MOST WIDELY USED METHOD FOR SEPARATION OF DIFFERENT TYPES OF DRUGS.A 20MICROLETERS SAMPLE IS SAFFICIANT TO SEPARATION OF DRUG.

1. Presented by
G.Krishnam Raju
M.Pharmacy
Pharmaceutical Analysis & Quality
Assurance 1st year 2nd semester
HT.NO:-13TK6S0401
SVS Group Of Institutions
SVS school of pharmacy

2. CONTENT:-
 INRODUCTION
 HPLC METHOD DEVELOPMENT STEPS
 CONCLUSION
 REFERENCE

3. Introduction:
ANALYTICAL METHOD DEVELOPMENT:
• Method development usually requires selecting the
method requirements and deciding on what type of
instrumentation to utilize and why.
• The wide variety of equipment, columns, eluent and
operational parameters involved makes HPLC method
development .
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4. There are several reasons for developing new methods
of analysis:
1. A suitable method for particular analyte in the
specific matrix is not available.
2. Existing methods may be too error or they may be
unreliable (have poor accuracy or precision)
3. Existing methods may be too expensive, time
consuming.
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5. HPLC method development generally follows the following
steps:
Step 1-selection of the HPLC method and initial system.
Step2-Selection of optimum conditions.
Step3-selectivity optimization.
Step4-system parameter optimization.
Step5-method validation.
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6. Step 3a
Initial HPLC
condition
Step 2
Sample
preparationStep 1
Method goals and
chemistry
Step 3b
Optimize HPLC
separation
Step 4
Standardization
Step 5
Method validation
Fig 2.2 Pie diagram showing the time that should be spent on different steps of the
method development to meet the commended timeline. The sequence of events and
percentage of time allocated is only suggestive.
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7. STEP1-SELECTION OF HPLC METHOD AND INITIAL CONDITIONS
SELECTION OF HPLC METHOD:
• When selecting an HPLC system it must have a high propability
of actually being able to analyse the sample.
• For example if the sample includes polar analytes then RP-
HPLC would offer both adequate retention and ressolution.
consideration must be given to the following ;
a. sample preparation
b. Types of chromatography
c. Column selection
d. Detector selection
e. Selection of mobile phase composition 28 January 2015 7

8. SAMPLE PREPARATION
Sample preparation is an essential part of HPLC
analysis, to provide a reproducible and homogenous
solution i.e. suitable for injection on to the column.
The aim of sample preparation is a sample that :
1. It is relatively free of interferences
2. Will not damage the column
3. Should compatible with the intended HPLC method
;
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9. TYPES OF CHROMATOGRAPHY
 Reversed phase is the choice for the majority of the
samples.
 But if acidic or basic analytes are present reversed
phase ion suppression (for weak acids and bases) or
reversed phase ion pairing (for strong acids and bases)
should be used.
 For low or medium polarity analytes normal phase
HPLC is used, particularly if the separation of isomers
is required.
 For inorganic anion or cation analysis ion exchange
chromatography is best.
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10.  Size exclusion chromatography would normally be
considered for analyzing high molecular weight
compounds.
 Gradient HPLC only a requirement for complex samples
with a large number of components (20-30) .
 Reversed phase HPLC is commonly used in peptide and
small protein analysis using an acetonitrile –water
mobile phase containing 1% trifluoroethanolic acid.
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11. COLUMNSELECTION
A column is chosen based on the
Knowledge of sample
On the expectation of how its components will interact
with the packing material.
 the properties of column packing material.
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12. Column selection
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1.Knowledge of the Sample: which influences the
choice of Column Bonded Phase characteristics
Knowledge of the sample
• Structure of sample components?
• Number of compounds present?
• Sample matrix?
• pKa values of sample components?
• Concentration range?
• Molecular weight range?
• Solubility?
• Other pertinent data?
Column Chemistry
(bonded phase, bonding
type, endcapping,
carbon load)

