Sys Rev Pharm 2021; 12(8): 450-454 Review Article

A multifaceted review journal in the field of pharmacy

E-ISSN 0976-2779 P-ISSN 0975-8453

A Review: Analytical Method Development and Validation

Ramole Rina, Mohini Baile*, Ashish Jain
Department of Quality Assurance, Shri. D. D. Vispute College of Pharmacy and Research Center, Mumbai, India

Article History: Submitted: 21.05.2021 Accepted: 04.06.2021 Published: 11.06.2021

ABSTRACT bustness. Validation should be done as per regulatory

guidelines such as ICH guidelines. This article was pre-
Development and validation of analytical method play
pared with an aim to review analytical method develop-
an essential role in the discovery, development and
ment and validation.
manufacturing of pharmaceuticals. Every year, number
of drugs entered into the market; hence it is mandatory
to develop newer analytical methods for such drugs. Keywords: Analytical method, Spectroscopy, UV-VIS
After the development, it becomes necessary to vali- spectroscopy, Chromatography, HPLC, Method devel-
date the new analytical method. Method development opment, Validation
is the process which proves that the analytical method
is acceptable for use. Validation of analytical method
gives information about various stages and parame- *
Correspondence: Mohini Baile, Department of Quali-
ters like accuracy, precision, linearity, Limit Of Detec- ty Assurance, Shri. D. D. Vispute College of Pharmacy
tion, Limit Of Quantification, specificity, range and ro- and Research Center, Mumbai, India, E-mail: b.mohini@
yahoo.co.in

INTRODUCTION Classical methods are divided into 3 main types are: a) Sep-
Analytical chemistry is a branch of chemistry which deals with aration of analyte, b) Qualitative analysis and c) Quantitative
identification of components (qualitative) and determination of analysis. Separation of analyte includes extraction, distillation,
quantity of components (quantitative) of substances or samples precipitation and filtration. Qualitative analysis includes boil-
or mixture. There are two types of analysis, one is qualitative anal- ing point, freezing point, colour, odour, density, reactivity and
ysis and another one is quantitative analysis. In qualitative analy- refractive index. Quantitative analysis includes gravimetric an-
sis, there is identification of components or analyte of mixture or alysis and volumetric analysis.
sample is carried out. In quantitative analysis, there is determina- Instrumental methods are divided into four main types are: a)
tion of amount of components or analyte of mixture or sample is spectroscopic methods, b) electrochemical methods, c) chro-
carried out (Kenkel J, 2003). Analytical data is required not only matographic methods and d) other techniques.
in chemistry but also in other sciences like biology, zoology, arts Spectroscopic methods include ultraviolet-visible spectros-
such as painting and sculpture, archaeology, space exploration copy, infrared spectroscopy, Raman spectroscopy, atomic
and clinical diagnosis. Important areas of application of analytical absorption spectroscopy and atomic emission spectroscopy,
chemistry are quality control in manufacturing industries, mon- x-ray spectroscopy and nuclear magnetic spectroscopy.
itoring and control of pollutants, clinical and biological studies,
Electrochemical methods include Potentiometry, Coulometry
geological assays, fundamental and applied research (Kissinger
and Voltametry.
PT, 2002).
Chromatographic methods include column chromatography,
ANALYTICAL METHOD paper chromatography, thin layer chromatography, high per-
Analytical method includes use of a specified technique and formance liquid chromatography, gas chromatography and
detailed-stepwise instructions which are used in qualitative, modern methods (LC-MS, GC-MS, LC-MS-MS, GC-MS-MS,
quantitative or structural analysis of a sample for one or more LC-NMR and GC-NMR).
analytes (Kissinger PT, 2002). Other techniques include x-ray methods, radioactivity, mass
Analytical methods are mainly classified into two types: Clas- spectrometry, optical methods (Refractometer, optical ro-
sical methods and Instrumental methods (Figure 1) . A meth- tation) and thermal methods (Thermogravimetry, differen-
od in which the signal is proportional to the absolute amount tial thermal analysis and differential scanning calorimetry)
of analyte is called classical method. A method in which the (Ravisankar P, et al., 2015; Jeffery GH, 1989).
signal is proportional to the analytes concentration is called
INTRODUCTION TO SPECTROSCOPY
instrumental method (Harvey D, 2000).
Spectroscopy is the study of interaction of electromagnetic
radiation with matter. These interactions involve absorption
and emission of radiation (energy) by the matter. Spectroscopy
are of two types, absorption spectroscopy and emission spec-
troscopy. The study of electromagnetic radiation absorbed by
the sample, in the form of spectra is called absorption spec-
troscopy (UV-visible, IR, NMR, microwave and Radiowave
spectroscopy). The study of electromagnetic radiation emitted
by the sample, in the form of spectra is called emission spec-
troscopy (flame photometry and fluorimetry). Spectroscopy
is useful for the study of atomic and molecular structure and
used in the analysis of a wide range of samples. Atomic spec-
troscopy is the study of interaction of electromagnetic radia-
Figure 1: Classification of Analytical Methods tion with atoms, changes in energy takes place at atomic level

