newer analytical method development of erlotinib

www.wjpps.com Vol 9, Issue 9, 2020.

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Durgadasheemi et al. World Journal of Pharmacy and Pharmaceutical Sciences

NEWER ANALYTICAL METHOD DEVELOPMENT OF ERLOTINIB

HYDROCHLORIDE AND STUDY OF IN VITRO DRUG-DRUG

INTERACTION WITH PROTON PUMP INHIBITORS

Nagaraj N. Durgadasheemi*1, Kavitha S. K.

2, Shilpashrre A. T.

3 and Syed Nizamuddin

4

1Department of Pharmaceutical Chemistry, R.R. College of Pharmacy, Bangalore, India.

2Department of Pharmacology, R.R. College of Pharmacy, Bangalore, India.

ABSTRACT

In the present work, newer analytical methods for the determination of

Erlotinib hydrochloride has been developed, stability of Erlotinib

hydrochloride is determined by performing stress degradation studies

and in vitro drug-drug interaction of Erlotinib hydrochloride with PPIs

was studied. A simple, precise RP-HPLC method was developed and

validated for estimation of Erlotinib hydrochloride using C18 column

maintained at constant temperature and UV wavelength was at 254 nm.

The mobile phase consists of acetonitrile (A) and Phosphate buffer (B)

(50:50 v/v). The isocratic mode was employed at flow rate of 1 ml/min

and injection volume of 20 µl. The method has been validated using

ICH guidelines. In vitro drug-drug interaction study was performed for

Erlotinib hydrochloride with three different PPI at pH 1.4, 2.4 and 4.5 in aqueous medium

and in plasma.The retention time of Erlotinib hydrochloride in mobile phase A and B (50:50

v/v) was found 5.31 min. The linearity of standard Erlotinib hydrochloride was obtained in

the range of 10-100 µg/ml. The % RSD for precision is less than 2%. After performing in

vitro drug-drug interaction study at pH 1.4, pH 2.4 and pH 4.5, the observed result is no shift

in the Rt of the Erlotinib hydrochloride in different pH and in combination with PPI in

aqueous medium and in rat plasma. The developed analytical conditions were with good

resolution within short analysis time. From the obtained results, it can be concluded that

Erlotinib hydrochloride does not form any stable complex with PPI. Therefore, mentioned

results may consider during monitoring and concurrent therapy of both drugs. Thus the

developed method can be used for the routine analysis of Erlotinib hydrochloride in

laboratories, clinical trails and quality control purpose.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 9, Issue 9 1842-1881 Research Article ISSN 2278 – 4357

Article Received on

07 July 2020,

Revised on 28 July 2020,

Accepted on 18 Aug. 2020

DOI: 10.20959/wjpps20209-17054

*Corresponding Author

Nagaraj N.

Durgadasheemi

Department of

Pharmaceutical Chemistry,

R.R. College of Pharmacy,

Bangalore, India.


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KEYWORDS: Erlotinib- Erlotinib hydrochloride, PPI- Proton pump inhibitors.

INTRODUCTION

Pharmaceutical Analysis: is the branch of chemistry involved in separating, identifying and

determining the relative amounts of the components making up a sample of matter. It is

mainly involved in the qualitative identification or detection of compounds and quantitative

measurements of the substances present in bulk and pharmaceutical preparation. Analytical

method is a specific application of a technique to solve an analytical problem. Analytical

instrumentation plays an important role in the production and evaluation of new products and

in the protection of consumers and the environment. This instrumentation provides the lower

detection limits required to assure safe foods, drugs, water and air.[1]

Traditionally, analytical

chemistry has been split into two main types, qualitative and quantitative: Qualitative

analysis seeks to establish the presence of a given element compound in a sample.

Quantitative analysis seeks to establish the amount of a given element or compound in a

sample.[2]

Chromatography is defined as the process by which solutes are separated by a dynamic

differential migration in system consisting of two or more phases, one of which moves

continuously in a given direction. The individual substances exhibit different mobilities by

reason of differences in adsorption, partition, molecular size, ion exchange or charge density.

The individual substances thus obtained can be identified by analytical method.[3]

The importance of chromatography is increasing rapidly in pharmaceutical analysis for the

exact differentiation, selective identification and quantitative determination of the

compounds. Another important field of application of chromatographic methods is the purity

testing of final products and the intermediates (detection of decomposition products and by

products). As the consequence of the above points, chromatographic methods are given

important place in testing of the standards of the drugs. Application of chromatographic

methods is also applied in food safety, biochemical, environmental and industrial fields. The

application of chromatography have grown extensively in the last fifty years owing not only

to the development of several new types of chromatographic techniques but also to the

growing need by scientists for getting better methods for characterizing complex mixtures.


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Types of Chromatography

The principles of different chromatography are based on the nature of interactions between

the solute and the stationary phase, which may arise from hydrogen bonding, vander waa’l

forces, electrostatic forces and hydrophobic forces. Different modes of chromatography are

(a) Adsorption chromatography

Normal phase chromatography

Reverse phase chromatography

Ion exchange chromatography

(b) Partition chromatography

Gas chromatography

Liquid-liquid partition chromatography

(c) Size exclusion chromatography (SEC)

(d) Affinity chromatography

(e) Hydrophobic interaction chromatography (HIC)

Reversed phase chromatography In 1960s, chromatography started modifying the polar

nature of the silanol group by chemically reacting silicons with organic silanes. The object

was to make silica less polar or non-polar so that polar solvents can be used to separate water

soluble polar compounds. Since the ionic nature of the chemically modified silica is now

reversed i.e., it is non-polar or the nature of the is reverted, the chromatographic phase

separation carried out with such silica is referred as reversed-phase chromatography. A large

number of chemically bonded silica based stationary phases are available commercially.

Silica based stationary phases are still more popular in reversed phase chromatography;

however other adsorbents based on polymer (styrene divinyl benzene co polymer) are slowly

growing ground. The less water-soluble (i.e., the more non-polar) sample compounds are

better retained by the reverse phase surface. The retention time decreases in the following

order: Aliphatic > induced dipoles > permanent dipoles >weak lewis bases (ether, aldehydes,

and ketones) > strong lewis bases (amines) > weak lewis acids (alcohols, phenols) > strong

lewis acids (carboxylic acids). Also the retention increases as the number of carbon atoms

increases.

