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Pharmaceutical Analysis Application Of Analytical Procedures Biology Essay

Pharmaceutical Analysis may be defined as the application of analytical procedures used to determine the purity, safety and quality of drugs and chemicals. The term “Pharmaceutical analysis” is otherwise called quantitative pharmaceutical chemistry. Pharmaceutical analysis includes both qualitative and quantitative analysis of drugs and pharmaceutical substances starts from bulk drugs to the finished dosage forms. In the modern practice of medicine, the analytical methods are used in the analysis of chemical constituents found in human body whose altered concentrations during disease states serve as diagnostic aids and also used to analyze the medical agents and their metabolites found in biological system.

In modern practice of pharmacy it is important that pharmacists have more than an appreciative analytical methodology. They must have a working knowledge of what they are to influence, which drugs may have on clinical laboratory methods, and monitor drug levels during therapy. When the quality of a drug product is challenged, the pharmacist is responsible for initiating steps to determine if indeed defective. This may be accomplished by calling and advising the drug manufacturer of the problem involving the product, analyzing the preparation in the laboratory, borrowing needed equipment from a clinical laboratory if necessary, sending a portion of the sample to solve problems relating to drug quality. Pharmacists who have reason to be live that a drug product is not of proper quality and appears to be defective, because of improper labeling, discoloration, the presence of cloudiness, crystals or precipitates in the products known to be solution, or for any other reason.

The term “quality” as applied to a drug product has been defined as the sum of all factors, which contribute directly or indirectly to the safety, effectiveness and

reliability of the product. These properties are built into drug products through research and during process by procedures collectively referred to as “quality control”.

Quality control guarantees with in reasonable limits that a drug products

Is free of impurities.

Is physically and chemically stable

Contains the amount of active ingredients as stated on the label and

Provides optimal release of active ingredients when the product is administered.


Modern pharmaceutical formulations are complex mixtures including, in addition to one or more medicinally active ingredients, a number of inert materials such as diluents, disintegrants, colors and flavors. To ensure quality and stability of the final product, the pharmaceutical analyst must be able to separate these mixtures into individual components prior to quantitative analysis. Moreover, comparison of the relative efficiency of different dosage forms of the same drug entity requires the analysis of the active ingredient in complex biological matrices such as blood, urine and tissue. Among the most powerful techniques available to the analyst for the resolution of these complex mixtures are a group of highly efficient methods collectively called Chromatography.

The components to be separated are distributed between two phases:

A stationary phase, an immobile, immiscible

A mobile phase, which percolates through the stationary bed.

Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through stationary phase. The phases are chosen such that components of the sample have differing solubility in each phase.

A mixture of various components enters a chromatographic system, and the different components are flushed through the system at different rates. These differential rates of migration as the mixture moves over adsorptive materials provide separation. Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column.

Based on stationary phase is solid are classified differently from above because of unique nature of their separation processes

Ion Exchange Chromatography

In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

Molecular Exclusion Chromatography (Gel permeation or Gel filtration)

The liquid or gaseous phase passes through a porous gel, which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

Affinity Chromatography

This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein.

Chromatographic Techniques

H.P.L.C. (High Performance Liquid Chromatography)

G.C. (Gas Chromatography)


High performance liquid chromatography is the fastest growing analytical technique for the analysis of drugs. Its simplicity, high specificity and wide range of sensitivity make its ideal for the analysis of many drugs in both dosage forms and biological fluids. This technique is based on the same methods of separation as that of classical column chromatography i.e. adsorption, partition, ion exchange and gel permeation, but it differs from the column chromatography in the fact that the mobile phase is passed through the packed column under high pressure. In classical open column chromatography, the mobile phase flows slowly through the column by mans of gravity with the diameter of particles (of solid support) in the range of 150 - 200µm. But in HPLC, the separation is about 100 times faster than the conventional liquid chromatography due to packing of particles in the range of 3-10µm. Thus HPLC is having advantages of improved resolution, faster separation, improved accuracy, precision and sensitivity.

