ANALYTICAL

METHOD
DEVELOPMENT OF
BULK DRUGS

Rishabh Nagar
M.Pharma.(Analysis)

Introduction:

The number of drugs introduced into the market is increasing

every year. These drugs may be either new entities or partial
structural modification of the existing one. It becomes necessary,
therefore to develop newer analytical methods for such drugs.

Analytical Method Development

Analytical method development plays important role in the

discovery, development, and manufacturing of pharmaceuticals.
The official analytical methods that result from these processes
are used by quality control laboratories to ensure the identity,
purity, potency, and performance of drug products.
(James et al, 1988 )

Basic criteria for new method development

of drug analysis :-

The drug or drug combination may not be official in any

pharmacopoeias,
A proper analytical procedure for the drug may not be available
in the literature due to patent regulations,
Analytical methods may not be available for the drug in the form
of a formulation due to the interference caused by the formulation
excipients.
Analytical methods for the quantitation of the drug in biological
fluids may not be available,
Analytical methods for a drug in combination with other drugs
may not be available,
The existing analytical procedures may require expensive
reagents and solvents. It may also involve extraction and
separation procedures and these may not be reliable.

There may not be a suitable method for a particular analyte in the

specific sample matrix.
Existing methods may be have error, artifact , or contamination
prone, or they may be unreliable (have poor accuracy or precision).

Existing methods may not provide adequate sensitivity or

analyte selectivity in samples of interest.
Newer instrumentation and techniques may have evolved that
provide opportunities for improved methods, including
improved analyte identification or detection limits, greater
accuracy or precision, or better return on investment.

Analytical Method Development is required

for :
Herbal Products
New process and reactions
New molecules
Active ingredients (Macro analysis)
Residues (Microanalysis)
Impurity Profiling
Component of Interest in different matrices

NEEDS:

Affordability and necessity based eg. academic and

personnel Laboratories.
Simple, easier, cost effectiveness method is preferred.
Need for alternative method of analysis for better accuracy
and precision.
Better recovery of bulk drugs.
Evaluation and rethought over the already existing methods.
New and innovative ideas are encouraged advances in
analytical field.
Broadens the scope of research and exploration of new
properties of drugs those can be exploited for their
estimation and quantitative determination.
Availability of solvents/ reagents costs issue.

GOALs:

To improve the degree of accuracy and precision

For better recovery of drugs.
Improvement of sensitivity and/or specifity.
Simpler and easier method.
Reduce the cost (cost of operation and running cost of HPLC are
high)etc.

Validation of developed Analytical method:

Validation of analytical procedures is the process of

determining the suitability of a given methodology for
providing useful analytical data.
J. Guerra, Pharm. Tech. March 1986
Method validation is the process of demonstrating that
analytical procedures are suitable for their intended use and
that they support the identity, strength, quality, purity and
potency of the drug substances and drug products.
G. Maldener , Chromatographia , July 1989

Analytical method development

requirement : Validation of analytical methods is a prerequisite for

prequalification of product dossiers

Non-compendial APIs are tested with methods
developed by the manufacturer
For compendial APIs the applicability of
pharmacopoeial methods to particular products must be
demonstrated (verification)
Analytical methods must be developed and validated
according to TRS 823, Annex 5, Validation of analytical
procedures used in the examination of pharmaceutical
materials ; ICH Q2 (R1)
To be used within GLP and GMP environments

PHYSICAL PARAMETRS OF BULK DRUGS

Structure
Molecular weight
Melting point/Boiling point
Solubility profile
pH and pka value
Refractive index
Optical rotation
Salt formation
Partition coefficient
Moisture content
Method of synthesis of bulk
drugs
Impurity profile
Related substance

METHOD CYCLE :

Development

Validation

Optimization

Method Development, Optimization, and Validation

Approaches:-

Method development usually requires selecting the method

requirements and deciding on what type of instrument to utilize and why
. In the development stage, decisions regarding choice of column,
mobile phase, detector(s), and method of quantization must be
addressed. In this way, development considers all the parameters
pertaining to any method.
During the Optimization stage, the initial sets of conditions that have
evolved from the first stages of development are improved or maximized
in terms of resolution, peak shape and area under curve, plate counts,
asymmetry, capacity, elution time, detection limits, limit of quantization,
and overall ability to quantify the specific analyte of interest.
In the Validation stage, an attempt should be made to demonstrate that
the method works with samples of the given analyte, at the expected
concentration in the matrix, with a high degree of accuracy and
precision.

