INTRODUCTION:

Pharmaceutical analysis is essential in the process of quality control as well as quality assurance of pharmaceutical chemicals and their formulations. It provides information on the identity, purity, content, and stability of starting materials, excipients, and active pharmaceutical ingredients. It also ensures the safety, efficacy, and quality of pharmaceutical products used for therapeutic purposes. Analytical methods are intended to establish the identity, purity, physical characteristics, and potency of the drugs that we use. Methods are developed to support drug testing against specifications during manufacturing and quality release operations, as well as during long-term stability studies. It may also support safety and characterization studies or evaluations of drug performance.^1-2

Figure A: Block Diagram of Typical HPLC Setup

HPLC is also known as high-pressure liquid chromatography. It is essential to form column chromatography in which the stationary phase is consist of small particles 2.99-49.9μm pickings contained in a column with a small pore 1.99-4.99mm one end of which is attached to a source of a pressurized liquid eluent. The three forms of high-performance liquid chromatography most often used are ion-exchange partition and adsorption. HPLC has benefits in lots of fields along with progressed resolution, quicker separation, progressed accuracy, precision, and sensitivity. For this reason, it has grown to be the most widely usedanalytical approach for the quantitative analysis of prescribed drugs, biomolecules, polymers, and different organic compounds.

HPLC is classed into:

1. Reverse phase chromatography

2. Normal phase chromatography

3. Ion exchange chromatography

4. gel permeation or size exclusion

5. liquid-solid chromatography

6. Liquid-liquid chromatography

In the literature review, various articles are available for method development and validation via HPLC. The present work is therefore focused on achieving the optimum chromatographic parameters covering all the points of method development and validation according to ICH guidelines.

Primary Chromatographic Descriptors:^2,3

The following principal parameters are usually used to file characteristics of the chromatographic column, system, and required separation:

1. Capacity factor or Retention factor (k)

2. Efficiency (Plate number, N)

3. Resolution (R)

4. Separation factor (Selectivity, α)

5. Tailing factor (T).

1. Capacity Factor or Retention Factor:

V_R– V₀ V’_R t_R– t₀

K = –––––––––– = –––––– = –––––––

V₀V₀ t₀

Retention factor (k) is the unit less measure of the retention of a particular compound/analyte in a particular chromatographic system at a given condition defined as

Where V_R is the analyte retention volume, V₀ is the volume of the liquid phase present in the chromatographic system, T_R is the analyte retention time, and T₀ is sometimes defined as the retention time of a non-retained analyte.

The retention factor is convenient since it is independent of the column dimensions and mobile phase flow rate. All other chromatographic conditions significantly affect analyte retention.

Fig. B: Retention Factor in Chromatography

2. Efficiency (Plate Number, N):

Efficiency is defined as “the measure of the degree of peak dispersion in a particular column, as such, it is very essential to the characteristic of the column”. Efficiency is expressed as the number of theoretical plates (N) and is calculated as,

N=16(tR /w)2

Where tR is the analyte retention time, and w is the width of the peak at the baseline.

Fig. C: Scientific Diagram of Efficiency Measurements

3. Resolution (R)

Resolution is defined as “a measure of the quality of separation of adjacent peaks/bands in a chromatogram; overlapping bands have small R values”. Two adjacent peaks’ width and retention time are used to calculate resolution.

R= 2(t2 – t1)/w1+w2

Where t1 and t2 are the retention time of first and second adjacent peaks respectively, and

w1 and w2 are widths of the baseline of the peaks. If reliability is poor, R is < 0.9999.

Fig. D: Determination of Resolution Between Two Peaks

4. Separation Factor (Selectivity):

Selectivity (α) is “the ability of the chromatographic system to discriminate between two different analytes present in the sample”. It is defined as” the ratio of corresponding capacity factors”.

α = k2/k1 = tR2 - t0 / tR1 - t0

Fig. E: Typical Chromatogram for Selectivity of HPLC

5. Tailing Factor (T):

The tailing factor (T) is “the measure of peak symmetry”. It resembles perfectly symmetrical peaks and its value increases as tailing becomes more profound. In some conditions values less than unity may be observed. As peak asymmetry increases, precision becomes less reliable.

