Method Development & Validation For Simultaneous Estimation of Netarsudil & Latanoprost by RP-HPLC

Method development & validation for simultaneous estimation of Netarsudil &
Latanoprost by RP-HPLC
1. INTRODUCTION
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Pharmaceutical Analysis assumes an extremely crucial part in the quality affirmation and
quality control of mass medications and their plans. Pharmaceutical investigation is a specific
branch of explanatory science which includes isolating, recognizing and deciding the relative
measures of segments in a specimen of issue. It is worried about the synthetic portrayal of
issue both quantitative and subjective.
In recent years, several analytical techniques have been evolved.
CLASSIFICATION OF HPLC ( 4-10)
Based on modes of chromatography:
• Normal phase chromatography
• Reverse phase chromatography
Based on principle of separation:
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography
• Size exclusion chromatography
• Affinity chromatography
• Chiral phase chromatography
Based on elution technique:
• Isocratic separation
• Gradient separation
Department of Pharmaceutical Analysis & Quality Assurance

VJ’s college of pharmacy Page 1
Based on the scale of operation:
• Analytical HPLC
• Preparative HPLC
Normal Phase - High Performance Liquid Chromatography (NP-HPLC)
NP-HPLC investigates the distinctions in the quality of the polar

communications of the analytes in the blend with the stationary stage. The more grounded the
analyte-stationary stage association, the more drawn out the analyte maintenance. Analyte
atoms contend with the versatile stage particles for the adsorption locales on the surface of
the stationary stage. The more grounded the versatile stage communications with the
stationary stage, the lower the distinction between the stationary stage associations and the
analyte cooperations, and along these lines the lower the analyte maintenance. Versatile
stages in NP-HPLC depend on non-polar solvents, (for example, hexane, heptanes, and so
forth.) with the little expansion of polar modifier (i.e., methanol, ethanol). Pressing materials
generally utilized as a part of NP-HPLC are typically permeable oxides, for example, silica
(SiO2) or alumina (Al2O3). Surface of these stationary stages is secured with the thick
populace of OH gatherings, which makes these surfaces exceedingly polar. Synthetically
changed stationary stages can likewise be utilized as a part of NP-HPLC. Silica adjusted with
trimethoxy glycidoxypropyl silanes (normal name: diol-stage) is run of the mill pressing
material with diminished surface extremity. Since NP-HPLC utilizes for the most part non-
polar solvents, it is the technique for decision for exceptionally hydrophobic mixes (which
may demonstrate extremely more grounded collaboration with non polar versatile stages),
which are insoluble in polar or watery solvents.
Reversed Phase - High Performance Liquid Chromatography (RP-HPLC)
Rather than NP-HPLC, RP-HPLC utilizes predominantly dispersive powers

(hydrophobic or vanderwal's associations). The polarities of portable and stationary stages are
turned around, with the end goal that the surface of the stationary stage in RP-HPLC is
hydrophobic and versatile stage is polar, where for the most part water-based arrangements
are utilized. RP-HPLC is by a long shot the most well known method of chromatography.
Just about 90 % of all investigations of low-sub-atomic weight tests are done utilizing RP-

HPLC. Dispersive powers utilized in this detachment mode are the weakest intermolecular
powers, in this way making the general foundation communication vitality in the
chromatographic framework low contrasted with other partition systems. This low foundation
vitality takes into account recognizing little contrasts in atomic collaborations of firmly
related analytes. Adsorbents utilized in this method of chromatography are permeable
unbending materials with hydrophobic surfaces. The lion's share of pressing materials
utilized as a part of RP-HPLC is synthetically altered permeable silica.
Adsorption chromatography:
The analyte interact with solid stationary surface and are displaced with eluent for active
sites on surface.
Partition chromatography:
This strategy comes about because of a thermodynamic circulation of analytes between

two fluid stages. Based on relative polarities of stationary and portable stage, parcel
chromatography can be partitioned in to typical stage and invert stage chromatography. In
typical stage chromatography, the stationary stage bed is unequivocally polar in nature (e.g.
silica gel) and the versatile stage is non polar, (for example, n-hexane or tetrahydrofuran).
Polar specimen are hence held on polar surface of the segment pressing longer than polar
material while in turn around stage chromatography, the stationary bed is non polar
(hydrophobic in nature, while the portable stage is polar fluid, for example, blend of water
and methanol or Acetonitrile).
Size exclusion chromatography:
This involves a solid stationary phase with controlled pole size. Solids are
separated according to molecular size, with the large molecule unable to enter the pores
eluted first.
Ion exchange chromatography (IEC):
IEC is based on the differences in affinities of the analyte ions for the oppositely
charged ionic center in the resin or adsorbed counter ions in the hydrophobic stationary
phase. Consider the exchange of two ions A+ and B+ between the solution and exchange

resin
E−: A•E + B+ ↔B•E + A+
The equilibrium constant for this process is shown in Eq. below:
K = ([A+][BE])/([AE][B+])
This essentially determines the relative affinity of both cations to the exchange
centres on the surface. If the constant is equal to 1, no discriminating ability is expected for
this system. The higher the equilibrium constant (provided that, it is greater than 1), the
greater the ability of cation B+ to substitute A on the resin surface. Depending on the charge
of the exchange centres on the surface, the resin could be either anion-exchanger (positive
ionic centers on the surface) or cation-exchanger (negative centres on the surface). Cross
linked styrene-divinyl benzene is the typical base material for ion exchange resin. Exchange
groups are attached to the Phenyl rings in the structure and the degree of cross linkage is
between 5 % and 20 %. The higher the cross linkage, the harder the material and the less
susceptible it is to swelling, but the material usually shows lower ion-exchange capacity.
Four major types of ion-exchange centres are usually employed:
• SO3-—strong cation-exchanger
• CO2-—weak cation-exchanger
• Quaternary amine—strong anion-exchanger
• Tertiary amine—weak anion-exchanger
Analyte retention and selectivity in ion exchange chromatography are strongly dependent
on the pH and ionic strength of the mobile phase.
Size exclusion chromatography (SEC):
SEC is the strategy for dynamic division of particles as indicated by their size. The
partition depends on the rejection of the atoms from the permeable space of pressing material
due to their steric deterrent. Hydrodynamic range of the analyte particle is the principle factor
deciding its maintenance. This is the main chromatographic division strategy where any
positive collaboration of the analyte with the stationary stage ought to be kept away from.

In SEC, the higher the molecular weight of the molecule, the greater its hydrodynamic
radius results in faster elution. At the same time, if an analyte molecule interacts (undesired)
with the stationary phase, thus increasing the retention of larger molecules, which may
conform separation of molecules based solely on their hydrodynamic radius. The adsorbent
pore size distribution plays the dominant role in the adsorbent ability to discriminate
molecules according to their molecular weight. Hydrodynamic radius of the polymer is also
dependent on the analyte interaction with the solvent. Polymer conformation and degree of
the salvation varies with the variation of the solvent properties.
1.3 INSTRUMENTATION OF HPLC
HPLC is an exceptional branch of Column Chromatography in which the versatile

stage is constrained through the section at rapid. Accordingly, the examination time is
decreased by 1-2 requests of greatness with respect to established Column Chromatography
and the utilization of significantly littler particles of the retentive or support winds up
noticeably conceivable expanding the section productivity considerably. The Basic HPLC
Instrumentation (5-9) was appeared fig.no.1

Figure .No.1.1 HPLC basic instrumentation
I. Solvent delivery system:
The most imperative part of HPLC in dissolvable conveyance framework is the pump,
since its execution specifically impacts the maintenance time, reproducibility and identifier
affectability. Among the few dissolvable conveyance frameworks, (coordinate gas weight,
pneumatic intensifier, responding and so forth.) responding pump with twin or triple
cylinders is generally utilized, as this framework gives less standard commotion, great stream
rate reproducibility and so on.
The pumping systems used in HPLC can be categorized in three different ways.
 The first classification is according to the eluent flow rate that the pump is capable of
delivering.
 The second classification is according to the construction materials.
 The final classification is according to the mechanism by which the pump delivers the
eluent.
Each of these classifications is considered below.
Pump classification according to flow rate:
When classified in terms of flow rate, pumps may be defined as micro bore or
preparative.

 Standard bore systems are the most commonly used pumping systems for analytical
HPLC because they provide reliable operation at flow rates ranging from 100 µl / min
to 10 µl / min.
 Micro bore systems are intended for use with column diameters ranging up to 2 mm.
The narrow column diameter and small size of the packing material causes relatively
low flow rates for the pumping system, from 1 to 250 µl / min as the minimum head
size for reciprocating pumps is around 25 µl, smooth, reliable operation at flow rates
less than 10 µl / min is difficult.
Pump classification according to materials of construction:
Pumps may also be classified according to the primary construction materials.

