Chapter 1
Chapter 1
CHAPTER-1. INTRODUCTION
1.1 INTRODUCTION
The pharmaceutical business remains an integral part of the health care cycle, doing
critical research and producing medications that sustain and restore human life.
Standards for quality, safety, and effectiveness are quite high for today's
pharmaceuticals intended for human consumption. Adequate procedures for product
quality control are necessary for the assessment of safety, effectiveness, and the
ongoing maintenance of these qualities in practice. Quantitative analysis may be used
to identify species in a sample based on their chemical makeup. One or more of these
species or analytes may be detected using a quantitative analysis to determine their
abundances.
Estimation methods for various formulation components include the ones listed
below.
Optical methods
• Amperometry
• Voltametry
• Potentiometry
• Conductometry
Methods of separation/chromatography
• Chromatography of Gas-Liquid
• Chromatography of Gases and Solids
• Chromatography of a liquid sample
• Chromatography, Liquid-Solid
• Chromatography on a thin layer
• Chromatography on paper
• Chromatography with gel permeation
The choice of the most efficient method for a particular analysis is one of the most
important decisions an analyst must make. He needs to be familiar with the theoretical
foundations, real-world applications, and the circumstances under which each
approach is dependable in order to do this. He must also be aware of any potential
interferences that may occur and be able to devise solutions to avoid them.
1. Chromatography
2. Electrophoresis
3. Mass spectroscopy
Chromatography
adsorbed to the stationary phase move more slowly than those that are not over the
course of the stationary phase. The Greek roots of the word chromatography-chromos
and graphy-mean, roughly, ''writing in colour.'' It was Tswett who first used a
chromatography column made of adsorbent powder and rinsed with a liquid solvent to
separate colour bands of plant pigments, hence the words ''adsorbent powder'' and
''mobile phase.'' This is transmitted down the tube's length in a stationary solid or
liquid phase.
Researchers in the late 1960s and early 1970s built on the theoretical foundations laid
by earlier chromatographic methods, most notably column chromatography, to create
the high performance liquid chromatography technique (HPLC). Similar to traditional
column chromatography, this method employs a number of different separation
mechanisms. These methods include things like adsorption, partitioning (including
reverse phase partition), ion exchange, and gel permeation. In high-pressure liquid
chromatography (HPLC), the mobile phase is pushed through a less porous, more
densely packed column. Improved resolution of the compounds being separated,
shorter separation periods, and better accuracy, precision, and sensitivity in
quantifying the substances being separated are the primary benefits of HPLC in
comparison to traditional (gravity feed) column chromatography.
1. Partition chromatography.
2. Normal–phase chromatography.
3. Displacement chromatography.
4. Reversed-phase chromatography (RPC)
5. Size-exclusion chromatography.
6. Ion-exchange chromatography.
7. Bio-affinity chromatography.
8. Aqueous normal-phase chromatography
stationary phase, while the liquid that moves over them constitutes the mobile phase.
The solubility of components in the phases and the molecular size of the sample
inform the distribution equilibrium under a specific dynamic solution.
As a result, during the static phase, the different parts move at different speeds,
creating physical separation between them. Spherical solid particles are jammed into a
tube made of stainless steel (or resin) to form the column. Liquid pumping keeps
mobile phase flowing steadily into the column's intake. The column's inlet is placed
close to the sample injector. The column is first loaded with mobile phase, and when
the sample's constituent parts move through it, they switch back and forth between the
stationary phase and the mobile phase.
Compounds that migrate quicker across the column are only those that are in the
mobile phase, whereas those that are more often found in the stationary phase travel
through the column more slowly. In this arrangement, the components are separated
on the column before eluting in a specified sequence from the output. A detector
affixed to the column's exit picks up every chemical that elutes from the column. The
instant the sample is entered, the separation process is recorded and graphed. This
diagram is known as a chromatogram. Both the retention time (or elution time) and
the ratio of compound concentration to peak area are affected by the compound's
specific properties.
Selectivity of HPLC
Affinity chromatography
Size exclusion chromatography (gel permeation and gel filtration
chromatography)
a supply of mobile phase with variable flow and pressure that can feed the
column. To purge the solvents of dissolved air and other gases, a degasser is
required.
A mobile phase pump that supplies the column with mobile phase. There can't be
any pulsations in the pumping system. Low-cost chromatography requires a
pump with a pressure range of at least 100 atm (1500 psi). However, a better
upper limit might be 400atm (600 psi). The generation of a modest flow rate of
0.5 to 2 ml per minute was all that was required for many analytical columns.
A sampling valve or loop at the top of the separation column is used to inject the
sample into the moving mobile phase.
A guard column or inline filter placed at the separation column's inlet can catch
any particles too tiny to pass through the main column's filtration system.
The HPLC separation packing required to achieve the separation is housed in the
separation column. Silica for adsorption chromatography, bonded phases for
liquid-liquid chromatography, ion-exchange functional groups attached to
stationary support for ion-exchange chromatography, gels with a specific porosity
for exclusion chromatography, and so on are all examples of suitable media for
these separation techniques.
