Human Journals

Review Article
July 2021 Vol.:21, Issue:4
© All rights are reserved by R. D. Chakole et al.

A Review on HPLC Method Development and Validation

Keywords: High-Pressure Liquid Chromatography (HPLC),
Method validation, Method development

ABSTRACT
P. M. Deshmukhe, M. S. Charde, R. D. Chakole*
HPLC is the most commonly used separation technique for
Post Graduate Department of Pharmaceutical detecting, separating, and quantifying drugs. To optimize
Chemistry the method, several chromatographic parameters were
Government College of Pharmacy, Vidyanagar, Karad, investigated, including sample pretreatment, mobile phase
Dist.: Satara Pin- 415124, Maharashtra, India. selection, column selection, and detector selection. The
purpose of this article is to go over the method
Submitted: 21 June 2021
development, optimization, and validation processes.
Accepted: 27 June 2021
Because of its advantages such as rapidity, specificity,
Published: 30 July 2021
accuracy, precision, and ease of automation, the HPLC
method can be used to analyze the majority of drugs in
multicomponent dosage forms. HPLC method development
and validation are critical in new drug discovery,
development, and manufacturing, as well as a variety of
[Link]
other human and animal studies. Validation of analytical
methods is required during drug development and
manufacturing to ensure that these analytical methods are
fit for their intended purpose. To meet GMP requirements,
pharmaceutical industries should have an overall validation
policy that details how validation will be carried out. This
article is primarily concerned with the optimization of HPLC
conditions.
 [Link]

INTRODUCTION:

High-Performance Liquid Chromatography has become one of the most powerful analytical
chemistry tools. It is capable of separating, identifying, and quantifying the compounds
present in any sample that can be dissolved in a liquid. High-performance liquid
chromatography (HPLC) is one of the most accurate analytical methods for both quantitative
and qualitative drug product analysis. [1] The principle is that a sample solution is injected
into a porous material column (stationary phase), and a liquid (mobile phase) is pumped at
high pressure through the column. The sample is separated based on differences in migration
rates through the column caused by different partitioning of the sample between the
stationary and mobile phases. Elution occurs at different times depending on the partition
behavior of different components. [2] A sample compound with a higher affinity for the
stationary layer will travel slower and for a shorter distance than a compound with a lower
affinity, which will travel faster and for a longer distance. [3] The purpose of the HPLC
method is to separate and quantify the main drug, any reaction impurities, all available
synthetic intermediates, and any degradants. High-Performance Liquid Chromatography has
become one of the most powerful analytical chemistry tools. It is capable of separating,
identifying, and quantifying the compounds present in any sample that can be dissolved in a
liquid. It is one of the most accurate analytical methods for quantitative and qualitative drug
product analysis, as well as determining drug product stability. [4]

HPLC principle:

The distribution of the analyte (sample) between a mobile phase (eluent) and a stationary
phase is the basis for the separation principle of HPLC (packing material of the column). The
molecules are retarded while passing through the stationary phase depending on the chemical
structure of the analyte.

Types of HPLC:

The phase system used in the process determines the type of HPLC used in the analysis.
Normal phase chromatography (NP-HPLC): This method separates analytes based on polarity
and is also known as normal phase HPLC (NP-HPLC). In NP-HPLC, a polar stationary phase
and a non-polar mobile phase are used. The polar stationary phase interacted with the polar
analyte and retained it. The interaction between the polar analyte and the polar stationary
phase prolongs elution time as analyte polarity increases. [5,6]

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

67
 [Link]

Classification of HPLC can be done as:

I. Preparative HPLC and analytical HPLC (based on the scale of operation)

II. Affinity chromatography, adsorption chromatography, size exclusion chromatography,

III. Ion exchange chromatography, chiral phase chromatography (based on the principle of
separation)

IV. Gradient separation and isocratic separation, (based on elution technique)

V. Normal phase chromatography and reverse-phase chromatography (based on modes of

operation). [9]

1. Size exclusion chromatography:

SEC, also known as gel permeation or gel filtration chromatography, is a technique for
separating particles based on their size. It is also capable of determining the tertiary and
quaternary structures of proteins and amino acids. This method is commonly used to calculate
polysaccharide molecular weight.