As a general rule the retention increases with an increase in the contact area between sample

molecule and stationary phase i.e., with an increase in the number of water molecule, which


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are released during the adsorption of a compound. Branched chain compounds are eluted

more rapidly than their corresponding normal isomers.

The solvent strength in reverse phase chromatography is reversed from that of adsorption

chromatography (silica gel) as stated earlier. Water interact strongly and highly with silanol

groups, so that, adsorption of sample molecules become highly restricted and they are rapidly

eluted. Exactly opposite applies in reversed phase system; water cannot wet the non-polar

(hydrophobic alkyl group such as C-18 of ODS phase) and therefore does not interact with

the bonded moiety. The elution time (retention time) in reversed phase chromatography

increases with increasing amount of water in the mobile phase.

Normal phase chromatography

In normal phase chromatography, the stationary phase is polar adsorbent. The mobile phase is

generally a mixture of non aqueous solvents. The silica structure is saturated with silanol

group at the end in normal phase separations. These OH groups are statistically distributed

over the whole of the surface. The silanol groups represent the active sites (very polar) in the

stationary phase.

High performance liquid chromatography

Liquid Chromatography (LC), which is one of the forms of chromatography is an analytical

technique that is used to separate a mixture in solution into its individual components. The

separation relies on the use of two different "phases" or "immiscible layers," one of which is

held stationary while the other moves over it. Liquid Chromatography is the generic name

used to describe any chromatographic procedure in which the mobile phase is a liquid. The

separation occurs because, under an optimum set of conditions, each component in a mixture

will interact with the two phases differently relative to the other components in the mixture.

HPLC is the term used to describe Liquid Chromatography in which the liquid mobile phase

is mechanically pumped through a column that contains the stationary phase.4 An HPLC

instrument, therefore, consists of an injector, a pump, a column, and a detector shown a

Solvents must be degassed to eliminate formation of bubbles. The pumps provide a steady

high pressure with no pulsating, and can be programmed to vary the composition of the

solvent during the course of the separation. Detectors rely on a change in refractive index,

UV-Visible absorption, or fluorescence after excitation with a suitable wavelength. The

different types of HPLC columns are described in a separate document. Liquid

Chromatography has come a long way with regard to the practical development of HPLC


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instrumentation and the theoretical understanding of different mechanisms involved in the

analyte retention as well as the development of adsorbents with different geometries and

surface chemistry. In the modern pharmaceutical industry, HPLC is a major analytical tool

applied at all stages of drug discovery, development and production. Fast and effective

development of rugged analytical HPLC methods is more efficiently undertaken with a

thorough understanding of HPLC principles, theory and instrumentation. In HPLC, a liquid

sample, or a solid sample dissolved in a suitable solvent, is carried through a

chromatographic column by a liquid mobile phase. Separation is determined by

solute/stationary-phase interactions, including liquid–solid adsorption, liquid–liquid

partitioning, ion exchange and size exclusion, and by solute/mobile-phase interactions with

their applications. In each case, however, the basic instrumentation is essentially the same.[5]

The LC methods based on the use of common UV (Ultraviolet), DAD (Diode Array

Detector) or ECD (Electron Channel Detector) are insufficiently selective and sensitive for

the determination of biological samples because of variety of complexity of matrix and the

small amounts of residues present. The lack of sensitive and selective LC detectors has been

overcome by combining LC with mass spectrometry. Liquid chromatography coupled to

mass spectrometry is the most powerful technique and preferred for the analysis of

compounds that are of low volatility, high polarity and thermal liability in nature.

HPLC is also an excellent way to remove potentially interfering molecules from the sample

such as salts, buffers and detergents. These types of molecules greatly influence the

efficiency of the ionization and the quality (and quantity) of data generated by the MS, which

is greatly dependent on a clean sample prior to ionization. Thus, LC is very effective in

separating analytes.


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Column: It is the main part of the HPLC. This may be referred as the structural and function

unit of HPLC. Column’s used in HPLC must posses the following properties:

It should with stand the operating pressure.

Materials of column should not be corroded.

It should be non reactive with mobile phase and solute.

Column Dimension

External diameter- 6-35 mm

Internal diameter- 2-5 mm

Length upto - 10-30 mm

Types of column

Analytical columns: These are mainly employed for getting quantitative and qualitative

information of the compounds to be analyzed. This is involved in conventional liquid

chromatography where less amount of the solute is used 10 mg.

Fig 2: Analytical columns.

Advantages

1. Good column efficiency

2. The separation is according to the need

Fast columns

One of the primary reasons for using these columns is to obtain improved sample out put. For

many columns increasing the flow rate through the stationary phase will adversely affect the

resolution and separation. There fore fast columns are designed to decrease time of the

chromatographic analysis without forsaking significant deviation in results. These columns

have the same internal diameter but much shorter length than most other columns, and they

are packed with smaller particles. Particle size 3.0 micrometer in diameter.


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Advantages

Increased sensitivity.

Decreased analysis time.

Decreased mobile phase usage.

Increased reproducibility.

Preparative column

These are improved analytical column in order to isolate purify and enrich the sample in the

quantitative range of 10-100mg from complex mixtures

The different types of preparative columns are

Semi preparative columns

Preparative columns

Micro preparative columns

Micro bore and small bore columns

Capillary columns

Column Preparation

Bottom portion of the column is packed with cotton wool or glass wool or may contain

asbestos pad, above which adsorbent is packed.

Whatman filter paper disc can be used.

After packing the column with the adsorbent, a similar paper disc is kept on the top so

that the adsorbent layer is not disturbed during introduction of sample.

Packing Techniques

Dry packing.

Wet / slurry packing.

Dry packing: Required quantity of adsorbent is packed in the column in dry form and the

solvent allowed to flow through the column till equilibrium is reached Disadvantages:

Air bubbles are entrapped and the column may not be uniformly packed.

Cracks appear in the adsorbent present in the column hence clear band of the separated

component may not be obtained

Wet / Slurry Packing The required qty. of the adsorbent is mixed with the mobile phase &

poured into the column.


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Advantages

– No entrapment of air bubbles.

– No crack in adsorbent.

