HPLC METHOD DEVELOPMENT AND VALIDATION: A REVIEW

Varma et al. World Journal of Pharmaceutical Research

www.wjpr.net │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │

405

HPLC METHOD DEVELOPMENT AND VALIDATION: A REVIEW

M. Mohan Varma, Ashok Thulluru, K. T. Sunil Kumar, G. Sai Kumar and *K. Pavani

Department of Pharmaceutics & Pharmaceutical Quality Assurance, Shri Vishnu College of

Pharmacy (Autonomus), Vishnupur, Bhimavaram-534 202, West Godavari District, Andhra

Pradesh, India.

ABSTRACT

High performance liquid chromatography the most precise techniques

commonly used for the qualitative and quantitative study of drug

substance. Drug research, drug production, and pharmaceutical

manufacturing all depend on the development and evaluation of

analytical methods. It involves evaluating a drug substance's purity and

toxicity. To refine the methods in the study of system production in

HPLC, a variety of chromatographic parameters were evaluated. We

create an effective mobile phase, column, column temperature,

wavelength, and gradient. Force degradation tests aid in the discovery

and validation of stability-indicating experiments, as well as the

identification of drug product and drug substance degradation

pathways. The aim of validating an empirical technique is to show that

it is suitable for intended purpose. In accordance with ICH guidelines,

the parameters accuracy, precision, specificity, limit of detection, limit

of quantitation, linearity, range, ruggedness, and robustness are specified here.

KEYWORDS: HPLC, Degradation, Impurities, Method developments, Validations.

INTRODUCTION

The qualitative and quantitative composition of the substance examined is determined by

analytical chemistry. To interpret the sample material, all aspects are necessary. Analytical

chemistry is classified into two types: quantitative and qualitative. A qualitative analysis

tells us about the nature of the sample by determining the presence or absence of certain

elements.

World Journal of Pharmaceutical Research SJIF Impact Factor 8.084

Volume 10, Issue 11, 405-426. Review Article ISSN 2277– 7105

*Corresponding Author

K. Pavani

Department of Pharmaceutics

& Pharmaceutical Quality

Assurance, Shri Vishnu

College of Pharmacy

(Autonomus), Vishnupur,

Bhimavaram-534 202, West

Godavari District, Andhra

Pradesh, India.

Article Received on

28 June 2021,

Revised on 18 July 2021,

Accepted on 08 August 2021

DOI: 10.20959/wjpr202111-21356



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The conventional and Physicochemical properties are used for the study of the sample in non-

instrumental. The instrumental methods of analysis are focused on measurements to

determine a chemical composition of certain physical properties of the substance using the

apparatus. Analytical procedures created using advanced instruments such as

spectrophotometers, HPLC, GC, and HPTLC therefore have broad variety of applications to

assure raw and finished materials quality and quantity.

Chromatography

Chromatography represents for two words: ―chromo‖- color and ―graphy‖ –writing.[1]

Chromatography is a process used to isolate the mixture components by continuous

distribution between two stages of the component. One stage pass (Mobile phase)

continuously over the other phase (stationary phase).

Type of chromatography

Absorption chromatography

If the phase is solid and mobile, it is considered an Adsorption chromatography. If the

phase is liquid or gaseous.

Examples: Thin layer chromatography, column chromatography, gas-solid chromatography.

Partition chromatography

The partition chromatography is the stationary and mobile phases are liquid.

Example: Paper partition chromatography, Gas-liquid chromatography.

Theory of chromatography

• The plate theory

According to the original martin and Synge model. The plate model suggests that the

chromatographic column has a high number of distinct layers, which are referred to as

"Theoretical Plates." The separate equilibrations of the sample between the stationary phase

and mobile phase occur in the plates. The analyte moves down the column by transfer of

equilibrated mobile phase from one plate to next plate.

