Introduction to HPLC and procedure for method development (1)

Isolating, recognizing, and determining the relative quantities of constituents in a

specimen are all part of pharmaceutical investigation, which is a subdivision of chemical
analysis. It is crucial for quality assurance and control in pharmaceutical formulations (2).
Pharmaceutical investigation entails both qualitative and quantitative characterisation of the
sample. Quantitative investigation displays the relative amounts of elements in the sample in
number terms, whereas qualitative analysis revealed the sample's chemical identity. The rapid
growth of pharmaceutical companies and medication production in many regions of the world
has resulted in a surge in demand for novel analytical techniques in the industry.

Chemical methods and instrumental methods are two types of analytical procedures.
Chemical methods emphasised theoretical implementation with an efficient reaction in
chemistry, as well as the precise proportion of chemical quantities required to complete the
reaction or create the appropriate amount of yield formed in the reaction. Titrimetric,
volumetric, and gravimetric approaches, for example.

Instrumental techniques are based on the proportion of some physical features of a

material or compound, such as optical or electrical, and are also used to determine the
concentration of a substance in a specimen.

Instrumental methods are now widely recognized due to their superior speed,
selectivity, precision, and specificity of analysis when compared to traditional methods.
They're really sensitive. As a result, they can provide exact and accurate information for even
small samples. Depending on the nature and kind of material, a suitable analysis method ( 3,
4) is used, either alone or in fusion. Instrumental techniques such as Spectroscopy (UV-
Visible Spectrophotometry, IR, Mass Spectroscopy, NMR, Nephelometry, Fluorimetry, and
Turbidimetry), Chromatography (GC, TLC, and HPLC), and Chromatography (GC, TLC,
and HPLC) enable accurate assay investigation of medications and formulations with
negligible substance, reagents, and time consumption. Various chromatographic techniques
are utilised in the pharmaceutical sector for a wide range of samples. The HPLC technique is
often utilised in those chromatographic procedures.

CHAPTER-1 page 1
LCMS Introduction:

LC/MS is a hyphenated technology that merges the dividing capability of HPLC with
mass spectrometry's detecting power.

Mass spectrometry is a broad systematic approach that involves the generation of

charged species, followed by their separation and identification.

The abbreviation LCMS (Liquid Chromatography-Mass Spectrometry) [Fig 1.01] is

used to refer to a wide range of applications. This lesson will look at the various instrument
accession procedures and the types of data that can be generated by such apparatus.

HPLCsystem

Figure 1.01: LCMS Diagram

1.1 Instrument Principles

CHAPTER-1 page 2
 The mass spectrometer is a device that separates ions in the gas phase based on their
m/z (mass to charge ratio) value.
These parathion of charged species are created by a number of ionisation techniques
in LC-MS and are used in mass spectrometry. Electro spray ionisation (ESI) and atmospheric
pressure chemical ionisation are two examples.
Under atmospheric pressure, the charged species are formed as gas phase ions in all
circumstances.
The gas phase ions are separated in the mass spectrometer using current field and/or
field of magnet to discriminate them.
The mass spectrometer additionally has an air ionisation chamber, a vacuum system,
and a detector in addition to the analyzer. Figure 1.02 shows the main elements of LC/MS
instrument.

1 2 3 4 5 6

Figure 1.02: Main elements of LC/MS equipment

Where:

1. The HPLC (5) eluent is scattered into the atmospheric pressure zone as the ion source.
2. Skimmer Cone: A cone with a smaller sampling gap that preferentially samples gas phase
ions and reduces the gas charge into the mass analyser device's vacuum system.

CHAPTER-1 page 3
3. Quadrupole: A device that separates ions based on their m/z as they move through the
center line of four side by side same distant rods using electric fields.
4. Collision Cell: Ions from the first mass investigator are quickened by a potential variation
and strike with impartial gas molecules like H2, N2, or Ar, fragmenting the analyte.
5. Detector: Once the ions have been created and isolated, they must be detected and
converted into an useable signal. Most modern mass spectrometer systems use reproduce,
dynode, photodiode, and multi channel plate (MCP) ion detection methods.
6. Vacuum system: In order to use in a expected and well organized manner, mass analyzers
require a high degree of vacuum. Most current LC-MS systems (6) use a vacuum system that
consists of two or more distinctive pushed vacuum cells splitted by baffles or opening plates
that vary in outline turn on the instrument seller.
Process
HPLC column partitioning, in which the reagents are partitioned differently between the
movable and unmoving phases (covered onto a hold up matter and fill into the column).
Some retention and partition procedures use Hydrophobic Interaction, Ion Exchange (7), Ion
Pair, Surface Localization, or other chromatography techniques.
After the separated species have been sprayed into an API, the majority of the eluent is
pushed to the trash.
Ions are sorted using a mass analyzer based on their mass to charge ratio. Quadrupole (seen
flip side), The most popular is time of flight (8), ion trap (9), and magnetic zone. Ions with a
given mass to charge ratio can be extracted from the mass analyzer, or they can be "scanned"
through all of the ion m/z results.
The detector counts the ions that emerge from the mass analyzer and may additionally
magnify the signal produced by each ion. Electron reproduce, dynode, photo diode, and
several-channel plate are all common detector types.
All mass analysis and detection takes place at a high vacuum level, which is achieved by
combining foreline (roughing) and turbo molecular pumps.