This prime analytical method was developed since the compounds, which are non-volatile or thermally unstable, cannot be analyzed by GLC. They include the inorganic salts (which cannot be converted into gases at the temperature the GC column in operating), natural products, (like carbohydrates, steroids, alkaloids, peptides, amino acids, antibiotics, nucleotides. etc) synthetic and naturally occurring compounds arising from research in the pharmaceutical, agriculture and food industries.

Non-volatile compounds can analyze by GLC after converting them into volatile compounds by dramatization, but this is a time consuming process.

Modern LC uses very small particles for packing. The small particles size results in more rapid approach to the distribution equilibrium and consequently smaller plate height, so that a given length of column includes large number of plates, which makes the column efficient and the peak narrow. But the close packing of these small particles reduces the flow rate of the mobile phase through the packed bed (the packing said to develop high back pressure) and in order to achieve a reasonable flow rate it is necessary to apply pressure to the mobile phase. So the designation put forth as high-pressure liquid chromatography.

Different Types of Principles: According to the phases involved, HPLC can be classified into several types, which are as follows:

Normal Phase Chromatography (NPC)

Reverse – Phase Chromatography (RPC)

Liquid – Solid Chromatography or adsorption HPLC

Liquid – Liquid Chromatography or Partition HPLC

Ion exchange Chromatography or Ion exchange HPLC

Size exclusion or gel permeation or steric exclusion HPLC

Ion pair HPLC

Affinity HPLC

Theoretical principles of HPLC:

a. Retention time: The time is required between the injection point and the peak maximum is called the retention time. It is denoted as the Rt. It is mainly useful for the qualitative analysis for the identification of compound.

b. Capacity factor: It represents the molar ratio of the compound in the stationary phase and the mobile phase. It is independent of column length and mobile phase flow rate. It is denoted as the “k”. It should be kept 1-10. If “k” values are too low it is likely that the solutes may be adequately resolved and for high ‘k’ values the analysis time is too long. It can be calculated by

tr – t0



tr = Retention time, t0 = Dead time.

c. Resolution: The degree of separation of one component from another is described by the resolution. It is generally denoted by 'Rs'. It is measured as the difference in retention time and the arithmetic mean of the two peak widths.

tr2 – tr1


0.5(w1 + w2)

tr2 = Retention time of first peak w1 = width of first peak

tr1 = Retention time of second peak w2 = width of second peak

d. Theoretical plates: It is important property of the column. It reflects its quality of separation and its ability to produce sharp, narrow peak and achieving good resolution of peak. ‘N’ denotes it.

3500 X L (cm)



L = length of the column - in cm, dp = diameter of the particle (µm)

It follows that if the exchange is fast and efficient, the theoretical plate will be small in size and there will be large number of plates in the column.

e. Height equivalent to theoretical plate (HETP): Number of plates directly proportional to the column length (L) and inversely proportional to the diameter of the particles (dp). The value of H is a criterion for the quality of a column. Lower the HETP, higher is the efficiency of the column.' Its value depends upon particle size, flow rate, viscosity of mobile phase.

H = L/N

L = Length of column, N = No. of theoretical plate

f. Tailing factor: Closer study of a chromatographic show that the Gaussian forms is usually not completely symmetrical. The graph spread out to a greater or lesser extent, forming a tail. It reduces the column plate number which intern influences the resolution. Tailing is mainly due to deteriorated column, overloading column, extra column-volumes, and incompatibility of sample with standard and/or mobile phase. Practically it can be calculated or determined at 10% of the total peak height. It must not be greater than 2.5.

Distribution of analytes between phases


A mobile A stationary



A term called the retention factor, k', is often used to describe the migration rate of an analyte on a column. It also called the capacity factor. The retention factor for analyte A is defined as;

k'A = tR - tM / tM


When define a quantity called the selectivity factor, which describes the separation of two species (A and B) on the column;

= k'B / k'A



Although the selectivity factor, describes the separation of band centers, it does not take into account peak widths. Another measure of how well species have been separated is provided by measurement of the resolution. The resolution of two species, A and B, is defined as

Baseline resolution is achieved when R = 1.5

It is useful to relate the resolution to the number of plates in the column, the selectivity factor and the ret1ention factors of the two solutes;

To obtain high resolution, the three terms must be maximised. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening, which may not be desirable. Instead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles.