– The bands eluted from the column will be uniform & ideal for separation.

Column Packing Material

Micro porous supports:

o 3-10 µm in diameter.

o Composed of silica, alumina, ion exchange resin

Pellicular supports

o 40 µm in diameter.

o Porous particles are coated onto an inert solid core such as glass bead.

Bonded phases

o Stationary phase is chemically bonded onto inert support.

For adsorption chromatography

o Adsorbents such as silica or alumina are available as micro porous forms.

o Pellicular systems generally have a high efficiency but low sample capacity & so micro

porous supports are preferred.

– Spherical shape gives good efficiency & flow properties.

Liquid- liquid partition system

– Stationary phase coated on to the inert support.

– Both micro porous & pellicular supports are used for supporting liq. Phase.

Disadvantages

Developing solvent may gradually wash off the liq. Phase with repeated use.

To overcome this problem bonded phases have been developed.

Ex. - silica, silicone polymer

Analytical method development

Methods are developed for new products when no official methods are available. Alternate

methods for existing (non-pharmacopoeial) products are developed to reduce the cost and

time for better precision and ruggedness. Documentation starts at the very beginning of the


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development process, a system for full documentation of the development studies must be

established. All data relating to these studies must be recorded in laboratory notebook or an

electronic database.[6]

Standard analyte characterization

All known information about the analyte and its structure is collected i.e., physical and

chemical properties, toxicity, purity, hygroscopic nature, solubility and stability.

The standard analyte (100% purity) is obtained. Necessary arrangement is made for the

proper storage (refrigerator, desiccators, and freezer).

When multiple components are to be analyzed in the sample matrix, the number of

components is noted, data is assembled and the availability of standards for each one is

determined.

Only those methods (MS, GC, HPLC etc.,) that are compatible with sample stability are

consid Requirements for analytical method development

The goals or requirements of the analytical method that need to be developed are considered

and the analytical figures of merit are defined. The required detection limits, selectivity,

linearity, range, accuracy and precision are defined.

Literature search and prior methodology

The literature for all types of information related to the analyte is surveyed. For synthetic

drugs, physical and chemical properties, solubility and relevant analytical methods are

colleceted. Books, periodicals, chemical manufacturers and regulatory agency compendia

such as USP / NF, AOAC and ASTM publications are reviewed. Chemical Abstracts Service

(CAS) automated computerized literature searches are convenient.

Choosing a method

Using the information in the literatures and prints, methodology is adapted. The methods

are modified wherever necessary. Sometimes it is necessary to acquire additional

instrumentation to reproduce, modify, improve or validate existing methods for in-house

analytes and samples.

If there are no prior methods for the analyte in the literature, from analogy, the

compounds that are similar in structure and chemical properties are investigated and are


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worked out. There is usually one compound for which analytical method already exist

that is similar to the analyte of interest.

Instrumental setup and initial studies

The required instrumentation is setup. Installation, operational and performance qualification

of instrumentation using laboratory standard operating procedures (SOPs) are verifiedered.

The analyte standard in a suitable injection/introduction solution and in known concentrations

and solvents are prepared. It is important to start with an authentic, known standard rather

than with a complex sample matrix. If the sample is extremely close to the standard (e.g.,

bulk drug), then it is possible to start work with the actual sample.Analysis is done using

analytical conditions described in the existing literature.

Optimization: During optimization one parameter is changed at a time and set of conditions

are isolated, rather than using a trial and error approach. Work has been done from an

organized methodical plan, and every step is documented (in a lab notebook) in case of dead

ends.

Documentation of analytical figures of merit

The originally determined analytical figures of merit limit of quantitation (LOQ), Limit of

detection (LOD), linearity, time per analysis, cost, sample preparation etc., are documented.

Evaluation of method development with actual samples

The sample solution should lead to unequivocal, absolute identification of the analyte peak of

interest apart from all other matrix components.

Determination of percent recovery of actual sample and demonstration of quantitative

sample analysis

Percent recovery of spiked, authentic standard analyte into a sample matrix that is shown to

contain no analyte is determined. Reproducibility of recovery (average +/- standard

deviation) from sample to sample and whether recovery has been optimized has been shown.

It is not necessary to obtain 100% recovery as long as the results are reproducible and known

with a high degree of certainty. The validity of analytical method can be verified only by

laboratory studies. Therefore, documentation of the successful completion of such studies is a

basic requirement for determining whether a method is suitable for its intended applications.


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Validation

Action of providing accordance with the principles of good manufacturing practice, that any

procedure, process, equipment material, activity or system actually leads to expected results.

In brief validation is a key process for effective Quality Assurance.[7]

Analytical method validation

Analytical monitoring of a pharmaceutical product or of specific ingredients within the

product is necessary to ensure its safety efficacy throughout all phases of its shelf life. Such

monitoring is in accordance with the specifications elaborated during product development.

All new methods developed must be validated.

Parameters for Method Validation

The parameters for method validation have been defined in different working groups of

national and international committees and are described in the literature. Unfortunately, some

of the definitions vary between the different organizations. An attempt at harmonization was

made for pharmaceutical applications through the ICH (4, 5), where representatives from the

industry and regulatory agencies from the United States, Europe and Japan defined

parameters, requirements and, to some extent, methodology for analytical methods validation.

The parameters, as defined by the ICH and by other organizations and authors, are

summarized below and are described in brief in the following paragraphs.[8]

Specificity

Selectivity

Precision

Repeatability

Intermediate precision

Accuracy

Linearity

Range

LOD

LOQ

Robustness

Ruggedness


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ANALYTICAL PERFORMANCE CHARACTERISTICS

Accuracy

Accuracy is the closeness of test results obtained by that method to the true value. In case of

assay of a drug substance accuracy may be determined by application of the analytical

method to an analyte of known purity (e.g. reference standard) or by comparison of the

results of the method with those of a second well characterized method, the accuracy of

which has been stated or defined. Accuracy is calculated as the percentage of recovery by the

assay of the known added amount of analyte in the sample, or as the difference between the

mean and the accepted true value, together with confidence intervals. The ICH documents

recommended that accuracy should be assessed using a minimum of nine determinations over

a minimum of three concentrations levels, covering the specified range (i.e., three

concentrations and three replicates of each concentration

Precision

Precision is the degree of agreement among individual test results when the method is applied

repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method

is usually expressed as the standard deviation or relative standard deviation (coefficient of

variation) of a series of measurements. Precision may be measure of either the degree of

reproducibility or of repeatability of the analytical method under normal operating conditions.