The pumps are classified as
 metallic
 Non-metallic, depending on the material used for the eluent flow path.
 The most commonly used material for HPLC pumping systems is 316 stainless steel,
because of its mechanical strength, corrosion resistance, good thermal stability and
malleability. Only a handful of HPLC solvents such as Hydrochloric acid will cause
damage to 316 stainless steel. Therefore pumps are also constructed from non-
metallic materials such as PEEK (poly ethyl ethyl ketone), Teflon (poly tetra fluoro
ethylene) and Ceramics.
Pump classification according to mechanism of eluent displacement:
The third classification of pumps is according to the mechanism by which the

liquid is forced through the chromatograph. The pumps are classified into two types.
They are
 syringe pumps and
 reciprocating-piston pump
HPLC systems are also provided an online degassing system which
continuously removes the dissolved gases from the mobile phase.
Solvent degassing system

 The constituents of the mobile phase should be degassed and filtered before use.
Several methods can be applied to remove the dissolved gases in the mobile phase.
They include
 heating and stirring,
 vacuum degassing with an aspirator,
 filtration through 0.45μm filters,
 vacuum degassing with an air-soluble membrane,
 Helium purging ultra signification or purging or combination of these methods.
II. Sample introduction system
Two means for analyte introduction on the column are injection into a flowing stream and a
stop flow injection. These techniques can be used with a syringe or an injection valve.
Automatic injector is a microprocessor-controlled version of the manual universal injector.
Injector
Injectors should provide the possibility of injecting the liquid sample within the range of
0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000 psi).
They should also produce minimum band broadening and minimize possible flow disturbances.
The most useful and widely used sampling device for modern LC is the micro sampling
injector valve. With these sampling valves, samples can be introduced reproducibly into
pressurized columns without significant interruption of flow even at elevated temperatures.
LOAD (the sample loop) INJECT (move the sample loop

In the mobile phase
Figure .No.1.2 Injection syste

III. Columns
The core of the framework is the section. Investigative section is the most critical
piece of the HPLC which chooses the productivity of detachment. The decision of regular
pressing material and versatile stages relies upon the physical properties of the medication.
The accompanying properties of the segment stationary stages assume an imperative part in
giving distinctive selectivity for divisions.
I) Particle estimate, ii) Particle shape, iii) Pore measure/Pore volume, iv) Specific surface
region,
v) End topping vi) % carbon stacking.
The accompanying are the most generally utilized segments with stationary stages for
detachment and measurement of wide assortment of medications .
I) Pure silica and cross breed silica segments.
ii) Silica based segments with various holding stages like C4, C6, C8, C18, C20 and holding
stages having useful gatherings like cyano, phenyl, naphthyl and amino.
iii) Silica based sections with polar installed stages inside chains of C8, C18, NH2.
iv) Hybrid silica based sections like C4, C6, C8, C18, C20 and holding stages having useful
gatherings like cyano, phenyl, naphthyl and Amino.
v) Strong cation trade (SCX) and solid anion trade (SAX) segments.
vi) Size Exclusion chromtography (SEC) or gel saturation chromatography (GPC) sections.
vii) Silica based stone monument segments.vii) Fused core silica columns with bonding
phases like C8, C18, CN, and phenyl.
viii) Metal oxide columns like zirconia based and alumina based.
ix) Chiral columns.
Column-packing materials:

Silica (SiO2.X H2O) is the most broadly utilized substance for the fabricate of pressing
materials it comprise of a system of siloxane linkages(Si-O-Si) in an inflexible three
dimensional structure containing entomb associated pores. Hence an extensive variety of
business items are accessible with surface regions running from 100 to 800 m2/g and
molecule sizes from 3 to 50 µm. The silonol bunches on the surface of silica give it a polar
character, which is abused in adsorption chromatography utilizing non-polar natural elutents.
Silica can be radically changed by response with organochlorosilanes or organoalkoxysilanes
giving Si-O-Si-R linkages with the surface. The connection of hydrocarbon fasten to silica
creates a non polar surface appropriate for turned around stage chromatography where blends
of water and natural solvents are utilized as eluents. The most well known material is octa
decyl silica (ODS) which contains C18chains, yet material with C2, C6, C8 and C22 chains
are likewise accessible. Amid produce, such materials can be responded with a little mono
practical silane (eg: trimethychlorosilane) to lessen additionally number of silanol bunches
staying at first glance (end topping). There is a tremendous scope of materials which have
middle of the road surface polarities emerging from the attaching to silica of other natural
mixes which contain gatherings, for example, phenyl, nitro, amino and hydroxyl. Solid
particle trade is likewise accessible in which sulphonic corrosive gatherings and quaternary
ammonium bunches are attached to Silica. The helpful pH extend for segments is 2 to 8, since
Siloxane linkages are severed beneath pH 2 while at pH esteems over 8 Silica may break
down. In HPLC, for the most part two sorts of segments are utilized, ordinary stage section
and turned around stage segment. Utilizing typical stage chromatography, especially of non-
polar and respectably polar medications can make superb partition and was initially trusted
that division of mixes in blends happens gradually by differential adsorption on a stationary
silica stage. In any case, it now appears that parcel assumes an essential part, with the mixes
cooperating with the polar silonol bunches on the silica or with bound water atoms. While in
ordinary stage, appears the entry of a moderately non-polar portable stage over a polar
stationary stage, turned around eliminate chromatography is conveyed utilizing a polar
versatile stage, for example, methanol, acetonitrile, water, cradle and so on finished a non
polar stationary stage. A scope of stationary stages (C18, C8, - NH2, - CN, - Phenyl and so
forth.) are accessible and exceptionally particular detachment can be accomplished.
The most well known brands of LC segments are Inertsil, Hypersil, X-land, X-connect,
Sun-fire, Atlantis, Aquity-BEH, Zorbax, Lichrosphere, Purosphere, Sperisorb, Luna,
Kromasil, ACE, YMC, Symmetry, Chiralcel and Chiralpak. These LC segments are provided
in various measurements, viz., lengths of 10 mm, 50 mm, 100mm, 150mm, 250mm, 300mm,
500mm and interior distances across of 2.1mm, 3.0mm, 4.0mm, 4.6mm. LC segments with
stationary stages having diverse molecule sizes like 5.0 µm, 4.0 µm 3.5 µm, 3.0 µm, 2.5 µm,
2.0 µm, 1.9 µm, 1.8 µm, 1.7 µm and 1.3 µm are accessible.
IV. Mobile phase

Versatile stages utilized for HPLC are commonly blends of natural solvents and water
or watery cushions.
The accompanying focuses ought to likewise be considered while picking a versatile stage:
 It is basic to set up that the medication is steady in the versatile stage for at any rate
the span of the investigation.
 Excessive salt focuses ought to be maintained a strategic distance from. High salt
fixations can bring about precipitation which can harm HPLC hardware. Lessen cost and
poisonous quality of the portable stage by utilizing methanol rather than acetonitrile at
whatever point conceivable.
Table No. 1.2 Physical properties of common HPLC solvents

UV Density Viscosity
RI Dielectric
Solvent MW BP Cut-off g / ml cP
o
(25 C) Constant
o o
(nm) (25 C) (25 C)
Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8
Dioxane 88.1 101 1.420 215 1.034 1.26 2.21

Ethanol 46.1 78 1.359 205 0.789 1.19 24.5
Ethyl acetate 88.1 77 1.372 256 0.901 0.450 6.02
Methanol 32.0 65 1.326 205 0.792 0.584 32.7
CH2Cl2 84.9 40 1.424 233 1.326 0.44 8.93
Isopropanol 60.1 82 1.375 205 0.785 2.39 19.9
n-propanol 60.1 97 1.383 205 0.804 2.20 20.3
THF 72.1 66 1.404 210 0.889 0.51 7.58
Water 18.0 100 1.333 170 0.998 1.00 78.5
V. Detectors
At the point when chromospheres are available, the wavelength of recognition for a
medication ought to be founded on its UV Range in the versatile stage and not in
unadulterated solvents. The most specific wavelength for identifying a medication is as often
as possible the longest wavelength greatest to maintain a strategic distance from impedance
from solvents, cradles and excipients. Different techniques for recognition can be helpful are
required in a few examples. The discovery of UV light absorbance offers both
accommodation and affectability for atoms.
• Solute specific detectors (UV-Vis, fluorescence, electrochemical, infra-red, radio

activity)
• Bulk property detectors (refractive index, viscometer, conductivity)
• Desolvation detectors (flame ionization etc.)
• LC-MS detectors
• Reaction detectors

Applications of HPLC in pharmaceutical research ( 9-13) :
• Separation: This can be accomplished using HPLC by utilizing the fact that, certain
compounds have different migration rates given a particular column and mobile phase. The
extent or degree of separation is determined by the choice of stationary phase and mobile
phase along with parameters like flow, temperature and gradient programme.
• Identification: For this purpose a clean peak of known sample has to be observed from
the chromatogram. Selection of column mobile phase and flow rate matter to certain level in
this process. Identification is generally by comparing with reference compound based on
retention time and also based on UV-Vis spectra in some cases. Identification can be assured
by combining two or more detection methods, where necessary.
• Quantification: Analyte concentrations are estimated by measuring the responses ( peak
areas) known reference standards followed by unknown samples. Quantification of known
and unknown components are done by various methods like - area normalization method,
internal standard method, external standard method and diluted standard method along with
relative response factors.
• Isolation: It refers to the process of isolation and purification of compounds using
analytical scale or preparative scale HPLC. Volatile buffers and solvents are preferred choice
as mobile phases as it reduces the effort on purification. Solute purity and throughput is the
key challenge in isolation and purification processes.
1.4) HPLC theory ( 14-16) :
System suitability parameters:
Elite fluid chromatography is characterized as a partition of blends of mixes because of
contrasts in their conveyance harmony between two stages, the stationary stage stuffed inside
sections and the versatile stage, conveyed through the segments by high weight pumps.
Segments whose dissemination into the stationary stage are higher, are held longer, and get
isolated from those with bring down circulation into the stationary stage. The hypothetical
and handy establishments of this strategy were set down toward the finish of 1960s and
toward the start of 1970s. The hypothesis of chromatography has been utilized as the reason
for framework appropriateness tests, which are set of quantitative criteria that test the
reasonableness of the chromatographic framework to recognize and measure sedate related
examples by HPLC at any progression of the pharmaceutical examination.Retention time
(tR), capacity factor k' and relative retention time (RRT):
The time elapsed between the injection of the sample components into the column
and their detection is known as the retention time (tR). The retention time is longer when the
solute has higher affinity to the stationary phase due to its chemical nature. For example, in
reverse phase chromatography, the more lyophilised compounds are retained longer.
Therefore, the retention time is a property of the analyte that can be used for its identification.
A non-retained substance passes through the column at a time t0, called the void time.
Retention factor is calculated as follows:
Figure .No.1.3. showing retention factor