It is expressed as,

T = w0.05 /2f0.05

Where, w0.05 is the width of the peak at 5 % height, f0.05= half of the peak width at 5% peak height. Ideally, the T value should be ≤ 1.999.

Fig. F: Schematic Diagram of Tailing Factor Chromatography

The Objective of the Research Study:

In the present-day situation, the introduction of a variety of drugs and numerous pharmaceutical formulations is growing at an excessive rate in the marketplace. It's far necessary to determine the one’s drugs and pharmaceutical dosage forms using the sensitive, particular, and correct analytical techniques to keep the high quality of the drug formulations.

1 To carry out an initial evaluation of the GTH drug.

2 To develop the RP-HPLC method for the estimation of GTH in bulk and formulation.

3 To validate the evolved RP-HPLC method for the estimation of GTH in bulk and formulation according to the ICH Q2 (R1) guidelines.

Strategy for Method Development in HPLC:

Three major steps are involved in strategy for method development:

· Consider analyte chemistry.

· Optimized selectivity.

· Use of Scouting gradient.

Consider Analyte Chemistry:^4-5

The interaction of an analyte with the HPLC stationary and mobile phase results in retention. Without this interaction, there may be no separation. Therefore, going return to a few chemistry fundamentals regarding your analytes will govern some of the selections you are making approximately the whole HPLC technique, including column choice and mobile phase design. Unique importance is the polarity of your analytes. Nonpolar analytes are ideal for retention and separation beneath Reversed-phase HPLC situations on hydrophobic stationary phases (consisting of C18, C8). Polar or ionizable analytes would require a greater polar stationary phase. For example, cyano or amino, or may maybe extraordinary mode of chromatography together with hydrophilic interaction liquid chromatography, normal phase, or mixed-mode to gain separation. For ionizable analytes, which can be tough to maintain below reversed-phase HPLC conditions when in their ionized form, it's far vital to properly alter the pH of the mobile phase to render these analytes new neutral to gain retention and avoid peak shape troubles along with tailing with bases. Understanding analyte pKa can be tremendously beneficial in designing a mobile phase. The mobile phase should be two pH units under or above this value relying on if the analyte is an acid or a base, respectively to render the analyte impartial. It could be useful to screen a variety of pH values and examine the impact on retention and resolution.

Optimize Selectivity:⁵

Of all of the chromatographic parameters that govern decision, selectivity has the greatest impact on HPLC. Each parameter (Specificity, Accuracy, Efficiency) can be altered through specific method parameters, consisting of Mobile phase composition, Temperature, or Column dimensions. However, the column stationary phase has the largest impact on selectivity. therefore, the second tip for HPLC method development is to screen columns with one-of-a-kind phase chemistries. This approach has numerous advantages, including fast finding the most excellent stationary phase in your separation, as well as the reality that orthogonal stationary phase chemistries may additionally reveal underlying impurity peaks that won't have been picked up if best a similar column chemistry had been attempted. Other approach parameters that may be used to affect selectivity in addition, Mobile phase pH, Solvent components, and Temperature.

Use A Scouting Gradient:⁶

A scouting gradient is a linear gradient from 5–10% C to 100% Cover a set time (15 min is usual). The elution strength is held at 100% C for a couple of minutes, to make sure that all sample additives were eluted. For reversed-phase gradient elution, water or buffer if there are ionizable analytes and acetonitrile are usually the eluents of desire. the use of risky buffers including ammonium formate will bring about a method this is well-matched with mass spectrometry for use in LC–mass spectrometry.

The chromatogram acquired can reveal plenty about the mobile phase composition, column chemistry, and mode of an evaluation required for the separation. If compounds are eluted over extra than 20% of the gradient time after the end of the gradient, then a more potent C solvent or a much less retentive column is needed for the evaluation.

Steps Concerned in Method Development:^7,8

· Understanding the Physicochemical Properties of the drug molecules.

· Choice of chromatographic conditions.

· Developing the approach of analysis.