Precision of an analytical method is determined by assaying a sufficient number of aliquots of

a homogenous sample to be able to calculate statistically valid estimates of standard deviation

or relative standard deviation (coefficient of variation). The ICH documents recommend that

repeatability should be assessed using a minimum of nine determinations covering the

specified range for the procedure.

Repeatability

The precision of the analytical method when repeated by the same analyst under set of

laboratory conditions, the only difference being the sample.The repeatability of a test

procedure is assessed by carrying out complete separate determinations on the separate

samples of the same homogeneous batch of the material and this will provide a measure of

the precision of the procedure under normal laboratory operating conditions.

Reproducibility: When the procedure is carried out by different analysts in different

laboratories using different equipments, reagents and laboratory setting, the reproducibility of

a test procedure is determined by evaluating the samples from the same homogeneous batch,


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the analytical data will provide information about the reproducibility of the test procedure

under validation.

Specificity

ICH documents defines specificity as the ability to assess unequivocally the analyte in the

presence of compounds that may be expected to present, such as impurities, degradation

products and matrix components. ICH documents state that when chromatographic

procedures are used, representative chromatograms should be presented to demonstrate the

degree of selectivity and peaks should be appropriately labeled. Peak purity tests (e.g., Using

diode array or mass spectrometry) may be useful to show that the analyte chromatographic

peak is not attributable to more than one component.

Detection Limit: Lowest amount of analyte in a sample that can be detected, but not

necessarily quantities as an exact value, under stated experimental conditions. The detection

limit is usually expressed as the concentration of analyte (e.g., percentage parts per million)

in the sample. For instrumental and non-instrumental methods detection limit is generally

determined by the analysis of samples with known concentration of analyte and by

establishing the minimum level at which the analyte can be reliably detected.

Quantitation Limit

It is the lowest amount of analyte in a sample that can be determined with acceptable

precision and accuracy under the stated experimental conditions. Quantitation limit is

expressed as the concentration of analyte (e.g. percentage, parts per billion) in the sample.

For instrumental and non-instrumental methods, the quantitation limit is generally determined

by the analysis of samples with known concentration of analyte and by establishing the

minimum level at which the analyte can be determined with acceptable accuracy and

precision.

Linearity and Range

Linearity of an analytical method is its ability to produce results that are directly, proportional

to the concentration of analyte in samples. The range of the procedure is an expression of the

lowest and highest levels of analyte that have been demonstrated to be determinable with

acceptable precision, accuracy and linearity. These characteristics are determined by

application of the procedure to a series of samples having analyte concentration spanning the


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claimed range of the procedure. When the relationship between response and concentration is

not linear, standardization may be provide by means of a calibration curve. ICH recommends

that for the establishment of linearity a minimum of 5 concentrations normally used.

Sensitivity

Sensitivity is the capacity of the test procedure to record small variations in concentration. It

is the slope of the calibration curve. A more general use of the term to encompass limit of

detection and or / limit of quantitation should be avoided.

Ruggedness

Degree of reproducibility of test results obtained by the analysis of the same samples under a

variety of conditions, such as different laboratories, different analysts, different instruments

etc., Normally expressed as the lack of influence on test results of operational and

environmental variables of the analytical method. Ruggedness is a measure of reproducibility

of test results under the variation in condition normally expected from laboratory to

laboratory and from analyst to analyst.

Determination of ruggedness: By analysis of aliquots from homogenous lots in different

laboratories, by different analysts, using operational and environmental conditions that may

differ but are still within the specified parameters of the assay. Degree of reproducibility of

test results is then determined as a function of the assay variables.

Robustness: Robustness of an analytical method is measure of its capacity to remain

unaffectedly small but deliberate variations in method parameters and provides an indication

of its reliability during normal usage.

System Suitability

According to USP system suitability are an integral part of chromatographic methods. These

tests verify that the resolution and reproducibility of the system are adequate for the analysis

to be performed. One consequence of the evaluation of robustness and ruggedness should be

that a series of system suitability parameters is established to ensure that the validity of the

analytical method is maintained whenever used. System suitability tests are based on the

concept that the equipment, electronics, analytical operations and samples constitute an

integral system that can be evaluated as a whole.[9,10]


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DRUG PROFILE

Structure

Fig 3: Erlotinib hydrochloride structure.

Drug profile

Molecular formula- C22H23N3O4.HCL

Molecular weight - 429.20

Chemical name: N-(3-ethynylphenyl)-6, 7 bis(2 methoxy ethoxy) quinazolin-4-amine

hydrochloride.

Characteristic -.White to off white powder

Solubility – Erlotinib hydrochloride is very slightly soluble in water, slightly soluble in

methanol and practically insoluble in acetonitrile, acetone, ethylacetate and hexane.

pKa- 5.42 at 25 °C

Melting Point- 223-228 °C

Category- Antineoplastic agent.

Erlotinib has recently been shown that to be potent inhibitor of JAK2V617F activity.

JAK2V617F is a mutant of tyrosine kinase JAK2, is found in most patients with

polycythemia vere (PV) and a substantial proportion of patients with idiopathic myelofibrosis

or essential thrombocythemia. The study suggests that Erlotinib may be used for treatment of

JAK2V617F-Positive PV and other myeloproliferative disorders.

In-vitro drug-drug interaction studies

Nowadays multiple drug therapy is a common and useful practice for the treatment of

diseases where two or more drugs are given at the same time or concurrently. The drugs may

exhibit effects independently or may interfere or interact with each other. The interaction may

be potentiation or antagonism of one drug by another. Sometimes multiple drug therapy is

beneficial to the patients and sometime it causes serious harmful effects. Thus the drug

interaction study is very much important in respect to both bio-pharmaceutics and


1857


pharmacology. However, to take any step to manage the interaction problems, the nature of

interaction should be known. We should know the possible interaction of a new drug prior to

use clinically. For the drugs which are being used conventionally, interaction studies are also

very important to detect the problems yet to be found out.[20]

Drug-drug interaction study by RP-HPLC method

High performance liquid chromatography is a chromatographic technique that can separate a

mixture of compounds and is used to identify, quantify and purify the individual components

of the mixture. Retention time and absorbance peak of one species in solution may be

changed due to the interaction with other species. In the present study, analysis of a

combination of Erlotinib hydrochloride and proton pump inhibitors (1:1 molar ratio) was

carried out using RP-HPLC at pH 1.4, 2.4 and 4.5.