The selectivity factor , can also be manipulated to improve separations. When is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then is increased by one of the following procedures:

Changing mobile phase composition

Changing column temperature

Changing composition of stationary phase


Most commonly used Methods in HPLC

Reverse Phase Chromatography

Normal Phase Chromatography


Retention by interaction of the stationary phase’s non-polar hydrocarbon chain with non-polar parts of sample.

Retention by interaction of the stationary phase’s polar surface with polar parts of the sample molecules.

Stationary Phase

bonded siloxane with non-polar functional groups like n- octadecyl (C18) or n- octyl (C8), ethyl, phenyl, -(CH2) n-diol, (CH2) n-CN molecules.

bonded siloxane with polar functional group like SiO2, Al2O3, -NH2, -CN, -NO2, - Diol

Mobile Phase

Polar solvents like methanol, acetonitrile, water or buffer (Sometimes with additives of THF or dioxane).

Nonpolar solvents like heptane, hexane, cyclohexane, chloroform, ethyl ether, and dioxane.


Separation of nonionic and ion forming nonpolar to medium polar substances (carboxylic acids hydrocarbons)

Separation of nonionic, nonpolar to medium polar substances.

Elution Order

Most polar components are eluted first.

Least polar components are eluted first.



a. Pumps: Pumps are required to deliver a constant flow of mobile phase at pressures ranging from 1 - 550 bar pumps capable of pressure up to 8000 psi provide a wide range of flow rates of mobile phase, typically from 0.01-10ml min-1. Low flow rates (10-100l min-1) are used with micro bore columns, intermediate flow rates (0.5-2ml min-1) are used with conventional analytical HPLC columns, and fast flow rates are used for preparative or semi preparative columns and for slurry packing techniques.

Mechanical pumps of the reciprocating piston type view a pulsating supply of mobile phase. A damping device is there fore required to smooth out the pulses so that excessive noise at high levels of sensitivity or low pressure does not detract from detection of small quantities of sample. This type of pump is extremely useful, however in that a constant volume of liquid is delivered, the actual value being set by adjustment of piston stroke. This means that the pressure shown on a gauge acts as indicator of working conditions. Thus, if the column becomes partially blocked, rice in pressure occurs until ultimately the relief wall operates. Similarly, leakage from column connection or pump walls will show up as lower pressures. In both cases suitable maintenance measures can be put in to operation immediately.

Dual – piston reciprocating pumps produce an almost pulse free flow because the two pistons are carefully faced so that as one is filling the other is pumping. These pumps are more expensive than single piston pumps but are of benefit when using a flow sensitive detector such as ultraviolet or refractive index detector.

b. Injection Systems: Injection ports are of two basic types, (A) those in which the sample with injected directly into the column and (B) those in which the sample is deposited before the column inlet and then swept by a valving action into the column by the mobile phase.

Modern injectors are based on injection valves, which allow the sample at atmospheric pressure to be transported to the high-pressure mobile phase immediately before the column inlet. With the injector in the LOAD position, the sample is injected from a syringe through a needle port into the loop. The lever is when turned through 600 to the INKJET position and the sample is swept into the flowing the mobile phase. If an excess of sample is flushed through the loop in the LOAD position, the volume injected is the volume of the loop, which is typically 10-20 l for analytical separations and 0.1-1ml for semi preparative or preparative separations this complete filling positions offers the analyst the highest reproducibility, and is capable of giving relative standard deviations of less than 0.2%. Precision of this order generally avoids the need for internal standard. Many of the injection valves also allow a partial filling procedure in which any selected volume less than the volume of the loop is injected into the loop. The precision off this technique depends on the precision of the syringe and, with care, relative standard deviation of about 1% can be achieved.

c. Columns: HPLC columns are made of high quality stainless steel, polish internally to a mirror finish. Standard analytical columns are 4-5 mm internal diameter and 10-30 cm in length. Shorted columns (3-6 cm) containing a smaller particles size packing material (3 or 5 m) produce similar or better efficiencies, in terms of the number of theoretical plates (about 7000), that those of 20 cm columns containing 10 m irregular particles and are used an short analysis time and highest throughput of samples are required. Micro bore columns of 1-2 mm internal diameter and 10-25 cm in length have certain advantages of lower detection limits and lower consumption of solvent, the latter being important if expensive HPLC – grade solvents are used. HPLC are also being carried out on the semi preparative scales by using columns of 7-10 mm or 20-40 mm internal diameter respectively.