· Sample preparation

· Method optimization

· Method validation

Fig. G: Flowchart Showing Selection Process for Phase

Method Development:^8,9

Optimized Chromatographic Conditions:

Column, Mobile phase, Wavelength, Flow rate, Temperature, Injection volume, Diluent, run timeare all optimized chromatographic conditions were developed.

Chemicals and Reagents: All chemicals and reagents used are HPLC grade. All solvents and solutions had been filtered via membrane filters 0.45µm pore size and degassed before use. Another essential parameter for HPLC method development is the solubility of the compounds in which it is dissolved. The solvent should be chosen based on the solubility of drug substances, their impurities, and degradation products. The solvent should be compatible with the mobile phase to get a better peak shape of the analyte. Consider the purity of solvents and only use HPLC grade solvents. In normal phase HPLC systems, non-polar solvents such as hexane, diethyl ether, dichloromethane, isopropyl alcohol, iso-octane, etc are used whereas reversed-phase HPLC requires polar solvents such as water, acetonitrile ethanol or methanol. The choice of the mobile phase is governed by the physical properties of the solvent. Factors that are considered to be essential for the selection of a particular solvent are polarity, miscibility with other solvents, chemical inertness, and toxicity. The polarity index indicates the ability of a solvent to elute a compound from the column.

Instruments: Analytical balance, HPLC, Filter Membrane, Sonicator, Vertex machine all are required in method development and validation.

Preliminary analysis of drug:

· Description: The API of the drug became observed for its color and texture.

· Solubility: The API of the drug was taken in test tubes and observed for solubility in water, acetonitrile, methanol, acetone, ethanol, buffer, DMSO, etc.

· Melting Point: The API of the drug was taken in a capillary tube and kept in a melting point apparatus and analysis became noted.

Preparation of Mobile Phase:^10,11

The mobile phase is prepared in a suitable diluent and had been filtered through a 0.45µm membrane filter and solutions had been sonicated for 15 min to degas thenpreparation of standard stock solutions and preparation Drug Product Stock Solution using diluent. The mobile phase selection is one of the critical parameters as it encourages the solute and the stationary phase interactions. Appropriate care must be taken while selecting the mobile phase the use of strong acids, strong bases, and halide solutions should be avoided. Buffers are usually employed in the mobile phase to obtain consistent chromatographic results. Buffers are employed to control the retention of ionic analytes. When the analyte is in ionic form, it usually attains polar and spends a shorter time on the stationary phase, and elutes quickly. To control the selectivity of the ionic analytes, the buffer pH plays a significant role. In general, when buffer pH increases, the acidic analytes get ionized and become more polar, and conversely, when buffer pH decreases, the basic analytes get ionized. The pH of the mobile phase selected should be at least 1.0 pH units from the analyte pKa value. This confirms that the analytes are either 100% ionized or 100% non-ionized and it helps in controlling peak shape and the run to run reproducibility. It always usesa buffer in the aqueous portion of the mobile phase and it increases the ruggedness of the method.

Selection of Analytical Wavelength:¹²

To analyze an appropriate wavelength for determination of drug solution in the mobile phase were scanned in UV- Visible Spectrophotometer within the range of 200- 400nm. The wavelength at which the molecule shows maximum absorption is the analytical wavelength which is called the lambda max of a particular drug.

Analytical Method Validation:^13-14

Method validation is the process of establishing that the performance characteristics of the analytical method are suitable for the intended application. Chromatographic methods need to be validated before first routine use. To obtain the most accurate results, all of the variables of the method should be considered, including sampling procedure, sample preparation, chromatographic separation, detection, and data evaluation, using the same matrix as that of the intended sample. The validity of an analytical method can only be verified by laboratory studies. All validation experiments used to make claims or conclusions about the validity of the method should be documented in the report.

Fig. H: Flowchart diagram displaying Steps for method development

Specificity:^15,16

Specificity is described as the capacity to degree accurately and especially the quantity of analyte of interest in the presence of other additives that are expected to be present within the sample matrix. Specificity is a measure of the degree of interference from such matters. it can consist of different active elements, excipients, impurities, and degradation products, making sure a peak reaction. Specificity is measured through the resolution, plate count, and tailing factor inside the analysis and documented for this reason.