The time passed between the infusion of the example parts into the section and
their recognition is known as the maintenance time (tR). The maintenance time is longer
when the solute has higher liking to the stationary stage because of its substance nature. For
instance, backward stage chromatography, the more lyophilised mixes are held longer.
Subsequently, the maintenance time is a property of the analyte that can be utilized for its
distinguishing proof. A non-held substance goes through the segment at once t0, called the
void time.
Maintenance factor is figured as takes after:
The limit factor portrays the thermodynamic premise of the partition and its definition is the
proportion of the measures of the solute at the stationary and versatile stages inside the
analyte band inside the chromatographic segment: Where Cs is the convergence of the solute
at the stationary stage and Cm is its focus at the versatile stage and phi is the proportion of
the stationary and versatile stage volumes all inside the chromatographic band. The Retention
Factor is utilized to think about the maintenance of a solute between two chromatographic
frameworks, normalizing it to the segment's geometry and framework stream rate. The

maintenance factor esteem ought to be in the middle of 1-20. The need to decide the void
time can be dubious now and again, because of the flimsiness of the elution time of the void
time marker, t0, along these lines, when the chromatogram is intricate in nature, and one
known part is constantly present at a specific maintenance time, it can be utilized as a
maintenance marker for different pinnacles. In such cases the proportion between the
maintenance time of any top in the chromatogram and the maintenance time of the marker is
utilized (tR (Peak)/tR (Marker)) and alluded to as the Relative Retention Time (RRT). RRT is
additionally utilized rather than the limit proportion for the recognizable proof of the analyte
and additionally to look at its degree of maintenance in two distinctive chromatographic
frameworks. The sharpness of a pinnacle with respect to its maintenance time is a measure of
the framework's productivity, computed as N, plate tally. Band-widening wonders in the
section, for example, whirlpool dispersion, sub-atomic dissemination, and mass-exchange
energy and additional segment impacts lessen the proficiency of the partition. The sharpness
of a pinnacle is pertinent to the furthest reaches of identification and breaking point of
evaluation of the chromatographic framework. The keener the top for a particular territory,
the better is its flag to-commotion; thus the framework is equipped for distinguishing lower
fixations. In this manner, the proficiency of the chromatographic framework must be set up
by the framework reasonableness test before the examination of low fixations that requires
high affectability of the framework, for example, the investigation of medication
debasements and corruption items.
Efficiency: Plate count N and peak capacity Pc:
The efficiency of the separation is determined by the plate count N when
working at isocratic conditions, whereas it is usually measured by Peak Capacity Pc when
working at gradient conditions. The following equation for the plate count is used by the
United States Pharmacopoeia (USP) to calculate N:
Figure. No.1.4. showing Number of Theoretical Plates

Where w is measured from the baseline peak width calculated using lines tangent to the peak
width at 50 % height. European and Japanese pharmacopoeias use the peak width at 50% of
the peak height, hence the equation becomes:
Peak capacity Pc is defined as number of peaks that can be separated within a

retention window for a specific pre-determined resolution. In other words, it is the runtime
measured in peak width units (34). It is assumed that peaks occur over the gradient
chromatogram. Therefore, peak capacity can be calculated from the peak widths in the
chromatogram as follows:
Where n is the quantity of crests at the section of the slope chose for the computation, tg.
Along these lines crest limit can be essentially the inclination run time isolated by the normal
pinnacle width. The more honed the pinnacles the higher is the pinnacle limit, consequently
the framework ought to have the capacity to determine more crests at the chose run time and
also recognize bring down fixations.
Another measure of the column's chromatographic efficiency is the height equivalent

to theoretical plate (HETP) which is calculated from the following equation:
HETP = (L/N)
Where L is column length and N is the plate count. HETP is measured in micrometer.
The conduct of HETP as capacity of direct speed has been portrayed by different
conditions. It is as often as possible called "The Van-Deemter bend", and it is as often as
possible used to depict and portray different chromatographic stationary stages' execution and
contrast them with each other. The lower are the estimations of HETP, the more productive is
the chromatographic framework, empowering the discovery of lower focuses because of the
upgraded motion to-commotion proportion of the considerable number of tops in the
chromatogram.

Peak asymmetry factor Af and tailing factor T:
 The chromatographic pinnacle is expected to have a Gaussian shape under perfect

conditions, portraying typical dissemination of the speed of the atoms populating the
pinnacle zone relocating through the stationary stage inside the section. Any deviation
from the typical circulation shows non-ideality of the dissemination and the relocation
procedure in this way may imperil the uprightness of the pinnacle's combination,
lessening the exactness of the quantitation. This is the motivation behind why USP
Tailing is a pinnacle's parameter quite often measured in the framework reasonableness
advance of the examination.
 The deviation from symmetry is measured by the asymmetry factor, Af or tailing factor
T. The calculation of asymmetry factor, Af is described by the following equation
Figure. No.1.5. showing Asymmetric Factor
Where An and B are areas in the even line parallel to the gauge, drawn at 10% of the
pinnacle stature. The count of following Factor, T, which is all the more generally utilized as
a part of the pharmaceutical business, as recommended by the pharmacopeia's, the place An
and B are areas in the flat line parallel to the benchmark, drawn at 5% of the pinnacle stature.
The USP recommends that following element ought to be in the scope of 0.5 up to 2 to
guarantee an exact and precise quantitative estimation.
Selectivity Factor α, and Resolution Factor Rs:
The division is an element of the thermodynamics of the framework.

Substances are isolated in a chromatographic section when their rate of movement varies,

because of their diverse dispersion between the stationary and portable stages. The selectivity
factor, α, and determination factor, Rs, measure the degree of partition between two adjoining
tops. The selectivity factor accounts just for the proportion of the maintenance factors, k', of
the two pinnacles (k'2/k'1), while the determination factor, Rs, represents the distinction
between the maintenance times of the two pinnacles with respect to their width.
FigureNo.1.6.showing Resolution Factor
The equation that describes the experimental measurement of the resolution factor, Rs, is as
follows:
Rs = ΔtR / 0.5 (W1 + W2)
Where tR is the retention time of peaks 1 and 2 respectively and w is their respective peak
width at the tangents' baseline. According to the pharmacopeia should be above 1.5 for an
accurate quantitative measurement.
The determination is a basic esteem when working with complex examples,

for example, tranquilizes pollutions and corruption items, or when the plan is intricate and
excipients may meddle with the quantitative estimations. In this way, it is a fundamental
piece of the framework appropriateness estimation arrange before the quantitative work of
these kinds of tests. The example utilized for the estimations of Rs amid the framework
appropriateness runs is in some cases called Resolution Solution, It more often than not
contains the segments that are the most hard to determine. The hypothetical depiction of the
Resolution Factor Rs condition is appeared in Equation. It includes some of the above
parameters, the plate count N, the selectivity α and the average of the two peaks' capacity
factors k':

Figure. No.1.7 showing selectivity
It can be plainly observed from this condition the plate check is the most affecting parameter
in the expansion of the chromatographic determination. Since the plate include increments
with the diminishment molecule distance across, it clarifies the lessening in molecule width
of the stationary stage material amid the most recent 3 many years of HPLC. This is likewise
the normal behind the current pattern in HPLC, the utilization of sub 2 micron molecule
sections and the improvement of an uncommonly outline of ultra execution HPLC
frameworks to oblige such segments.
1.5) ANALYTICAL METHOD DEVELOPMENT (17-21)
Techniques are created for new items when no official strategies are accessible. Exchange
techniques for existing (Non-Pharmacopeias) items are created to lessen the cost and time for
better exactness and toughness. Trial runs are led, strategy is advanced and approved. At the
point when substitute technique proposed is planned to supplant the current system, near
research facility information including merits/faults ought to be made accessible.
Steps involved in method development

Documentation starts at the very beginning of the development process. A system for full
documentation of development studies must be established. All data relating to these studies
must be recorded in laboratory notebook or an electronic database.
1. Analyte standard characterization

a) All known information about the analyte and its structure is collected i.e., physical and
chemical properties.
b) The standard analyte (100 % purity) is obtained. Necessary arrangement is made for the
proper storage (refrigerator, desiccators and freezer).
c) 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.
d) Only those methods (spectroscopic, MS, GC, HPLC etc.,) that are compatible with sample
stability are considered.
2. Method requirements
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.
3. Literature search and prior methodology
The writing for a wide range of data identified with the analyte is overviewed.
dissolvability profile (dissolvability of Drug in various solvents and at various pH
conditions), logical profile (Physico-compound properties, Eg: pKa, softening point,
debasement pathways, and so on) and strength profile (affectability of the medication towards
light, warm, dampness and so on) and significant expository strategies, books, periodicals,
concoction producers and administrative organization compendia, for example, USP/NF, are
investigated.
4. Choosing a method
a) Utilizing the data in the written works and prints, strategy is adjusted. The strategies are
adjusted wherever vital. Once in a while it is important to secure extra instrumentation to
replicate, alter, enhance or approve existing techniques for in-house analytes and tests. In the
event that there are no earlier strategies for the analyte in the writing, from relationship, the
intensifies that are comparable in structure and compound properties are explored and are
worked out.
b) There is usually one compound for which analytical method already exist that is similar to
the analyte of interest.
5. Instrumental setup and initial studies
The required instrumentation is setup. Establishment, operational and execution capability of

instrumentation utilizing research center standard working methodology (SOP's) are
confirmed. Continuously new consumables (e.g. solvents, channels and gases) are utilized.
For instance, strategy improvement is never begun on a HPLC section that has been utilized
before. The analyte standard in an appropriate infusion/presentation arrangement and in
known focuses and solvents are readied. It is essential to begin with a bona fide, known
standard as opposed to with a mind boggling test grid. On the off chance that the example is
greatly near the standard (e.g., mass medication), at that point it is conceivable to begin work
with the genuine specimen.
6. 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.
7. Documentation of analytical figures of merit
The originally determined analytical figures of merit are limit of quantitation (LOQ), limit of
detection (LOD), linearity, time per analysis, cost, sample preparation etc., are documented.
8. 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.
9. Determination of percent recovery of actual sample and demonstration of
quantitative sample analysis
Percent recuperation of spiked, credible standard analyte into a specimen lattice that is
appeared to contain no analyte is resolved. Reproducibility of recuperation (normal +/ -
standard deviation) from test to test and whether recuperation has been enhanced or not has
been appeared. It isn't important to acquire 100 % recuperation as long as the outcomes are
reproducible and known with a high level of sureness. The legitimacy of explanatory strategy
can be confirmed just by research facility ponders.