2. Ion exchange chromatography:

Retention in ion-exchange chromatography is based on the attraction of solute ions to

charged sites bound to the stationary phase. Ions with the same charge are not allowed. This
chromatography technique is widely used in water purification, ligand-exchange
chromatography, protein ion-exchange chromatography, high-pH anion-exchange
chromatography of carbohydrates and oligosaccharides, and other applications. [5,6]

3. Bio-affinity chromatography:

Separation based on reversible protein-ligand interactions. Ligands are covalently attached to

solid support on an abio-affinity matrix, which keeps proteins that interact with the column-
bound ligands in place. [5]

4. Normal phase chromatography:

The mobile phase in normal phase chromatography is non-polar, while the stationary phase is
polar. As a result, the polar analyte is retained by the station phase. The increased polarity of
solute molecules increases adsorption capacity, resulting in a longer elution time. In this
chromatography, a stationary phase of chemically modified silica (cyanopropyl, aminopropyl,

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

68
 [Link]

and diol) is used. [7] As an example, A typical column has an internal diameter of
approximately 4.6 mm and a length ranging from 150 to 250 mm. Polar compounds in the
mixture that are passed through the column will stick to the polar silica for a longer period
than non-polar compounds. As a result, the non-polar ones will move faster through the
column. [10]

5. RP-HPLC (Reversed-Phase HPLC):

The stationary phase of RP-HPLC is non-polar, and the mobile phase is polar or moderately
polar. The principle of hydrophobic interaction underpins RP-HPLC [9]. The non-polar
stationary phase will retain analytes that are relatively less polar in a mixture of components
for a longer period than those that are relatively more polar. As a result, the most polar
component elutes first. [11]

Instrumentation of High-Performance Liquid Chromatography (HPLC):

In analytical separation, a high-pressure flow of a liquid through a column containing the

stationary phase is used. Stationary phase: This can be a solid (LSC) or a liquid (L) (LLC). A
mixture of compounds injected at one end separates as the compounds pass through the
column. Separated compounds are detected electronically as they elute at the other end of the
column. High-Performance Liquid Chromatography is a more versatile technique than gas
chromatography. There is a greater variety of mobile and stationary phases to choose
from.[7]

Figure No. 1: Flow Diagram of HPLC Instrumentation

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

69
 [Link]

The HPLC technique has the characteristics listed below:

❖ High resolution, small diameter, stainless steel, and glass column

❖ Quick analysis

❖ Significantly higher mobile phase pressure

❖ Mobile phase flow rate control.

HPLC has many advantages, including:

❖ Simultaneous Analysis

❖ High Resolution

❖ Extreme Sensitivity

❖ Excellent repeatability

❖ Limited sample size

❖ The analysis condition is moderate.

❖ It is simple to fractionate and purify the sample. [8]

HPLC Method Development:

Analytical method development and validation are critical steps in the discovery,
development, and manufacturing of pharmaceuticals. These techniques are used to ensure the
identity, purity, potency, and performance of pharmaceutical products. When developing
methods, there are numerous factors to consider. In the case of UV detection, they first gather
information about the analyte's physiochemical properties (pKa, log P, solubility) and
determine which mode of detection would be suitable for analysis. The majority of the
analytical development effort is spent validating an HPLC–method for indicating stability.
The purpose of the HPLC method is to separate and quantify the main active drug, any
reaction impurities, all available synthetic intermediates, and any degradants. [12,13]

HPLC method development:

The following is a step in HPLC method development:

❖ Recognizing the Physicochemical Properties of Drug Molecules

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

70
 [Link]

❖ Choosing chromatographic conditions

❖ Creating an analytic approach.

❖ Preparation of Samples

❖ Method Improvement

❖ Validation of methods

Figure No. 2: Steps involved in HPLC Method development

1. Recognizing the Physicochemical Properties of Drug Molecules:

The physicochemical properties of a drug molecule are critical in method development. To

develop a method, one must first investigate the physical properties of the drug molecule,
such as solubility, polarity, pKa, and pH. A compound's polarity is a physical property. It

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

71
 [Link]

assists an analyst in determining the solvent and mobile phase composition. The polarity of
molecules can be used to explain molecular solubility. Polar solvents, such as water, and
nonpolar solvents, such as benzene, do not mix. In general, like dissolves like, which means
that materials with similar polarities dissolve in each other. The solubility of the analyte
influences the choice of mobile phase or diluents. The analyte must be soluble in diluents and
not react with any of its constituents. The pH and pKa values are important in the
development of HPLC methods. The pH value is defined as the inverse of the logarithm to
base 10 of the hydrogen ion concentration.

pH equals -log10[H3O+].