Types of Column Pac kings as per I.P

LC-1 (ODS) : Octadecyl Silane – Chemically bonded to porous silica or

Ceramic micro particles 3 - 10m in diameter.

LC-2 (OS) : Octyl Silane – Chemically bonded with totally porous silica 3

LC-3 : porous silica particles 3-10m in diameter.

LC-4 : very finely- divided silica gel consists of porous spherical

Particles either chemically bonded Nitrile or CN groups.

LC-5 : very finely divided silica gel chemically modified at the

Surface by the introduction of octylsilyl groups.

LC-6 : Trimethyl silane (TMS), chemically bonded to porous silica particles, 3-10m in diameters.

LC-7 : Rigid, Spherical styrene-divinyl benzene co-polymers,

d. Detectors:



Solvent Requirements



Any with chromophores

UV-grade non UV absorbing solvents

Has degree of selectivity and useful for many HPLC applications


Fluorescent compounds

UV-grade non UV absorbing solvents

Highly selective and sensitive, often used to analyze derivitized compounds

Refractive index

Compounds with different RI than mobile phase

Cannot run mobile phase gradients

Limited sensitivity


Charged or polar compounds

Mobile phase must be conducting

Excellent for ion exchange compounds


Readily oxidized or reduced compounds, specially biological samples

Mobile phase must be conducting

Very selective and sensitive


Broad range compounds

Must use volatile solvents or volatile buffers

Highly sensitive. Many modes available. Needs trained person

HPLC method development:

The wide variety of equipment, columns, eluent and operational parameters involved makes high performance liquid chromatography (HPLC) method development seem complex.

The main objective of method development is to obtain a good separation with minimum time and effort. Based on the goal of separation, the method development is preceded. The steps involved are

Information on sample, define separation goals

Need for special HPLC procedure, sample pretreatment, etc.

Choose detector and detector settings

Choose LC method, preliminary run;Estimate best separation conditions

Optimize separation conditions

Check for problems or requirement for special procedure

Validation for release to routine laboratory

Necessary consideration in developing of HPLC method:

Keep it simple

Try the most common columns and stationary phases first

Thoroughly investigate binary mobile phases before going on to ternary

Think of the factors that are likely to be significant in achieving the desired resolution.

Mobile phase composition, for example, is the most powerful way of optimizing selectivity whereas temperature has a minor effect and would only achieve small selectivity changes. pH will only significantly affect the retention of weak acids and bases.

Method development in HPLC

In developing HPLC method for the quantitative analysis of multicomponent formulation the following general requirements should be fulfilled.

The identity of the component to be analyzed should be established.

Separation of specific components should be achieved.

Sample preparation should be reproducible.

Standard of known purity should be available and accuracy will be directly related to the degree of purity of standards used in determination.

A stationary phase that separates the component in reproducible manner.

There must be a constant flow of mobile phase.

Sample application or injection should be reproducible.

Steps Involved In Development of HPLC Method

Literature survey:

Here a detailed account of all analytical methods developed for the drug is collected to avoid duplication of the method developed. Details about the structure of the drugs and their physicochemical properties are also collected.

Selection of chromatographic method:

First reversed phase should be tried.

If not successful normal phase should be taken into consideration.

For ion exchange or ion pair chromatography, first ion suppression by pH control and reversed phase chromatography should be tried.

Selection of stationary phase:

Matching the polarity of sample and stationary phase and using a mobile phase of different polarity achieve a successful separation.

Selection of mobile phase:

Reversed phase bonded packing, when used in conjunction with highly polar solvents; approach is ideal and is a universal system for liquid chromatography. Mobile phase may be either single liquid or combination of liquids, which are compatible with sample, column and instrument.