Detection of MS

CHAPTER-1 page 4
 Contains compounds that can be identified (structural elucidation via spectral
interpretation combined with elemental composition from accurate mass analysers is
possible)
 Apt to be emotionally affected (fempto-gram amounts have been detected by certain mass
analyzer types)
 Difficult to please" (certain analyzer and experiment combination scan lead to highly
selective and sensitive analysis of a wide range of analytes).
 the process of ionization is known as ionization
 Electron- or atom-exchange processes, also called ionisation, manipulate the internal
structure of atoms or molecules to form ions. In LC-MS, charged molecules like protons
can interact with the molecule to which you are applying the charge.
 Strong electric fields are used in LC/MS systems to form these ions in the vapour or
condensed phase. Ionization or dissolution at sub atmospheric pressure is known as
Atmospheric Pressure Ionization (AP-I) (API).
 Electro spray Ionization (ESI) - condensed phase ionization
 APCI–gas phase ionization
 AERIAPAP (APPI)–gas-phase ionization

Figure 1.03: Diagram for Ionization

Figure 1.03 shows the diagram of ionization.

Ionization of the Atmospheric Pressure (API)

CHAPTER-1 page 5
The dissolver expulsion and ionisation processes are integrated in Atmospheric Pressure
Ionization (API), which takes place in the ion source.

All interface types have two main considerations in common:

1. Desolvation of the analyte molecule: To form gas phase analyte ions, the dissolver
fragments must be eliminated from the eluent of HPLC.

2. Substance fragments Charging: Ions must be produced in order for the substance (or
substance by-product) to be sent from the interface into the mass spectrometer, where they
will be sieved from other heaps and then determined.

Electro spray ionisation (ESI) (10) and APCI are the two major forms of ionisation employed
in API LC/MS (APCI). The primary processes employed in API ionisation techniques are
depicted in the schematic image below [Fig 1.04].

Figure 1.04: Ion production process

CHAPTER-1 page 6
 In Electro spray ionization, substance ions are formed in the movable phase prior to
approaching the API interface. In the API interface for APCI, ions are produced via
charge transfer mechanisms in the gas phase.
 electro spray ionizations
 Electro spray ionization (ESI) [Fig 1.05] employs liquid phase (liquid) charge
detachment and ion escape processes to produce vapour phase analyte ions. Before
spraying the analyte molecules into the Electro spray interface, they must first be
ionized. This also indicates that analytes are ionized in the HPLC column before or after
partitioning in the column.
 Electro spray ionization uses three key processes to separate ions from the HPLC eluent
into the gas phase of the mass spectrometer. To manufacture charged droplets, one of
these processes must be taking place at the tip of the capillary.
 the drops turning into a puddle
 Ion production in the gas phase from small/highly charged droplet

Figure 1.05: Diagram for Electro spray Ionizations

Within the contact, hot drying gas dissolved sprayed eluent droplets.

Small solvated droplet or gas phase analyte molecule ([M+H]+) with a more
number of fundamental charges than the initial sprinkled bubbles.

Chemical Ionization of Atmospheric Pressure (APCI) [Fig 1.06]

CHAPTER-1 page 7
 To produce vapour phase analyte ions, APCI involves substance dissolvation and
charge shift processes in the vapour phase.
The eluent is injected into the interface in APCI via a capillary that is comparable to
the ESI source. However, instead of applying a potential to the capillary, the liquid exits from
the capillary into a heated zone, surrounded by a flow of inert, nebulizing gas.

Figure 1.06: Diagram for APCI process

APPI (Atmospheric Pressure Photo Ionisation)

It is a complementary technique to ESI and APCI that was designed to expand the scope of
ionizable substances at air pressure.

Using APPI, it is possible to ionize difficult-to-ionize mixtures, including low- and non polar
mixtures (APPI has been used in the investigation of polycyclic aromatic hydrocarbons).

The ionization process in APPI is done by subjecting a droplet aerosol to light irradiation.
When a molecule absorbs a photon, it produces a molecular radical ion. Only when their
radiating Photon (of energy) exceeds the molecule's ionization potential (IP) is this process
conceivable.

According to APPI [Fig 1.07], species that have an electrical charge may develop in either an
ionic or non-ionic mode.

CHAPTER-1 page 8
 i

Lamp

Figure 1.07: Diagram for APPI process

Mass Analyzers
It is a type of mass analyzer [Fig 1.08].
The most basic kind of LC/MS mass analysis is performed using aparathion or filtration of
analyte ions or fragments of analyte ions in the API interface or in the regions between the
API interface and the high vacuum region of the mass analyzer (products of collision-
produced separation etc.).

Figure 1.08: Mass analyzer

There are numerous main categories of mass analyzers used in regular liquid chromatography
mass spectrometric investigation, and they all vary in how they differentiate species on a
mass-to-charge basis:
Ions are separated using electrostatic potentials that are provided to the elements of mass,
which are used to “select” ions based on their m/z.

CHAPTER-1 page 9
Mass spectrometers that use Time of Flight separate ions by applying a wide range of flying
times to quicken ions as they fly along a long path.
The magnetic zone mass analyzers use magnetic fields to guide the stream of ions towards
the detector, thus selecting ions of interest.
The relative abundance of each mass is graphed in terms of m/z ratio. This illustrates the
results of the mass spectrum [Fig 1.09] of the analyte.
Relative abundance

m/z

Figure 1.09: Diagram for Mass Spectrum

Quadrupole
It is a four-pole structure.
Electric fields are used in quadruple mass analyzing devices to distinguish ions according on
their m/z ratio as they pass through a lode (or pillar). To create an electrostatic zone interior
of the instrument, ion detachment is accomplished by applying regulated voltages to the mass
investigator rods.
As long as x and y, which control the point of an ion from the middle of the rods, remain
constant, the ion will be able to pass through the quadrupole [Fig 1.10] without hitting the
rods, remain < r0.