Selection of extraction method

In bioavailability and bioequivalence studies there are 4 methods of extraction procedures:

Solid phase extraction

Liquid-liquid extraction

Protein precipitation

Hybrid extraction technique

Liquid –liquid extraction

Liquid- liquid extraction is based on the differential solubility and partitioning equilibrium

i.e, the partition phenomenon. It requires two immiscible phases that is aqueous and organic

phase but both phases must be immiscible. Analyte is removed from the matrix selectively by

choosing a suitable extraction solvent and buffering the sample, if required, LLE provides

efficient removal of analyte with desired specificity/selectivity required for intended bio-

analysis. In the reverse phase the stationary phase is non polar and the mobile phase is polar.

In such case the most polar compound is eluted first and the most non polar solvent is eluted

at the last. In Reverse phase chromatography a partition mechanism is typically used for the

separation by non polar differences. The normal solvents used in Liquid-Liquid Extraction

are Tertiary Butyl Methyl Ether, Ethyl acetate, Diethyl ether, Dichloro methane. The purpose

of the present study is to investigate in vitro complex formation and to study the nature and

strength of complexes which could be formed due to interaction of Erlotinib hydrocshloride

with different proton pump inhibitors like pantaprazole, omeprazole and rabeprazole.


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MATERIALS AND METHODS MATERIALS

Table 1: Materials.

Sl. No Type

1 UFLC

2 Analytical balance

3 Vertex shaker

4 Ultra sonicator

5 Micropipettes

6 Refrigerator

7 Analytical Columns

8 Hot air oven

9 Syringe

10 Ultra centrifuges

11 Ependroff tubes

12 Hand gloves

13 Pipette

Instrumentation

Shimadzu HPLC model SPD-M20A was used for method development and validation. PDA

detector is used. It is a multichannel detector contains an ideal sensor for an entire spectrum

in a uv/vis dispersive spectrophotometer. These are useful in both research and quality

assurance laboratories and provides users most advanced level of sensitivity. Eclipse plus C18

columns are designed for superior peak with basic compounds and deliver high efficiency

and excellent peak shape with all sample types. Eclipse plus C18 is especially useful for the

separation of acidic, basic, and other highly polar compounds by reverse-phase liquid

chromatography (250 × 4.6 mm, particle size is 5 µm). The binary mobile phase consisted of

a mixture of A and B which was filtered through a membrane filter 4.5 µm. The solvents

were degassed before running at a flow rate of 1 ml/min. The column temperature was

ambient at 30 ºC. The 20 µl volume of sample was injected and peaks were detected at 240

nm.

Reagents and Pharmaceutical Preparations

Table 2: Drugs and Reagents.

SL. No Drugs Reagents

1 Erlotinib Hydrochloride Methanol

2 Pantaprazole Acetonitrile

3 Omeprazole Potassium dihydrogen ortho phosphate

4 Rabeprazole Orthophospheric acid


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Preparation of buffer solutions

pH 1.4 Buffer

The buffer was prepared by mixing 6.57 g of potassium chloride with 119.0 ml of 0.1 M

hydrochloric acid and diluted up to 1000 ml with milli Q water. Then pH was adjusted to 1.4

with hydrochloric acid. 250 ml of 0.1 M hydrochloric acid was prepared by mixing 2.25 ml

of 37% hydrochloric acid with milli Q water.

pH 2.4 Buffer

It was prepared by mixing 6.7 ml of orthophosphoric acid with 50.0 ml of 4% v/v solution of

2 M sodium hydroxide and diluted to 1000 ml with demineralized water. pH was adjusted to

2.4 with sodium hydroxide. 100 ml of 2 M sodium hydroxide was prepared by dissolving 8.0

g of sodium hydroxide in demineralized water and standardized with oxalic acid.

pH 4.5 Buffer: Dissolve 5.4 gm of sodium acetate and 3.35 ml of glacial acetic acid and

dilute with water to 100 ml.

Selection of analytical column

Selection of coloum plays an important role in the method development. For most of the

samples short columns (10-15 cm) are recommended to reduce method development time.

Such columns afford shorter retention time. In this work non polar column: C18 was used. It

was used for the determination of Erlotinib HCl because of good separation.

Selection of mobile phase

The selection of ideal mobile phase is done by altering the ratio of the buffers, composition of

the mobile phase solvents in the different ratio and also by passing them through the

coloumn. Based on the literature survey and other experimental parameters finally the best

suitable mobile phase for the drug Erlotinib hydrochloride is selected for the method

development.

Mobile phase: Acetonitrile (50%): Buffer (50%)

Buffer preparation: Accurately weighed 1.9 g of potassium di-hydrogen orthophosphate

transferred in 500 ml of milli Q water and 1 ml of tri-ethyl amine was added and adjusted the

pH to 2.4 by dilute ortho phosphoric acid solution and made the volume upto 1000 ml with

purified water.


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Mobile phase preparation

Acetonitrile and phosphate buffer were mixed in the ratio of 50: 50 v/v and sonicated for 15

min for degassing. The solution was filtered through 0.45 µm membrane filter.

Preparation of standard stock solutions

Standard stock solution of Erlotinib hydrochloride of 1000 µg/ml was prepared by dissolving

100 mg of standard drug in 20 ml of methanol in a 100 ml volumetric flask. The above

solution was sonicated for 10 minutes and the volume was made upto 100 ml with the

methanol. The standard stock solution (10 ml) was diluted to 100 ml with the mobile phase to

get the final concentration of 100 µg/ml. Series of dilution were made to get concentration

range 10 -100 µg/ml.