For identification purposes, specificity is demonstrated by the ability to discriminate between compounds of closely related structures, or by comparison to known reference materials. For assay and impurity tests, specificity is illustrated by the resolution of the two closest eluting compounds in the sample. The compounds are usually major components or active ingredients and an impurity. If the impurities are present in the sample, it must be demonstrated and proved that the assay is unaffected by the presence of spiked materials which may include impurities or excipients. If impurities are absent, the test results are compared to a second well-characterized and developed procedure. For the assay, the two results are compared. For impurity tests, the impurity profiles are compared.

Specificity is measured and documented in a separation by the resolution, plate count efficiency, and tailing factor. Specificity can also be evaluated with modern photodiode array detectors that compare spectra collected across a peak mathematically as an indication of peak homogeneity ICH also uses the term specificity, and divide it into two separate categories: identification and assay tests.

The selectivity of an analytical method is its ability to measure accurately and specifically the analyte of interest in the presence of components that may be expected to be present in the sample matrix. If an analytical procedure can separate and resolve the various components of a mixture and detect the analyte qualitatively the method is called selective. It has been observed that there were no peaks of diluents and placebo at the main peaks. Hence, the chromatographic system used for the estimation ofdrugs was very selective and specific. Specificity studies indicated that the excipients did not interfere with the analysis.

The chromatogram showed a neat baseline, without any interference of excipients and high resolution of the separated compounds was observed. This indicates that the proposed method was specific.

In the case of the assay, demonstration of specificity requires that the procedure is unaffected by the presence of impurities or excipients. In practice, this can be done by spiking the drug substances or product with appropriate levels of impurities or excipients and demonstrating that the assay is unaffected by the presence of these extraneous materials. If the degradation product impurity standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure.

The chromatogram of blank, standard, and test samples was compared to justify the specificity of the target analyte. It has been observed that there were no peaks of diluents and placebo at the main peaks. Specificity studies indicated that the excipients did not interfere with the analysis. There was no interference of excipients in the chromatogram. This indicates that the proposed method is specific.

Linearity and Range:¹⁷

Linearity is the ability of the method to elicit test results that are directly proportional to analyte concentration within a given range. Linearity is generally reported as the variance of the slope of the regression line. The range is the interval between the upper and lower levels of the analyte that has been demonstrated to be determined with precision, accuracy, and linearity using the method as written. The range is normally expressed in the same units as the test results obtained by the method. The ICH guidelines specify a minimum of five concentration levels and certain minimum specified ranges.

Linearity is decided using a series of concentrations specifically 5 to 6 injections of five or more standards whose concentrations span 80-120 percent of the expected concentration range. The response is without delay proportional to the concentration of analyte present inside the sample or proportional to the properly-defined mathematical calculation. A linear regression equation became applied to the consequences that have an intercept now not substantially different from zero. If a massive non-zero intercept is obtained, it should be established that no effect on the accuracy is seen inside the method.

For the assay, the minimum specified range is from 80-120 % of the target concentration. For an impurity test, the minimum range is from the reporting level of each impurity to 120 % of the specification.

The peak response is directly proportional to the concentration of the drug and it was observed to be linear. The correlation coefficient was observed to be 0.999 is well within the accepted standards.

Accuracy:^18,19

Accuracy is the measure of exactness of an analytical method or the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. It is measured as the percent of analyte recovered by assay, by spiking samples in a blind study. For the assay of the drug substance, accuracy measurements are obtained by comparison of the results with the analysis of standard reference material or by comparison to a second, well-characterized method. For the assay of the drug product, accuracy is evaluated by analyzing synthetic mixtures spiked with known quantities of components.

To document accuracy, the ICH guideline on methodology recommends collecting data from a minimum of nine determinations over a minimum of three concentration levels covering the specified range (for example, three concentrations, three replicates each). The data should be reported as the percent recovery of the known, added amount, or as the difference between the mean and true value with confidence intervals. Percent recoveries of the results suggest that the recoveries are well in the acceptance range, consequently, the method was discovered to be correct.