Figure 1.10: Diagram for Mass Spectrum Quadrupole

CHAPTER-1 page 10
 When an ion is made to vibrate with a path whose magnitude exceeds r0, it collides
with a rod and is emitted, then pushed to waste. An unstable or collisional trajectory is what
this is called. Figure 1.11 shows the collisional energy diagram.

Figure 1.11: Collisional energy diagram

Quadrupole mass analyzer
Advantages Disadvantages
 Reproducibility  Low resolution
 Massdiscrimination.Peakheigh
 Low cost
tvs.massresponsemustbe'tuned'

ETA (Estimated Time of Arrival) (TOF) (11)

In comparison to innumerable other common mass examined appliance, the basic concepts of
mass study employing time-of-flight mass investigator [Fig 1.12] are rather simple.
In the ion source, ions are removed (or created) in small cracks or packets and then
accelerated. The ions then ‘drift' or ‘fly' down a predetermined length (‘d') of evacuated tube.
Once the ions are outside of the accelerating voltage area, the fast at which they move
through the tube is determined by their mass (m) and charge (c) (z). This mass spectrometer
is handy since it detects all ions (nearly) simultaneously. Because studying the mass period of
total ions is done quickly, the instrument's inherent sensitivity is boosted.

Figure 1.12: Diagram for Time-of-flight

Time-of-flight (TOF)

CHAPTER-1 page 11
 Advantages Disadvantages
 Highiontransmission  Fastdigitizersusedi
nTOFcanhavelimit
 HighestpracticalmassrangeofallM
eddynamicrange
Sanalyzers
 Detectionlimit

Mass Analyzer with Ion Trap [Fig 1.13]

Ion trap mass spectrometers work by trapping ions and modulating them with applied
DC and RF fields. The lectrostatic field gives non-selected ions path, causing them to exit the
trap. It is feasible to fragment selected ions by stuffing the trap with an inert gas. When
structural information is necessary, this is useful.
The system offers numerous unique features, such as the ability to do numerous
production scans with excellent sensitivity (MSn). Due to the differing crash administration
in the systems (collision energy/gas), the spectra obtained with an ion trap mass investigator
may differ dramatically from those obtained with a triple quadrupole system.

Figure 1.13: Diagram for Time-of-flight Ion Trap Mass Analyser

Table 1.01 gives the advantages and disadvantages of ion trap mass analyzer.
Table 1.01: Ion Trap Mass Analyser

CHAPTER-1 page 12
 Advantages Disadvantages
 Highsensitivity  Producesveryunusualspectraiftheionsarestor
 MultipleProductIonscanc edinthetraptoolong.
n
apability(MS)  Easilysaturated
 Highresolution  Poorforlowmasswork(below100Da)
 GoodforDDAanalyses  Poordynamicrange(exceptthemostmodernde
vices)andhencemayhavelimitedquantitativeuse

Tandem Mass Spectrometry (MS/MS) [Fig 1.14]

To put it another way, MS/MS can be described as a combination of two or more
individual MS studies. Our goal is to either grow more structurally solid as a result of
breaking apart the ions that were initially isolated through the first investigation, or to acquire
better discernment and sensitivity in quantitative reasoning by employing both the first and
second investigators.
Multiple analysers (of the same or different kinds) can be linked together to do
MS/MS analysis, or an ion trap can be used to do consecutive fragmentations of trappedions.
Multiple ion generation and filtration in a single instrument is referred to as MS n
(should read MS to then). Instruments are typically made up of multiple quadrupoles
combined with a collision cell. Fragmenting the emergents from the first analyser before
secondary mass filtering is a typical design. Other massanalysing device combinations are
feasible, such as quadrupoles with timeo ff light, or quadrupoles with magnetic sector
instruments.

IonBridge
Q1Static
Q2
Collisioncell Q3Scanning i

Figure 1.14: Diagram for Tandem Mass Spectrometry

Detectors [Fig 1.15]

CHAPTER-1 page 13
After passing through the mass analyser, the ions must be identified and converted into a
useful signal. A mass spectrometer's detector is essential to obtaining signals from either
secondary electrons that are amplified or a current (generated by moving charges). An ion
detector system can be divided into 2 types: passive and active.
Beer-sniffing tubes: Ions do not exist in one particular location and thus affect detector
locations in a spectrometer over time, creating point detectors.
Array detectors: Ions are spatially determined, and total ions come at the same time (or
almost at the same time) and are rerecorded along a plane with a bank of detectors.

Figure 1.15: Diagram for Detectors

1.3 High pressure/performance liquid chromatography (HPLC)

From the last twenty five years, HPLC has been the most important component in
pharmaceutical and biomedical research methodologies. HPLC is the most efficient
systematic technique for API (12) or sample testing during the research, Evolution and
synthesizing processes for drug allowance, evaluation, and recognisation. HPLC is a kind of
column chromatography in which a fluid (referred to as the mobile phase) is injected via the
column and a test mixture or substance (referred to as the stationary phase) is transported
through the column with chromatographic stuffed substance (called as the stationary phase) at
a stable high compulsion. Figure 1.16 depicts a systematic diagram of an HPLC.