13. Packing material:-
 Most HPLC separations are performed on bonded
phase HPLC columns
 Octadecyl-derivatized silica gel columns are the most
widely used bonded phase columns in the reverse phase
mode.
 Commonly used polar bonded phases certain diol,cyano
or amino functional groups.
 Silica based packing materials are used in about 75% of
all HPLC separations performed today.
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14. • due to
 the physical stability
 the availability of bonded phase
 and to the high efficiency of silica based HPLC columns
• Many new resin packings have been introduced in recent
years particularly for biochemical analysis.
• The most well known resin based packings are formed by
the co polymerization of polysterene and divinyl benzene.
• Other packing materials such as alumina, titania and
zirconia have also been employed for bio polymer analysis.
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15. COLUMN DIMENSIONS
Effect on chromatography
Column Dimension
• Short (30-50mm) - short run times, low backpressure
• Long (250-300mm) - higher resolution, long run times
• Narrow ( 2.1mm) - higher detector sensitivity
• Wide (10-22mm) - high sample loading
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16. DETECTOR SELECTION
Consideration must given to the following:
Do the analytes have chromophores to enable UV detection ?
Is more selective or sensitive detection required?
What detection limits are necessary ?
Will the sample require chemical derivatization to enhance
detect ability and /or improve the chromatography.
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17. DETECTOR SELECTION
A HPLC detector will have a number of performance characteristics that
need to be specified and known before a particular detector can be
chosen for a specific application and are listed as follows.
1. Dynamic range
2. Response index or linearity
3. Linear dynamic range
4. Detector response
5. Detector noise level
6. Detector sensitivity, or minimum detectable concentration.
7. Total system dispersion
8. Pressure sensitivity
9. Flow arte sensitivity.
10. Operating temperature range.
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18. THE UV DETECTOR
 Limited to the detection of those substances that absorb
light in the UV wave length range.
 UV detectors detects all sample components that contain
chromophores.
 Specifications:
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S.NO CHARACTERISTIC FIXED WAVELENGTH
UV DETECTOR
DIODE ARRAY
DETECTOR
1 Sensitivity
(solute benzene)
5×10ˉ⁸gml 1×10ˉ⁷gml
2 Linear dynamic
range
5×10ˉ⁸ to
5×10ˉ⁴gml
10ˉ⁸to
5×10ˉ⁴gml
3 Response index 0.98 - 1.02 0.97- 1.03

19. THE FLUORESCENCE DETECTOR
 It can detect eluted solutes on the basis of fluorescence
,but it can also provide their fluorescence spectra.
 Fluorescence and electro chemical detectors should be
used for trace analysis.
 Specifications:
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S.No CHARACTERISTIC FLUOROSCENCE
DETECTOR
1. Sensitivity
(solute anthracene)
1×10ˉ⁹gml
2 Linear dynamic range 1×10ˉ⁹ to 5×10ˉ⁶gml
3 Response index 0.96 – 1.04

20. ELECTRICAL CONDUCTIVITY DETECTOR
 It is usually employed with an ion suppressor column to
allow salts and buffers to be used in the mobile phase
without affecting the detector output.
 Specifications:
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S.NO CHARACTERISTIC CONDUCTIVITY
DETECTOR
1. Sensitivity(sodium
chloride)
5×10ˉ⁹gml
2. Linear dynamic range 5×10ˉ⁹to 1×10ˉ6gml
3. Response index 0.97-1.03

21. REFRACTIVE INDEX DETECTOR
 It is one of the least sensitive LC detectors and is used
circumstances where other detector s are inappropriate.
 For preparative HPLC it is preferred because it can
handle concentration without overloading the detector
 Specifications:
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S.NO CHARACTERISTIC RI DETECTOR
1. Sensitivity(solute
benzene)
1×10ˉ⁶g/ml
2. Linear dynamic range 1×10ˉ⁶ -1×10ˉ⁴g/ml
3. Response index 0.97-1.03