Therefore documentation of the successful completion of such studies is a basic requirement

for determining whether a method is suitable for its intended applications.
1.6) METHOD DEVELOPMENT PROCEDURE (18)
The wide variety of equipment’s, columns, eluent and operation preparations

involved high performance liquid chromatography (HPLC) method development seems
complex. The processes influenced by the nature of analytes and generally follow the
following steps
Steps:
 Step 1 - Selection of the HPLC method and initial system

 Step 2 - Selection of initial conditions
 Step 3 - Selectivity optimization
 Step 4 - System optimization
 Step 5 - Method validation.
Contingent upon the general prerequisites and nature of the example and analytes, some of these means
won't be important amid HPLC examination. For instance, a palatable partition might be found amid
stage 2, hence stages 3 and 4 may not be required. The degree to which technique approval (stage 5) is
examined will rely upon the utilization of the end investigation; for instance, a strategy required for
quality control will require more approval than one produced for an erratic examination. The
accompanying must be considered when building up a HPLC technique:
HPLC method development (19)
Step 1 - selection of the HPLC method and initial system.
When building up a HPLC strategy, the initial step is dependably to counsel the writing to find
out whether the partition has been beforehand performed and assuming this is the case, under what
conditions - this will spare time doing pointless exploratory work. While choosing a HPLC framework, it
must have a high likelihood of really having the capacity to dissect the specimen; for instance, if the
example incorporates polar analytes at that point turn around stage HPLC would offer both sufficient
maintenance and determination, though typical stage HPLC would be substantially less plausible.
Thought must be given to the accompanying:

Sample preparation:
 Does the sample require dissolution, filtration, extraction, preconcentration or clean up,
 Is chemical derivatization required to assist detection sensitivity or selectivity
Types of chromatography:
1.Reverse stage is the decision for the larger part of tests, however in the event that acidic or fundamental
analytes are available at that point turn around stage particle concealment (for feeble acids or bases) or
invert stage particle blending (for solid acids or bases) ought to be utilized. The stationary stage ought to
be C18 fortified.
2. for low/medium extremity analytes, typical stage HPLC is a potential hopeful, especially if the
partition of isomers is required. Carbon reinforced stages are simpler to work with than plain silica for
typical stage partitions. For inorganic anion/cation examination, particle trade chromatography is ideal.
Measure rejection chromatography would typically be considered for dissecting high sub-atomic weight
mixes.
Segment measurements:
For most examples (unless they are exceptionally perplexing), long sections (25 cm) are prescribed to
improve the segment effectiveness. A stream rate of 1-1.5 ml/min ought to be utilized at first. Pressing
molecule size ought to be 3 or 5 μm.
Identifiers:
Thought must be given to the accompanying:
• Do the analytes have chromospheres to empower UV recognition
• Is more specific/touchy discovery required?
• What detection limits are necessary
• Will the sample require chemical derivatization to enhance detect ability and/or improve the
chromatography?
Step 2 - selection of initial conditions.

This step determines the optimum conditions to adequately retain all analytes; that is, ensures
no analyte has a capacity factor of less than 0.5 (poor retention could result in peak overlapping) and no
analyte has a capacity factor greater than 10–15 (excessive retention leads to long analysis time and
broad peaks with poor detectability). Selection of the following is then required.
Mobile phase solvent strength:
The solvent strength is a measure of its ability to pull analytes from the column. It is generally
controlled by the concentration of the solvent with the highest strength; for example, in reverse phase
HPLC with aqueous mobile phases, the strong solvent would be the organic modifier; in normal phase
HPLC, it would be the most polar one. The aim is to find the correct concentration of the strong solvent.
With many samples, there will be a range of solvent strengths that can be used within the aforementioned
capacity limits. Other factors (such as pH and the presence of ion pairing reagents) may also affect the
overall retention of analytes.
Step 3 - selectivity optimization:
The aim of this step is to achieve adequate selectivity (peak spacing). The mobile phase and
stationary phase compositions need to be taken into account. To minimize the number of trial
chromatograms involved, only the parameters that are likely to have a significant effect on selectivity in
the optimization must be examined. To select these, the nature of the analytes must be considered. Once
the analyte types are identified, the relevant optimization parameters may be selected. Note that the
optimization of mobile phase parameters is always considered first as this is much easier and convenient
than stationary phase optimization.
Step 4 - system parameter optimization:
This is used to find the desired balance between resolution and analysis time after satisfactory
selectivity has been achieved. The parameters involved include column dimensions, column-packing
particle size and flow rate. These parameters may be changed without affecting capacity factors or
selectivity.
Step 5 - Method validation:
Proper validation of analytical methods is important for pharmaceutical analysis when ensure of
the continuing efficacy and safety of each batch manufactured relies solely on the determination of
quality. The ability to control this quality is dependent upon the ability of the analytical methods, as

applied under well-defined conditions and at an established level of sensitivity, to give a reliable
demonstration of all deviation from target criteria.
Analytical methods should be used within good manufacturing practice (GMP) and good
laboratory practice (GLP) environments, and must be developed using the protocols set out in the
international conference on harmonization (ICH) guidelines (Q2A and Q2B). The US food and drug
administration (FDA) and US Pharmacopoeia (USP) both refer to ICH guidelines. The most widely
applied validation characteristics are accuracy, precision (repeatability and intermediate precision),
specificity, detection limit, quantitation limit, linearity, range, robustness and stability of analytical
solutions. Method validation must have a written and approved protocol prior to use.
1.7 .STABILITY INDICATING METHOD
It is basic that the explanatory strategies produced for estimation of the virtue and pollutions are
sufficiently competent to isolate all the coveted and undesired segments and without any impedances
from the definition lattice. At the point when explanatory techniques can unequivocally and precisely
measure without missing any contaminations, without underestimation or over estimation, and
distinguish every conceivable polluting influence and degradants those can shape amid strength ponders
with sufficient affectability and precisely mirror the nature of medication substances and medication
items (figured results of medications), those strategies are called security showing techniques.
A soundness showing examine strategy ought to precisely quantify the dynamic fixings, without
impedance from debasement items, process contaminations, excipients, or other potential polluting
influences. In the event that an industry utilizes a non-soundness demonstrating explanatory system for
discharge testing, at that point an investigative technique able to do subjectively and quantitatively
checking the contaminations, including debasement items, should supplement it. Systematic
methodology for security investigations of test ought to be solidness demonstrating. Because of security
testing a re-trial for the dynamic substance or a time span of usability for the pharmaceutical item can be
built up, and capacity conditions can be suggested.
The ICH (International meeting on Harmonization) rule QIA on Stability Testing of New Drug
Substances and Products underscores that the testing of those highlights which are helpless to change
amid capacity and are probably going to impact quality, wellbeing as well as viability must be finished
by approved solidness demonstrating testing strategies. It is additionally specified that constrained
deterioration examines (stretch testing) at temperatures in 10 °C increases over the quickened
temperatures, extremes of pH, under oxidative and photolytic conditions ought to be completed on the
medication substance and medication item in order to build up the natural dependability attributes and
debasement pathways to help the appropriateness of the proposed investigative methodology.

1.8 ANALYTICAL METHOD VALIDATION (16-19)
As per ICH Guidelines Method Validation can be characterized as "Establishing archived confirm,
which gives a high level of confirmation that a particular action will reliably deliver a coveted outcome
or item meeting its foreordained determinations and quality attributes".
A test for a noteworthy segment requires an alternate approach and acknowledgment criteria than a
technique for a follow debasement. A last technique might be performed at various locales around the
globe. Contrasts in HPLC instrumentation, research center gear and reagent sources and varieties in the
abilities and foundation of work force may require particular highlights in the HPLC strategy. Also, the
improvement of various plans of a similar medication with changing qualities or physical structures may
require adaptability in technique methodology.
Technique approval think about incorporate framework reasonableness, linearity, exactness,

precision, specificity, strength, point of confinement of identification, breaking point of measurement and
soundness of tests, reagents, instruments.
1. System Suitability
Before the examination of tests of every day, the administrator must build up that the HPLC
framework and method are equipped for giving information of worthy quality. This is refined with
framework reasonableness tests, which can be characterized as tests to guarantee that the technique can
produce aftereffects of worthy exactness and Precision. The prerequisites for framework reasonableness
are typically created after strategy improvement and approval have been finished.
Table No.1.3 System Suitability Parameters and their recommended limits

Parameter Recommendation
Capacity Factor (K’) The peak should be well-resolved from other peaks and the
void volume generally K>2
Repeatability RSD ≤ 2%
Relative Retention Not essential as the resolution is stated
Resolution(Rs) Rs of > 2 between the peak of interest and the closest eluting
Tailing Factor(T) T≤2

Theoretical Plates(N) In general should be > 2000
2. Linearity
The linearity of a technique is a measure of how well an adjustment plot of reaction versus focus
approximates a straight line. Linearity can be evaluated by performing single estimations at a few analyte
fixations. The information is then prepared utilizing a direct slightest squares relapse. The subsequent
plot incline, capture and relationship coefficient give the coveted data on linearity.
3. Precision
The precision of estimation is characterized as the closeness of the deliberate an incentive to the
genuine esteem. In a technique with high precision, a specimen (whose "genuine esteem" is known) is
broke down and the deliberate esteem is indistinguishable to the genuine esteem. Normally, exactness is
spoken to and dictated by recuperation thinks about. There are three approaches to decide exactness:
1. Repeatability
2. Intermediate precision and
3. Reproducibility
Repeatability is the precision of a method under the same operating conditions over a short period of
time.
Intermediate precision is the agreement of complete measurements (including standards) when the same
method is applied many times within the same laboratory.
Reproducibility examines the precision between laboratories and is often determined in collaborative
studies or method transfer experiments.
4. Accuracy
The exactness of estimation is characterized as the closeness of the deliberate an incentive to the
genuine esteem. In a technique with high precision, an example (whose "genuine esteem" is known) is
investigated and the deliberate esteem is indistinguishable to the genuine esteem. Normally, exactness is

spoken to and controlled by recuperation considers. There are three approaches to decide exactness:
Comparison to a reference standard
1. Recovery of the analyte spiked into blank matrix or
2. Standard addition of the analyte.
It should be clear how the individual or total impurities are to be determined. e.g.,Weight /
weight or area percent in all cases with respect to the major analyte.
5. Specificity / selectivity
The terms selectivity and specificity are regularly utilized reciprocally. As indicated by ICH,
the term particular for the most part alludes to a strategy that delivers a reaction for a solitary
analyte just while the term specific alludes to a technique which gives reactions to various
substance elements that could possibly be recognized from each other. On the off chance that
the reaction is recognized from every other reaction, the strategy is said to be particular.
Since there are not very many strategies that react to just a single analyte, the term selectivity
is typically more fitting. The analyte ought to have no obstruction from different incidental
segments and be all around settled from them. An agent chromatogram or profile ought to be
created and submitted to demonstrate that the superfluous pinnacles either by expansion of
known mixes or tests from pressure testing are standard settled from the parent analyte.
6. Robustness: The idea of heartiness of an explanatory system has been characterized by