CHAPTER-1 page 14
 Figure 1.16: HPLC systematic diagram

The following are the main components of an HPLC apparatus:

Data Handling Device and Microprocessor Control for HPLC Solvent push dose
Column Detector Data administration appliance and logic circuit Control for HPLC Solvent
Pump Injector Column Detector

1.3.1 Solvent for HPLC

The solvent reservoir is another name for this section. This is where the movable
phase is kept. Highly purified solvents such as HPLC grade solvents and water with a
resistivity of 18.2Mcm at room temperature (25°C) are used to prepare the mobile phase.

1.3.2 Pump

Because it affects reproducibility and retention time, the pump is an important

component of HPLC apparatus. With a constant flow rate, the HPLC pump delivers mobile
phase to the column, which is pressurised by the solvent reservoirs (5000 pounds per square
inch or more). Removing other gases and diffused air from the dissolver is imperative to the
success of the pump. While it is important to have a good pump, the following characteristics
should be avoided in order to avoid problems when switching solvents: a pump that has
regular stream delivery with no build-up of end pressure, the ability to change worn
components easily, solvent suitability, rust resistance, and low dead volume.

1.3.3 Injector
Injection ports are used to inject samples into HPLC. A testing loop and an injection pipe
may be found on an injection port. After being decomposed into the moving action, or with a

CHAPTER-1 page 15
selected diluent, the instance was injected into the testing loop via an injection pipe. In order
to introduce the specimen into the mobile process flow, the pipe rotor is utilized to open the
curve and then close the pipe. The loop volume ranges from 10 litres to over 500 litres. The
samples were immediately fed into a sophisticated HPLC apparatus.

1.3.4 Column
Critical components in HPLC are one of the most preferred columns because the sample
mixture is separated through the column. Since of its porosity structure, exterior
characteristics, and particle form, silica gel is widely utilized to heap columns. Because of its
usually predictable and reproducible chromatographic action, silica is used to separate a
variety of chemical substances. Silica also has a higher surface activity that is quickly altered
by water and other solvents. There are two types of HPLC columns. A shield column is the
first, while an actual column is the second. Analytical columns are typically 5cm, 10cm,
15cm, and 25cm in length and packed with particles with a diameter of 3, 5, or 10m. The
interior diameter of a column is usually 4.6mm. The guard column is a disposable top of the
primary analytical column that protects the analytical column. It extends the analytical
column's life and protects it from impurities and particulates in solvents. The most widely
utilised material is octadecyl-silica, which is a reverse phase C18 column. HPLC is offered,
as well as C8, C6, C8, C8, C4, cyano, and amino columns.

1.3.5 Detectors

Without a detector, it's nearly impossible to carry out HPLC analysis. One of the HPLC
ingredients that releases a reaction and then indicates a peak on the chromatogram is used to
elute the compound. To identify the chemicals that are eluted from the column, it is located
behind the static phase. Coarse and fine-tuning settings were utilised to determine the overall
band width and height of the peaks. The fine-tuning and coarse settings can be adjusted to
change the detection and sensitivity characteristics in various situations. In HPLC detection,
the benefits are that they can differentiate between all of the chemical components in a
mixture. Non-disastrous operational simplicity and accuracy; and non-disastrous. Some of the
most often used HPLC detectors are as follows:

1.3.5.1 Refractive index detectors

The refractive index detector measures the index of refraction difference in the middle of the
eluent that passes all over the stream cell and the uncontaminated movable phase. It's referred

CHAPTER-1 page 16
to as a universal detector. It functions on an isocratic basis. As a result, it is especially sharp
to replace in pressure, stream, and normal temperature, and it cannot be utilized for gradient
extraction. This detector comes in handy when it comes to identifying nonionic substances.
UV and fluorescence detectors are unable to detect these substances because they do not
absorb ultraviolet light or emit fluorescence.

1.3.5.2 UV (ultraviolet) detectors

The UV-visible absorbance detector is the most frequent type of detector utilized in HPLC
nowadays. UV detectors are utilized in HPLC to notice and recognize mixtures by displaying
a spectrum in the visible or UV range (between 190 and 600 nm). A emit lamp with a
wavelength of 190-380nm is utilized in UV detectors to use deuterium as a light source.
When components are identified at wavelengths greater than 380 NM, a tungsten light is
utilised in addition. The light from the lamp scatters according to its wavelength when it is
focused on the grating. The diffraction grating's angle is tuned to test the desired wavelength.
The brightness then goes via the reflector, dividing into two beams, one of which travels
along the flow cell and the other via the reference-side light-receiving section. The difference
in light intensity in the middle of the brightness from the standard cell and brightness from
the stream cell can be quantified. As a result of the procedure, the absorbance was obtained.
The UV detector detects all of the components with excellent sensitivity. Figure 1.17 shows
a schematic of the UV detector.

Figure 1.17: shows a diagrammatic representation of a UV-VIS detector optical

apparatus

Fixed wavelength: only detects one wavelength, which is commonly 254nm.

Wave length variation: The UV detector is capable of detecting a wide variety of

wavelengths, but one at a time.

CHAPTER-1 page 17
Diode array: The brightness from the origin is sucked by the stream cell and then scattered
by a diffraction grating into different wavelengths. The dispersed light is detected using a
PDA (Photodiode Array). The CPU scans each photodiode, which gathers a different narrow
wavelength band. The readings are displayed on the monitor. Figure 1.18 shows a schematic
diagram of a diode array detector.

Figure 1.18: PDA optical system is depicted in diagrammatic form.