450 Systematic Review Pharmacy Vol 12, Issue 8, Mar Apr, 2021
 Rina R : A Review: Analytical Method Development and Validation

(e.g. atomic absorption spectroscopy and flame photometry). Molecu- • Ion exchange chromatography
lar spectroscopy is the study of interaction of electromagnetic radiation • Molecular exclusion chromatography
with molecules, changes in energy takes place at molecular level (e.g.
2. Based on chromatographic bed shape
ultraviolet and infrared spectroscopy) (Chatwal GR and Anand SK,
2002). • Column chromatography
UV-VIS spectroscopy • Planar chromatography
In UV-visible spectroscopy, the amount of light absorbed at each wave- • Paper chromatography
length of UV and visible region of electromagnetic spectrum is meas- • Thin layer chromatography
ured. This absorption spectroscopy uses electromagnetic radiations • Displacement chromatography
between 200 nm to 800 nm and is divided into the ultraviolet (UV,
3. Techniques by physical state of mobile phase
200-400 nm) and visible (VIS, 400-800 nm) regions (Kumar S, 2006).
• Gas chromatography
The principle of UV-Visible spectroscopy is based on the absorption of
ultraviolet light or visible light by sample or chemical substance which • Liquid chromatography
results in the production of different spectra. When a molecule absorbs • Affinity chromatography (Luxminarayan L, et al., 2017).
UV radiation, the electron present in that molecule undergo excitation, HPLC
this causes transition of electron within a molecule from a lower level
to a higher electronic energy level and the ultraviolet emission spectra HPLC stands for high performance liquid chromatography or
arise from the reverse type of transition. Most commonly used solvents high-pressure liquid chromatography. HPLC can separate, identify and
in UV spectroscopy are water, methanol, ethanol, ether, chloroform, quantify the compounds present in any sample which can be dissolved
carbon tetrachloride, cyclohexane and dichloroethane. Applications of in liquid (Chawla G and Chaudhary KK, 2019).
UV spectroscopy are detection of functional groups, detection of con- The main principle of liquid chromatography is adsorption. It is a chro-
jugation, detection of geometrical isomers and detection of impurities matographic technique in which mobile phase is liquid. Sample is in the
(Chatwal GR and Anand SK, 2002). form of liquid solution. Sample is injected into a column of a porous
material (stationary phase) and a liquid phase (mobile phase). Sample
Instrumentation of UV-Visible spectroscopy
move through the column with mobile phase by high pressure deliv-
A. Radiation sources: Most commonly used radiation sources are ered by a pump. Sample components travel according to their affinity
tungstan lamp, hydrogen discharge lamp, deuterium lamp, xenon dis- towards the stationary phase. The component which has more affinity
charge lamp and mercury arc (Figure 2). towards the stationary phase travels slower. The component which has
less affinity towards the stationary phase travels faster. The components
are separated from each other (Vidushi Y and Meenakshi B, 2017). The
most common solvents used for HPLC are n-hexane, methylene chlor-
ide, chloroform, methyl-t-butyl ether, Tetrahydrofuran (THF), Isopr-
opanol (IPA), Acetonitrile (MeCN or CAN), Methanol (MeOH) and
water (McPolin O, 2009). Fundamental chromatographic parameters
are efficiency (number of theoretical plates), retention factor, selectiv-
ity, resolution and pressure (Ravisankar P, et al., 2015). Applications of
Figure 2: UV-Visible spectroscopy HPLC are chemical separation, purification and identification. Other
applications of HPLC include pharmaceutical applications, environ-
B. Wavelength selector: The monochromator is used to disperse the mental applications, forensics, clinical, food and flavour (Figure 3)
radiation according to the wavelength. The basic elements of a mono- (Malviya R, et al., 2010).
chromator are an entrance slit, a dispersing element and an exit slit.
C. Sample cell: In UV-Visible spectroscopy sample containers are used
to hold liquid sample are called as cells or cuvettes. Cuvettes are made
from quartz.
D. Photo detector: Most commonly used detectors in UV spectropho-
tometer are barrier layer cell, photocell and photomultiplier tube.
E. Readout device: The output from the detector is suitably amplified
and then displayed on a readout device (Chatwal GR and Anand SK,
2002).
INTRODUCTION TO CHROMATOGRAPHY
Chromatography is a physicochemical method for separation of mix- Figure 3: HPLC system
ture of compounds. Chromatography is a method of separation of mix-
Instrumentation of HPLC
ture of compounds into individual components between two phases,
a stationary phase and a mobile phase (Luxminarayan L, et al., 2017). Components of the HPLC system:
Chromatography is classified as follows: A. Solvent reservoir, mixing system and degassing system
1. Based on interaction of solute to stationary phase B. High pressure pump
• Adsorption chromatography C. Sample injector
• Partition chromatography D. Column
E. Detector