Selection of suitable detector:

Detector is the eye of HPLC system and measures the compounds after their separation on the column. There are basically two types of detectors: bulk property detectors and solute property detectors. Detectors, in order of their popularity are UV, fluorescent, conductivity, polarimeter and refractive index detectors. UV detector is the first choice because of its convenience and applicability in case of most of the samples. The latest version of equipments is available with photo diode- array detectors (PAD or DAD).

The response obtained from a given detector will vary according to the nature of solute molecule. With a UV detector the response is related to the both concentration and molecular extinction coefficient of the component at the wavelength of detection.

Sampling and sample preparation:

The sample should be homogeneous. It should be completely soluble and the solvent used to dissolve the sample should be initial mobile phase or any solvent miscible with mobile phase.

Chromatographic separation:

After achieving a resolution with optimized solvent system, to obtain reproducible results following criteria must be satisfied.

Monitoring flow rate.

Keeping the solvent composition intact.

Solvent system must be covered before storage.

Monitoring column temperature.

Measurement and calibration:

The various approaches used for quantitative analysis:

Peak height method:

Peak height measurements represent a simple, satisfactory method for calculating detector response in the absence of mechanical or electronic signal integrators. In practice, a baseline is drawn from the leading edge of the chromatography peak to the trailing edge. The vertical distance from the peak apex to the predetermined baseline represents the peak height. For quantitative purpose

The peak of interest must be symmetrical

All parameters that affect peak width must be held constant

b) Peak area method:

Computing electronic integrators are the simplest and most popular method for the determination of chromatographic peak areas. Quantitative measurements based on peak areas can be performed by several methods such as,

Triangulation: The peak is converted into a triangle and area is determined by ½ x base x height. A variant of this is to calculate the area by multiplication of peak height times the width at half height.

Cut and weigh method: Peaks are cut out and weighed.

Use of planimeter: It provides a numerical value for the area contained within a perimeter of the figure traced out by a planimeter.

Retention time method: This method involves arbitrary area determinations by multiplying peak height times retention time


Validation is documented evidence, which is completed to ensure that an analytical method is accurate, reproducible and robust over the specific range. The quality of the analytical data is a key factor in the success of a drug development program. The process of method development and validation has a direct impact on the quality of these data.

Method validation:

Method validation is the process to confirm that analytical procedure employed for a specific test is suitable for its intended use. Method needs to be validated or revalidated

Before their introduction into routine use

Whenever the conditions changes for which the method has been validated , e.g., instrument with different characteristics

Whenever the method is changed, and the change is outside the original scope of the method.

Purpose of Validation:

Enable the scientists to communicate scientifically and effectively on technical matter.

Setting the standards of evaluation procedures for checking compliance and taking remedial action.

Economic: Reduction in cost associated with process sampling and testing.

As quality of the product cannot always be assured by routine quality control because of testing of statistically insignificant number of samples.

Retrospective validation is useful for trend comparison of results compliance to CGMP/CGLP.

Closure interaction with Pharmacopoeial forum to address analytical problems.

International Pharmacopoeial harmonization particularly in respect of impurities determination and their limits.

Depending on the use of the assay, different parameters will have to be measured during the assay validation. ICH and several regulatory bodies and Pharmacopoeia have published information on the validation of analytical procedures.

The goal of the validation process is to challenge the method and determine the limit of allowed variability for the conditions needed to run the method. The following statistical parameters are to be determined to validate the developed method.

Correlation coefficient(r):

When the changes in one variable are associated or followed by changes in the other, it is called correlation. The numerical measure of correlation is called the coefficient of correlation and is defined by the relation.

(x – x’) (y –y’)


√ (x –x’) 2 (y –y’) 2

Regression equation :

Regression equation= I + aC

Y2 – Y1

a = slope = ---------------

X2 – X1

I = Intercept = regression – a C

As a percentage of mean absorbance.