1.3.5.3 Fluorescence Detectors

A fluorescence detector is utilized to examine mixtures with fluorescence characteristics. The

usage of this detector is limited due to the fact that only a few substances have fluorescence
qualities. It is the most sensitive detector of the HPLC detectors now available. This detector
can identify a single analyte in a flow cell. For heavy UV absorbing materials, illumination
perceptivity is 10 to 1000 times larger than UV detector perceptivity. Due to its spectroscopic
and chemical perticularity, it is particularly selective for analysing chemicals from
complicated matrix. It's most commonly used to measure samples that contain certain
fluorescent species.

1.3.5.4 Electrochemistry-based detectors

Electrochemical detectors detect molecules that undergo oxidation or reduction processes. As

the sample passes between the electrodes, they calculate the difference in electrical potential.

1.3.5.5 Evaporative Light Scattering Detector (ELSD)

The components that do not absorb UV rays are analysed using ELSD detectors. The mobile
phase solvent is pushed through a nebulizer from an HPLC. The mobile phase evaporates and
small particles remain after passing through a heated funnel. These particles scatter light,
which is detected by a photomultiplier, when they travel across a narrow light beam. The

CHAPTER-1 page 18
detector's response is determined by particle size and number. It gives a flat baseline even
with gradient elution.

1.3.6 Microprocessor control and data handling device

Microcomputers are used to combine the signals after they have been seen. The integrator
reports the retention time, peak area, and peak height, making qualitative and quantitative
analysis easier. Using various chromatography software, the obtained data is evaluated and
transformed to a presentable format.

1.4 HPLC applications

1. The pharmaceutical industry

Prepare massive amounts of pure materials.

Complex molecule separation

Quantitative and qualitative research

To look for trace contaminants in the purified chemicals

Pharmaceutical product self-life determinations

2. The environment

It's used to monitor air quality and test drinking water.

To detect trace amounts of pollutants in pesticides and waste oil, such as PCBs.

3. Disease and disorder clinical diagnosis

4. Forensic examination

5. The food processing industry

It is used in the food sector to detect, separate, and analyse additives, preservatives, proteins,
vitamins, and amino acids for quality checks and analysis.

6. Steroid determination in urine, sweat, and blood

It's used in bioinformatics and DNA fingerprinting, for example.

1.5 Approaches to the development of analytical method

CHAPTER-1 page 19
The reverse phase chromatographic separation technique is the most widely used analytical
technology in the pharmaceutical (13) business. Reverse phase chromatography is widely
utilised for medicinal substance assay and impurity profiling. In a Quality by Design (QbD)
(14) setting, the importance of technologies such as HPLC and UPLC is expanding. The
method's requirements are usually determined by the drug's stage of development. The
emphasis in the early stages is on quick turnaround and high throughput, whereas in the latter
stages, the emphasis is on high throughput and low turnaround time. Pharmaceutical
manufacture must be a simple procedure that is also sturdy, tough, and technically simple.
The following parameters must be closely assessed while developing simple, robust, and
tough analytical procedures.

 Literature gathering
 Chemical design pH and pKa values of substance
 Diluent choice/Sample dispersible
 Column choice
 Detector choice
 Mobile phase choice

1.6.1 Gathering of literature

Before beginning the technique creation process, the same or similar types of
pharmaceuticals can be found in pharmacopoeias such as IP, USP, JP, EP, and
chromatographic publications and copyright, among others. If the needed technique is
useable, assess its applicability to satisfy or adjust needs, such as solve probable adulterants
during the preparation phase, one of which is adulterations, as well as process adultarations
and deterioration. Gather all contaminants, normal, and test mixtures, as well as data on
physico-chemical effects, at each step.

1.6.2 Chemical make-up

Beginning substances, byproducts, middle products, adultarants, and deterioration

results, also their polarity, are compared to the structure of the medicinal ingredient (whether
they are polarised more or < the compound of interest). It is feasible to determine if a particle
is neither acidic nor basic, acidic, or alkali depend on the functional group of the molecule.
The pH value of the movable phase can be elected depend on the compound's nature. If the
chemical is acidic, acidic mobile phases are preferred. A low pH and a basic mobile step are

CHAPTER-1 page 20
desirable if the chemical is basic. A neutral mobile step is ideal for neutral substances. The
elution of a substance is determined by its polarity. Under reverse phase circumstances, a
molecule with a higher hydrophobicity will be preserved for a longer period of time. There is
a chance that non-aqueous conditions will be required. If a substance is ionic, it is hydrophilic
(less hydrophobic) in nature and hence has a shorter retention time. For the separation of
these types of chemicals, HELLIC columns are used.

1.6.3 Compound pH and pKa values

The nature and polarity of a substance can be determined using pH and pKa values. When the
movable phase pH is equal to the pKa of the substance, the chemical is 50% ionised. The
transfer in degree of ionization is approximately 90percent if pH is one part away from the
substance's pKa result; it is approximately 99% if pH is two parts away from the substance's
pKa value; and it is approximately 99.9% if pH is 3 parts away from the analyte's pKa value.
The rule of two pHs, which is used to estimate the degree of ionisation, was thus devised.
Most pH-related alterations occur within 2 units of the pKa value. During this range, the
compound either has an ionic or non-ionic structure, and the chemical retention duration
remains constant regardless of pH.

1.6.4 Sample solubility/diluent choice

In movable phase results, movable phase-organic combination, and water-organic

combination, the solubility of all components in pharmacological compounds is investigated.
The movable phase is a secure alternative for test diluents since it detaches baseline noise,
negative peaks, and ghost peaks. All of the components must be totally soluble to obtain a
simple solution.