Method Validation Parameters (ICH guideline)

Various parameters are used to validate the methods:

Linearity
Range
Accuracy
Precision

Repeatability

Intermediate Precision

Reproducibility

Limit of Detection
Limit of Quantitation
Specificity
Ruggedness
Robustness

Definition

Linearity :

Ability of an assay to elicit a direct and proportional response to changes in analyte

concentration.

By Visual Inspection of plot of signals vs. analyte concentration

By Appropriate statistical methods
Linear Regression (y = mx + c)
Correlation Coefficient, y-intercept (c), slope (m)

Acceptance criteria: Linear regression r 2 > 0.95 (min 5 concentration require )

Range :Acceptable range having linearity, accuracy, precision.

For Drug Substance & Drug product Assay

80 to 120% of test Concentration

For Content Uniformity Assay

70 to 130% of test Concentration

For Dissolution Test Method

+/- 20% over entire Specification Range

For Impurity Assays

From Reporting Level to 120% of Impurity Specification for Impurity Assays
From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods

Accuracy :Closeness of the test results obtained by the method to the true value.

It Should be established across specified range of analytical procedure.

It Should be assessed using a minimum of 3 concentration levels, each in

triplicate (total of 9 determinations)

Should be reported as:

Percent recovery of known amount added
The difference between the mean assay result and the accepted value

Precision :Expresses the closeness of agreement between a series of measurements

obtained from multiple sampling of the same homogenous sample
Is usually expressed as the standard deviation (S), variance (S2) or coefficient
of variation (RSD) of a series of measurements
Precision may be considered at three levels
Repeatability (intra-assay precision)
Intermediate Precision (variability within a laboratory)
Reproducibility (precision between laboratories)

Repeatability

Express the precision under the same operating conditions over a short interval of
time.

Also referred to as Intra-assay precision

Should be assessed using minimum of 9 determinations

(3 concentrations/ 3 replicates) or Minimum of 6 determinations at the 100% level.

Intermediate Precision

Express within-laboratory variations.

Expressed in terms of standard deviation, relative standard deviation (coefficient of
variation) and confidence interval.
Depends on the circumstances under which the procedure is intended to be used.
Studies should include varying days, analysts, equipment, etc.

Reproducibility

It is an Ability reproduce data within the predefined precision

Determination: SD, RSD and confidence interval

Repeatability test at two different labs.

LOD
Lowest amount of analyte in a sample that can be detected but not necessarily
quantitated.
Estimated by Signal to Noise Ratio of 3:1.
LOQ
Lowest amount of analyte in a sample that can be quantified with suitable
accuracy and precision.
Estimated by Signal to Noise Ratio of 10:1.
LOQ & LOD Estimated by :-

1.

Based in Visual Evaluations

- Used for non-instrumental methods

2.

Based on Signal-to Noise-Ratio

- 3:1 for Detection Limit
- 10:1 for Quantitation Limit

3.

Based on Standard Deviation of the Response and the Slope

Y=b
X+a

Statistical estimate of LOD & LOQ

LOD

= 3.3

S LOQ
bl / b

bl

LOD

LOQ

= 10

bl / b

Specificity & Selectivity:-

Ability of an analytical method to measure the analyte free from interference due
to other components.