The typical length of a column is between 10 and 30 centimeters, but shorter,
faster columns are just 3 to 8 centimeters in length and have an interior diameter
of 4 to 5 millimeters. Preparative chromatography typically uses particles with a
diameter of 3 to 5 nm, but can use particles as large as 10 nm.
The bare-bones set of instruments is rounded out by a detector coupled to a data-
handling device.
1. UV visible photometers
2. Refractive index detector
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Chapter-1 INTRODUCTION
3. Flourimetric detector
4. Conductivity detector
5. Amperometric detector
6. PDA
There is currently no universal detector available for HPLC that has sufficient
sensitivity. Therefore, deciding on a detector is task-specific.
where tw1 and tw2 are located at the point where the tangents meet the baseline. The
tangent lines for a Gaussian peak that is symmetrical in shape are drawn at a distance
of 0.60 times the height of the peak.
Capacity factor: It's a metric for determining how effectively the column retains
sample molecules during isocratic separation. Solvent makeup, separation age, and
separation temperature all have a role.
K=tr-t0/t0
where, tr = Band separation time
Column efficiency: Theoretical plates number is the term for this concept. A peak's
band widening is quantified. Lower band spread results in a greater number of
theoretical plates. It's a sign of a well-performing column and overall system health. N
values may be used to characterise the performance of a column.
Where,
Ve = elution volume, retention time or retention distance (mL, sec, or cm)h = peak
height
Peak Asymmetry:
b = The 10 percent of the peak's height corresponding to the distance from its midway
to its leading edge.
a = distance from the peak's front to its center (described as a proportion of the peak's
total height)
Peak tailing:
Where,
Selectivity:
It measures relative retention of two components. Selectivity is the function of
chromatographic surface ( column), melting point and temperature.
α= K2/K1 = V2-V0/V1-V0
Measuring the heights of peaks or the areas of peaks only gives you a response in
terms of the detector signal.
The answer should depend on how much of the target component is present or how
much mass there is to account too. This may be achieved by means of a calibration
procedure.
The proportion of the overall area that each individual peak takes up is known as the
normalised peak area. In order to determine the relative quantities of trace impurities
or degradation products in a purified material, it is common practice to calculate the
response factor for each component.
Each standard has its own response factor (RF), which may be calculated as follows.
To run most samples in HPLC without substantial preparation, the external standard
technique can be utilised.
The analyte is separated from the internal standard, which is a distinct component.
The internal reference point should be selected such that it behaves similarly to the
example item. Samples that need extensive processing or cleaning before analysis are
a prime candidate for the use of an internal standard.
Calibration plots for quantitative analysis may be generated using the standard
addition approach. It is more often used in trace analysis. In order for the standard
addition method to work, the response before spiking additional analytes must be
sufficiently large to establish an acceptable signal-to-noise ratio (10).(Vogel’s 2003).
1. Sample size: The size of the sample used is a major drawback of high-
performance liquid chromatography. Analysis typically requires just a few
microliters of sample, which might be problematic for scarce or costly
materials. However, sample size may be increased by the use of sample
concentrators or through an increase in the detector's sensitivity.
2. Resolution: High-performance liquid chromatography (HPLC) separates
compounds based on their physical and chemical properties. High-
performance liquid chromatography is widely used in chemistry and biology
for chemical separation and identification (HPLC). Gas chromatography (GC),
mass spectrometry (MS), and our very own GC-UV solution are all tools we
advocate for use in such circumstances.
3. Detection limit: Another drawback is that HPLC is not as sensitive as other
analytical methods like GC or UV, hence its detection limit is less.
4. Column lifetime: HPLC columns are consumables that will need to be
changed at some point. Columns have different lifespans depending on their
types, the sample matrix, and the circumstances of the sample. Cleaning and
storing columns properly may prolong their useful lives, but doing so is
laborious and time-consuming.
1.8 VALIDATION
Definition
Validation is ''established recorded proof that offers a high degree of confidence that a
certain method will consistently generate a product of predefined specifications and
quality characteristics,'' according to the US Food and Drug Administration.
Why Validate?
• Rapid introduction
• To increase the productivity
• To target and reduce the number of controls
• To reduce product cost
1. Assurance of quality.
2. Government regulation.
1. Verification of Equipment
2. Validation of Process
3. Confirmation of Cleaning
4. Verification of Analytical Methods
5. Checking the utilities and certifying the facility
The process of assessing whether or not a certain analytical technique may be utilized
for its intended purpose is known as method validation. Only comprehensive
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analytical testing can provide the assurance that a pharmaceutical product is pure,
stable, safe, and effective. The quality control process is not complete without
validating the analytical methods used.