1.6.5 Selecting Columns

A number of different column-styles are offered in the business market place. To

guarantee consistency, we must look at lot to lot and batch to batch reproducibility before
choosing a column. All internal diameter, surface region, particle size, and carbon load must
be tested in order to ensure system compatibility. The non-polar column and the polar period
of mobility were utilized in the RP-chromatographic analysis. Columns C18, C8, C4, cyano,
phenyl, and amino can be used to counteract a more polar mobile phase, i.e. C18, C8, C4,

CHAPTER-1 page 21
cyano, phenyl, and amino. Mobile phase polarity is lower in an NP-chromatographic analysis.
Normal phase chromatography uses normal phase, cyano, phenyl, silica, and chiral columns.

For very good resolution and retention, start with C8 or C18 columns for reverse phase
chromatography. The ideal type of phases for development of early quick methods are C8
and C18, which are both C8/C18 phase complexes. This figure shows the polarity and
hydrophobicity of columns, as shown in Figure 1.19.

Figure 1.19: Shows the polarity order of different static phases.

1.6.6 Selecting a Detector

In liquid chromatography, the detector is a crucial component. For precise

determination and selective separation, it should be carefully chosen. The most important
component is determined by the progress of a feature separation that may be inspected and
registered automatically. For solutes of different orders of magnitude, a satisfactory detector
response is linear. It should be very reproducible, dependable, and stable. It should have a

CHAPTER-1 page 22
low dead volume to reduce extra-column band broadening. The suitable detection technology
must be chosen depends on the type of the molecule and its makeup [Table 1.02].

Table 1.02: Detector selection depending on chemical application

Detection technique Molecule structure and nature

UV Compounds containing chromophores are

referred to as chromophores.

Fluorescence A characteristic of certain substances is used

for fluorescence.

Electrochemistry-based detectors When it comes to chemicals that are easily

oxidised

RI detectors It's a detector that you can take with you

everywhere you go. It's employed for
chemicals that don't need a lot of sensitivity
and don't have a chromophore. For gradient
elution, however, it is unsuccessful.

Evaporative Light Scattering Detectors These are superior to the RI detectors used in
(ELSD) gradient elution because they have a higher
sensitivity. A universal detector is another
name for it.

When a compound lacks chromophores, alternative detectors such as RI, GC, or ELSD are
used. Record the spectra of all the molecules in a component whether the detecting
technology is a UV-Visible spectrophotometer or a photodiode array (PDA). Select the
product's absorption limit by considering all of the product's elements. Other issues, such as
component intermediates, degradations, and process-related contaminants, must be addressed.
supplies of raw materials If any component acts differently, conduct a dual wavelength
analysis and compare data from different scanning ranges. Peak purity with all elements must
be ensured, and the purity threshold must be greater than the purity angle.

CHAPTER-1 page 23
1.7 VALIDATION OF ANALYTICAL METHOD

Validation is a critical component of any material presented to international regulatory

agencies. Unless using methods published in any recognised standard references or the
applicable pharmacopoeia, procedures must be validated. All testing analytical methods (9)
must be evaluated for system suitability in actual use conditions and must be accurately
recorded. All analytical techniques must be validated, according to ICH recommendations
(15) for addressing Q2 (R1), which represents analytical processes for validation. Excluded
methods should be validated. The main goal of the International Conference on
Harmonization is to obtain agreement between the European Community, the United States
of America (EC) and Japan, the three biggest geographic markets, on the technical
requirements for product registration. According to the United States Pharmacopoeia (USP)
(16) and the United States Food and Drug Administration (US FDA), the current method
validation guidance documents used by the United States Pharmacopoeia (USP) and the
United States Food and Drug Administration (US FDA) both refer to the International
Conference on Harmonisation (ICH) guidelines. Linearity, specificity, reliability, detection
limit, range, quantitation limit, exactness, and resilience are some of the most commonly used
typical validation characteristics for many types of tests. Analytical method validation (17)
also considers the applicability of the system and the stability of analytical solutions.

1.7.1.1 Selectivity
Both terms selectivity and precision are utilized interchangeably, as in "selectivity is
precision with selection." Selectivity is a term that refers to a process that produces a
response for a wide range of possible chemical entities, whereas specificity is a process that
produces a response for a single analyte. the answer stands out from the rest Selectivity is a
better term to use, because few analytical procedures respond to a single analyte. Specificity
is defined as the capability to measure the analyte of interest with complete certainty while
simultaneously excluding all other potentially interfering substances from the sample matrix.

CHAPTER-1 page 24
The measure of how intense other interferences are is called specificity. The peak response is
a result of a single analyte, which means there are no other components present. Tailing
factor, resolution, and plate count are used to calculate it. The resulting spectra are also
compared to determine peak homogeneity using a contemporary PDA device, which
calculates peak homogeneity by measuring the spectra obtained over a peak. To accurately
measure the purity of a sample, make sure that the concentration of the sample is in the
acceptable range.

1.7.2 Precision

It refers to the precision of the analytical process, as well as the consistency of the expected
point of reference and the result calculated. The proportion of component recovered by test or
spiking in a blind sample is the metric. components or contaminants. For the test of a drug
product, It is calculated by using known concentrations of analytes in combination with
predetermined formulations. A drug sample is tested using a comparison method or a method
which is highly similar to the second. Quantification of impurities is measured using
impurity-spiked samples.