Precision:¹⁹

Precision is the measure of the degree of repeatability of an analytical method under normal operation and is normally expressed as the percent relative standard deviation for a statistically significant number of samples. According to the ICH, precision should be performed at three different levels: repeatability, intermediate precision, and reproducibility. Repeatability is the result of the method operating over a short time interval under the same conditions of inter-assay precision. It should be determined from a minimum of nine determinations covering the specified range of the procedure, for example, three levels, three repetitions each, or from a minimum of six determinations at 100 % of the test or target concentration. Intermediate precision is the results from within lab variation due to random events such as different days, analysts, equipment, etc. In determining intermediate precision, experimental design should be employed so that the effects of the individual variables can be monitored.

Limit of Detection:²⁰

The limit of detection is defined as the lowest concentration of the analyte in the sample that can be detected, though not necessarily quantitated. Limit tests specify whether an analyte is above or below a certain value. LOD may be calculated based on the standard deviation of the response and the slope(S) of the calibration curve at levels approaching the LOD according to the formula: LOD = 3.3(S.D/A). The standard deviation of the response is determined on basis of the standard deviation of the blank, that is on the residual standard deviation of the regression line. Or it can also be determined by the standard deviation of the y-intercept of the regression lines. It is mandatory to document and support the method used to determine LOD. An appropriate number of samples should be analyzed to validate the developed method.

Limit of Quantitation:²¹

The limit of quantitation (LOQ) is defined as the detection of the lowest amount of analyte present in the sample. It is the parameter that gives the actual concentration of an analyte in a sample which can be determined with acceptable precision and accuracy under the stated operating conditions of the method during analysis. The calculation of LOQ is based on the standard deviation (S.D) of the response and the slope (A) of the calibration curve. The formula to calculate is; LOQ = 10(S.D/A). The standard deviation of the response is determined based on the standard deviation of the blank (S.D), the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines of the plot. Similar to LOD, the method used to determine LOQ should also be documented and supported. An appropriate number of samples should be analyzed at the limit to validate the method developed.

Robustness:^22,23

Robustness is defined as the capacity of a method to remain unaffected by small but deliberate variations in method parameters. The robustness of a method is evaluated via various method parameters which include the percentage of organic solvent, pH, ionic strength, or temperature, and determining the effect on the results of the method. The robustness evaluation should be considered during the development phase and depends on the type of study under study. It should show the reliability of analysis concerning deliberate variations in method parameters. A robustness test was carried out by small variation in the chromatographic conditions at a concentration equal to the standard concentration anda % change in the results was calculated. Here robustness was performed by a change in mobile phase ratio, mobile phase flow rate, and wavelength of the detector.

To assess the robustness of the RP-HPLC method, a few parameters had been deliberately varied. The parameters covered a variety of flow rates and slight variations of the wavelength changes. Standard concentration was analyzed beneath those experimental situations. It was found that there had been no marked changes in chromatograms, which established that the evolved technique become robust.

System Suitability:^24,25

System suitability tests are an integral part of chromatographic methods. These tests are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples constitute an integral system that can be evaluated as a whole. The purpose of the system suitability test is to ensure that the complete testing system (including instruments, reagents, columns, analysts) is suitable for the intended application.

System suitability is the checking of a system to ensure system performance before or during the analysis of unknowns. Parameters such as plate count, tailing factors, resolution, and reproducibility %RSD retention time and area for six repetitions are determined and compared against the specifications set for the method. These parameters are measured during the analysis of a system suitability "sample" that is a mixture of main components and expected by-products.

Similar to the analytical method development, the system suitability test strategy should be revised as the analysts develop more experience with the assay. In general, consistency of system performance and chromatographic suitability.

Chromatograms were studied for different parameters such as Asymmetry; Retention time, and Theoretical plates to see whether they comply with the recommended limit or not.

Table 2: System Suitability Test Parameters

Parameter	System Suitability
Theoretical Plates (N)	To be greater than 2000
Tailing Factor (T)	Generally, should be less than 2
Resolution (R)	Usually, more than 2
Repeatability	RSD ≤ 1% for n ≥ 5 is acceptable.
Capacity Factor (k)	Should be greater than 2

Ruggedness:²⁶

Ruggedness, according to the USP, is the degree of reproducibility of theresults obtained under a variety of conditions, expressed as % RSD. Theseconditions include different laboratories, analysts, instruments, reagents, days, etc. In the guideline on definitions and terminology, the ICH did not addressruggedness specifically. This apparent omission is a matter of semantics, however, as ICH chose instead to cover the topic of ruggedness with precision, asdiscussed previously.