22. MOBILE PHASE SELECTION
 The organic phase concentration required for the mobile
phase can be estimated by gradient elution method.
 Gradient can be started with 5-10 % of the organic phase
in the mobile phase and the organic phase concentration
can be increased up to 100% within 30-45%.
 The elution strength of a mobile phase depends upon its
polarity, the stronger the polarity higher is the elution.
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23.  Ionic samples(acidic or basic) can be separated if they
are present in undissociated form. Dissociation of ionic
samples may be suppressed by the selection oh pH.
 If the retention times are too long an increase of the
organic phase concentration is needed.
 When tailing or fronting is observed, it means that the
mobile phase is not totally compatible with the solutes.
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24. SELECTION OF INITIAL SYSTEM
It could based on :
 assessment of the nature of the sample and analytes
together with literature data.
Experience
Expert system software and
Empirical approaches
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25. STEP 2 : SELECTION OF INITIAL CONDITIONS
• This step determines the optimum conditions to adequately
retain all analytes ; i.e.
 Ensures no analyte has a capacity factor of less than 0.5(poor
retention could result in peak overlapping).
 No analyte has a capacity factor greater than 10-15 (excessive
retention leads to long analysis time and broad peaks with poor
detectability).
Determination of initial conditions:
• The recommended method involves performing two gradient
runs differ in only in the run time.
• A binary system based on either aceto nitrile/ water or
methanol/water should be used.
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26. STEP 3: SELECTIVITY OPTIMIZATION
 The aim of this step is to achieve adequate selectivity.
 The mobile phase and stationary phase compositions
need to be taken in to account.
 To select these the nature of the analytes must be
considered.
 Once the analyte types are identified the relevant
optimization parameters may be selected.
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27. STEP 4: SYSTEM PARAMETER OPTIMIZATION
 This is used to find the desired balance between
ressolution and analysis time after satisfactory selectivity
has been achieved.
 The parameters involve include column dimensions,
column packing particle size and flow rate.
 This parameters may be changed without affecting
capacity factors or selectivity.
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28. TYPES OF OPTIMIZATION
Two types :
 Manually and
 By using soft wares.
By Manual : separation then can be optimized by change in the
initial mobile phase composition and the slope of the
gradient according to the chromatogram obtained from the
preliminary run.
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29. By usINg soft wares :
 Chemometrics in HPLC Optimization
 Chemo metric protocols available for the development and optimization of HPLC methods:
 Experimental Design (ED)
 Factorial design
 Plackett-Burman design
 D-optimal design
 Two-level full factorial design
 Central composite design
 Box-Behnken design
 Doehlert design
 Multi-Criteria Decision Making (MCDM)
 Overlay plots
 Pareto optimality
 Utility function
 Derringer’s desirability function
2928 January 2015