451 Systematic Review Pharmacy Vol 12, Issue 8, Mar Apr, 2021
 Rina R : A Review: Analytical Method Development and Validation

F. Data recording system 4) Residues (Micro analysis)

1. Solvent reservoir, mixing system and degassing system: Solvent 5) Impurity profiling
reservoir stores the solvent (mobile phase). These are glass or stain- 6) Degradation studies
less-steel containers. The most common type of solvent reservoir is
7) Herbal products (Chauhan A, et al., 2015)
glass bottle (Jena AK, 2011). In addition to delivery of mobile phase,
the pump must mix solvents with high accuracy and high precision. Steps involved in method development:
Two types of mixing unit are low pressure mixing and high pressure 1) Standard analyte characterization:
mixing (Agilent Technologies, 2016). Degassing system removes en- • All the known information about analyte and its structure is collected
trapped air bubbles from the solvent. Degassing is done by degasser for example physical and chemical properties.
techniques are ultra-sonication and filtration (Jena AK, 2011).
• The standard analyte with 100% purity is received. Proper storage
2. High pressure pump: The role of pump is to force a liquid and give condition is arranged such as freezer, refrigerator and desiccators.
a specific flow rate. Flow rate is expressed in millilitres per minute
• Estimation of multiple components from the sample matrix are ana-
(ml/min). Normal flow rate is 1-2 ml/min. Pump pressure range is
lyzed, the number of components are considered, data is compiled and
6000-9000 psi (400-600 bar) (Chawla G and Chaudhary KK, 2019).
the availability of standards is determined for each component.
Commonly used pump types are constant pressure pump, syringe type
pump and reciprocating piston pump (Hamilton RJ, Sewell PA, 1982). • Those methods (Spectroscopic, HPLC, GC, MS, etc.) are considered
only, which are suitable with sample stability (Ravisankar P, et al.,
3. Sample injector: The liquid sample is introduced into the mobile
2014).
phase by sample injector. Sample valve come between the pump and
the column (Jena AK, 2011). An injector (auto sampler) is able to in- 2) Method requirements: Requirement of analytical methodology is
ject the sample into the continuous flowing mobile phase stream that necessary to establish the analytical figures of advantage such as linear-
carries the sample into the HPLC column. Typical sample volumes are ity, precision, accuracy, Limit Of Detection, Limit Of Quantification,
5-20 microliters (µl) (Chawla G and Chaudhary KK, 2019). Two types specificity, selectivity and range etc. are marked (Ravisankar P, et al.,
of injector are manual injector and automatic injector (Hamilton RJ, 2014).
Sewell PA, 1982). 3) Literature survey and prior methodology: All types of information
4. Column: Column is a place where actual separation of components (Physical properties, chemical properties, solubility, manufacturing,
takes place. Column is made up of stainless steel. It is 5-25 cm long related analytical methods etc.) regarding the analyte are obtained
and 2-4.6 cm internal diameter (Chawla G and Chaudhary KK, 2019). by doing literature survey by referencing books, journals, pharmaco-
poeias etc. Chemical Abstract Service (CAS) automated computerized
5. Detector: The detector can detect the individual component that
literature searches are also helpful for literature survey (Ravisankar P,
elute from the column and convert the data into an electrical signal
et al., 2014).