1.7.3 Accuracy

Under normal operational conditions, it defines how reproducible an analytical methodology

is. Precision is commonly stated as a percent RSD when a statistically significant number of
samples are considered. In accordance with ICH standards, precision is carried out in three
stages. They are described as having intermediate accuracy, reproducibility, and repeatability.
The repeatability of an analytical method performed under the same conditions within a short
time period is referred to as an analytical method's effect. To have precision, at least 9
determinations (concentrations 3 times) are performed using the analytical technique's
defined range, or at least 6 determinations (concentrations of 100 percent) are performed
using the target concentration. Reproducibility refers to the level of uniformity among
laboratories. The outcomes found in random events, such as within-lab variation, facilities,
various days and so on, reflect intermediate precision. Confidence intervals, coefficient of
variance, relative standard deviation, and standard deviation should all be included in the
documentation research.

1.7.4 LOD (detection limit)

CHAPTER-1 page 25
An amount of analyte which can be detected but not quantified in a specimen that has been
quantified properly is the smallest that can be detected. A determination of whether an
analyte is above or below a certain threshold level is a type of limit test. If you have a S/N
ratio of 3 or 2:1, you'll use the LOD calculation.

1.7.5 Limit of Quantification (LOQ)

Under the given conditions of the technology, the minimal concentration of an analyte in a
specimen that can be detected with sufficient exactness and reliability is found. The LOQ,
which is calculated using the S/N ratio of 10:1, is widely employed. It is calculated by
calculating the lowest level of reliability and exactness that can be achieved for the analyte,
and measuring known concentrations of the analyte. When you calculate LOD and LOQ
values, you can use the linear regression method to obtain estimates for them. The device
response, y, is assumed to be linearly related to the nominal concentration, x, over a certain
concentration range. It's possible to display it in a variety of ways.

y=a+bx

The letter ‘a' represents an interception in this example.

When b is the slope of the line, and

x is the explanatory variable,

y is the dependent variable.

this model can estimate LOD, as well as LOQ.

LOD=3 Sa /b,

LOQ=10 Sa /b,

Where b denotes the steepness of the curve.

The standard deviation of the response is denoted by Sa.

Use this approach in any context. When research is conducted in a setting where there
is no background noise, it is most productive.

CHAPTER-1 page 26
1.7.6 Range/Linearity

The term linearity refers to the test results being directly proportional to the concentration of
the analyte in the specimen. Most commonly, linearity is defined as the slope of the
regression line's regression equation's standard deviation. The range of analytical methods are
defined as the distance between the lowest and highest amounts (concentration) of the analyte
for which the exactness, reliability, and linearity of the method have been exhibited. It is
possible for ranges to be given in the same units as the procedure results. There are five
concentration levels recommended by the International Conference on Harmonization, as
well as minimum ranges (ICH). When screening tests have a minimum specific range of 80 to
120 percent of the target concentration, this is known as a minimum specific range screening
test. When screening tests have a minimum specific range of 80 to 120 percent of the target
concentration, this is known as a minimum specific range screening test. To ensure the
correct reading, a range needs to be specified that reflects the total number of harmful or
powerful contaminants that need to be tracked.

1.7.7 Ruggedness

Ruggedness is defined as the degree of reproducibility of results obtained under various

settings, as measured by percent RSD, according to the United States Pharmacopeia
(percentage of the relative standard deviation). Instruments, analyzers, laboratories,
experimental periods, and chemical reagents are among the circumstances listed above. The
ICH guideline on definitions and nomenclature included no mention of toughness. Instead of
covering the toughness topic, an ICH prefers to discuss reproducibility (precision).

1.7.8 Robustness

It's described as the ability to remain unaffected by tiny changes in the method's analytical
parameters in the face of adversity. Process variables like pH, percent organic solvent,
temperature, or ionic strength affect the results, and those variables can be adjusted to
manipulate the results. Designers should keep in mind the principles of the International
Conference on Harmonization, and they should ensure that their systems are as robust as
possible early in the design process (ICH). It is important to evaluate various equipment, such
as analyzers, as well as the stability of analytical solutions before making any decision. if the
results of a process or further measurements are sensitive to changes in analytical technique

CHAPTER-1 page 27
parameters, the procedure or measurements should be followed under acceptable analytical
technique specification changes.

1.8 Assay method indicating stability

Patients who use a pharmaceutical medicine for a specific ailment expect it to be effective
and safe. The product must retain its potency, purity, identity, and quality for the duration of
its market availability, as required by international pharmaceutical regulatory organisations.
Due to the use of the marketing application, agencies want to see data on product stability in
order to help support the product's suggested expiration date. So the agency needs to conduct
stability studies in order to do so. The ICH requires medicinal compound stability studies to
be conducted using hydrolytic, oxidative, thermal, and photolytic stress tests.

1.8.1 Degradation under duress

The forced degradation research is a crucial component of method development; these

investigations are carried out to define intrinsic stability features. Deliverables for stress
degradation must be focused on development activities rather than degradant isolation and
identification. It's a process that involves degrading pharmaceuticals and other compounds
under more severe settings than accelerated settings. As a result, degradation products are
produced in order to measure the analyte's stability. Forced degradation investigations are
carried out if a stability indicating approach is required. Degradation studies must be done
whenever methodologies, formulations, or processes are changed. Hydrolytic (acidic and
basic degradation), thermal, oxidative, and photolytic degradation were applied to the drug
product or drug material in order to establish a stability indicating approach. As far as stress
studies for all of the pharmaceuticals being looked at are concerned, they are all done in the
same conditions, but only with time and temperature varying from drug to drug.

 Thermolytic deterioration,
 Hydrolytic deterioration,
 Oxidative deterioration and
 Photolytic deterioration

are the most well-known investigations of forced deterioration.