• The rate theory

A most realistic description of the processes at work inside a column takes. The time it takes

for the solute to reach equilibrium between the stationary and mobile phases. The pace of

elution therefore influences the band shape of a chromatographic peak. It is also affected by



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the different path available to solute molecule as they travel between particles of stationary

phase.

Phase of chromatography

A) Based on modes of chromatography

• Normal phase chromatography

The mobile phase in normal phase chromatography is non-polar, whereas the stationary phase

is polar. Hence, the station phase retains the polar analyte. The polarity of solute molecules

increases their adsorption capability, due to longer elution time. This is because of lower

affinity between the non-polar compounds and they stationary phase. Polar compound in the

mixture being passing through the column. These techniques can be used to separate, identity

and it is not generally used for pharmaceutical applications.[2]

• Reverse phase chromatography

In the reverse phase column, chemical compounds are commonly used in many fields such as

pharmaceuticals, the petroleum industry, forensics, and clinical science to isolate chemical

compounds into their individual parts for purification. Non-polar hydrophobic packing with

the functional group octyl or octa decyl silane or C18, C8, C4. The stationary phase and the

mobile phase is polar solvent. In this mode, the polar compound gets eluted first and non-polar

for a longer time, compounds are kept. Since most drugs and pharmaceuticals are polar in

nature, they are not retained for longer periods and are therefore eluted more quickly.[3]

Based on principle of chromatography

• Ion exchange chromatography

Stationary stage Contains ion groups like NR3+, SO3 which is communicating with the ionic

groups of the molecules in the study. This is sufficient for the separation of charged

molecules. Shift in pH and the concentration of salt will modulate the retention.

• Ion pair chromatography

The techniques are often referred to reverse phase ion pair chromatography or soap

chromatography. It can be used for the isolation of the ion compounds and this process can

also replace ion exchange in chromatography. Strong acid and basic compounds can be

distinguished by reverse phase mode by from of ion pairs (columbic association species

shaped between two ions with opposite electrical charge) adequate counter ions.



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• Affinity chromatography

This technique is very much used relevant biochemical separation interactions. This is

stationary step comprises unique group of molecules. The sample can be absorbed if any

steric and charged. The necessary conditions have been achieved. This technique can be used

isolate proteins, enzymes and complex antibodies blends.

• Size exclusion chromatography

The molecules are distinguished by their molecular masses. The biggest molecules are first

eluted and the smallest last. This approach is commonly used where a combination of

involves at least 10 % molecular mass difference.[4,6]

Method development

Analytical method development is a process of proving that the chromatography method

development is sufficient for the intended to use in the development and manufacturing of the

drug substance and drug product. The analytical approach is intended to assess the identity,

purity, physical character and the potency of the substance that we use. These are many steps

involved in method development of HPLC is follows:

• The Physicochemical properties of drug molecules.

• Set up HPLC conditions

• Sample preparation

• Method optimization

• Validation of developed method

The physicochemical properties of drug molecules

The Physicochemical characteristics of a therapeutic molecule are critical in method growth.

For method growth, physical properties such as solubility, polarity, pKa and pH of the drug

molecule must be observed. Polarity is the physical property of the substance. It allows the

observed to decide on the solvent and the structure of the mobile phase. The solubility of the

molecules can be clarified by the polarity by the molecules. pH and pKa play an important role

in the development of the HPLC system. The pH value is defined as the negative value of the

logarithm at base 10 of the hydrogen ion concentration.

The selection of a suitable pH for ionizable analytes also leads in symmetrical and sharp

HPLC peaks. To achieve low detection limits, low relative standard deviations between



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injections, and reproducible retention times, sharp, symmetrical peaks are important in

quantitative analytics.[7,8]

pH = - log10[H3O+]

Set up HPLC conditions

A buffer is a partially neutralized acid that resists pH changes. Salts such as sodium citrate or

sodium lactate is typically used to partially neutralize the acid.

Buffer capacity

Buffer capacity is measure of the efficiency of a buffer in resisting change in pH.