the ICH as "a measure of its ability to stay unaffected by little, yet think varieties in
technique parameters". A decent practice is to shift essential parameters in the technique
methodicallly and measure their impact on partition. The variable strategy parameters in
HPLC procedure may includes stream rate, segment temperature, test temperature, pH and
versatile stage sythesis.
7. Limit of detection: Cutoff of location (LOD) is the least convergence of analyte in an example
that can be identified, however not really qualtitated, under the expressed trial conditions. With UV
locators, it is hard to guarantee the recognition accuracy of low level mixes because of potential
progressive loss of affectability of identifier lights with age or commotion level variety by finder
producer. At low levels, confirmation is required that the LOD and LOQ limits are achievable with
the test technique each time. With no reference standard for a given contamination or intends to

guarantee perceptibility, unessential peak(s) could "vanish/show up." A rough technique to assess
the achievability of the superfluous pinnacle recognition is to utilize the rate asserted for LOD from
the region checks of the analyte. A few methodologies for deciding the LOD are conceivable,
contingent upon whether the methodology is a non-instrumental or instrumental.
•Based on visual evaluation
•Based on signal-to-noise
•Based on the standard deviation of the response and the slope
The LOD may be expressed as:
LOD = 3.3 σ / S
Where,
σ = Standard deviation of Intercepts of calibration curves
S = Mean of slopes of the calibration curves

The slope S may be estimated from the calibration curve of the analyte.
8. Limit of quantitation
Limit of quantitation (LOQ) is the lowest concentration of analyte in a sample that can be
determined with acceptable precision and accuracy under the stated experimental conditions. Several
approaches for determining the LOQ are possible depending on whether the procedure is a non-
instrumental or instrumental.
• Based on visual evaluation
• Based on signal-to-noise Approach
• Based on the standard deviation of the response and the slope
The LOQ may be expressed as:
LOQ = 10 σ / S
Where,
σ = Standard deviation of Intercepts of calibration curves

S = Mean of slopes of the calibration curves
The slope S may be estimated from the calibration curve of the analyte
Table No.1.4: Characteristics to be validated in HPLC
Characteristics Acceptance Criteria
Accuracy/trueness Recovery 98-102% (individual)
Precision RSD < 2%
Repeatability RSD < 2%
Intermediate Precision RSD < 2%
Specificity / Selectivity No interference
Detection Limit S/N > 2 or 3
Quantitation Limit S/N > 10
Linearity 2
Correlation coefficient R > 0.999
Range 80 –120 %

2. AIM, OBJECTIVE AND PLAN OF WORK
Aim
The main aim of the present study is to develop an accurate, precise, sensitive, selective,
reproducible and rapid analytical technique for simultaneous estimation of Netarsudil,
Latanoprost in bulk and Pharmacuetical dosage form.
Objective and Plan:
Following are the objectives of the present work:
 To develop a new stability indicating HPLC method for simultaneous

estimation of ` Netarsudil and Latanoprost and to develop the validated method
according to ICH guidelines.
 To apply the validated method for the simultaneous estimation of Netarsudil
and Latanoprost pharmaceutical formulation

3. DRUG PROFILE (16-17)
Netarsudil- DRUG PROFILE
Description;
Netarsudil is a drug for the treatment of glaucoma . Netarsudil A Rho kinase inhibitor with
norepinephrine transport inhibitory activity that reduces production of aqueous . As of
December 18, 2017 the FDA approved Aerie Pharmaceutical's Rhopressa (netarsudil
ophthalmic solution) 0.02% for the indication of reducing elevated intraocular pressure in
patients with open-angle glaucoma or ocular hypertension. Acting as both a rho kinase
inhibitor and a norepinephrine transport inhibitor, Netarsudil is a novel glaucoma medication
in that it specifically targets the conventional trabecular pathway of aqueous humour outflow
to act as an inhibitor to the rho kinase and norepinephrine transporters found there as opposed
to affecting protaglandin F2-alpha analog like mechanisms in the unconventional uveoscleral
pathway that many other glaucoma medications demonstrate.
Structure;
Fig 1.1: Structure of Netarsudil
CAS Number : 1254032-66-0

Molecular Weight : 453.542,Monoisotopic: 453.205241741
Molecular Formula : C28H27N3O3

Appearance : Powder
Physical State : Liquid
Solubility : 0.000277 mg/ml.
Storage ; Refrigerator.
IUPAC Name : (4-((1S)-1-(Aminomethyl)-2-(isoquinolin-6-
ylamino)-2-oxoethyl)phenyl)methyl 2,4- dimethylbenzoate Netarsudil
Indication:
Netarsudil is indicated for the reduction of elevated intraocular pressure (IOP) in patients
with open-angle glaucoma or ocular hypertension.
Pharmacodynamics
Aqueous humour flows out of the eye via two pathways: 1) the conventional trabecular
pathway and 2) the unconventional uveoscleral pathway. And, although it has been shown
that the conventional trabecular pathway accounts for most aqueous outflow due to various
pathologies, most medications available for treating glaucoma target the uveoscleral pathway
for treatment and leave the diseased trabecular pathway untreated and unhindered in its
progressive deterioration and dysfunction.
Mechanism of action:
The medical condition glaucoma is a leading cause of progressive visual impairment and
blindness across the world with primary open-angle glaucoma (POAG) being the major type
of glaucoma. Elevated intraocular pressure (IOP) resulting from increased resistance to
aqueous humor outflow is considered a major risk for the development and progression of
POAG, , but various clinical studies have demonstrated that the reduction and tight control of
IOP can delay or prevent POAG and the vision loss associated with it. Ordinary physiological
IOP results from aqueous humor produced by the ocular ciliary body and its outflow through
two main outflow pathways: the conventional (trabecular) and the unconventional
(uveoscleral) pathways.
Absorption:
The systemic exposure of netarsudil and its active metabolite, AR-13503, after topical ocular
administration of netarsudil opthalmic solution 0.02% once daily (one drop bilaterally in the
morning) for eight days in 18 healthy subjects demonstrated no quantifiable plasma

concentrations of netarsudil (lower limit of quantitation [LLOQ] 0.100 ng/mL) post dose on
Day 1 and Day 8. Only one plasma concentration at 0.11 ng/mL for the active metabolite was
observed for one subject on Day 8 at 8 hours post dose .
Metabolism:
After topical ocular dosing, netarsudil is metabolized by esterases in the eye to its active
metabolite, netarsudil-M1
Route of elimination:
Clinical studies assessing the in vitro metabolism of netarsudil using corneal tissue from
humans, human plasma, and human liver microsomes and microsomal S9 fractions
demonstrated that netarsudil metabolism occurs through esterase activity. Subsequent
metabolism of netarsudil's esterase metabolite, AR-13503, was not detectable. In fact,
esterase metabolism in human plasma was not detected during a 3 hour incubation.
Dosageforms:Opthalmic solution
Brandnames: RHEOPRESSA

Latanoprost- DRUG PROFILE
Description;
Latanoprost is a prodrug analog of prostaglandin F2 alpha that is used to treat elevated
intraocular pressure (IOP). It was initially approved by the FDA in 1998. Latanoprost is the
first topical prostaglandin F2 alpha analog used for glaucoma treatment. It has been found to
be well-tolerated and its use does not normally result in systemic adverse effects like other
drugs used to treat elevated intraocular pressure, such as Timolol. Another benefit latanoprost
is that it can be administered once a day.
Structure;
Fig 1.2: Structure of Latanoprost
CAS Number 130209-82-4

Molecular Weight Average: 432.5928
Monoisotopic: 432.28757439
Molecular Formula C26H40O5
Appearance Powder

Physical State Liquid

Solubility 0.0129 mg/mL
Storage Refrigerator
IUPAC Name isopropyl (Z)-7-((1R,2R,3R,5S)-3,5-
dihydroxy-2-((3R)-3-hydroxy-5-
phenylpentyl)cyclopentyl)-5-heptenoate
Indication:
Latanoprost is indicated for the reduction of elevated intraocular pressure in patients who
have been diagnosed with open-angle glaucoma or ocular hypertension. Latanoprost may be
combined in a product with Netarsudil, a rho kinase inhibitor, for the same indications. In
addition to the above indications, the Canadian monograph for this drug also approves
latanoprost for the treatment of elevated intraocular pressure as a result of angle-closure
glaucoma that has been treated with peripheral iridotomy or laser iridoplasty.
Pharmacodynamics ;
Latanoprost effectively decreases intraocular pressure by increasing uveoscleral outflow. A
decrease in intraocular pressure has been measured within 3–4 hours post-administration,
reaches a maximum decrease at 8–12 hours, and can be maintained for a period of 24 hours.
Between 3 to 10% of patients taking latanoprost have experienced iris pigmentation after
about 3-4 months of latanoprost use. Patients should be notified of this risk before initiating
treatment. It may occur in both patients with light-colored irides (green-brown or blue/grey-
brown) or dark-colored (brown) irides, but is less pronounced in the latter group. This drug
may also cause other ocular effects including infrequent conjunctival hyperemia,
pigmentation of periocular tissues, eyelash changes, hypertrichosis, and ocular irritation.
Mechanism of action:
Elevated intraocular pressure leads to an increased risk of glaucomatous visual field loss. The
higher the intraocular pressure, the higher the risk of damage to the optic nerve and loss of
visual field. Latanoprost selectively stimulates the prostaglandin F2 alpha receptor and this
results in a decreased intraocular pressure (IOP) via the increased outflow of aqueous humor,
which is often implicated in cases of elevated intraocular pressure. Possible specific
mechanisms of the abovementioned increased aqueous outflow are the remodeling of the
extracellular matrix and regulation of matrix metalloproteinases. These actions result in
higher tissue permeability related to humor outflow pathways, which likely change outflow
resistance and/or outflow rates.
Absorption:
This drug is rapidly absorbed in the cornea as an isopropyl ester prodrug and is then activated
by the process of hydrolysis. A small amount of this drug is systemically absorbed. The Cmax
of latanoprost in the systemic circulation is reached after 5 minutes and is measured to be 53
pg/mL. The Cmax in the aqueous humor is attained within 2 hours after administration. and
has been estimated to be 15-30 ng/mL.
Metabolism:

After corneal uptake, this prodrug is hydrolyzed and activated by esterases to become a
pharmacologically active drug. The small portion of this drug that is able to reach the
circulation is found to be metabolized by the liver to the 1,2-dinor and 1,2,3,4-tetranor
metabolites through fatty acid beta-oxidation.
Route of elimination:
After hepatic beta-oxidation, the metabolites of latanoprost are primarily found to be excreted
by the kidneys. About 88% of the latanoprost dose is recovered in the urine after topical
administration. About 15% of a dose is reported to be excreted in the feces.
Dosage forms:
Opthalmic solution form
Brand names: XALATAN

4. LITERATURE REVIEW (18-23)
Analytic Author Drugs Column Mobile phase λmax

al name
Method
RP- Pratikeshk Latanoprost Supelco analytical acetonitrile as an organic 210nm
HPLC umar C18 (250 mm × modifier and water of 60:40
method Patel1 4.6 mm i.d., 5 μm (v/v).
particle size)
RP- R.V Latanoprost Waters symmetry 0.05% (v/v) trifluoro acetic acid
HPLC Rele* , and Timolol C18 column (250 in water : 0.05% (v/v) 210nm
method Maleate mm x 4.6 mm, 5 trifluoroacetic acid
µm) in acetonitrile (40:60 v/v)
RP- Agarwal Latanoprost, buffer: acetonitrile (40: 60 v/v), gradient

HPLC Ankit * Timolol and Inertsil C18, 300 x 254,210
method Benzalkoniu 3.9mm, 5µ nm
m chloride
LC Alessio latanoprost octylsilica (C8) . ethyl acetate and isopropanol -

method Zammataro column 60:40 (v/v)
RP- U.satyanar latanoprost Waters Xterra RP 10mM Ammonium formate PH 210nm

HPLC ayana1 18(250 x4.6mm,5 adjusted to 3.5 with formic acid
method µ and Acetonitrile.
LC Jigar Latanoprost, reverse phase A mixture of phosphate buffer 295nm&

method Mehta timolol&be cyano column of pH 3.2, acetonitrile and 210nm
et,al. nzalkonium {Hypersil BDS CN methanol was used as the mobile
chloride (250 × 4.6 mm), 5 phase.
μm}

5. MATERIALS AND METHODS
Materials:
 Netarsudil and Latanoprost pure drugs (API), Combination Netarsudil and
Latanoprost Opthalmic solution (ROCKLATAN), Distilled water, Acetonitrile,
Phosphate buffer, , Methanol, Potassium dehydrogenate ortho phosphate buffer,
Ortho-phosphoric acid.All the above chemicals and solvents are from Rankem
Instruments:
 Electronics Balance-Denver
 pH meter -BVK enterprises, India
 Ultrasonicator-BVK enterprises
 WATERS HPLC 2695 SYSTEM equipped with quaternary pumps,Photo Diode
Array detector and Auto sampler integrated with Empower 2 Software.
 UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm
and 10mm and matched quartz cells integrated with UV win 6 Software was used
for measuring absorbances of Netarsudil andLatanoprost solutions.
Methods:
Diluent: Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water
taken in the ratio of 50:50 as diluent.
Preparation of Standard stock solutions: Accurately weighed 5mg of Netarsudil, 1.25mg

of Latanoprost and transferred to individual 50ml volumetric flasks separately. 3/4 th of
diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up
with diluents and labeled as Standard stock solution 1 and 2. (100µg/ml of Netarsudil and
25µg/ml of Latanoprost)
Preparation of Standard working solutions (100% solution): 1ml from each stock
solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent.
(10µg/ml Netarsudil of and 2.5µg/ml of latanoprost)

Preparation of Sample stock solutions: 10 vails were weighed and was transferred into a
10ml volumetric flask, 5ml of diluents was added and sonicated for 10 min, further the
volume was made up with diluent and filtered by HPLC filters (100µg/ml of Netarsudil and
25µg/ml of Latanoprost)
Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock
solution was transferred to 10ml volumetric flask and made up with diluent.(10µg/ml of
Netarsudil and 2.5µg/ml of Latanoprost)
Preparation of buffer:
0.01N KH2PO4 Buffer: Accurately weighed 1.36gm of Potassium dihyrogen Ortho

phosphate in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and
degas to sonicate and finally make up the volume with water then PH adjusted to 3.48 with
dil. Orthophosphoric acid solution.
Validation:
System suitability parameters:
The system suitability parameters were determined by preparing standard solutions of

Netarsudil (10ppm) and Latanoprost (2.5ppm) and the solutions were injected six times and
the parameters like peak tailing, resolution and USP plate count were determined.
The % RSD for the area of six standard injections results should not be more than 2%.
Specificity: Checking of the interference in the optimized method.We should not find
interfering peaks in blank and placebo at retention times of these drugs in this method. So this
method was said to be specific.
Precision:
Preparation of Sample stock solutions: 10 vails were weighed and was transferred into a 10
ml volumetric flask, 5ml of diluents was added and sonicated for 10 min, further the volume
was made up with diluent and filtered by HPLC filters(100µg/ml of Netarsudil and 2.5µg/ml
of Latanoprost)

Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock
solution was transferred to 10ml volumetric flask and made up with diluent.(10µg/ml of
Netarsudil and 2.5µg/ml of Latanoprost)
Linearity:
Preparation of Standard stock solutions: Accurately weighed 5 mg of Netarsudil, 1.25 mg

of Latanoprost and transferred to individual 50 ml volumetric flasks separately. 3/4 th of
with diluents and labeled as Standard stock solution 1 and 2. (100µg/ml of Netarsudil and
2.5µg/ml of Latanoprost).
25% Standard solution: 0.25ml each from two standard stock solutions was pipetted out
and made up to 10ml. (2.5µg/ml of Netarsudil and 0.625µg/ml of Latanoprost)
50% Standard solution: 0.5ml each from two standard stock solutions was pipetted out and
made up to 10ml. (5µg/ml of Netarsudil and1.25µg/ml of Latanoprost)
and made up to 10ml. (7.25µg/ml of Netarsudil and1.875µg/ml of Latanoprost)
and made up to 10ml. (10µg/ml of Netarsudil and 2.5µg/ml of Latanoprost)
and made up to 10ml. (12.5µg/ml of Netarsudil and 3.75µg/ml of Latanoprost)
150% Standard solution: 1.5ml each from two standard stock solutions was pipettede out
and made up to 10ml (15µg/ml of Netarsudil and 5µg/ml of Latanoprost)
Accuracy:
Preparation of Standard stock solutions: Accurately weighed 5 mg of Netarsudil, 1.25 mg

of Latanoprost and transferred to individual 50 ml volumetric flasks separately. 3/4 th of
with diluents and labeled as Standard stock solution 1 and 2.

Preparation of 50% Spiked Solution: 0.5ml of sample stock solution was taken into a 10ml
volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made
up to the mark with diluent.
Preparation of 100% Spiked Solution: 1.0ml of sample stock solution was taken into a
10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and
made up to the mark with diluent.
Preparation of 150% Spiked Solution: 1.5ml of sample stock solution was taken into a
10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and
made up to the mark with diluent.
Acceptance Criteria:
The % Recovery for each level should be between 98.0 to 102

Robustness: Small deliberate changes in method like Flow rate, mobile phase ratio, and
temperature are made but there were no recognized change in the result and are within range
as per ICH Guide lines.
Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase
minus, mobile phase plus, temperature minus (25°C) and temperature plus(35°C) was
maintained and samples were injected in duplicate manner. System suitability parameters
were not much affected and all the parameters were passed. %RSD was within the limit.
LOD sample Preparation: 0.25ml each from two standard stock solutions was pipetted out
and transferred to two separate 10ml volumetric flasks and made up with diluents. From the
above solutions 0.1ml each of Netarsudil, Latanoprost, solutions respectively were
transferred to 10ml volumetric flasks and made up with the same diluents
LOQ sample Preparation: 0.25ml each from two standard stock solutions was pipetted out
and transferred to two separate 10ml volumetric flask and made up with diluent. From the
above solutions 0.3ml each of Netarsudil, Latanoprost, and solutions respectively were
transferred to 10ml volumetric flasks and made up with the same diluent.
Degradation studies:
Oxidation:

To 1 ml of stock solution of Netarsudil and Latanoprost, 1 ml of 20% hydrogen peroxide

(H2O2) was added separately. The solutions were kept for 30min at 60 0 c. For HPLC
study, the resultant solution was diluted to obtain 10µg/ml&2.5µg/ml solution and 10µl
were injected into the system and the chromatograms were recorded to assess the stability
of sample.
Acid Degradation Studies:
To 1 ml of stocks solution Netarsudil and Latanoprost, 1 ml of 2N Hydrochloric acid was
added and refluxed for 30mins at60 0 c. The resultant solution was diluted to obtain
10µg/ml & 2.5µg/ml solution and 10µl solutions were injected into the system and the
chromatograms were recorded to assess the stability of sample.
AlkaliDegradationStudies:
To 1 ml of stock solution Netarsudil and Latanoprost, 1 ml of 2N sodium hydroxide was
added and refluxed for 30mins at 60 0 c. The resultant solution was diluted to obtain
10µg/ml & 2.5µg/ml solution and 10µl were injected into the system and the chromatograms
were recorded to assess the stability of sample.
DryHeatDegradationStudies:
The standard drug solution w a s placed in oven at 105°C for 6h to study dry heat
degradation. For HPLC study, the resultant solution was diluted to 10µg/ml & 2.5µg/ml
solution and 10µl were injected into the system and the chromatograms were recorded to
assess the stability of the sample.
PhotoStabilitystudies:
The photochemical stability of the drug was also studied by exposing the 100µg/ml &
25µg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200-Watt
hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to
obtain 10µg/ml & 2.5µg/ml solutions and 10µl were injected into the system and the
chromatograms were recorded to assess the stability of sample.
NeutralDegradationStudies:

Stress testing under neutral conditions was studied by refluxingthedruginwaterfor6h r s

atatemperature of 60º.For HPLC study, the resultant solution was diluted to 10µg/ml & 2.5
µg/ml solution and 10µl were injected into the system and the chromatograms were recorded
to assess the stability of the sample.