1.8.1.1 Thermolytic degradation

CHAPTER-1 page 28
 This test was carried out by melting a substance to determine its melting point. If this
is the case, the sample will be strained at 70 °C or 40 °C below its melting point at a
temperature of 150 °C. This component undergoes strain at a temperature of 105°C, having a
melting point greater than 150°C. To achieve the desired deterioration, heat the sample to a
high temperature (10 percent to 20 percent ).

1.8.1.2 Degradation by hydrolysis

The breakdown produced by hydrolysis is called hydrolytic degradation. A simple and

acidic hydrolysis process is one type of hydrolysis process, while an acidic and simple
hydrolysis process is the other. Acidic and basic conditions as well as water should be present
for more hydrolytic reactions to occur. Be sure to test the drug molecule of interest for its
ability to dissolve in water, as hydrolysis is done in water. Molecules that are hydrophobic
won't dissolve in water, so you need to dissolve them with a co-solvent. Co-solvents like
acetonitrile and methanol are frequently used. For acidic degradation, the samples were
treated with a suitable concentration of hydrochloric acid and refluxed for 12 hours, or until a
degradation of 1-20% was achieved. It was neutralised before injecting the stress solution.
The sample was treated with an appropriate strength sodium hydroxide and refluxed for 12
hours, or until a degradation of about 1-20% was achieved. It was neutralised before injecting
the stressed solution.

1.8.1.3 Degradation due to oxidation

Treated the sample with hydrogen peroxide (H2O2) or a mixture of KMnO4 and K2Cr2O7 at
room temperature, and then analysed the solution.

1.8.1.4 Photolysis induces chemical degradation

A UV light illuminates the sample with an average of 1.2 million Lux hours and 200 Watt
Hrs/Sq. Meter for an exposure time of one hour. The sample is then collected and analysed at
regular intervals.

1.9 Study of system suitability

CHAPTER-1 page 29
 The analytical validation of the method and evidence of correct analytical system
function must be provided prior to beginning laboratory studies. The system's suitability is
determined by a replication assessment of the standard or reference solution. The system
precision value defines the upper limits for each of the tailing factor, theoretical plates, RSD,
and resolution parameters, but if these limits are not exceeded, the system is suitable.

1.9.1 Resolution [Fig 1.20]

Chromatography peak distance calculation uses Resolution. In quantitative estimations,

resolving the finest peaks is the main objective. Affinity for the stationary phase governs the
separation between closely spaced peaks. Increasing the length of the column, changing the
static phase particle size, and changing the polarity of the mobile phase is all effective at co-
eluting the co-eluting chemicals. It is deemed complete if the resolution is equal to or greater
than 1.5.

Figure 1.20: Resolution

2 ( t 2−t 1 )
Rs=
W 1 +W 2

The peak widths W1 and W2 are obtained from the tangent points where the baseline
intersects, and the retention times for peak2 and peak1 are also noted.

1.9.2 Asymmetry or Tailing Factor (AS) [Fig 1.21]

There are numerous factors which influence the shape of the chromatographic peak,
which results in an asymmetrical shape. Peak deformation causes peak tailing or peak
fronting. When a component has more than one retention mechanism, peak tailing occurs.

CHAPTER-1 page 30
When the stationary phase has been capped, or the pH of the movable phase has been
adjusted, the number of discrete components can be decreased.

Figure 1.21: Asymmetry or Tailing factor (AS)

Asymmetry factor

A s=B/ A

Peak widths A and B represent ten percent of the peak height of the tallest peak

Since Ideal peak, AS =1. 0, AS (or equivalent amount) must be between 0.90 and 1.10.

When AS is greater than or equal to 1.2, trailing is apparent

Tailing is calculated by applying the USP formula that includes widths A and B, which are
each at 5% of peak height, as shown.

A +B
T=
2A

For the specification of tailing, T ≤2.00 is acceptable.

1.9.3 Precision

Replica injections of compound preparation are compared to verify the precision

requirements. There are a total of six copies. If the required number is larger than 2,
compound data injections are utilized to determine the percent RSD. As long as the required

CHAPTER-1 page 31
value is less than 2.0, five replicate injections of data are used to determine the percentage of
random variation.

1.9.4 Theoretical plates

This assumes that chromatography columns have many independent layers, known as
theoretical plates. These imagined layers have movable and stationary phases that balance
each other out. This molecule moves the equilibration movable phase from one plate to the
next in the column by moving the equilibration mobile phase. Plate count is a measure of
how effective the column is (N). Plates should not be fewer than 2000 in total.

[ ]
2

( t R ) or N=5.54 ( R )
[ ]
2 t
N=16 W1
W
2

Where, W is the peak width at baseline,

t R is the retention period, and

W (1/2) is the peak width at half height.

1.9.5 Retention factor (k) [Fig 1.22]

One-and-a-half the ratio of time spent in the mobile phase to time spent in the stationary

phase is known as the retention factor.

' t R −t M
k=
tM

CHAPTER-1 page 32
 Figure 1.22: Retention factor (k′)

Ideally, k' is greater than or equal to 2.0.

When the k' value is high, the ketonate is higher in the compound holding the column.

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development, 2nd edition, 1997, pp. 1-3.

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3. Willard, H.H., Merritt, L.L. Jr., Dean, J.A., Settle, F.A. Jr., Instrumental methods of
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4. B.K.Shar., Instrumental methods of chemical analysis, 28 th edition, pp.2086-385,

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5. Gritti Fabrice, Guiochon Georges. The van Deemter equation: Assumptions, limits,
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12. World Health Organization. Active Pharmaceutical Ingredient. Geneva, Switzerland,
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