Sample preparation

Twenty tablets of Erlocip, each containing 150 mg of Erlotinib were powdered. The

powdered drug equivalent to 300 mg was accurately weighed and transferred into 100 ml

volumetric flask and 100 ml of methanol was added. The solution flask was sonicated for 10

minute to complete dissolution of the drug in the methanol and filtered through a 0.45 µm of

nylon filter and made up to the volume with methanol. Filtered solution (10 ml) was taken in

a 100 ml volumetric flask and made up to the volume with the mobile phase to get the final

concentration 300 µg/ml. Further dilutions were made to get final concentration range (10 to

100 µg/ml). The amount of Erlotinib in the formulation was determined by applying values of

peak area to regression equation of the calibration graph.

Optimization of chromatographic conditions

Trail 1: Mobile phase with a composition of methanol and acetonotrile (50:50 v/v) and C18 is

used as column to get the desired peak. The observed chromatogram shows negative peak

with less peak response.

Trail 2: Mobile phase with a composition of acetonitrile and phosphate buffer 7.4 (50: 50

v/v) and C18 is used as column to get the desired peak. The observed chromatogram shows

broad peak with less peak response.

Trail 3: Mobile phase with a composition of acetonitrile and phosphate buffer 3.0 (50: 50

v/v) and C18 is used as column to get the desired peak. The observed peak was good with

good resolution.


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Chromatographic conditions

The mobile phase containing mixer of acetonitrile and phosphate buffer(50: 50 v/v) was

selected as optimum composition for good peak at the flow rate 1 ml/min and UV detection

was carried out at 254 nm. The pump mode was isocratic and run time was 10 minutes. All

determination was performed at constant temperature.

Analytical Method Validation

Validation of an analytical method is the process to establish by laboratory studies that the

performance, characteristic of the method meets the requirements for the intended analytical

application. Performance characteristics are expressed in terms of analytical parameters as

below.

Linearity

The linearity of the analytical method is determined by mathematical treatment of test results

obtained by analysis of samples with analyte concentrations across the claimed range.

Linearity studies were performed by taking serial dilutions of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 ml

from the stock solution (100 µg/ml) into different 10 ml volumetric flasks and made up to the

volume with the mobile phase A: B (50:50 v/v). The final concentrations of Erlotinib were

10, 20, 30, 40, 50, 60 µg/ml. The solutions were injected into chromatographic system,

chromatograms were obtained and peaks were noted for each drug concentrations. The

linearity graph was plotted by taking peak areas v/s concentrations of standard Erlotinib. Area

is plotted graphically as a function of analyte concentration. 20 l of each solution was

injected and chromatograms were obtained.

Calculation: Y = mx + c

Precision

The precision of the analytical method was studied by analysis of multiple sampling of

homogeneous sample. The precision expressed as % RSD. Method reproducibility was

demonstrated by repeatability and intermediate precision. The repeatability (within-day in

triplets) and intermediate precision (for 3 days) was carried out at 3 concentration levels for

compound and the average % RSD values for the determination of Erlotinib hydrochloride

was calculated.

% RSD = Standard deviation/Mean X 100


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Robustness

The robustness of the method was determined by making slight changes in the

chromatographic conditions. The robustness of the proposed HPLC method was assessed for

peak resolution and symmetric factor. The parameters investigated are:

Apparent pH of the mobile phase (± 0.2)

Mobile phase flow rate (± 0.2)

The robustness of the method shows that there were no marked changes in the

chromatographic parameters, which demonstrates that the method developed is robust.

Ruggedness The ruggedness of an analytical method is determined by analysis of aliquots

from homogenous lots by different analysts using operational and environmental conditions

that may differ but are still within the specified parameters of the assay. The assay of

Erlotinib-HCl was performed in different condition, different analyst.

Limit of detection (LOD)

The LOD is defined by the USP as the lowest concentration of analyte that can be detected,

but not quantified. This value must be lower than or equal to the reporting level or reporting

threshold, as defined in ICH Q3B guidelines, which is based on maximum daily dose for any

drug product. Limit of detection can also find by the visual method by injecting the lower

concentration until detection of peak. Limit of detection can be calculated using following

equation according to ICH guidelines:

LOD = 3.3 × N/S where N is the standard deviation of peak areas of the drug and S is the

slope of the corresponding calibration curve.

Limit of quantification (LOQ)

The LOQ is the lowest concentration in a sample that may be measured within an acceptable

level of accuracy and precision, under the selected experimental conditions. It can be

calculated using the equation according to ICH guidelines.

LOQ = 10 × N/S

Where N is the standard deviation of peak areas of the drug and S is the slope of the

corresponding calibration curve.

Stability studies for Erlotinib hydrochloride

Acid induced degradation: It was performed by adding 20 ml of stock solution (1 mg/ml) of

Erlotinib hydrochloride to 10 ml each of methanol and 0.1 M hydrochloric acid and refluxing


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the mixture at 60 ºC for approximately 6 hrs. The solution was then left to reach ambient

temperature and then diluted to 100 ml with mobile phase so as to get concentration 200

μg/ml. From this solution, 10 ml was diluted to 100 ml to get final concentration 20 μg/ml.

Finally 20 μl solution is injected and the flow rate is maintained at 1 ml/min.

Alkaline induced degradation : Base induced stress degradation was performed by adding

20 ml of stock solution (1 mg/ml) of Erlotinib hydrochloride to 10 ml of methanol and 0.1 M

NaOH and refluxing the mixture at 60° C for approximately 6 hrs. The solution was then left

to reach ambient temperature then diluted to 100 ml with mobile phase so as to get

concentration 200 μg/ml. From this solution 10 ml was diluted to 100 ml to get final

concentration 20 μg/ml.

In vitro drug-drug interaction study

Preperation of stock solutions for proton pump inhibitors

Standard stock solution of pantaprazole, omeprazole and rabeprazole of 1000 µg/ml were

prepared by dissolving 100 mg of each pantaprazole, omeprazole and rabeprazole in 20 ml of

methanol in a 100 ml volumetric flask separately. The above solution was sonicated for 10

minute and the volume was made upto 100 ml with the methanol. The standard stock solution

(10 ml) was diluted to 100 ml with the different buffers like 1.4, 2.4 and 4.5 to get the final

concentration of 100 µg/ml for all the PPI. Series of dilution were made to get concentration

range 50 µg/ml.