Choosing an appropriate pH for ionizable analytes frequently results in symmetrical and

sharp peaks in HPLC. In quantitative analysis, sharp, symmetrical peaks are required to
achieve low detection limits, low relative standard deviations between injections, and
reproducible retention times. [14,15]

2. Choosing Chromatographic Conditions:

During initial method development, a set of initial conditions (detector, column, mobile
phase) is chosen to generate the sample's first "scouting" chromatograms. In most cases,
reversed-phase separations on a C18 column with UV detection are used. At this point, a
decision should be made whether to develop an isocratic or a gradient method.

2.2.1 Selection of Column:

A column is, of course, the first and most important component of a chromatograph. A
properly chosen column can produce a good chromatographic separation, resulting in
accurate and reliable analysis. An incorrectly used column can frequently produce confusing,
insufficient, and poor separations, resulting in invalid or difficult-to-interpret results. [9] The
column is the heart of an HPLC system. During method development, changing a column
will have the greatest impact on analyte resolution. The best column for an application must
take into account stationary phase chemistry, retention capacity, particle size, and column
dimensions. The hardware, matrix, and stationary phase are the three main components of an
HPLC column. Silica, polymers, alumina, and zirconium are some of the matrices used to
support the stationary phase. The most common matrix for HPLC columns is silica. Silica
matrices are strong, easily derivatized, produced in consistent sphere sizes, and do not
compress under pressure. The best column for an application must take into account

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

72
 [Link]

stationary phase chemistry, retention capacity, particle size, and column dimensions. The
hardware, matrix, and stationary phase are the three main components of an HPLC column.
Silica, polymers, alumina, and zirconium are some of the matrices used to support the
stationary phase. The most common matrix for HPLC columns is silica. Silica matrices are
strong, easily derivatized, produced in consistent sphere sizes, and do not compress under
pressure. Most organic solvents and low pH systems are chemically stable to silica. One
disadvantage of a silica solid support is that it dissolves above pH 7. In recent years, silica-
supported columns for use at high pH have been developed. The silica's nature, shape, and
particle size aid in effect separation. A smaller particle results in an increased number of
theoretical plates. The type of stationary phase determines whether a column can be used for
normal or reverse-phase chromatography. A polar stationary phase and a non-polar mobile
phase are used in normal phase chromatography. Polar compounds elute later than non-polar
compounds in general. The most common reverse phase columns and their applications are
listed below. Propyl (C3), Butyl (C4), and Pentyl (C5) phases are useful for ion-pairing
chromatography (C4) and peptides containing hydrophobic residues, as well as other large
molecules. When compared to C8 or C18 phases, C3–C5 columns generally retain non-polar
solutes less well. Zorbax SB-C3, YMC-Pack C4, and Luna C5 are a few examples. These
columns are less resistant to hydrolysis in general than columns with longer alkyl chains.
Octyl (C8, MOS) phases have a wide range of applications. This phase is less retentive than
the C18 phases, but it is still beneficial for pharmaceuticals, nucleosides, and steroids. The
first and most important step in method development is the selection of the stationary
phase/column. It is impossible to develop a robust and reproducible method without the
availability of a stable, high-performance column. Columns must be stable and reproducible
to avoid problems caused by irreproducible sample retention during method development.
Separation selectivity for specific components varies between columns manufactured by
different manufacturers as well as between column production batches manufactured by the
same manufacturer. The main ones are column dimensions, silica substrate properties, and
bonded stationary phase characteristics. Due to a variety of physical properties, silica-based
packing is preferred in the majority of today's HPLC columns. [16]

2.2.2 Selection of Chromatographic mode:

Chromatographic modes are determined by the molecular weight and polarity of the analyte.
All case studies will concentrate on reversed-phase chromatography (RPC), which is the most
commonly used mode for small organic molecules. Ionizable compounds (acids and bases)

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

73
 [Link]

are frequently separated by RPC using buffered mobile phases (to keep the analytes from
becoming ionized) or ion-pairing reagents. [17]

2.2.3 Optimization of Mobile phase:

❖ Buffer Selection:

Various buffers, including potassium phosphate, sodium phosphate, and acetate, were tested
for system suitability parameters and overall chromatographic performance. Following
sequential trials with various buffers, it was determined that potassium dihydrogen phosphate
was suitable for the effective separation of all peaks. Potassium dihydrogen phosphate buffer
concentrations of 0.02M, 0.05M, and 0.1 M were tested. The change in buffer concentration
did not result in significant changes in the elution pattern or resolution, but the 0.05M
concentration increased the method's sensitivity. [18]

❖ Effect of pH:

If analytes are ionizable, the appropriate mobile-phase pH must be chosen based on the
analyte pKa so that the target analyte is either ionized or neutral. The ability to change the pH
of the mobile phase is one of the most powerful tools in the "chromatographer's toolbox,"
allowing for simultaneous changes in retention and selectivity between critical pairs of
components. [18]

❖ Effect of organic modifier:

In reverse phase HPLC, selecting the organic modifier type is relatively simple. The most
common options are acetonitrile and methanol (rarely THF). Gradient elution is typically
used with complex multicomponent samples because it may be impossible to elute all
components using a single solvent strength between k (retention factor) 1 and 10 under
isocratic conditions. [18]

2.2.4 Selection of detector and wavelength:

Following the chromatographic separation, the analyte of interest is detected using

appropriate detectors. UV detectors [32], fluorescence detectors, electrochemical detectors,
refractive index (RI) detectors, and mass spectrometry (MS) detectors are examples of
commercial detectors used in LC. The detector used is determined by the sample and the
purpose of the analysis. In the case of multicomponent analysis, the absorption spectra may
have shifted to longer or shorter wavelengths than the parent compound. As a result, the UV

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

74
 [Link]

spectra of the target analyte and impurities must be taken and overlaid, and the spectra must
be normalized due to the different amounts present in the mixture. A wavelength must be
chosen so that an adequate response is obtained for the majority of analytes. [18,19]

3. Creating an analytic approach:

The first step in developing an analytical method for RP-HPLC is to select various
chromatographic parameters such as mobile phase, column, a flow rate of mobile phase, and
pH of the mobile phase. All of these parameters are chosen based on trials, and they are then
compared to the system suitability parameters. Typical system suitability parameters include,
for example, a retention time of more than 5 minutes, a theoretical plate count of more than
2000, a tailing factor of less than 2, a resolution of more than 5, and a percent R.S.D. of the
area of analyte peaks in standard chromatograms of no more than 2.0 percent. In the case of
simultaneous estimation of two components, the detection wavelength is usually an isobestic
point. Following that, the drug's linearity is investigated to determine the concentration range
at which the drug follows the linear pattern. The laboratory mixture is also analyzed to
determine the practicability of the developed method for simultaneous estimation. Following
that, the marketed formulation is analyzed by diluting it up to the concentration range of
linearity.[20,21]

4. Sample preparation:

Sample preparation is an important part of HPLC analysis because it ensures that the solution
is reproducible and homogeneous enough to be injected onto the column. The goal of sample
preparation is to create a sample aliquot that is relatively free of interferences, will not
damage the column, and is compatible with the intended HPLC method, which means that the
sample solvent will dissolve in the mobile phase without affecting sample retention or
resolution. Sample preparation begins with sample collection and continues with sample
injection onto the HPLC column. [22]

5. Method optimization:

Identify the method's "weaknesses" and optimize the method using experimental design.
Understand how the method performs under different conditions, with different instrument
setups, and with different samples. [23]

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

75
 [Link]

6. Validation:

Validation is the examination and provision of objective evidence that the specific
requirements for specific intended use are met. A method of assessing method performance
and demonstrating that it meets a specific requirement. In other words, it understands what
your method is capable of delivering, especially at low concentrations. [24]

Method Validation:

The process of validating an analytical method is the process of establishing, through

laboratory studies, that the method's performance characteristics meet the requirements for
the intended analytical application. Any new or modified method must be validated to ensure
that it is capable of producing reproducible and reliable results when used by different
operators using the same equipment in the same or different laboratories. The type of
validation program required is entirely dependent on the specific method and its proposed
applications. 13 Method validation results can be used to assess the quality, reliability, and
consistency of analytical results; it is an essential component of any good analytical practice.
The use of equipment that is within specification, working properly, and properly calibrated
is critical to the method validation process. Validation or revalidation of analytical methods is
required. [25]

• Before they are put into routine use; • When the conditions for which the method has been
validated change; • When the method is changed,