CONCLUSION:

The reasons for the development of HPLC methods for drug analysis are: 1) When there is no official analytical method for analysis of drug or drug combination available in the pharmacopeias. 2) When there is no analytical method for the existing drug in the literature due to patent regulations. 3) When there are no analytical methods for the formulation of the drug due to the interference caused by the formulation excipients. 4) Analytical methods for the quantitation of the analyte in biological fluids are found to be unavailable. 5) The existing analytical procedures may need costly reagents and solvents. It may also involve complicated extraction and separation procedures. When developing an HPLC method, the first step is always to consult the chromatographic literature to find out if anyone else has done the analysis, and how they did it. This will at least give an idea of the conditions that are needed and may save one from having to do a great deal of experimental work. When there are no authoritative methods are available, new methods are being developed for the analysis of novel products. To analyze the existing either pharmacopoeial or non-pharmacopoeial products novel methods are developed to reduce the cost besides time for better precision, accuracy, and sensitivity. These methods are optimized and validated through trial runs. Alternate methods are proposed and put into practice to replace the existing procedure in the comparative laboratory data with all available merits and demerits. Method development encompasses many stages and can take months to complete, depending on the complexity and goals of the method.

Table 3: Validation of Analytical Procedures

Type of analytical procedure	Identifi cation	Testing for impurities	ASSAY Dissolution (measurement only) content/ potency
Characteristics	Identifi cation	quantitative limit
Accuracy	-	+ -	+
Precision Repeatability Inter day Precision	- -	+ - + -	+ +
Specificity	+	+ +	+
Detection Limit	-	- +	-
Quantitation Limit	-	+ -	-
Linearity	-	+ -	+
Range	-	+ -	+

High-performance liquid chromatography (HPLC) has proven to be one of the major analytical techniques used in the qualitative and quantitative analysis of drugs worldwide. The majority of drugs prescribed in official pharmacopeias are being analyzed HPLC. Method development and validation are the essential part of the drug development program in pharmaceutical industries and are associated with various steps such as pre-formulation, formulation, production, quality control, quality assurance, and marketing of pharmaceutical products. Analytical method development is a complicated process that takes time from a few hours to months. This review paper covers the basic practical aspects of HPLC and gives guidance on how to develop HPLC analytical methods. This review provides a brief introduction, needs, and various steps involved in HPLC analytical method development.

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Received on 21.06.2022 Modified on 05.11.2022

Asian J. Pharm. Ana. 2023; 13(2):143-151.

DOI: 10.52711/2231-5675.2023.00024

Detector	Types of Molecules	General Use, Comments
RI (Refractive Index Detector)	Carbohydrates, Polymers	Molecules that do not have a UV chromophore.
UV/Vis, PDA	Organic molecules, Biomolecules, except carbohydrates.	PDAs are typically used for any molecule that absorbs light between 190 and 800nm when the molecule in the mixture absorbs at different wavelengths or when λ maxis unknown. PDAs can be used for determining peaks.
Fluorescence	Aromatic compounds which Exhibit fluorescence.	Generally used for applications that requires Extremely high sensitivity.
ELSD (Evaporative Light Scattering Detector)	Carbohydrates, Polymers	Molecules that do not have a UV Chromophore can be used with gradients, generally more sensitive than an RI detector.
MS(Mass Spectrometer)	Organicmolecules, Biomolecules	Can be used to detect and determine the mass of any molecule that can be ionized and is within the Mass range of the specific MS.
MS/MS	Organic molecules, Bio molecules	As above but allows more detailed structural studies to be performed.
ECD (Electrochemical Detector)	Carbohydrates are many other organic molecules whose redox potential is different from the mobile.	Can provide extra sensitivity and sensitivity for molecules that are not readily detected by devices.
Conductivity	Cations and Anions	Used for ion chromatography.