• Buffering Capability increases with the molar concentration (molarity) the buffer solution

of salt / acid increases.

• The nearest are buffered pH is to the pKa and the higher pKa is Capacity Buffering.

• Buffering power is expressed as sodium molarity Hydroxide required to increase pH by

1.0

In the reverse phase chromatography (RPC) method development of ion analytes,

consideration of the effect of pH on analyte retention, the type of buffer to be used, and its

concentration, solubility in the organic modifier and its effect on detection are important. In

terms of buffering species, ionic strength and pH, an improper choice of buffer will result in

low or Ir-reproducible retention and tailing in the reverse phase.[9,11]

pH of buffer

Usually, the choice of buffer is dictated by the desired pH. The standard pH range for inverted-

phase silica-based packaging is pH 2 to 8. It is critical that the buffer has a pKa close to the

desired pH because the buffer regulates pH at its pKa best. The rule is to pick a buffer with a

pKa value < 2 units of the target mobile phase pH value.[12]

Buffer concentration

Generally, for small molecules, a buffer concentration of 10- 50mM is sufficient. Generally,

with a buffer, no more than 50 percent organic can be used. This would depend on both the

particular buffer and its concentration. The most common buffer systems for reversed phase

HPLC are phosphoric acid and its sodium or potassium salt.[13]



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Table 1: Buffers and Its pH ranges.

Buffer pka Useful pH

Range

Ammonium Acetate 4.8

9.2

3.8-5.8

8.2-10.2

Ammonium Format 3.8

9.2

2.8-4.8

8.2-10.2

KH2PO4/Phosphoric acid 2.1 1.1-3.1

KH2PO4/K2PO4 7.2 6.2-8.2

Potassium Acetate /Acetic acid 4.8 3.8-5.8

Borate (H3BO3/Na2BO7 10H20) 9.2 8.2-10.2

Ammonium

hydroxide/Ammonia

9.2 8.2-10.2

Trifluoroacetic acid ˂2 1.5-2.5

Potassium formate/Formic acid 3.8 2.8-4.8

Detector selection

Detector is a very critical component of the HPLC method selection of the detector depends

on the chemical quality of the analytes, the possible disturbance, the detection limit is

required, the availability and/or the expense of the detector. UV-Visible detector is a flexible,

dual-wavelength HPLC absorbance detector. This detector provides the high sensitivity

needed for routine UV-based applications for low- level impurity detection and quantitative

analysis. The Detector provides superior optical detection for water analytical HPLC, Preparative

HPLC, or LC/MS system solutions. High chromatographic and spectral sensitivity are provided

by its integrated software and optics innovations. Chromatographic and spectral accuracy,

stability and reproducibility refractive indexes make this detector the perfect solution for

evaluating components with or without limited UV penetration. The Multi-wavelength

Fluorescence Detector measures low target chemical concentrations using fluorescence

detection with great sensitivity and selectivity.[14,15]

Column selection

The column is at the basis of the HPLC technique. Changing the column would have the

greatest impact on the resolution of analytes during the production of the process. A properly

chosen column can produce a successful chromatographic separation and provide precise and

reliable analysis. Sometimes, an incorrectly used column can cause uncertainty, difficulties,

and weak separations that can lead to results that are invalid or difficult to interpret.

Generally, modern reverse-phase HPLC columns are made by packing column housing with

spherical silica gel beads that are coated with a stationary.[16]



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Column temperature

Column temperature control is critical for long-term reproducibility, as temperature can

influence selectivity. A target temperature of 30–40 ° C is usually adequate for good

reproducibility. The use of high temperatures can be beneficial for a variety of reasons.