(Chawla G and Chaudhary KK, 2019). Types of detector used are of
two types, specific detectors and bulk property detectors. Specific de- 4) Selecting a method: The methodology is developed by using the in-
tectors include UV-VIS detector, photo diode array detector, fluor- formation obtained from the literature. The method is being revised
escence detector and mass spectrometric detector. Bulk property de- where necessary. Few times, there is a need to include extra instrumen-
tectors include refractive index detector, electrochemical detector and tation to reproduce, modify, validate or improve available methods for
light scattering detector (Hamilton RJ, Sewell PA, 1982). samples and analytes.
6. Data recording system: The output is recorded as a series of peaks If there is no any established method for analyte in the literature, then
and the area under the peak can be calculated automatically by the such compounds are searched which are identical in chemical proper-
computer linked to the display (Malviya R, et al., 2010). ties and structure of analyte (Ravisankar P, et al., 2014).
Analytical method development 5) Proper instrumental arrangement and initial studies:The necessary
equipment must be set up. Installation Qualification (IQ), Operational
Analytical method development is the activity of selecting an accurate
Qualification (OQ) and Performance Qualification (PQ) are verified by
assay procedure to find out the composition of a formulation. Develop-
using Standard Operating Procedures (SOP’s). Every time new things
ment of analytical method is the process which is used to prove that an
(e.g. solvents, filters and gases) are used. For example, method develop-
analytical method is suitable for use in laboratory. Analytical methods
ment is never initiated with previously used HPLC column. The ana-
must be used inside GMP and GLP environments and should be de-
lyte solution, standard solutions of known concentrations and solvents
veloped by using the given protocols and acceptance criteria in the ICH
are prepared. It is necessary to begin with a genuine, known standard
guidelines Q2 (R1) (Chauhan A, et al., 2015).
instead a complex sample matrix. If the sample is very close to the stan-
The requirements for method development are as follows: dard (active drug), after that it is probable to begin work with the actual
1) Qualified analysts sample (Ravisankar P, et al., 2014).
2) Instruments-qualified and calibrated 6) Optimization: A single parameter during optimization is changed at
3) Documented methods a time and the set of terms is different, instead of using a trial and error
approach. There is work has been done from the systematic plan and
4) Reliable reference standards each case is documented in a lab notebook (Ravisankar P, et al., 2014).
5) Sample selection and integrity 7) Proper documentation of analytical figures of merits: The initially
6) Change control (Chauhan A, et al., 2015) determined analytical figures of merit are Limit Of Detection (LOD),
Analytical method development is useful for: Limit Of Quantification (LOQ), linearity, evaluation time, expenses,
1) New process and reactions sample preparation etc. are documented (Ravisankar P, et al., 2014).
2) New molecule development 8) Evaluation of method development along with actual samples:
The prepared solution for analyte needs to be specific, absolute iden-
3) Active ingredients (Macro analysis) tification of the peak interest of the medicament apart from all the dif-