6. RESULTS AND DISCUSSION
Method development: Method development was done by changing various, mobile phase
ratios, buffers etc.
Trial 1:
Chromatographic conditions:
Mobile phase : Acetonitrile and 0.01N KH2po4 (60:40)
Flow rate : 1 ml/min

Column : Ascentis 150 C18 (4.6 x 150mm, 2.7µm)
Detector wave length : 220nm
Column temperature : 30°C
Injection volume : 10mL

Run time : 10.0 min
Diluent : Water and Acetonitrile in the ratio 50:50
Results : In this trial only netarsudil peak was eluted latanoprost
peak is not eluted and baseline disturbance observed . So, Further Trial is carried out
Fig 6.3 Trial chromatogram 1

Trial 2:
Mobile phase : 0.01% OPA: Acetonitrile (60:40)

Column : Water C18 (4.6 x 150mm, 5µm)

Run time : 6 min
Results : In this trial Both peaks are eluted but Netarsudil peak is
splitted and baseline disturbance observed , so further trial is carried out.

Trial 3:
Mobile phase : 0.01N Kh2po4: Acetonitrile (50:50)

Flow rate : 1ml/min
Column : BDS 150 C18 (4.6 x 150mm, 5µm)

Run time : 10 min
Results : Both peaks were eluted with good shapes but the retention
time of both peaks are long and resolution is not satisfactory . So, further trial is carried out.
Fig. No.6.7 Trial chromatogram 3

Trial 4:
Mobile phase : 0.1%OPA: Acetonitrile (60:40)
Column : Agilent 150 C18 (4.6 x 150mm, 5µm)
Injection volume : 10µL
Run time : 10 min
Results : Both peaks were eluted with good peak shapes but the
retention time of Latanoprost peak is too long, so further trial is carried out.

Optimized method:
Mobile phase : 60% 0.1N KH2PO4: 40% Acetonitrile

Flow rate : 1ml/min
Column : Agilent 150 C18 (4.6 x 150mm, 5µm)

Run time : 5min
Results : Both peaks have good resolution, tailing factor, theoretical
plate count and resolution.

Fig 6.5 Optimized Chromatogram
Observation: Netarsudil and Latanoprost were eluted at 2.875min and 4.106min respectively
with good resolution. Plate count and tailing factor was very satisfactory, so this method was
optimized and to be validated.
Systemsuitability:All the system suitability parameters were within the range and
satisfactory as per ICH guidelines
Table:6.1 System suitability parameters for Netarsudil and Latanoprost
S
no
Netarsudil Latanoprost
In RT(min) USP Tailing RT(min) USP Tailing Resolution

j Plate Plate
Count Count
1 2.218 8000 1.26 2.621 10693 1.30 3.9
2 2.219 8590 1.28 2.622 9703 1.39 3.9
3 2.219 8585 1.26 2.623 9512 1.29 3.9

4 2.219 7987 1.27 2.624 9273 1.22 3.8
5 2.220 8540 1.26 2.626 9631 1.25 3.8

6 2.220 7785 1.26 2.626 10117 1.20 3.9
Fig 6.11System suitability Chromatogram
Discussion: According to ICH guidelines plate count should be more than 2000, tailing factor
should be less than 2 and resolution must be more than 2. All the system suitable parameters
were passed and were within the limits.
Validation:
Specificity:

Figure No. 6.12.Chromatogram of blank.
Figure No. 6.13Chromatogram of placebo

Figure No. 6.14 Optimized Method
Discussion: Retention times of Netarsudil and Latanoprost were 2.875min and 4.106 min
respectively. We did not found and interfering peaks in blank and placebo at retention times
of these drugs in this method. So this method was said to be specific.

Linearity:
Table 6.2: Linearity table for Netarsudil and Latanoprost.
Conc Conc
Peak area Peak area
(μg/mL) (μg/mL)
0 0 0 0
2.5 169159 0.625 27932
5 319482 1.25 54561
7.5 475186 1.875 81928
10 630141 2.5 109126
12.5 789603 3.125 132978
15 951690 3.75 163564
1000000
900000 f(x) = 62951.69 x + 4328.14
R² = 1
800000
700000
600000
500000
400000
300000
200000
100000
0
0 2 4 6 8 10 12 14 16
Fig No. 6.15 Calibration curve of Netarsudil

180000
160000
f(x) = 43162.77 x + 510.88
140000 R² = 1
120000
100000
80000
60000
40000
20000
0
0 0.5 1 1.5 2 2.5 3 3.5 4
Fig No. 6.16Calibration curve of Latanoprost
Discussion:Six linear concentrations of Netarsudil (2.5-15µg/ml) and Latanoprost (0.625-

2.5µg/ml) were injected in a duplicate manner. Average areas were mentioned above and
linearity equations obtained for Netarsudil was y = 62952x + 4328.1 and of Latanoprost was
y = 43163x + 510.88 Correlation coefficient obtained was 0.999 for the two drugs.

Fig. No.6.17Linearity 25% ChromatogramofNetarsudil and Latanoprost
Fig No.6.18Linearity 50% ChromatogramofNetarsudil and Latanoprost





Precision:
System Precision:
Table 6.3 System precision table of Netarsudil and Latanoprost
S. No Area of Netarsudil Area of Latanoprost
1. 629278 108432
2. 635873 108606
3. 636722 109178
4. 639643 108536
5. 624980 106479
6. 629571 107342
Mean 632678 108096
S.D 5577.6 992.0
%RSD 0.9 0.9


Fig 6.23 System precision chromatogram
Discussion: From a single volumetric flask of working standard solution six injections were
given and the obtained areas were mentioned above. Average area, standard deviation and %
RSD were calculated for two drugs.% RSD obtained as 0.9%and 0.9% respectively for
Netarsudil and Latanoprost .As the limit of Precision was less than “2” the system precision
was passed in this method.
Repeatability:
Table 6.4 Repeatability table of Netarsudil and Latanoprost
Area of Area of
S. No
1. 624225 107101
2. 634399 108254
3. 627653 107843
4. 629255 108647
5. 624494 107638
6. 629887 108123
Mean 628319 107934
S.D 3798.3 536.0
%RSD 0.6 0.5


Fig No.6.24Repeatability chromatogram
Discussion: Multiple sampling from a sample stock solution was done and six working
sample solutions of same concentrations were prepared, each injection from each working
sample solution was given and obtained areas were mentioned in the above table. Average
area, standard deviation and % RSD were calculated for two drugs and obtained as 0.6% and
0.5% respectively for Netarsudil and Latanoprost. As the limit of Precision was less than “2”
the system precision was passed in this method.
Intermediate precision (Day_Day Precision):

Table 6.5 Intermediate precision table of Netarsudil and Latanoprost
S. No Area of Netarsudil Area ofLatanoprost
1. 611382 99883
2. 607686 98757
3. 616092 97387
4. 611120 97025
5. 611792 98600
6. 614048 97823
Mean 612020 98246
S.D 2854.7 1046.3
%RS
D 0.5 1.1


Fig: 6.25Inter Day precision Chromatogram

Discussion: Multiple sampling from a sample stock solution was done and six working
sample solutions of same concentrations were prepared, each injection from each working
sample solution was given on the next day of the sample preparation and obtained areas were
mentioned in the above table. Average area, standard deviation and % RSD were calculated
for two drugs and obtained as 0.5% and 1.1% respectively for Netarsudil and Latanoprost. As
the limit of Precision was less than “2” the system precision was passed in this method.
Accuracy:
Table 6.6 Accuracy table of Netarsudil
Amount
Amount Spiked recovered Mean
% Level % Recovery
(μg/mL) %Recovery
(μg/mL)
5 5.021 100.43
50% 5 5.091 101.82
5 5.058 101.15
10 9.994 99.94
100.46%
100% 10 9.913 99.13
10 9.936 99.36

15 15.168 101.12
150% 15 15.090 100.60
15 15.085 100.57
Table 6.6: Accuracy table of Latanoprost
Amount
Amount Spiked recovered Mean
% Level % Recovery
(μg/mL) %Recovery
(μg/mL)
1.25 1.252 100.19
50% 1.25 1.263 101.05
1.25 1.237 98.92
2.5 2.531 101.22
100% 2.5 2.489 99.55 100.20%
2.5 2.461 98.44
3.75 3.143 100.57
150% 3.75 3.172 101.49
3.75 3.136 100.34
Discussion: Three levels of Accuracy samples were prepared by standard addition method.
Triplicate injections were given for each level of accuracy and mean %Recovery was
obtained as 100.46% and 100.20% for Netarsudil and Latanoprost respectively.


Fig No.6.26Accuracy 50% ChromatogramofNetarsudil and Latanoprost


Fig No.6.27Accuracy 100% ChromatogramofNetarsudil and Latanoprost


Fig No. 6.28Accuracy 150% Chromatogram ofNetarsudil and Latanoprost
Sensitivity:
Table 6.7 Sensitivity table of Netarsudil and Latanoprost
Molecule LOD LOQ
Netarsudil 0.08 0.25

Latanoprost 0.04 0.12

Fig. No.6.29 LOD Chromatogram of Standard
Fig. No. 6.30 LOQ Chromatogram of of Standard
Robustness:
Table 6.8 Robustness data forNetarsudil and Latanoprost.

S.no Condition %RSD of %RSD of
1 Flow rate (-) 0.9ml/min 0.7 1.1
2 Flow rate (+) 1.1ml/min 1.2 1.7
3 Mobile phase (-) 65B:35A 0.6 0.4
4 Mobile phase (+) 55B:45A 1.2 1.6
5 Temperature (-) 25°C 1.7 0.8
6 Temperature (+) 35°C 1.0 0.9
Discussion: Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min),
mobile phase minus (65B:35A), mobile phase plus (55B:45A), temperature minus (25°C)
and temperature plus(35°C) was maintained and samples were injected in duplicate
manner. System suitability parameters were not much affected and all the parameters
were passed. %RSD was within the limit.