Selectivity describes the ability of an analytical method to differentiate various

substances in a sample
Original term used in USP
Also Preferred by IUPAC
Also used to characterize chromatographic columns

Degree of Bias (Used in USP)

The difference in assay results between the two groups
- the sample containing added impurities, degradation products, related

chemical compounds, placebo ingredients

- the sample without added substances

Robustness :-

Definition: Capacity to remain unaffected by small but deliberate

variations in method parameters

Determination: Comparison results under differing conditions with

precision under normal conditions

Examples of typical variations in LC

Influence of variations of pH in a mobile phase

Influence of variations in mobile phase composition
Different columns (different lots and/or suppliers)
Temperature
Flow rate

Ruggedness :-

Degree of reproducibility of test results under a variety

of conditions
Different Laboratories
Different Analysts
Different Instruments
Different Reagents
Different Days

Expressed as %RSD

Techniques:

Chromatographic Methods - HPLC, GC, TLC,

GC/MS,LCMS, etc.
Spectrophotometric Methods UV/VIS, IR, NIR, AA,
NMR, XRD,MS
Electrochemical techniques:-Potentiometry,
Polarography ,Conductometric titration.

UV-Visible spectrophotometry:A molecule which has either n , pie or sigma valance electrons,
absorb the characteristics radiations and undergoes transition
from ground state to exited state.

Almost all organic compounds absorb in the UV range(180nm380nm)

Solution in appropriate solvent is prepared in a given
concentration range and then scanned in the UV range to obtain.
Lambda-max(nm)
Presence of auxochrome and chromophores is identified by
examining the structure which is must for absorption in UVrange.
If conjugate double bond is present ,a better peak is obtained.
Determination whether and how a chromophore can be
derivatised or auxochrome may be introduced to obtained better
peak values.

Introduction To Hplc Methods Of Analysis For Drugs

In Combination

HPLC defined as a separation technique in which pressure

is applied to the column forcing the mobile phase through
at much higher rate. It is based upon number of theoretical
plates available for different component to be separated.
Most of the drugs in multi component dosage forms can be
analyzed by HPLC method because of the several
advantages like rapidity, specificity, accuracy, precision and
ease of automation in this method. HPLC method
eliminates tedious extraction and isolation procedures.
Some of the advantages are:
Speed (analysis can be accomplished in 20 minutes or less),
Greater sensitivity (various detectors can be employed),
Improved resolution (wide variety of stationary phases),

Easy sample recovery, handling and maintenance,

Instrumentation tends itself to automation and quantitation (less
time and less labour),
Precise and reproducible,
Suitable for preparative liquid chromatography on a much larger
scale.
Reusable columns (expensive columns but can be used for many
analysis),
Ideal for the substances of low volatility.
There are different modes of separation in HPLC. They are
normal phase mode, reversed phase mode, reverse phase ion pair
chromatography, affinity chromatography and size exclusion
chromatography.

HPTLC

(High Performance Thin Layer Chromatography) is a well known and versatile separation
method which shows a lot of advantages in comparison to other separation techniques .

Layer of Sorbent
100m
Efficiency
High due to smaller particle size generated
Separations
3 - 5 cm
Analysis Time
Shorter migration distance and the analysis time is greatly reduced
Solid support
Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase
modes
Development chamber
New type that require less amount of mobile phase
Sample spotting
Auto sampler
Scanning
Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and
quantitatively and the scanner is an advanced type of densitometer

Features of HPTLC

Simultaneous processing of sample and standard - better analytical

precision and accuracy less need for Internal Standard
Several analysts work simultaneously
Lower analysis time and less cost per analysis
Low maintenance cost
Simple sample preparation - handle samples of divergent nature
No prior treatment for solvents like filtration and degassing
Low mobile phase consumption per sample
No interference from previous analysis - fresh stationary and mobile
phases for each analysis - no contamination
Visual detection possible - open system
Non UV absorbing compounds detected by post-chromatographic
derivatization

Parameters involved during

Analytical method development :1.
2.
3.
4.
5.
6.
7.
8.
9.

Optimization of Mobile Phase .

Selection of Buffers
Selection of Ion pair reagent
pH of buffer/mobile phase
Composition of Mobile Phase
Selection of Column
Optimization of Analyte signal
Objectives of Separation( Analysis time, Resolution, k factor
(Capacity factor), Peak height, Asymmetry, Theoretical Plates)
9. Flow rate

The parameters that are affected by the

changes in chromatographic conditions are:

Resolution (Rs),

Capacity factor (k),

Selectivity (a),
Column efficiency (N) and
Peak asymmetry factor (As).