Method I Drug-drug interaction study using aqueous medium by RP-HPLC method

High-performance liquid chromatography is a chromatographic technique that can separate a

mixture of compounds and is used to identify, quantify and purify the individual components

of the mixture. Retention time and absorbance peak of the species in solution may be changed

due to the interaction with other species. In the present study, analysis of Erlotinib

hydrochloride with proton pump inhibitors were carried out, using HPLC (Shimadzu, Japan)

at pH 1.4, 2.4 and 4.5 with a concentration of 50 μg/ml where combination of Erlotinib

hydrochloride with proton pump inhibitors was 1:1 molar ratio (50 μg/ml). The studies were

repeated twice.


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Procedure

Stock solutions of Erlotinib hydrochloride was prepared in methanol of 100 µg/ml, these

stock solutions are diluted with pH 1.4, 2.4 and 4.5 a buffers to get final stock of 50

µgm/ml.

Stock solutions of proton pump inhibitors were prepared in methanol to get concentration

100 µg/ml, these stock solutions are diluted with pH 1.4, 2.4 and 4.5 artificial buffers to

get final stock of 50 µg/ml.

These stock solutions were mixed (1:1) ratio in 10 ml volumetric flask by taking 5 ml

each solutions and sonicated for 15 minutes. After sonication these solutions are filtered

using 0.45 µm membrane filter and injected to HPLC for analysis of sample. Based on the

difference in retention time of Erlotinib hydrochloride without PPI and with PPI,

interactions of drug is judged and reported.

Method II

Extraction of plasma from rat blood:

Blood samples (2 ml) are collected in evacuated glass tubes from healthy rats (6 animals) by

retro artery route method, which does not injected with any other medicaments. The blood

was centrifuged at 1500 rpm for 10 mins and the supernatant plasma was separated using

micropipette. The separated plasma is deproteinated using methanol. The supernatant

obtained was filtered through a 0.45 µm membrane filter. Plasma thus obtained was mixed in

the ratio of 1:1 ratio with drug solutions.

Procedure

Blood from rat was collected from retroarticcular route using capillary tube.

Around 2 ml of rat blood was collected in pre-coated sodium citrate blood collection tube.

Collected blood is centrifuged at 1500 rpm for 10 mins, plasma layer was separated.

Seperated plasma layer is collected in new eppendorf tubes.

Collected plasma is mixed with drug stock solution prepared from artificial buffer at pH

1.4, 2.4 and 4.5 in 1:1 ratio.

250 μl of drug solution and 250 μl of plasma are mixed using micropipette and placed in

eppendorf tubes.

To the 500 μl mixture, add 4.5 ml of methanol, this is centrifuged at 1500 rpm for 10

mins.


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After centrifugation the protein present in serum is precipitated and the above supernatant

layer is collected, filtered and placed in petridish.

This solution is allowed to dry for 4 hours, and then methanol is evaporated.After drying,

the remaining drug on petridish is redissolved in 1 ml of methanol. This solution is

filtered using 0.45 µm filter and injected for hplc analysis.

Based on the difference in retention time of Erlotinib hydrochloride without PPI and with

PPI, drug-drug interactions are judged and reported.

RESULTS AND DISCUSSION

Determination of Erlotinib hydrochloride

In this study, HPLC conditions were optimized to obtain, an adequate separation of eluted

compounds. Initially, various mobile phase compositions were tried to elute title

ingredient. Mobile phase and flow rate selection was based on peak parameters (height,

capacity, theoretical plates, tailing or symmetry factor), run time, resolution. The system

with acetonitrile and potassium orthophophate buffer (50: 50 v/v mobile phase) with 1

ml/min flow rate is used. The optimum wavelength for detection was 254 nm at which

better detector response for the drug was obtained. The average retention time for

Erlotinib hydrochloride was found 5.3 minutes and various system suitable parameters

were recorded.

Retention time

The average retention time for Erlotinib hydrochloride was found 5.3 ± 0.02 min.


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Fig 4: Chromatogram of Erlotinib hyrdrochloride (20 µg/ml).

Fig 5: Chromatogram of Erlotinib hydrochloride 30 µg/ml.


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The linearity was evaluated from 10-100 µg/ml of the standard solution concentration. A

graph was plotted to concentration in µg/ml on X- axis and area on Y-axis. The detector

response was found to be linear with a correlation coefficient of 0.999 and y intercept is

13318.76. A graph of the calibration curve was shown in figure 2 and the linearity results

were shown in Table 4.

Table 3: Linearity data of Erlotinib hydrochloride by RP-HPLC method.

Sl.No Concentration in µg/ml Peak area of analyte

1 10 797458

2 20 1545456

3 30 2298648

4 40 3121354

5 50 3825798

Fig 6: Linearity curve of Erlotinib hydrochloride.

Table 4: Linear regression data for the calibration of Erlotinib hyrdrochloride

Parameters

Calibration range (µg/ml ) 10-100

Detection limit ( µg/ml) 0.36 µg/ml

Quantitation limit (µg/ml) 1.98 µg/ml

Regression equation (Y) y = 76725x + 13319

Slope (b) 6725

Intercept (a) 13318.76

Correlation coefficient 0.999

PRECISION

Intraday precision

The intra-day precision was performed for Erlotinib in the morning, afternoon and evening (9

am, 12 noon and 3 pm), the % RSD was found 1.5%. And the % RSD found less than 2 %

which indicates that the proposed method was good precise (Table 5).


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Interday precision

Inter-day precision was carried out in three different days. The % RSD for first day, second

and third was found to be 1.75%. The %RSD was found less than 2%, which confirms the

developed method was precise (Table 5).

Fig 7: Chromatogram of Erlotinib hydrochloride 50 µgm/ml intraday precision studies.


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Fig 8: Chromatogram of Erlotinib hydrochloride 50 µgm/ml inter day precision studies.

Table 5: Results of Intraday precision

Times Amount of drug

(µg/ml)

Intra-day

Days

Inter-day precision

% precision ±

SD % RSD

% precision

±SD %RSD

9.00 am 50 101.7050799

1.500639

1st day 101.699

1.745 12.30 noon 50 99.41475404 2nd

day 100.087

3.00 pm 50 98.88016603 3rd

day 98.212


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LOD

LOD is a lower concentration of drug detected in the developed method. The limit of

detection for Erlotinib hydrochloride was found by visual evaluation and based on the

standard deviation of response and slope. The limit of detection for Erlotinib hydrochloride

was found 0.36 µg/ml.