The following are typical parameters recommended by the FDA, USP, and ICH. [25,26]

1. Specificity

2. Linearity & Range

3. Precision

I. Method precision (Repeatability)

II. Intermediate precision (Reproducibility)

4. Accuracy (Recovery)

5. Solution stability

6. Limit of Detection (LOD)

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

76
 [Link]

7. Limit of Quantification (LOQ)

8. Robustness

9. Range

10. System suitability

1. Specificity:

In strategy approval, selectivity and specificity are sometimes used interchangeably to

represent the same concept. Specificity is the ability to evaluate the analyte unequivocally in
the vicinity of parts that may be required to be displayed. The specificity of a test system is
controlled by contrasting test results obtained from an investigation of tests containing
contaminations, debasement items, or placebo fixings with those obtained from an
investigation of tests without contaminations, debasement items, or placebo fixings. [27,28]

2. Linearity and range:

The ability of an analytical procedure to produce test results that are directly proportional to
the concentration of analyte in the sample (within a given range) is referred to as linearity. A
linear relationship should be evaluated across the analytical procedure's range. The proposed
procedure is used to demonstrate it directly on the drug substance by dilution of a standard
stock solution of the drug product components. Linearity is typically expressed as the
confidence limit around the regression line's slope. 16-18 The ICH guidelines recommend a
minimum of five concentrations for the establishment of linearity. 19 The interval between
the upper and lower levels that have been demonstrated to be determined with precision,
accuracy, and linearity using the method is referred to as the range of an analytical
method.[29]

3. Precision:

It denotes the degree of agreement (degree of scattering) between a set of measurements

obtained from multiple samplings of the same homogeneous sample under the specified
conditions. Precision is a measure of how reproducible the entire analytical method is. [30] It
is made up of two parts: repeatability and intermediate precision. The variation experienced
by a single analyst on a single instrument is referred to as repeatability. It does not distinguish
between variation caused by the instrument or system and variation caused by the sample
preparation process. Repeatability is determined during validation by analyzing multiple

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

77
 [Link]

replicates of an assay composite sample using the analytical method. The value of recovery is
computed. The variation within a laboratory, such as different days, different instruments,
and different analysts, is referred to as intermediate precision. [31] The relative standard
deviation is then used to express the precision.

4. Accuracy:

The closeness of a measured value to the true or accepted value is defined as accuracy. In
practice, accuracy denotes the difference between the mean value discovered and the true
value. It is calculated by applying the method to samples containing known amounts of
analyte. To ensure that there is no interference, these should be compared to standard and
blank solutions. The accuracy is then calculated as a percentage of the analyte recovered by
the assay based on the test results. It is frequently expressed as the recovery of known, added
amounts of analyte by assay.[32]

5. Solution stability:

The stability of standards and samples is established during validation under normal
conditions, normal storage conditions, and sometimes in the instrument to determine if
special storage conditions, such as refrigeration or light protection, are required.[33]

6. Limit of Detection (LOD):

The detection limit of a single explanatory method is the most basic measure of analyte in an
example that can be recognized but not quantitated as an accurate quality.[34]

7. Limit of Quantification (LOQ):

The quantitation limit of an individual expository system is the smallest amount of analyte in
an example that can be quantitatively determined with appropriate accuracy and exactness
[35,36]. The quantitation limit is a quantitative test parameter for low levels of mixtures in
test lattices, and it is particularly useful for determining polluting influences and/or
corruption items.[37]

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

78
 [Link]

8. Robustness:

The robustness of an analytical procedure is a measure of its ability to remain unaffected by

small but deliberate variations in method parameters, and it indicates its dependability under
normal conditions. [38]

9. Range:

The method's range is the range of an analyte's upper and lower levels that have been
determined with acceptable precision, accuracy, and linearity. It is normally expressed in the
same units as the test results and is determined on a linear or nonlinear response curve (i.e
where more than one range is involved, as shown below). [39]

Figure No. 3: Range determination

10. System Suitability:

Liquid chromatographic methods include system suitability tests as a standard procedure.