452 Systematic Review Pharmacy Vol 12, Issue 8, Mar Apr, 2021
 Rina R : A Review: Analytical Method Development and Validation

ferent matrix parts (Ravisankar P, et al., 2014). Steps in method validation

9) Determination of percentage recovery of actual sample and dem- 1) Develop a validation protocol, an operating procedure or a valida-
onstration of quantitative sample analysis: The percent recovery of tion master plan for the validation.
spiked, genuine standard analyte into a sample matrix that do not have 2) Define the scope, purpose and applications of the method.
analyte is estimated. Ability to reproduce recovery from sample to sam-
ple has been optimized. If the results are reproducible then it is not re- 3) Define the performance parameters and its acceptance criteria.
quired to obtain 100% recovery. The verification of validity of analytic- 4) Define validation experiments.
al method is done only by laboratory study. Therefore, documentation 5) Verify related performance characteristics of equipment.
of such successful studies is a basic requirement to determine a method 6) Qualify materials, ex. Standards and reagent.
is satisfactory for its desired application (Ravisankar P, et al., 2014).
7) Perform pre-validation experiments.
VALIDATION
8) Adjust method parameters or/and acceptance criteria if required.
Validation is a concept developed in the United States in 1978. The
9) Perform full internal (and external) validation experiments.
concept of validation has been broaden over the years to achieve many
activities like from analytical methods used to control quality of drug 10) Develop SOPs for implementing the method in the routine.
substances and drug products up to computerized systems for clinical 11) Define criteria for revalidation.
trials, process control or labelling. Validation is best seen as a necessary 12) Define type and frequency of system suitability tests and/or Ana-
and prime part of cGMP. lytical Quality Control (AQC) checks for the routine.
The word validation means evaluation of validity or the act of proving 13) Document validation experiments and results in the validation
effectiveness. Validation is a team work involving people from different (Lavanya G, et al., 2013).
branches of plants.
Parameters (components) of method validation
Method validation is a “process of establishing documented evidence”
that provides a high level of guarantee that the product (equipment) 1) Accuracy
will meet the requirements of the desired analytical applications (La- 2) Precision
vanya G, et al., 2013). 3) Linearity
Importance of validation 4) Limit of detection
• Assurance of quality 5) Limit of quantitation
• Minimal batch failure 6) Specificity
• Reduction in rejections 7) Range
• Improved efficiency and productivity 8) Robustness
• Increased output 1) Accuracy: Accuracy is defined as the closeness of the test results to
• Reduced testing in process and in finished goods (Lavanya G, et al., the true value.
2013). 2) Precision: Precision is defined as the measurement of closeness of
Types of validation agreement for multiple measurements on the same sample.
There are four types of validation: The precision is expressed as the relative standard deviation.
1) Equipment validation %RSD = Standard deviation/Mean ×100
a. Design Qualification 3) Linearity: Linearity is the ability of analytical procedure to obtain
a response that is directly proportional to concentration (amount) of
b. Installation Qualification
analyte in the sample.
c. Operational Qualification
Linearity is expressed as the confidence limit around the slope of the
d. Performance Qualification regression line.
2) Process validation 4) Limit Of Detection (LOD): LOD is defined as lowest amount (con-
a. Prospective validation centration) of analyte in a sample that can be detected or identified, not
b. Retrospective validation quantified. LOD is expressed as a concentration at a specified signal:
noise ratio, usually 3:1.
c. Concurrent validation
LOD = 3.3 × S/ SD
d. Revalidation
5) Limit Of Quantitation (LOQ): LOQ is defined as lowest amount
3) Analytical method validation
(concentration) of analyte is a sample that can be quantified. For LOQ,
4) Cleaning validation (Lavanya G, et al., 2013) ICH has recommended a signal: noise ratio 10:1.
Types of analytical procedures to be validated LOQ = 10 × S/SD
• Identification tests 6) Specificity: Specificity is defined as the ability of an analytical meth-
• Quantitative tests for impurities content od to measure the analyte clearly in the presence of other components.
• Limit tests for the control of impurities This definition has following implications:
• Quantitative tests of the active moiety in samples of drug (Lavanya a. Identification
G, et al., 2013) b. Purity tests
c. Assay