Peak purity

Resolution- Rs, of two neighboring peaks is defined as the

ratio of the distance between maxima of two peak. It is the
difference between the retention times of two solutes divided by
their average peak width. For baseline separation, the ideal value
of Rs is 1.5. It is calculated by using the formula,
where,
Rt1 and Rt2 are the retention times of components 1
and 2 and
W1 and W2 are peak width of components 1 and 2.

Capacity Factor (k):

Capacity factor is the ratio of the

reduced retention volume to the dead volume.
Capacity factor, k, is defined as the ratio of the number of
molecules of solute in the stationary phase to the number of
molecules of the same in the mobile phase. Capacity factor is a
measure of how well the sample molecule is retained by a column
during an isocratic separation. The ideal value of k ranges from
2-10. Capacity factor can be determined by using the formula,

Where, tR = retention volume at the apex of the peak (solute) and

t0 = void volume of the system.

Capacity Factor (k') changes are typically

due to:

Variations in mobile phase composition

Changes in column surface chemistry (due to aging)
Changes in operating temperature.
In most chromatography modes, capacity factor (k')
changes by 10 percent for a temperature change of 5 C.

Adjusting Capacity Factor (k')

Good isocratic methods usually have a capacity factor (k') in the range
of 2 to 10 (typically between 2 and 5). Lower values may give
inadequate resolution. Higher values are usually associated with
excessively brood peaks and unacceptably long run times.

If the analyte fall outside their specified windows run the initial column
test protocol to compare the results obtained with a new column.

Capacity Factor (k') values are sensitive to:

solvent strength
composition
purity
temperature
column chemistry
sample

Selectivity(a):
The selectivity (or separation factor), a, is a measure of relative retention of two components in a
mixture. Selectivity is the ratio of the capacity factors of both peaks, and the ratio of its adjusted
retention times. Selectivity represents the separation power of particular adsorbent to the mixture of
these particular components.

This parameter is independent of the column efficiency; it only depends on the nature of the
components, eluent type, and eluent composition, and adsorbent surface chemistry. In general, if the
selectivity of two components is equal to 1, then there is no way to separate them by improving the
column efficiency.

The ideal value of a is 2. It can be calculated by using formula,

a= V2 V1 / V1 V0 = k1/ k2

Where, V0 = the void volume of the column,

V1 and V2 =the retention volumes of the
second and the first peak respectively.

Column Efficiency/ Band broadening: Efficiency,

N, of a column is measured by the number of
theoretical plates per meter. It is a measure of band
spreading of a peak. Similar the band spread, higher is
the number of theoretical plates, indicating good
column and system performance. Columns with N
ranging from 5,000 to 100,000 plates/meter are ideal for
a good system. Efficiency is calculated by using the
formula
Where,
tR is the retention time and W is the peak
width.

Peak asymmetry factor (Tf):Peak asymmetry factor, Tf, can be used as a criterion of column performance. The peak half width, b,
of a peak at 10% of the peak height, divided by the corresponding front half width, a, gives the
asymmetry factor.
For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.

Peak purity:
The null hypothesis these spectra are identical can in this case
(purity) with two sided significance. During the purity test the
spectrum taken at the first peak slope is correlated with the
spectrum of peak maximum [r(s,m)] and the correlation of the
spectra taken at the peak maximum with the one from the down
slope or peak end [r(m,e)] which is used as a reference spectra
for statistical calculation. An error probability of 1% only be
rejected if the test value is greater than or equal to 2.576

REFERENCES :

Snyder, L.R.; Kirkland, J.J.; Glajch , J.L. Practical

HPLC Method Development, 2nd ed. John Wiley &
Son: New York, 1997.
WHO guidelines for Analytical Method Development.
FDA, Analytical Procedures and Methods Validation:
Chemistry, Manufacturing, and Controls , Federal
Register (Notices) 65 (169) 52,77652,777 (30 August
2000).
An Introduction to Analytical method development of
formulation (http://www.pharmainfo.net)

Thank

You