Standard Deviation:

S = √ (X- X!) 2/N – 1

Where, X = observed values

X! = Arithmetic mean = X/N

N = Number of deviations

For practical interpretation it is more convenient to express ‘S' in terms of percent of the approximate average of the range of analysis is used in the calculation of 'S'. This is called co-efficient of variation (C.V) or percent relative standard deviation (%RSD).

C.V OR %RSD = 100* S/ X!

It is customary to use probability limits (P) 0.05 level (95% of the readings will be within the calculated limits ± x 100 where x = t.s/√n, t value is 1.965 from Students table) and 0.01 level (99% of the readings will be within the limits ± x100 where y = t.s/√n, t value is 3.499 from students Table for six determinations).

% Range of error at p = 0.05 level = ± x 100

% Range of error at p = 0.01 level = ± x100



It is the closeness of agreement between the actual value of the drug and the measured value. Spike and recovery studies are performed to measure accuracy: a known sample is added to the excipients and the actual drug value is compared to the value found by the assay. Accuracy is expressed as the bias or the % error between the observed value and the true value (assay value/actual value x 100%).


Use a minimum of 3 spiking concentrations in the excipient solution. Prepare 2 samples of each concentration. Test the 6 samples in triplicate on one run Measure expected vs. average measured value. Calculate the % recovery = bias.


It is the closeness of agreement between the values obtained in an assay. It is expressed as the coefficient of variation (% CV). CV is the standard deviation of the assay values divided by the concentration of the analyte. Several types of precision can be measured: intra-assay precision (repeatability) is the % CV of multiple determinations of a single sample in a single test run; inter-assay precision (also called intermediate precision) measures the % CV for multiple determinations of a single sample, controls and reagents analyzed in several assay runs in the same laboratory.

Reproducibility is the precision between laboratories usually in collaborative studies and not directly relevant to assay validation in a manufacturing facility.

i) Intra-assay:


Prepare three dilutions of the sample (high/medium/low conc.s in the range).

Test 10 replicates of each dilution of the sample.

Calculate the average and standard deviation for each point on the curve.

Calculate the CV for each point on the curve.

ii) Inter-assay:


Prepare three dilutions of the sample (high/medium/low conc. in the range).

Test triplicates of each dilution of the sample in three different assays.

Do for day-to-day variations

Do for lot-to-lot variations of assay materials

Do for technician-to-technician variation.

Calculate the average and standard deviation for each point on the curve for each individual test.

Calculate the CV for each point on the curve between the assay runs.


It is the capacity of an assay to remain unaffected by deliberate changes to various parameters of the method and gives an indication of its reliability during normal assay conditions. The variations could be in room or incubator temperature or humidity, variations in incubation times, minor variations in pH of a reagent, etc.

Under each of these conditions, the accuracy and precision or other assay parameter can be measured to see what variations can be tolerated in the assay conditions.


It is the ability of an assay to obtain test results, which are directly proportional to the concentration of an analyte in the sample. The determination of this parameter will identify the range of the analytical assay. It can be measured as slope of the regression line and its variance or as the coefficient of determination (R2) and correlation coefficient (R).


Determining the coefficient of correlation R for dilutions of the sample over the range claimed for the assay.

Prepare 6 to 8 sample dilutions across the claimed range

Test each dilution in triplicate for 3 runs

Record expected values, actual values, and % recoveries for each run

Analyze each set of dilutions as a linear curve and calculate R for each assay.


Calculate the accuracy and precision at each dilution. Range is the highest and lowest concentration with satisfactory accuracy and precision.

If the validation study for an analytical test is well planned it should be possible to design the protocol to consider many of the parameters in a single series of tests, for instance: selectivity (specificity) linearity, range, accuracy and precision for a potency test.


It is a measure of the highest concentration of an analyte that can be measured with acceptable accuracy and precision. It is the upper limit of the linearity determination. If the relationship between response and concentration is not linear, the range may be estimated by means of a calibration curve.

SELECTIVITY (also termed specificity):

It is the ability of an analytical assay to measure the analyte in a sample in the presence of the other components expected to be present in the product. This parameter is measured for identity tests, for content or potency tests, and for purity tests to ensure that the assay provides an accurate statement of the identity, potency or purity of a product. Selectivity (specificity), like accuracy, is expressed as the bias or the % error between the measured and known value.