They are used to ensure that the chromatographic system's detection sensitivity, resolution,
and reproducibility are adequate for the analysis. The tests are based on the idea that the
equipment, electronics, analytical operations, and samples to be analyzed are all part of a
whole that can be evaluated as a whole. To determine the suitability of the used method,
factors such as peak resolution, the number of theoretical plates, peak tailing, and capacity
were measured.[40]

CONCLUSION:

The development of analytical methods for drug identification, purity evaluation, and
quantification has received a lot of attention in the field of pharmaceutical analysis in recent
years. This review provides a general overview of HPLC method development and
validation. A general and very simple approach to developing HPLC methods for compound
separation was discussed. Before developing an HPLC method, it is critical to understand the

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

79
 [Link]

physicochemical properties of the primary compound. The composition of the buffer and
mobile phase (organic and pH) has a significant impact on separation selectivity. Finally, the
gradient slope, temperature, and flow rate, as well as the type and concentration of mobile-
phase modifiers, can be optimized. The optimized method is validated using various
parameters (e.g., specificity, precision, accuracy, detection limit, linearity, and so on)
following ICH guidelines.

ACKNOWLEDGMENTS:

The authors of this review article would like to express their gratitude to the Principal of the
Government College of Pharmacy in Karad for allowing us to present this paper. We are also
grateful to the AICTE for providing a research fellowship to help us complete our research
and turn it into publications.

REFERENCES:

1. Rao BV, Sowjanya GN, Ajitha A, Rao Uma MV. A review on stability-indicating HPLC method
development, World journal of pharmacy and pharmaceutical sciences.2015; 4(8):405-423.
2. Rajan HV. Development and validation of HPLC method - A Review. International Journal of current
research in pharmacy. 2015;1(2):55-68.
3. Kumar V, Bharadwaj R, Gupta G, Kumar S. An Overview on HPLC Method Development, Optimization and
Validation process for drug analysis. The Pharmaceutical and Chemical Journal. 2015; 2(2):30-40.
4. B.V. Rao, G.N. Sowjanya1, A. Ajitha, V.U.M. Rao, Review on stability indicating hplc method development,
World Journal of Pharmacy and Pharmaceutical Sciences, 4(8) (2015) 405-423.
5. Arpino, Patrick.1992: Combined liquid chromatography-mass spectrometry. Part III. Applications of
thermospray”. Mass Spectrometry Reviews, 11: 3. doi:10.1002/mas.1280110103.
6. Arpino, Patrick.1989: Combined liquid chromatography-mass spectrometry. Part I. Coupling by means of a
moving belt interface”. Mass Spectrometry Reviews 8: 35. doi:10.1002/mas.1280080103.
7. Gupta V, Jain AD, Gill NS, Gupta K. Development and validation of HPLC method - a review. International
Research Journal of Pharmaceutical and Applied Sciences. 2012; 2(4):17-25.
8. Sonia K, Nappinnai M. Development and validation of HPLC and UV-visible spectrophotometric method for
the pharmaceutical dosage form and biological fluid –review. European Journal of Biomedical and
Pharmaceutical sciences. 2016; 3(3): 382-391.
9. Sánchez MLF. Chromatographic techniques, European RTN Project, GLADNET, retrieved on 05-09-2013.
10. HPLC – Chemiguide. May 2, 2007. [Link]
11. Mcpolin Oona. An Introduction to HPLC for Pharmaceutical Analysis. Mourne Training Service. 11-12.
12. United States Pharmacopoeia and National Formulary, (24th) Asian Edition, The United States
Pharmacopoeia Convention Inc. U.S.A.2126.
13. Sankar SR, Text book of Pharmaceutical Analysis. 5th Edition 2006. Rx publications, Tirunelveli. 2006;13-
1,2.
14. [Link]
15. Buffers and pH Buffers: available from: [Link].
16. M.S. Charde, A.S. Welankiwar, J. Kumar, Method development by liquid chromatography with validation,
International Journal of Pharmaceutical Chemistry, 04 (02)(2014) 57-61.
17. M.W. Dong, Modern Hplc for practicing scientists, John Wiley & Sons, New Jersey, 2006.
18. C.K. Kaushal, B. Srivastava, A process of method development: A chromatographic approach, J. Chem.
Pharm. Res. 2(2) (2010) 519-545.