453 Systematic Review Pharmacy Vol 12, Issue 8, Mar Apr, 2021
 Rina R : A Review: Analytical Method Development and Validation

7) Range: The range of the method is the interval between upper level 7. Kumar S. Spectroscopy of organic compounds. Cosmic rays.
and lower level of analyte that have been determined with acceptable 2006; 10: 4.
accuracy, precision and linearity. It is determined on either a linear or 8. Luxminarayan L, Sharma N, Viswas A, Khinchi MP. A review on
nonlinear response curve and expressed in the same unit as the test chromatography techniques. Asian Journal of Pharmaceutical Re-
results are expressed. search and Development. 2017; 1-8.
8) Robustness: Robustness is defined as the measurement of capacity of
9. Chawla G, Chaudhary KK. A review of HPLC technique covering
analytical procedure to remain unaffected by small variations in meth-
its pharmaceutical, environmental, forensic, clinical and other ap-
od parameters (Vidushi Y and Meenakshi B, 2017).
plications. Int J Pharm Chem Anal. 2019; 6(2): 27-39.
CONCLUSION
10. Vidushi Y, Meenakshi B. A review on HPLC method development
This article gives an idea that how to develop a method, what is valid- and validation. Res J Life Sci. 2017; 2(6): 178.
ation, importance of validation, types of validation, how to perform
validation process and its parameters to prove that the method is suit- 11. McPolin O. An introduction to HPLC for pharmaceutical analy-
able for its intended use. The primary objectives of development of sis. Lulu. 2009.
analytical methods are for identification, purification and eventually 12. Malviya R, Bansal V, Pal OP, Sharma PK. High performance liquid
to qualification any necessary drug etc. The development of analytical chromatography: a short review. J Glob Pharma Technol. 2010;
methods helps in understanding the critical process parameters and to 2(5): 22-26.
reduce their effects on precision and accuracy. Validation is a necessary
13. Jena AK. HPLC: highly accessible instrument in pharmaceutical
technique in the Pharma sector and that used to ensure that quality
industry for effective method development. Pharm Anal Acta.
work is done in the process which supports the development of medi-
2011; 3.
cine and products.
14. Agilent Technologies. The LC Handbook Guide to LC Columns
REFERENCES
and Method Development. Agilent Technologies. 2016; 16.
1. Kenkel J. Analytical Chemistry for Technicians. Lewis Publishers.
15. Hamilton RJ, Sewell PA. Introduction to high performance liquid
2003.
chromatography. Springer. 1982; 1-12.
2. Kissinger PT. Instant Notes: Analytical Chemistry. Clin Chem.
16. Chauhan A, Mittu B, Chauhan P. Analytical method development
2002; 48(12): 2303.
and validation: a concise review. J Anal Bioanal Tech. 2015; 6(1):
3. Harvey D. Modern analytical chemistry. McGraw-Hill. 2000. 1-5.
4. Ravisankar P, Navya CN, Pravallika D, Sri DN. A review on step- 17. Ravisankar P, Gowthami S, Rao GD. A review on analytical meth-
by-step analytical method validation. IOSR J Pharm. 2015; 5(10): od development. Indian J Res Pharm Biotech. 2014; 2(3): 1183.
7-19.
18. Lavanya G, Sunil M, Eswarudu MM, Eswaraiah MC, Harisudha
5. Jeffery GH. Textbook of quantitative chemical analysis. Longman. K, Spandana BN. Analytical method validation: An updated re-
1989. view. Int J Pharm Sci Res. 2013; 4(4): 1280.
6. Chatwal GR, Anand SK. Instrumental Methods of Chemical
Analysis. Himalaya Publishing House. 2002.

454 Systematic Review Pharmacy Vol 12, Issue 8, Mar Apr, 2021