Firstly, operating at a temperature greater than ambient reduces the viscosity of the mobile

phase and thus the total back pressure of the column. Lower system pressures allow higher

flow speeds and hence faster analysis. Selectivity patterns can also be influenced by the

temperature, as analytes may react appropriately to different temperatures.[17]

Mobile phase

Solvent type

The solvent form (methanol, acetonitrile, and tetrahydrofuran) will influence selection. The

preference between methanol and acetonitrile can depend on the solubility of the analyte as

well as the buffer used. Tetrahydrofuran is the least polar of these three solvents, frequently

responsible for significant shifts in selectivity and is typically compatible with the lower

wavelength detection required for the majority of pharmaceutical compounds.

pH of mobile phase

When that sample is eluted in a mobile phase of 100% organic, there is no distinction since

the sample is eluted in an empty volume. This is due to the fact that the sample is not

retained; but retention is observed when the strength of the mobile phase solvent decreases to

allow the equilibrated competition of the solvent molecules between the bonded and the

mobile phase. When the separation is complicated, that is, several components have to be

separated, then when the solvent intensity is lowered and there is always no resolution

between two closest peaks, another organic solvent is distinct.

It could be appropriate to attempt polarity or even a combination of two organics to effect

separation. In addition, mobile phase optimization can be enhanced in combination with

bonded phase optimization.

Separation techniques

Isocratic separations

Isocratic, constant eluent composition implies the conditions of balance in the column, as

well as the precise velocity of the things flowing because of it. Constant; the analyte-eluent

and analyte-stationary-phase correlations are also constant during the entire run. This



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makes isocratic variations more predictable, while separation power (the number of

compounds that may be resolved) is not very high. The peaks potential is low; the longer the

part stays on the column, the larger is the resulting slopes.

Gradient separation

Gradient separation significantly reduces the separation capacity of the device, primarily due

to a drastic improvement in the apparent efficiency (decrease in the maximum width). The

situation in which the tail of the chromatographic region is still under the control of a greater

eluent composition contributes to a decrease in the width of the peaks. Gradient elution

isused for multi - component complex Samples since it may not be necessary to procure all

components eluted between k (retention factor) 1 and 10 using a single solvent power under

isocratic conditions This leads to a general problem of elution where no one number of

conditions is successful in eluting all components from a column within a sufficient timeline

while also reaching the resolution of each component.[16,18,19]

Based on scale of operation

Analytical HPLC:- Where only the samples are evaluated. Sample recovery is not done

because only very small samples are used.

Preparative HPLC: - Where the individual compound fractions can be collected by means

of a fractional collector. Samples gathered shall be reused.

Based on type of analysis

Qualitative analysis:- The compound is identified, the presence of impurities is detected, the

number of components is determined, etc. The retention time values are performed.

Quantitative analysis:- The quantity of individuals or multiple components in a mixture is

determined. The peak area of standard and sample is compared for this purpose.

Sample preparation for method development

The drug material being studied should be soluble in solution (dilution). The preparation of

solutions in amber flasks should be carried out during the initial production of the process

before it is established that the active component are involved. Under typical experimental

conditions, the is stable at room temperature and does not deteriorate. The sample solution

should be filtered using a 0.22 or 0.45 μm pore filter is commonly preferred for the

elimination of particulate matter. Sample preparation is an essential step in the production of

the system to be examined by the researcher.[12]



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Method optimization

The experimental conditions should be designed to obtain the various partitions and

sensitivity after proper separation. Test stability will be accomplished by a planned / systemic

study of parameters including pH (if ionic), mobile phase components and ratio, gradient, flow

rate, temperature, sample quantity, injection volume and solvent form elements.