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

80
 [Link]

19. [Link], A. Kumar, S.D. Kumar, V.S. Bheemidi, Development and Validation of Analytical Methods for
Pharmaceuticals, J Anal Bioanal Techniques. 2(5) (2011) 1-4.
20. Sethi PD. Introduction – High Performance Liquid Chromatography, 1st edn, CBS Publishers, New Delhi.
2001; 1-28.
21. Julia T, Mena AJ, Aucoin MG, Kamen AA. Development and validation of a HPLC method for the
quantification of baculovirus particles. J Chromatogr B. 2011; 879: 61-68.
22. Santhosh G, Nagasowjanya G, Ajitha A, Uma Maheswara Rao Y. HPLC method development and
validation: an overview. International Journal of Pharmaceutical Research & Analysis. 2014; 4(2): 274-280.
23. Kayode J, Adebayo. Effective HPLC method development. Journal of Health, Medicine and Nursing.2015;
12: 123-133.
24. Gad S. Pharmaceutical manufacturing handbook of regulations and quality. John wiley and sons; 2006.
25 T. Bhagyasree, N. Injeti, A. Azhakesan, U.M.V. Rao, A review on analytical method development and
validation, International Journal of Pharmaceutical Research & Analysis, Vol 4 (8) (2014) 444-448.
26. V. Kumar, R. Bharadwaj, G.G., S. Kumar, An Overview on HPLC Method Development, Optimization and
Validation process for drug analysis, The Pharmaceutical and Chemical Journal, 2(2) (2015) 30-40.
27. Nevado JJB [Link]. Reliable and Sensitive SPE-HPLC-DAD Screening of Endocrine Disruptors Atrazine,
Simazine and their Major Multiresidues in Natural Surface Waters: Analytical Validation and Robustness Study
Perfomance. J Chromatograph Separat Techniq 2014; 5: 215.
28. Nia Y [Link]. Determination of Ti from TiO2 Nanoparticles in Biological Materials by Different ICP-MS
Instruments: Method Validation and Applications. J Nanomed Nanotechnol.2015; 6:269.
29. A. Shrivastava, V.B. Gupta, HPLC: Isocratic or Gradient Elution and Assessment of Linearity in Analytical
Methods, J Adv Scient Res, 3(2) (212) 12-20.
30. Weston A, Brown PR. HPLC and CE Principles and Practice. Academic press California; 1997.
31. Ngwa G. Forced Degradation Studies. Forced Degradation as an Integral part of HPLC Stability Indicating
Method Development. Drug Delivery Technology. 2010; 10(5).
32. Mohamad T, Mohamad MA, Chattopadhyay M. Particle size role, Importance and Strategy of HPLC
Analysis An update. International Archives of BioMedical and Clinical Research. 2016; 2(2): 5-11.
33. V. Kumar, R. Bharadwaj, G.G., S. Kumar, An Overview on HPLC Method Development, Optimization and
Validation process for drug analysis, The Pharmaceutical and Chemical Journal, 2(2) (2015) 30-40.
34. Subramanian Natesan [Link]. Improved Rp- Hplc Method for the Simultaneous Estimation of Tranexamic
Acid and Mefenamic Acid in Tablet Dosage Form. Pharm Anal Acta 2011, 2:115
35. Vishnu P [Link]. Simultaneous Estimation of Atorvastatin, Ezetimibe and Fenofibrate in Pharmaceutical
Formulation by RP-LC-PDA. Pharm Anal Acta 2010, 1:111
36. Lalit V. Sonawane and Sanjaykumar B. Bari Development and Validation of RP-HPLC Method for the
Simultaneous Estimation of Amoxicillin Trihydrate and Bromhexine Hydrochloride from Oily Suspension.
Pharm Anal Acta 2010, 1:107
37. Laxman Sawant [Link]. Quantitative HPLC Analysis of Ascorbic Acid and Gallic Acid in Phyllanthus
Emblica. J Anal Bioanal Techniques 2010, 1:111
38. ICH Q2A. Text on Validation of Analytical Procedures, International Conference on Harmonization.
Geneva; 1995.
39. A Guide to Validation in HPLC. [Link]
40. Validation of Compendial Procedures, United State Pharmacopeia, USP 36 NF, 27 (2) (2010).

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

81
 [Link]

[Link]
Image
Associate professor
Author -1
[Link] of pharmacy karad

[Link]. .[Link]
Image
[Link] pharmaceutical chemistry
Author -2
[Link] of pharmacy karad

[Link]
Image
Assistant professoer
Author -3
[Link] of pharmacy karad

Citation: R. D. Chakole et al. [Link], 2021; Vol. 21 (4): 66-82.

82