Fig No. 6.31Flow minus Chromatogram ofNetarsudil and Latanoprost.


Fig No. 6.32Flow plus Chromatogram ofNetarsudil and Latanoprost.


Fig No. 6.33Mobile phase minus Chromatogram ofNetarsudil and Latanoprost.


Fig No.6.34Mobile phase Plus ChromatogramofNetarsudil and Latanoprost.

Fig No. 6.35Temperature minus Chromatogram of Netarsudil and Latanoprost. .


Fig No. 6.36Temperature plus Chromatogram of Netarsudil and Latanoprost

Assay:(ROCKLATAN), bearing the label claim Netarsudil 0.2mg,Latanoprost 0.05mg.Assay

was performed with the above formulation. Average % Assay for Netarsudil and Latanoprost
obtained was 98.91% and 99.65%respectively
Table 6.9 Assay Data of Netarsudil
S.no Standard Area Sample area % Assay
1
629278 624225 98.27
2
635873 634399 99.87
3
636722 627653 98.81
4
639643 629255 99.06
5
624980 624494 98.31
6
629571 629887 99.16
Avg
632678 628319 98.91
Stdev
5577.6 3798.3 0.60
%RSD
0.9 0.6 0.6
.
Table 6.10 Assay Data of Latanoprost
S.no Standard Area Sample area % Assay
1
108432 107101 98.88
2
108606 108254 99.95
3
109178 107843 99.57
4
108536 108647 100.31

5
106479 107638 99.38
6
107342 108123 99.83
Avg
108096 107934 99.65
Stdev
992.0 536.0 0.5
%RSD
0.9 0.5 0.5
Fig 6.37Chromatogram of working standard solution
Fig No.6.38 Chromatogram of working sample solution

6.8. Degradation data
Type of Netarsudil Latanoprost

degradation AREA %RECO % AREA %RECOVE %
VERED DEGRAD RED DEGRADED
ED
Acid 608519 95.80 4.20 102690 94.81 5.19
Base 594165 93.54 6.46 101982 94.16 5.84
Peroxide 606959 95.55 4.45 103275 95.35 4.65
Thermal 622703 98.03 1.97 105632 97.53 2.47
Uv 629320 99.07 0.93 107204 98.98 1.02
Water 631873 99.07 0.93 107449 99.20 0.80
Degradation chromatograms
Acid degradation chromatogram

Fig.6.25 acid
Base degradation chromatogram

Fig.6.26 base
Peroxide degradation chromatogram
Fig.6.27 peroxide
Thermal degradation chromatogram

Fig.6.28 thermal
Uv degradation chromatogram
Fig.6.29 UV

Water degradation chromatogram
Fig.6.30 water

7. SUMMARY AND CONCLUSION
7.1 Summary Table
Parameters
Netarsudil Latanoprost LIMIT
Linearity 2.5-15µg/ml 0.625-3.75 µg/ml
Range(µg/ml)
Regressioncoefficient 0.999 0.999
Slope(m) 62952 43163
R< 1
Intercept(c) 4328.1 510.88
Regression equation y = 62952x + y = 43163x + 510.88
(Y=mx+c) 4328.1
Assay (% mean 98.91% 98.65% 90-110%
assay)
Specificity Specific Specific No
interference
of any peak
System precision 0.9 0.6 NMT 2.0%
%RSD
Method precision 0.9 0.5 NMT 2.0%
%RSD
Accuracy%recovery 100.46% 100.20% 98-102%
LOD 0.08 0.04 NMT 3

LOQ 0.25 0.12 NMT 10
FM 0.7 1.1
Robustness FP 1.2 1.7 %RSD
MM 0.6 0.4 NMT
MP 1.2 1.6 2.0
TM 1.7 0.8
TP 1.0 0.9

Conclusion
A simple, Accurate, precise method was developed for the simultaneous estimation of the
Netarsudil and Latanoprost in Tablet dosage form. Retention time of Netarsudil and
Latanoprost were found to be 2.875min and 4.106min. %RSD of the Netarsudil and
Latanoprost were and found to be 0.9 and 0.9 respectively. %Recovery was obtained as
100.46% and 100.20% for Netarsudil and Latanoprost respectively. LOD, LOQ values
obtained from regression equations of Netarsudil and Latanoprost were 0.08, 0.25 and 0.04,
0.12 respectively. Regression equation of Netarsudil is y = 62952x + 4328.1and y = 43163x +
510.88 of Latanoprost. Retention times were decreased and that run time was decreased, so
the method developed was simple and economical that can be adopted in regular Quality
control test in Industries.

Referances
1. MacNair J.E., Patel K.D. And Jorgenson J.W., Ultra pressure reversed phase capillary
Liquid Chromatography: Isocratic and gradient elusion using columns packed with 1μm
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Separation Science Re-Defined, LCGC Supplement, p. 8-11(MAY 2005).
4. Jerkovich A.D., Mellors J.S., and Jorgenson J.W., The use of micrometer-sized particles
in ultrahigh pressure liquid chromatography. LCGC. 2003; 21(7): 600–610.
5. MacNair J.E., Lewis K.C. And Jorgenson J.W., Ultrahigh-pressure reversed-phase
liquid chromatography in packed capillary columns. Anal. Chem. 1997; 69: 983–989.
6. Van Deemter JJ, Zuiderweg EJ, Klinkenberg A.Longitudinal diffusion and resistance to
mass transfer as causes of non ideality in chromatography. Chem. Eng. Sci. 1956; 5:
271–289.
7. Lars Y. and Honore H.S., On-line turbulent-flow chromatography–high-performance
liquid chromatography–mass spectrometry for fast sample preparation and quantitation
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8. McLoughlin D.A., Olah T.V., and Gilbert J.D., A direct technique for the simultaneous
determination of 10 drug candidates in plasma by Liquid Chromatography atmospheric
pressure chemical ionization mass spectrometry interfaced to a prospect solid-phase
extraction system. J. Pharm. Biomed. Anal. 1997; 15: 1893–1901.
9. Website information: Acquity columns. www.waters.com/acquitycolumns

10. Goodwin L, White SA, Spooner N. Evaluation of ultra-performance liquid
chromatography in the bioanalysis of small molecule drug candidates in plasma.J.
Chromatogr. Sci. 2007; 45(6): 298–304.
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tomorrow's HPLC technology today as published in LPI- June 2004.
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Massachusetts, USA, Journal of Liquid Chromatography & Related Technologies, 2005;
28: 1253–1263.
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Fang, et al. Applications of Ultra-Performance Liquid Chromatography to Traditional
Chinese Medicines. J.Chromatogr Sci., 2008; 48: 18-21.
16. https://www.drugbank.ca/drugs/DB13931
17. https://www.drugbank.ca/drugs/DB00654
18. Pratikeshkumar Patel1 et al. Bioanalytical Method Development and Validation for
Latanoprost Quantification in Pharmaceutical Opthalmic Microemulsion Formulation
by RP-HPLC, J Anal Bioanal Tech 6:284.
19. R.V Rele* et al. Simultaneous RP HPLC determination of Latanoprost and Timolol
Maleate in combined pharmaceutical dosage form, J. Chem. Pharm. Res., 2011,
3(1):138-144.
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Method Development & Validation For Simultaneous Estimation of Netarsudil & Latanoprost by RP-HPLC

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Method Development & Validation For Simultaneous Estimation of Netarsudil & Latanoprost by RP-HPLC

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Method development & validation for simultaneous estimation of Netarsudil &

In recent years, several analytical techniques have been evolved.

CLASSIFICATION OF HPLC ( 4-10)

Based on modes of chromatography:

• Normal phase chromatography

• Reverse phase chromatography

Based on principle of separation:

• Ion exchange chromatography

• Size exclusion chromatography

• Chiral phase chromatography

Based on elution technique:

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Based on the scale of operation:

Normal Phase - High Performance Liquid Chromatography (NP-HPLC)

NP-HPLC investigates the distinctions in the quality of the polar

Reversed Phase - High Performance Liquid Chromatography (RP-HPLC)

Rather than NP-HPLC, RP-HPLC utilizes predominantly dispersive powers

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This strategy comes about because of a thermodynamic circulation of analytes between

Size exclusion chromatography:

Ion exchange chromatography (IEC):

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E−: A•E + B+ ↔B•E + A+

The equilibrium constant for this process is shown in Eq. below:

• Quaternary amine—strong anion-exchanger

• Tertiary amine—weak anion-exchanger

Size exclusion chromatography (SEC):

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1.3 INSTRUMENTATION OF HPLC

HPLC is an exceptional branch of Column Chromatography in which the versatile

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Figure .No.1.1 HPLC basic instrumentation

I. Solvent delivery system:

Pump classification according to flow rate:

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Pump classification according to materials of construction:

Pumps may also be classified according to the primary construction materials.

Pump classification according to mechanism of eluent displacement:

The third classification of pumps is according to the mechanism by which the

Solvent degassing system

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II. Sample introduction system

LOAD (the sample loop) INJECT (move the sample loop

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v) End topping vi) % carbon stacking.

I) Pure silica and cross breed silica segments.

ix) Chiral columns.

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IV. Mobile phase

Table No. 1.2 Physical properties of common HPLC solvents

Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8

Dioxane 88.1 101 1.420 215 1.034 1.26 2.21

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Ethanol 46.1 78 1.359 205 0.789 1.19 24.5

Ethyl acetate 88.1 77 1.372 256 0.901 0.450 6.02

Methanol 32.0 65 1.326 205 0.792 0.584 32.7

CH2Cl2 84.9 40 1.424 233 1.326 0.44 8.93

Isopropanol 60.1 82 1.375 205 0.785 2.39 19.9

n-propanol 60.1 97 1.383 205 0.804 2.20 20.3

THF 72.1 66 1.404 210 0.889 0.51 7.58

Water 18.0 100 1.333 170 0.998 1.00 78.5

• Solute specific detectors (UV-Vis, fluorescence, electrochemical, infra-red, radio

• Bulk property detectors (refractive index, viscometer, conductivity)