LOQ

The limit of quantitation was calculated by using the calibration curve of sample. The

standard deviation of Y- intercept of regression line was used as the standard deviation. The

limit of quantitation of Erlotinib hydrochloride was found to be 1.98 µg/ml.

Robustness

Developed method was found to be robust by changing the temperature. There was no

considerable change in the peak areas and retention time. The parameters like tailing factor

and retention time showed adherence to the limit. %RSD for Erlotinib hydrochloride at 28 ºC

was found to be 0.014 and 16 ºC was found to be 0.43. The proposed method is superior with

less retention time, high theoretical plate count and good resolution with the mobile phase

composition.

Ruggedness

The ruggedness of the method was performed by different analysts. The solutions were

analysed in triplicate and the %RSD was calculated. The % RSD of analyst 1 and analyst 2

for Erlotinib hydrochloride was found 0.72 and 0.67. Ruggedness % RSD is less than 2%.

Table 6: Robustness and Ruggedness study of Erlotinib hydrochloride.

S. No. Robustness Ruggedness

18 ºC 28 ºC Analyst 1 Analyst 2

Amount of drug 60 (µg/ml) 50 50 50 50

%RSD 0.014 0.43 0.72 0.67

Stress degradation study of Erlotinib hydrochloride

Acid induced degradation

Acid induced stress degradation was performed by adding 20 ml of stock solution (1 mg/ml)

of Erlotinib hydrochloride to 10 ml each of methanol and 0.5 M and 1 M hydrochloric acid

and refluxing the mixture at 60 ºC for approximately 6 hrs. The % degradation for 0.5 M and

1 M hydrochloric acid was found 25.59 and 3.41 (table no7).


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Alkali induced degradation

Base induced stress degradation was performed by adding 20 ml of stock solution (1 mg/ml)

of Erlotinib hydrochloride to 10 ml each of methanol and 0.5 M and 1M sodium hydroxide

and refluxing the mixture at 60 °C for approximately 6 hrs. The % degradation for 0.5 M

sodium hydroxide and 1 M sodium hydroxide was found to be 53.11 and 43.21 (table no7).

Fig 9: Chromatogram of Untreated Erlotinib hydrochloride 20 microgram sample.


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Fig 10: Chromatogram of Erlotinib hydrochloride with 0.5 M HCL.

Fig 11: Chromatogram of Erlotinib hydrochloride sample with 0.5 M NAOH.


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Fig. 12: Chromatogram Erlotinib hydrochloride sample with 1 M NAOH.


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Fig 13: Chromatogram of Erlotinib hydrochloride with 1M HCl.

Table 7: Degradation of Erlotinib hydrochloride and retention time of degradation

products.

Sl.

No Condition

Rt time of drug

and degradation

products

Peak

Area Concentration

%

Degradation

1

Untreated

stock solution

(20µgm/ml)

5.31 24464489 20 -

2

Acid hydrolysis

0.5 M HCL 4.66 18203967 20 25.59

1 M HCL 3.410 21630070 20 3.410

3.

Base hydrolysis

0.5 M NAOH 4.66 10002613 20 59.11

1M NAOH 4.747 35036540 20 43.21


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IN VITRO DRUG-DRUG INTERACTION STUDY

In vitro drug-drug interaction study in aqueous medium

In the present study, analysis of Erlotinib hydrochloride with proton pump inhibitors was

carried out, using HPLC. The pH at 1.4, 2.4 and 4.5 with a concentration of 50μg/ml was

maintained, where combination of Erlotinib hydrochloride with proton pump inhibitors was

1:1 molar ratio (50 μg/ml). The study was repeated twice.

From the obtained results, it shows that in vitro drug-drug interaction studies at pH 1.4, pH

2.4 and pH 4.5 there is no change in the peak shift or gradual change in the retention time of

Erlotinib hydrochloride with the combination of PPI and without PPI. So there is no possible

interactions seen during our experiment at in vitro conditions in aqueous medium.

Fig 14: Chromatogram of of Erlotinib hydrochloride at pH 1.4.


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Fig 15: Chromatogram of In vitro drug-drug interaction of Erlotinib hydrochloride with

Pantaprazole at pH 1.4.


1877


Fig 16: Chromatogram of In vitro drug-drug interaction of Erlotinib hydrochloride with

Rabeprazole at pH 1.4


1878


Fig 17: Chromatogram of Erlotinib hydrochloride at pH 2.4.

Fig18: Chromatogram of In vitro drug-drug interaction Erlotinib hydrochloride at pH

2.4 with pantaprazole Summary and Conclusion.


1879


Analytical method development and validation

In the present work we have selected anticancer drug, literature survey revealed that a

very few analytical methods were reported on Erlotinib hydrochloride, therefore our

intrest was to develop newer, simple analytical method and to validate it as per ICH

guidelines.

In this method C18 column was used. The selected mobile phase was acetonitrile and

phosphate buffer (50:50 v/v). The drug was injected and found retention time 5.32

minutes at flow rate 1.0 ml/min.

After developing method, it is validated by using different parameters such as linearity,

precision, LOD, LOQ, robustness and ruggedness. The developed method showed

linearity with correlation coefficient of r2

=0.999. In interday and intraday precision, the

%RSD values are in good precise in the developed method.

Based on the obtained results the proposed RP-HPLC method is proven to be suitable as

well as found to be simple, precise and economical for the determination and can be

routinely adopted technique for the quantification of Erlotinib hydrochloride.

In vitro drug-drug interaction studies

It indicated that there is no possible interaction between Erlotinib hydrochloride and PPI from

the obtained results and above discussion it can be concluded that Erlotinib hydrochloride

does not form any stable complex with PPI. Therefore, mentioned results may consider

during monitoring and concurrent therapy of both drugs.

It can be concluded that all the developed methods have a good approach for obtaining

reliable results and were found to be suitable for the routine estimation of Erlotinib

hydrochloride.

ACKNOWLEDGEMENT

The present study would not have been possible without the assistance, help &

encouragement of many peoples, who have either directly & indirectly helped me

suceessfully complete, thank you everyone.


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