Analytical methods development and validation play important roles in the discovery, development, and
manufacture of pharmaceuticals. The official test methods that result from these processes are used by quality
control laboratories to ensure the identity, purity, potency, and performance of drug products. This review gives
information regarding various stages involved in development and validation of analytical methods like LC, HPLC,
High performance liquid chromatography (HPLC); Liquid-
liquid extraction (LLE); UV detector; Mass Spectrometry; NMR;
limit of detection (LOD); Limit of quantitation (LOQ)
Analytical method development
Analytical chemistry deals with methods for identification,
separation, and quantification of the chemical components of natural
and artificial materials . The choice of analytical methodology is
based on many considerations, such as: chemical properties of the
analyte and its concentration , sample matrix, the speed and cost of
the analysis, type of measurements i.e., quantitative or qualitative and
the number of samples. A qualitative method yields information of the
chemical identity of the species in the sample. A quantitative method
provides numerical information regarding the relative amounts of one
or more of the analytes in the sample.
The steps of method development and method validation depend
upon the type of method being developed. However, the following
steps are common to most types of projects:
• Method development plan definition
• Background information gathering
• Laboratory method development, it includes various stages
namely sample preparation, specific analytical method,
detection and data processing
• Generation of test procedure
A well-developed method should be easy to validate. A method
should be developed with the goal to rapidly test preclinical samples,
formulation prototypes, and commercial samples. There are five
common types of analytical methods, each with its own set of validation
• Identification tests
• Potency assays
• Quantitative tests for impurities
• Limit test for the control of impurities
• Specific tests
The first four tests are universal tests, but the specific tests such as
particle-size analysis and X ray diffraction are used to control specific
properties of the active pharmaceutical ingredient (API) or the drug
The most widely used methods for quantitative determination of
drugs and metabolites in biological matrices such as blood, serum,
plasma, or urine includes Gas chromatography (GC), High-performance
liquid chromatography (HPLC) [5,6], Thin layer chromatography
(TLC), combined GC and LC mass spectrometric (MS) procedures
such as LC-MS [7,8], LC-MS-MS [9,10], GC-MS [11,12], and GC-MSMS,
techniques like NMR is used for structure identification.
Chromatography in different forms is the leading analytical method
for separation of components in a mixture. The chromatographic
procedure for the separation of substances is based on differences in
rates of migration through the column arising from different partition
of the compounds between a stationary phase (column packing) and a
mobile phase transported through the system . Chromatographic
methods can be classified according to the physical state of the mobile
phase into the following basic categories: gas chromatography (GC),
supercritical fluid chromatography (SFC) and liquid chromatography
(LC). The technique was originally developed by the Russian botanist
M.S. Tswett in 1903 [14,15].
Today TLC is rapidly becoming a routine analytical technique due
to its advantages of low operating costs, high sample throughput and the
need for minimum sample preparation. The major advantage of TLC is
that several samples can be run simultaneously using a small quantity
of mobile phase unlike HPLC thus reducing the analysis time and cost
per analysis [16,17]. An enhanced form of thin layer chromatography
(TLC) is called as High performance thin layer chromatography
(HPTLC) [18,19]. A number of enhancements can be made to the
basic method of thin layer chromatography to automate the different
steps, to increase the resolution achieved and to allow more accurate
Liquid chromatography can be categorized on the basis of the
mechanism of interaction of the solute with the stationary phase as:
adsorption chromatography (liquid-solid chromatography), partition
chromatography (liquid-liquid chromatography), ion-exchange
chromatography (IEC), size exclusion chromatography (SEC) and
Early work in liquid chromatography was based on highly polar
stationary phases, and nonpolar solvents served as mobile phases,
this type of chromatography is now referred to normal-phase liquid
chromatography (NPLC) . Chromatography on bare silica is an
example of normal-phase chromatography. In reversed-phase high
performance liquid chromatography (RP-HPLC), the stationary phase
is nonpolar [21,22], often a hydrocarbon, and the mobile phase is
relatively polar . In RP-HPLC, the most polar component is eluted
first, because it is relatively most soluble in the mobile phase.
The definite break-through for liquid chromatography of low
molecular weight compounds was the introduction of chemically
modified small diameter particles (3 to 10μm) e.g., octadecyl groups
bound to silica in the late 1960s. The new technique became rapidly a
powerful separation technique and is today called high performance
liquid chromatography (HPLC).
HPLC-UV diode-array detection (DAD) [24,25] and HPLC-MS
techniques take advantage of chromatography as a separation method
and DAD or MS as identification and quantification methods. The
HPLC equipment consists of a high-pressure solvent delivery system,
a sample auto injector, a separation column, a detector (UV or DAD) a
computer to control the system and display results.
Ultra performance liquid chromatography (UPLC) is a recent
technique in liquid chromatography, which enables significant
reductions in separation time, solvent consumption and analysis time
as compared to the conventional HPLC [26,27].
The purpose of sample preparation is to create a processed sample
that leads to better analytical results compared with the initial sample.
The prepared sample should be an aliquot relatively free of interferences
that is compatible with the HPLC method and that will not damage the
column . The main sample preparation techniques are liquid-liquid
extraction (LLE) [29,30] and solid-phase extraction (SPE) . In these
methods the analyte of interest was separated from sample matrix, so
that as few potentially interfering species as possible are carried through
to the analytical separation stage.
