variations of column temperature. (± 5°C of the specified column
temperature, Normal column temperature is 30°C).

Influence of variations in mobile phase composition. (± 5% of specified composition
of organic phase).

Influence of variations in membrane filter and centrifuge (The difference in % of
impurities between centrifuge and filter the different portions of the same test sample
and inject it)
8. Solution stability:
Sample and standard solutions in duplicate should be tested over at least 48 hours period
(Initial, 24 hours & 48 hours) under room temperature (25°C ± 2°C) and refrigerator
conditions (2°C-8°C), and potency of
Standard and % impurities of sample should be determined by comparing the results against
the freshly prepared standard.
9. Revalidation criteria:
Revalidation shall be done whenever changes as follows.

Analytical method.

Manufacturing composition.

Manufacturer Change
7. MATERIALS AND METHOD:
7.1
Source of data:
Data will be obtained from Pubmed, Science Direct, Medline, various related website and
other Internet facilities, literature search, and related articles from library of Krupanidhi
College of Pharmacy and natco lab.
7.2 Method of collection of data:
Data will be collected from the following step wise experimental procedures proposed in the
study:
1. HPLC conditions
a. HPLC column (lot, age, brand)
b. Mobile-phase composition (pH ± 0.05 unit, percent organic ± 2%)
c. HPLC instrument (dwell volume, detection wavelength ± 2 nm, column temperature ± 5◦C,
flow rate)
2. Sample preparation
a. Sample solvent (pH ± 0.05 unit, percent organic ± 2%)
b. Sample preparation procedure (shaking time, different membrane filters)
c. HPLC solution stability
Built-in Robustness in Method procedure:
• Weighing error
• Dilution error
• Sonication
• Mobile phase as sample solvent
• Buffer
• Isocratic method.
This is to verify that the method performance is not affected by typical changes in normal
experiments. Therefore, the variation in method conditions for robustness should be small
and reflect typical day-today variation.
7.3 Does the study require any investigation of intervention to be conducted
on patients or other humans or animals? If so please describe briefly?
NO
7.4 Has ethical clearance been obtained from your institute in case of as
above?
Not applicable
8. REFERENCES
1. Information on the antifolate drug pemetrexed.
URL: http://www.pemetrexed.org/
2. Cancer. [Online]. 2008 [cited 2011 May 23];[1].
URL:http://www.who.int/topics/cancer/en/
3. Are the number of cancer cases of increasing or decreasing in the world? [Online]. 2008
[cited 2011 May 23];[1].
URL:http://www.who.int/features/qa/15/en/index.html
4. Cancer - A Genetic Disease, Classification of Cancer Types, Benign or Malignant Tumor,
Type of Cell - Type of Tissue, Site of Origin. [Online]. 1984 [cited 2011 May 23];[1].
URL:http://medicine.jrank.org/pages/1984/Cancer.html
5. Classification of Cancer, Cancer Staging. [Online]. 2002 [cited 2011 May 23];[1].
Available from
URL:http://www.oncologychannel.com/staging.html
6. Sriguru Bandana, Nandha NP, Vairale AS, Sherikar AV, Nalamothu V. Development and
validation of stability indicating HPLC method for the estimation of 5-Fluorouracil and
related substances in topical formulation. J Res Pharm Sci 2010;1(2):78-85.
URL: http://ijrps.pharmascope.org/downloads/Volume%201/Issue%202/56338.pdf
7. Prakash Sarsambi S, Kalyan Chakravarthy, Purushotham Rao K. Spectrophotometric
Estimation of Gemcitabine in bulk drug and its Formulation, Int J Anal Chem 2011;1(2):1894.
URL: http://www.ijpijournals.org/jac_volume_1/jac_issue_2/jac10.pdf
8. Renaud Respaud, jean- François Tournamille, Cecile Croix, Helene laborie, Claire Elfakir
et al., Development of an ion-pairing reversed phase liquid chromatography method using a
double detection analysis (UV and evaporative light scattering detection) to monitor the
stability of Alimta® pemetrexed preparations: Identification and quantification of L-glutamic
acid as a potential degradation. J Pharm Biomed Anal 2011 Jan 25;54(2):411-6.
URL: http://www.ncbi.nlm.nih.gov/pubmed/20869830
9. Monique Den Brok W.J, Bastiaan Nuijen, Michel Hillenbrand J.X, Christian Lutz, Hans-G
opitz H et al., LC-UV Method development and validation for the investigational anticancer
agent imexon and identification of its degradation products J Pharm Biomed Anal 2005 Jul
15;38 (4):686-94.
URL:http://www.ncbi.nlm.nih.gov/pubmed/15967296
10. Olivia Roth, Odile Spreux-Varoquaux, Stephane Bouchet, Philippe Rousselot, Sylvie Cas
taigne, Sophie Rigaudeau et al., Imatinib assay by HPLC with photodiode-array UV detection
in plasma from patients with chronic myeloid leukemia: Comparison with LC-MS/MS. Clin
Chim Acta 2010 Feb;411(3-4):140-6.
URL: http://www.ncbi.nlm.nih.gov/pubmed/19853594
11.Rama chandra Reddy Y, Srinivasan R, Nandan,Vijaya Bharathi D, Nagaraju B, Saidu Re
ddy S et al.,LC and LC–MS/MS study of forced decomposition behaviour of anastrozole
establishment of validate stability-indicating analytical method or impurities estimation in
low dose anastrozole tablets. J Pharm Biomed Anal 2009 Oct 15;50(3):397-404.
URL: http://www.ncbi.nlm.nih.gov/pubmed/19541446
12. Hui Li, Wenhong Luo, Qingyu Zeng, Zhexuan Lin, Hongjun Luo and Yuan Zhang.
Method for the determination of blood Methotrexate by high performance liquid
chromatography with online post column electrochemical oxidation and fluorescence
detection. J Chromatogr B Analyt Technol Biomed Life Sci 2007 Jan 1;845(1):164-8.
URL: http://www.ncbi.nlm.nih.gov/pubmed/16890029
13.Srinivasu P, Senlin Zhou, James Rake, Gareth Shackleton, Thomas H, Corbett et al.,
High-performance liquid chromatographic method for the estimation of the novel
investigational anti-cancer agent SR271425 and its metabolites in mouse plasma. J
Chromatogr B Biomed Sci Appl. 2001 Aug 5;759(1):175-8.
URL: http://www.ncbi.nlm.nih.gov/pubmed/11499623
14. Renuga Devi T.S, Gayathri.S, Estimation of paclitaxel drugs by HPLC method. Dear
pharma chemical 2010;2(2):109-115.
URL:http://derpharmachemica.com/vol2-iss2/DPC-2010-2-2-109-115.pdf
15. Leanne Embree, Karen Gelmon A, Anthonly Tolcher W, Norma Hudon J, Jean Heggie R,
Carole Dedhar et al., Validation of a high-performance liquid chromatographic assay method
for quantification of total vincristine sulfate in human plasma following administration
of vincristine sulphate liposome injection. J Pharm Biomed Anal 1997 Dec;16(4):675-87.
URL: http://www.ncbi.nlm.nih.gov/pubmed/9502163
16. Deanna W L, Thomas Burke G. Simple and versatile high-performance liquid
chromatographic method for the simultaneous quantitation of the lactone and carboxylate
forms of camptothecin anticancer drugs. J chromatogr B Biomedical Sci and Appl 1997
march 28;691(1):161-171.
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