Method validation

The methods were validated in conjunction with the International Conference on

Harmonization (ICH) recommendations for the validation of analytical methods. Validation

is necessary for any new or updated medication to ensure that it can produce reproducible,

consistent, and effective outcomes. Validation of the analytical method is the mechanism by

which laboratory experiments have defined the performance requirements of the method. Meet

the criteria of the planned analytical application. The guidelines of the USP, ICH, FDA, etc.

can also provide a basis for validation of pharmaceutical methods.[21,27]

Scope of process validation

Validation is one of the broadest and most complicated areas when it is included in all product

manufacturing levels. In various fields, the scope of validity is as follows:

• Analytical

• Instrument Calibration

• Process Utility services

• Raw materials

• Packaging materials

• Equipment

• Facilities

• Manufacturing operations

• Product Design

• Cleaning

• Operators

Imoprtance stages in validation

Stage 1

This includes the pre-validation qualification stage covering all exercises that define and

improve product studies, pilot batch testing formulation, scale-up analysis, exchange of

product studies, Innovation for groups on a market scale, setting the conditions for stability,



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In-process, completed pharmaceutical formulations, and maintenance of Equipment, master

records, and process limit approval.[28]

Stage 2

This contains the validation step of the procedure. Its purpose is to ensure that any installed limit of a

critical process parameter is not exceeded. is significant and that satisfactory products can be

generated even when the worst- case Situations.[28]

Stage 3

It is also referred to as the validation maintenance stage, requiring continuous review of all

archives relating to the procedure, including validation of the review reports, to ensure that

no changes, failures, errors and modifications to the production process have been made and

that all standard operating procedures (SOP's) involving change control procedures have be

detected. At this phase, the approval team, which includes representatives from all main

departments, guarantees that no improvements or deviations have occurred that would require

re-qualification and revalidation.[28]

Type of process validation

• Prospective validation

• Concurrent validation

• Retrospective validation

• Re-validation

Prospective validation

Prospective validation is described as documented proof that a device does what it aims to do

on the basis of techniques. The goal of the prospective validation is to show or to show that the

procedure functions under a pilot product trail validation master plan or protocol.[29]

The number of analytical procedures is not restricted in terms of their applicability.

A thorough explanation of the methodology

Equipment/facilities list is a summary of the main processing stages to be evaluated to be

used in conjunction with its calibration state (including computation, observing/recording

apparatus).

Finished dose forms for administration.

List of analytical techniques, as suitable.



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Sampling design.

Techniques for recording and assessing outcomes.[31]

Concurrent validation

In-process tracking of essential processing steps and end- product testing of output current

may provide documented evidence to demonstrate that the manufacturing process is in a state

of control. Except that the operating entity will sell the product during the qualification runs,

it is similar to the prospective, to the public as the market price.[29]

Retrospective validation

Historical evidence can be used to validate such processes. The measures involved include a

specific protocol to be prepared and the results of the data review to be published, leading to a

conclusion and a recommend. The basis for retrospective validity is True in-process

requirements for those characteristics shall be compatible with the final product requirements

of the drug product.[29]

Batch size/strength/producer/year/period.

File masters for production and packaging.

Current particulars for active ingredients/finished materials.[31]

Re-validation

It is a validated phase or element that has been reproduced. Changes of the source of the

active raw material producer are among the changes in re validation.[31]

Modifications in crude materials.

Modifications in the source of active crude material producer.

Alteration of packing material.

Modifications inside the plant/facility.[31]

Type of equipment validatio

Installation qualification (IQ)

Operational qualification (OQ)

Performance qualification (PQ)

Installation Qualification (IQ)

IQ ensures that the installation compliant with all crucial processing, packaging system, and

ancillary items. It verifies that the equipment has been established or installed in compliance



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with the manufacturer's recommendation in a Systematic manner and position in the

surrounding appropriate for its intended.[32]

Installation situations like wiring, functionality, utility and so forth.

Calibration, preventative protection, cleansing plans.

Security features.

Supplier documentation, prints, illustrations, and hand operated.

Product details.

Enlist the spare components.[33]

Operational qualification (OQ)

OQ was performed to offer a high level of assurance that the instrumentation will function as

planned.[32]

Dirty material specifics.

Technique for carrying out the process

Material managing necessities.

Management of process transformation

Short-term balance and capability of the techniques.[33]

Performance qualification (PQ)

PQ verifies that the device is repeatable and that a quality item is produced continuously.[32]

True product, procedure parameters, and process set up in OQ.