After the chromatographic separation, the analyte of interest is
detected by using suitable detectors. Some commercial detectors used
in LC are: ultraviolet (UV) detectors , fluorescence detectors,
electrochemical detectors, refractive index (RI) detectors and mass
spectrometry (MS) detectors. The choice of detector depends on the
sample and the purpose of the analysis.
The UV detectors are the most common HPLC detectors since they
are robust, cheap, easy to handle, and since many solutes absorb light
in this frequency range [33,34]. The ordinary UV detector measures
the absorbance at one single wavelength at the time. A diode-array
detector (DAD) can measure several wavelengths at the same time, and
since no parts are moved to change wavelength or to scan, there are no
mechanical errors or drift with time.
DAD detectors have been proposed for various applications, such
as preliminary identification of a steroidal glycoside in seed ,
peptide mapping , assay of sulfamethazine in animal tissues , or
identification of pesticides in human biological fluids .
HPLC with a mass spectrometer detector (LC-MS) [39,40] showed
superior sensitivity and selectivity compared to HPLC-UV methods
Mass Spectrometry: Mass spectrometry (MS) is a widely used
detection technique that provides quantitative and qualitative
information about the components in a mixture . In qualitative
analysis it is very important to determine the molecular weight of
unknown compound and MS is capable of that. MS is also more sensitive
than an UV detector for quantification. An MS detector consists of
three main parts: the ionization source where the ions are generated,
the mass analyzer, which separates the ions according to their massto-
charge ration (m/z), and the electron multiplier (detector). There
are several types of ion sources, which utilize different ionization
techniques for creating charged species. Three popular ionization
techniques are: electrospray ionization (ESI) , atmospheric pressure
chemical ionization (APCI) and matrix-assisted laser desorption
(MALDI). Electrospray is the most widely used ionization technique
when performing LC-MS [44-47].
NMR: Nuclear magnetic resonance (NMR) spectroscopy is a very
powerful tool to determine the structure of compounds [48,49]. This
nondestructive spectroscopic analysis can reveal the number of atoms
and their connectivity’s, and thus the conformations of the molecules.
Near infrared (NIR) spectroscopy is a quick, non-destructive
method that is amenable for spot analysis application. In the last two
decades, it has been increasingly used in pharmaceutical analysis .
“Validation of an analytical method is the process by which it is
established by laboratory studies, that the performance characteristics
of the method meet the requirements for the intended analytical
application - “.
The methods were validated according to International Conference
on Harmonization (ICH) guidelines for validation of analytical
procedures [52,53]. Validation is required for any new or amended
method to ensure that it is capable of giving reproducible and
reliable results, when used by different operators employing the same
equipment in the same or different laboratories . The type of
validation program required depends entirely on the particular method
and its proposed applications.
Typical analytical parameters used in assay validation include:
Accuracy: Accuracy is a measure of closeness between the
measured and real value .
Precision: Precision of an analytical procedure expresses the
closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the
prescribed conditions of repeatability , intermediate precision
Specificity: Specificity is the ability to measure the desired analyte
in the presence of components which may be expected to be present.
Typically these might include impurities, degradants, matrix, etc .
Detection limit: The detection limit of an individual analytical
procedure is the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact value.
– Standard Deviation of the Response and the Slope 
Quantitation limit: The quantitation limit of an individual
analytical procedure is the lowest amount of analyte in a sample which
can be quantitatively determined with suitable precision and accuracy.
The quantitation limit is a parameter of quantitative assays for low
levels of compounds in sample matrices, and is used particularly for the
determination of impurities and/or degradation products.
Linearity: The linearity of an analytical procedure is its ability to
obtain test results which are directly proportional to the concentration
of analyte in the sample. Test results should be evaluated by appropriate
statistical methods, for example, by calculation of a regression line by
the method of least squares.
Range: The range of an analytical procedure is the interval between
the upper and lower concentration of analyte in the sample for which it
has been demonstrated that the analytical procedure has a suitable level
of precision, accuracy and linearity.
Robustness: The robustness of an analytical procedure is a measure
of its capacity to remain unaffected by small, but deliberate variations
in method parameters  and provides an indication of its reliability
during normal usage.
Only specificity is needed for an identification test. However, the
full range of specificity, accuracy, linearity, range, limit of detection
(LOD) , limit of quantitation (LOQ) , precision, and robustness
testing is needed for more-complex methods such as quantitative
Recent development in pharmaceutical and biotechnological field
generates a cumulative demand for analytical methods. Rapid and
accurate quantification of the substrate and drug product is important in
the process development. Improvements in analytical instrumentation
leads to development of new techniques like isocratic and gradient
RP-HPLC, which evolved as the primary techniques for the analysis of
nonvolatile APIs and impurities. These analytical methods are critical
elements of pharmaceutical development so it is very important to
develop efficient and accurately validated analytical methods to develop
safe and effective drugs.
Skoog DA, West DM, Holler FJ (1996) Fundamentals of analytical chemistry.
(8thEdn), Fort Worth: Saunders College Pub.
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