30. STEP 5 METHOD VALIDATION
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31.  The objective of validation of an analytical procedure
is to demonstrate that it is suitable for its intended
purpose.
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32.  A brief description of types of tests considered in this
document is provided below:-
Identification tests are intended to ensure the
identity of analyte in a sample. This normally achieved
by comparing the properties of sample with that of
the reference standard.
Testing for impurities can be either a quantitative test
or a limit test for the impurity in a sample.
Assay procedures are intended to measure the analyte
present in a given sample.
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33. • Typical validation characteristics which should be
considered are listed below:-
1.Accuracy
2. Precision
a. repeatability
b. intermediate precision
3. specificity
4. Detection limit
5. Quantitation limit
6. Linearity
7. range.
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34. ACCURACY
 The accuracy of an analytical procedure expresses the
closeness of agreement between the value which is
accepted either as a conventional true or an accepted
reference value and the value found.
 Accuracy should be assessed using a minimum of 9
determinations over a minimum of 3 concentration
levels covering the specified range
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35. 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.
 Linearity should be evaluated by visual inspection of a
plot of signals as a function of analyte concentration or
content.
 For the establishment of linearity, a minimum of 5
concentrations is recommended.
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36. PRECISION
 The precision of an analytical procedure expresses the
closeness of agreement between the measurements
obtained from multiple sampling of the same
homogenous sample under the prescribed conditions.
repeatability
 Precision intermediate precision
reproducibility
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37. Repeatability:- Expresses the precision under the
same operating conditions over a short interval of time .
Repeatability is also termed intra- assay precision.
• a) a minimum of 9 determinations covering the specified
range for the procedure (e.g., 3 concentrations/3
replicates each);
or
• b) a minimum of 6 determinations at 100% of the test
concentration.
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38. Intermediate precision:- intermediate precision
expresses within – laboratories variations: different days,
different analysts, different equipment e.t.c.
Reproducibility:- reproducibility expresses the precision
between laboratories.
• The standard deviation, relative standard deviation
(coefficient of variation) and confidence interval should
be reported for each type of precision investigated.
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39. DETECTION LIMIT
 The detection limit of individual analytical procedure is
the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact value.
 Several approaches for determining the detection limit are
possible, depending on whether the procedure is a non-
instrumental or instrumental. Approaches other than those
listed below may be acceptable
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40. Based on Visual Evaluation
 Visual evaluation may be used for non-instrumental
methods but may also be used with instrumental methods.
 The detection limit is determined by the analysis of
samples with known concentrations of analyte and by
establishing the minimum level at which the analyte can
be reliably detected.
Based on Signal-to-Noise
 This approach can only be applied to analytical procedures
which exhibit baseline noise.
 Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known
low concentrations of analyte.
 A signal-to-noise ratio between 3 or 2:1 is generally
considered acceptable for estimating the detection limit.
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41. Based on the Standard Deviation of the Response
and the Slope
 The detection limit (DL) may be expressed as:
DL = 3.3 σ /s
Where, σ = the standard deviation of the response
S = the slope of the calibration curve
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42. 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.
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43. Based on Visual Evaluation
 Visual evaluation may be used for non-instrumental
methods but may also be used with instrumental methods.
 The detection limit is determined by the analysis of
samples with known concentrations of analyte and by
establishing the minimum level at which the analyte can
be reliably detected.
Based on Signal-to-Noise
 This approach can only be applied to analytical procedures
which exhibit baseline noise.
 Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known
low concentrations of analyte.
 A signal-to-noise ratio between 10:1 is generally
considered acceptable for estimating the detection limit.
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44. Based on the Standard Deviation of the Response
and the Slope
 The quantitation limit (QL ) may be expressed as:
QL = 10 σ /s
Where, σ = the standard deviation of the response
S = the slope of the calibration curve
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45. RANGE
 The range of an analytical procedure is the interval
between the upper and lower concentration of the analyte
in the sample for which it has been demon started that the
analytical procedure has a suitable level of precision,
accuracy and linearity.
 The following minimum specified ranges should be
considered:
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46. for the assay of a drug substance or a finished (drug)
product: normally from 80 to 120 percent of the test
concentration;
 for content uniformity, covering a minimum of 70 to 130
percent of the test concentration, unless a wider more
appropriate range, based on the nature of the dosage form
(e.g., metered dose inhalers), is justified;
for dissolution testing: +/-20 % over the specified range;
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47. ROBUSTNESS
The robustness of an analytical procedure is a
measure of its capacity to remain unaffected by small,
but deliberated variations in method parameters and
provides an indication of its reliability during normal
usage.
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48. CONCLUSION
 The method development and validation are continuous
and interrelated processes that are conducted throughout
the drug development process.
 The analytical validation verifies that a given method
measures a parameter as intended and establishes the
performance limits of the measurement.
 Reproducible quality HPLC results can only be obtained if
proper attention has been paid to the method
development, validation and system’s suitability to carry
out the analysis.

49. references
 Instrumental methods of chemical analysis by
Gurdeep.R.Chatwal, Sham.K Anand, p.no 2.624-2.639.
 D. H. Shewiy, E. Kaale, P. G. Risha, B. Dejaegher, J. S.
Verbeke, Y. V. Heyden, J. Pharmaceut. Biomed. Anal 2012,
66, 11–23.
 M. D. Rockville, General Tests, Chapter 621 – Chromatography
System Suitability, United States Pharmacopeial Convention
(USP), USP 31 (2009):
 Kasawar GB, Farooqui M. Development and validation of a
stability indicating RP-HPLC method for the simultaneous
determination of related substances of albuterol sulfate and
ipratropium bromide in nasal solution. J Pharmaceut Biomed
Anal 2010; 52:19–29
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50. Thank you
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