Guarantee of technique ability as built up in OQ.

Process repeatability, prolonged process stability.[33]

Cleaning validation

Cleaning validation is a form of recorded evidence that has a high level of confirmation that

can clean a system or equipment uniformly specification criteria have also been determined.

The approval for cleaning is a reported procedure that demonstrates the effectiveness and

consistency of the cleaning of pharmaceutical machinery for production Cleaning validation

protocol:

The goal of the validation procedure.

Equipment details.

The interval between the end of production and the start of the cleaning techniques.

The number of cleaning cycles that must be conducted on a continuous basis.

On a regular basis, inspect the apparatus.



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Clearly defined sampling areas.

Where applicable, information on percent recovery.

Analytical techniques including LOD and LOQ.[34-35]

Importance of validation

The cost of quality has reduced.

Rejection decreased.

Yield increases.

There have been less complaints regarding process-related difficulties.

New equipment starts up quickly and realistically.

Increased worker consciousness of the process.[36]

Validation parameters

Typical analytical performance characteristics which can be tested during validation methods

are as follows:

• Accuracy

• Precession

• Repeatability

• Linearity

• Detection Limit

• Quantitation Limit

• Specificity

• Robustness

• System Suitability Parameters

• Resolution

• Capacity factor

• Column Efficiency

• Stability Studies

• Accuracy

Accuracy is the similarity of the assessed value to the actual or authorised price Accuracy means

the difference between the mean value observed. It is calculated by attaching the procedure to

samples to which known concentrations of analyte have been applied. To confirm that there

is no participation, these may be compared to standard and blank solutions. The specificity is



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then determined from the test results as a percentage of the analyte recovered by the test. It

can also be expressed as recovery by an assay of known, added quantities of analyte.[37]

• Precision

The precision of an analytical method is the degree of consistency between the Individual test

results acquired by applying the technique to repeated sampling of a homogeneous population

trail. Precision is an indicator of the reproducibility of the whole analytical process. It is

classified into two parts: Repeatability and intermediate precision.[38]

• Repeatability

The difference seen by a single analyst in a single device is referred to as reproducibility.It

makes no distinction between changes to the equipment or its own technique and sample

preparation. Repeatability is performed during validation by testing several replicates of the

assay. Analytical technique is used to create a composite sample. The value of the recovery

is defined.[39,40]

Linearity

The linearity of an analytical approach is its ability to produce test results that are directly

proportional to the sample analyte concentration. Statistical methods can be used to test

results. Linearity is generally expressed as the confidence level on the slope of the regression

line.[41,42]

Detection limit

The detection limit (DL) or the detection limit (LOD) of an individual process is the lowest

concentration of analyte in a sample that can be identified but not generally quantified as an

absolute utility. In analytical procedures that exhibit baseline noise, the LOD can be

dependent on the signal-to- noise (S / N) ratio (3:1) which is typically expressed as the

quantity of the analyte in test.

The signal-to-noise ratio is determined by:

s = H/h

Where,



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H = height of the peak corresponding to then component.

h = absolute value of the largest noise fluctuation from the baseline of the chromatogram of a

blank solution.[43,44]

Limit of quantitation

The Limit of Quantitation (LOQ) or Quantitation Limit of an independent analytic technique is

the lowest volume of analyte in a sample that can be quantitatively measured with sufficient

precision and accuracy. For analytical processes such as HPLC that show baseline noise, the

LOQ is normally determined based on the calculation of the S / N ratio (10:1) and is typically

validated by the injection criteria that have this S / N ratio and therefore provide a suitable

percent corresponding standard deviation It can be deter-mined by following formula,

LOQ = 10 × δ/S

Where,

δ = standard deviation of response.

S = Mean of slopes of the calibration curves45.

Specificity

Specificity is the capability to measure the analyte distinctively in the presence of materials

that might be expected to be present, such as impurities, degradation products and excipients.