Method development is ongoing and occurs in tandem with the maturation of the
pharmaceutical product. With respect to time, money, and efficiency, the concept of
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Chapter-1 INTRODUCTION
With the help of more trials, the standards for system suitability that will be applied to
upcoming analytical sample sets may be better specified. System suitability tests are a
set of routine inspections that assess the overall functioning of the instrument,
software, reagents, and analysts.3. The ultimate system suitability parameters may be
determined by assessments of method robustness carried out under statistical
experiment design. The objective is to determine which factors are most important
when evaluating a method's or system's potential utility.
right thing in the right range for the right samples. By doing so, the analyst may learn
about the method's characteristics and determine its capabilities. In the endnotes, you
will find a collection of useful references and strategies for validating your methods.
The following criteria are used to carry out the assay procedure validations..
SPECIFICITY:
Specificity can be used for identification test, purity test, assays ,etc.
PRECISION:
Definition
The degree to which multiple measurements made from the same homogenous
material under controlled conditions agree with one another is considered a measure
of an analytical technique's accuracy. The precision of an analytical method is often
evaluated by using the variance, standard deviation, or co-efficient of variation of a
series measurement.
System precision
By comparing the peak responses of six replicate injections of the same standard
solution performed using the recommended method, the system's precision may be
determined. After calculating the RSD percentage, we know that it must be less than
or equal to 2%.
Method precision
The peak response of six duplicate injections of six distinct weighed sample solutions
prepared according to the suggested technique is measured to determine the method's
accuracy. The %RSD is determined, and it must be less than or equal to 2%.
Determination
ICH Requirements
ACCURACY
Determination
ICH Requirements
The ICH guidelines suggest testing accuracy with nine determinations across three
concentration levels that span the intended range (i.e., three concentration and three
duplicates of each concentration).
LINEARITY
Definition
Analytical methods are said to be linear within a specific range if they reliably yield
test results that are proportional to the concentration (quantity) of analyte in the
sample.
.Determination
At least five concentrations are often used to determine whether or not an analytical
technique is linear. A plot of signals vs. analyte concentration is visually inspected to
make the first determination. Appropriate statistical methods are employed to
establish test results if a linear relationship is suspected (i.e., by computing the
regression line using the least squares approach).
Definition
Lower Detection Limit (LOD) is the minimum substance concentration that a given
analytical technique can identify (but not necessarily measure). Below or above LOD
merely means that the sample is not at that level.
Determination
ICH Requirements
Methods for comparing the signal strength of samples containing known and blank
concentrations of analyte are detailed in the ICH. Figure out the smallest analyte
concentration at which a positive result is still feasible.
Definition
Determination
ICH Requirements
Methods for comparing signals measured from samples with a known low
concentration of analytes to those measured from blank samples are provided in the
ICH papers.
RANGE
Definition
It has been shown that analytical procedures are sufficiently specific, accurate, and
linear throughout a range of analyte concentrations in a sample. The range represents
the space between the sample's greatest and lowest analyte concentrations.
Determination
Analytical precision, accuracy, and linearity are tested using samples with both low
and high concentrations of the analyte to guarantee they are within the method's
acceptable range.
ROBUSTNESS
Definition
Analytical procedures may be judged by their robustness based on how well they hold
up under typical conditions, which can be affected by modest but deliberate
modifications to the parameters of the analysis.
Determination
By carrying out the assay while deliberately varying some parameters (such as the
flow rate by 10%, the mobile phase ratio by ±2, the pH of the mobile phase through
±0.2, the wave length detection by ±5 nm, and the temperature by ±1 to 50 degrees),
the robustness of the method is evaluated to ensure that the results are unaffected by
the changes in the parameters listed above.
RUGGEDNESS
Definition
Analytical robustness is defined as the degree to which results from repeated analyses
of the same samples remain consistent when performed in different laboratories by
different analysts using different instruments using different lots of reagents using
different elapsed assay times using different temperatures using different days, etc.
Determination
After proper preparation in line with the test method, the solution stability of the drug
substance or drug product can be evaluated. It is common practise for laboratories to
use auto samples for overnight runs, during which the sample will remain in solution
for several hours. Particular care must be taken with drugs that can be broken down
by water or light or that stick to glass.
1. Capacity factor
2. Precision\Injection repeatability
3. Relative retention
4. Resolution
5. Tailing factor
K’ = ( tR-tO/tf )
The capacity factor, a gauge of where the peak of interest falls in respect to the empty
volume, determines how long the non-retained components take to extract.
The performance of the HPLC (including the pumping, column, and ambient
conditions) is reflected in the injection accuracy, which is recorded as RSD (relative
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standard deviation) when the samples are analysed. Note that this does not account for
differences in sample preparation or manufacturing.
α = K’1/K’2
The distance between two peaks may be quantified by analysing their relative
retention. As long as the resolution (Rs) is specified, this is not a required option.
4. Resolution (Rs)
Rs = (tR2-tR1)/(1/2)(tw1+tw2)
Tailing factor
T = Wx/2f
Increases in peak tailing reduce the accuracy of quantitation because the integrator has
a harder time identifying when the peak terminates and, thus, calculating the area
beneath the peak . The analyst has provided integrator variables to provide precise
"
determination of the region around the peak of interest. Integrator accuracy suffers if
it is uncertain when an upslope or downslope starts.
N = 16(tR/tw) 2 = L/H
L- Length of column.