It is the lowest amount of the analyte in a sample that can be detected but not necessarily be quantitated as an exact concentration or amount.


Prepare a standard concentration of the product in the appropriate solution.

Prepare a blank solution without any sample (zero concentration).

Perform the assay at least 3 times in duplicate according to the SOP

Measure the OD values for the sample and blank.

Calculate the average OD for the sample and blank.

Calculate and standard deviation of the blank

Calculate the LOD as 3 x st dev of the blank

OD of sample/concentration of sample


It is the lowest amount of an analyte that can be measured quantitatively in a sample with acceptable accuracy and precision. The LOQ is a parameter for tests measuring impurities in a drug product.

The following table is based on the ICH document on analytical assay validation. It indicates what type of parameter must be validated for different types of tests. In addition to the above parameters which are common to both physico-chemical tests and bioassays, there have been several suggestions that additional measurements are important for bioassays partly because of their duration, complexity, and long term storage of biological samples and control and reference material.

These include: front-to-back test which determines whether the parameters for early samples on a large test are the same as later samples (because they have been prepared at a different time in comparison to the controls); freeze-thaw stability

Which uses samples and controls which have been frozen and thawed repeatedly to determine any effects of freezer storage on test results; and lot-to-lot precision which measures the precision of an assay with different lots of cell lines, serum or other highly variable component by the test? The latter is an important test of potency assay precision.

Table 2. Criteria for Validation of the Method




Recovery (98-102%)

Precision, Reputability

RSD < 2%

Intermediate precision

RSD < 2%


No Interference


S/N > 2 or 3


S/N > 10


Correlation Coefficient(r)>0.99




>24h or >12h


The sample is analyzed quantitatively in HPLC by either peak height or peak area measurements. Peak areas are proportional to the amount of material eluting from the column as long as the solvent flows at constant rate. Peak heights are proportional to the amount of material only when peak width is constant. Once the peak height or peak area is measured, there are five principles evaluation methods for quantifying sample.

a. Calibration by standards: Calibration curves for each component from pure standard, by using, identical operating conditions for standard and samples.

The concentration of sample is read from its curve if the curve is linear


X= concentration of sample

K= proportional constant (slope of curve)

b. Internal standard method: In this method a known quantity of the internal standard is injected and peak area ratio Vs concentration is ascertained. Then a quantity of the internal standard is added to the raw sample prior to any sample pretreatment or separation operations. The material selected for the internal standard must be completely separated from the adjacent sample components, and should not interfere with sample components.

Peak area of sample component


Peak area of internal standard

Sample concentration = Peak area ratio x concentration of standard

c. Area normalization: The technique used to evaluate the absolute purity of the sample .the procedure is to total up the areas under all peaks and then calculated the percentage of the total area that is contributed by the compound of interest. For this method the entire sample must be eluted, all components must be separated and each peak must be completely resolved

d. External standard method: It employs a separate injection of fixed volume of sample and standard solution. The peaks are integrated and concentration is calculated

Peak area of sample.

Sample concentration = ----------------------- x concentration of standard

Peak area of standard

e. Standard addition method: The chromatogram· of the unknown is recorded, the known amount of sample is added and the chromatogram is repeated using same condition. The increase in the peak area the original concentration will be computed by interpretation.

Importance of polarity in HPLC: The relative distribution of a solute between two phases is determined by the interactions of the solute species with each phase. In both normal phase and reversed phase HPLC, the eluting power or solvent strength of the mobile phase is mainly determined by its polarity. The relative strengths of these interactions are determined by the polarity of the sample and the mobile and stationary phase.

Polarity is a term that is used in chromatography as an index of the ability of compounds to interact with one another. It is applied very freely to solutes, stationary and mobile phase. If the polarities of stationary phase and the mobile phase are similar, it is likely that the interactions of solute with each phase may also be similar, resulting in poor separation. Retention of solutes altered by changing the polarity of the mobile phase. Successful chromatographic separation requires a proper balance of intermolecular force among three participants in separation process i.e. Analyte, Mobile phase and Stationary phase.

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