Specificity analyses only the desired element without interfering by other organisms that may

be present; Segregation is not necessarily required. etc. The peak purity value must be more

than 0.999.[45,46]

Robustness

It is described as the measurement of an analytical method's ability to remain unchanged by

minor but intentional changes in the parameters of the method (e.g. pH, structure of the

mobile phase, temperature and instrumental settings) and gives an indicator of its efficiency

during regular use. Robustness determination is a structured procedure of changing a

parameter and assessing the influence on the method by monitoring the suitability of the

device and/or analysis of the parameter about the samples.[47,48]

System suitability parameters

The examination of the elements of a system is known as system suitability determination of

an analytical system to show that a system performance meets the standard requirement a

procedure. To provide a quantitative system suitability test report, these parameters can be



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measured experimentally: The number of theoretical plates (efficiency), the capacity factor,

the separation (relative retention), the resolution, the tailing factor, and the relative standard

deviation are all factors to consider (precision).They are used to confirm that the resolution

and detection sensitive and reproducibility of the chromatographic method is necessary to

carry out the analysis.[50]

Resolution

Resolution is the capacity of the column in 2 individual peaks or chromatographic zones to

distinguish 2 drugs and is increased by increasing column length, decreasing particle size and

increasing temperature, modifying the elution or stationary phases. It can be defined in terms

of the apex separation ratio of two peaks by the average tangential width of the peaks. It is

determined by using the following resolution method.[51]

Figure 1: Determination of resolution between two peaks.

Capacity factor

The capacity factor, k ', is described as the ratio between the number of solvent molecules in

the stationary phases and the number of solvent molecules in the mobile phases. The capacity

factor is a measure of how the sample molecule is maintained during an isocratic separation

by a column or TLC layer. An ideal the value of k ' is between the capacity factor is determine

by using the formula,

Column efficiency

It is a calculation of the distribution of a peak band. The narrower spread of the band, the

larger the number of theoretical surfaces, which suggests good output of the column and



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system. For a decent system, columns ranging fro 5,000 to 100000 plates / meter with N are

suitable. Efficiency is determined by using the formula

Figure 2: Number of theoretical plates.

Peak asymmetry factor (As) and tailing factor

Under optimal conditions, the chromatographic peak was considered to have a Gaussian

shape. However, there is often a divergence from standard normal distribution in functional

situations, suggesting non-uniform migration and non-uniform distribution systems. This has

therefore been suggested by regulatory organizations like USP and EP as one of the criteria

of device suitability. Asymmetry factor is calculated by,

Asymmetry factor= B/A

B= Peak half width,

A= Front half width

Good columns produce peaks with as value of 0.95 to 1 % (Exactly symmetrical peaks have an

as value of 1.0%)



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Figure 3: Determination of Tailing and Asymmetric factor.

Peak purity

Analysis of the peak purity (or peak homogeneity) of the main peak to establish the presence

of impurities under the peak. It is an integral part of the validation of the method.[52,53]

Stability studies

The stability of standards and samples is defined during validation under normal conditions,

storage circumstances and often in the equipment to determine if particular storage

conditions, such as refrigerated or light safety, are necessary.[53]

CONCLUSION

This analysis discusses the general HPLC process construction and streamlined method

validation technique. The standard procedure for improving pharmaceutical compound

separation methods has been described. Before the production of the HPLC process,

knowledge of pKa, pH and solubility of the primary compound is extremely important. PH

information can help to distinguish the optimization quality of other impurities products in

the mixture (i.e. synthetic by products, metabolites, products for degradation, etc.). Buffer

selection and composition (organic and pH) of the mobile process play a major role in the

selectness of the separation. Changes in temperature, gradient level, flow rate as well as type

and concentration of mobile phase changes can be carried out for final optimization process.

The optimization approach is tested in compliance with the guidance of the ICH with different

parameters.



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HPLC METHOD DEVELOPMENT AND VALIDATION: A REVIEW