Analytical Method Develop

8/3/2019 Analytical Method Develop

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PERD Centre, Ahmedabad

ANALYTICAL METHOD DEVELOPMENT

BY

BHUPENDRASINH F. CHAUHAN

Research Scholar


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Drug synthesis,Separation

Raw Drug quality control &Impurities profiling

Preformulation of Drug &

Dosage Form Development

Preclinical and Clinical Studies,

BE/BA Studies

Med. Chem. Dept.

Q C Dept.

F & D Dept.

Pharmacology Dept.

Use of Analytical Method

in Pharma Industries


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Separation and Analysis

Qualitative analysis What are components A, B and C ?

Quantitative analysis What is the concentration of

components A, B and C ?


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The process of finding a set of conditions that

adequately separates and enables the

quantification of the analyte with acceptableaccuracy, precision, sensitivity, specificity, cost and

speed.

Method Development


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GC

UV-Visible

Spectrophotometer

GC-MS

HPLC

LC-MS

HPTLC


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What is HPLC ?

H: High

P: Performance (Pressure)

L: Liquid

C: Chromatography


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HPLC Basic Instrumentation


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Results obtained by HPLC


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Chromatogram containing three peaks

Qualitative analysis (identification) and

Quantitative analysis (determination)

Can be performed using the information contained in the

chromatogram

Chromatogram


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Identification

What is component

Component A elutes at specific Rt

Component A is identified


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DeterminationWhat is the concentration of component A?

Peak area (or height) is proportional to the concentration (or

amount) of the component.

The concentration of component A (caffeine) is determined

by comparing the peak area with that of the standard

caffeine peak.


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HPL Chromatographic separation is based oninteraction and differential partition of the sample

between the mobile phase and the stationary phase.

1. Reverse phase

2. Normal phase

3. Ion exchange

4. Ion pair

5. Chiral

6. Size exclusion

Separation techniques


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Normal Phase Vs Reverse Phase


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Mobile phase

Isocratic Gradient

Isocratic: Isocratic is the one in which the mobile phaseremains the same throughout the separation.

Gradient: It involves changing the relative amounts of

usually two, but occasionally three or four mobile phaseduring a chromatographic separation.


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External standard

The external standard method is more appropriate for samples as follow:

1. Sample with single target concentration and narrow concentration range.

2. Simple sample preparation procedure.

3. Increased baseline time for detection of potential extraneous peaks, e.g.,

impurities test.

Internal standard

The internal standard method is more appropriate for samples as follow:

1. Complex sample preparation procedure, e.g., multiple extraction.

2.L

ow concentration sample (sensitivity being an issue), e.g.,pharmacokinetics studies.

3. Wide range of concentrations expected in the sample for analysis, e.g.,

pharmacokinetics studies.

Reference Standard


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Trial and error

Literature review on similar compounds

Tools for method fine tuning

(e.g. Dry lab, ChromSword AUTO)

Few strategies to determine initial method

Method Development Approach


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Step 1: Define method objectives.

Step 2: Understand the chemistry of the analytes/ drug product.

Step 3: Develop initial conditions to achieve minimallyacceptable separations.

Step 4: Sample preparation procedure for the drug product.

Step 5: Final method optimization/robustness.Identify the weaknesses of the method and optimize the

method through experimental design.

Step 6: Method validation.

Steps to Develop Method


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Step 1: Define the Objective

Analytical Requirements

Linearity

Precision

Accuracy

Sensitivity

Assay reproducibility

Robustness

Preparative Requirements

Recovery

Product purity

Capacity

Costs

Scale up Process throughput

Speed

Analytical vs. Preparative


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Step 2: Chemistry of the Analytes

CHASM

Charge

Positive/negative

Hydrophobicity/hydrophilicity Affinity

Affinity for solid and mobile phase

Solubility & stability pH, ionic strength, organic solvents

Molecular weight


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Step 3: Develop initial conditions

Retention parameters

Column efficiency parameters

Peak symmetry parameters

Condition for Separation


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Retention parameters

tR: retention time (the time between the injection pointand the maximum detector response for

correspondent compound)

vR: retention volume (tR x eluent flow rate)

k: capacity factor t0: the time required for the component not retained

by the column to pass through the column


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Column Efficiency

N = 16 (tR/ W4W )2 N = 25 (tR/ W5W )

2 N = 5.545 (tR/ W0.5)2

The number of theoretical plates N is given by:


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Peak symmetry

S: symmetry factor, T: tailing factor


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Degree of separation


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Condition for Good Separation

Rs = -1

:Capacity term increases retention time

:Selectivity term increases time intervalbetween peaks

N : Column efficiency produces narrow peaks


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Parameters and selectivity


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Most analysts focus too much on the chromatographic conditionsand neglect the other two components of the method

(i.e., sample preparation, Integration).

Achieving good quality results can be translated into a simple

term:

Instrumentation: Selectivity, Resolution, Sensitivity, LOD,

Precision, Accuracy

Sample preparation: Speed, Size, Ease of use, Cost, Reliability,Ruggedness

Integration: Repeatability, Reliability, Accuracy.

Cleaner Sample =Better Results

Common Mistake..


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Many extractions practices are based on classical

methodologies of liquid-liquid or liquid-solid extraction

using different practices.

CLASSI

CAL Protein precipitation

Liquid-liquid extraction

Membrane extraction

Soxhlet

NEW

TE

CHN

OLOGIE

S Solid phase extraction

Solid phase micro extraction

Supercritical fluid extraction

Microwave-assistedextraction

Extraction Techniques


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Protein Precipitation

Probably the most popular and usually the first choice forsample preparation used in the pharmaceutical industry.

1. Pipette 200 Ql of plasma

2. Pipette 400-600 Ql of MeCN/MeOH

3. Vortex

4. Centrifuge

5. Collect supernant

6. Inject

It is faster method but the samples are crude and dirty. Not

useful for very low concentration.


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Liquid-Liquid Extraction

Solvent extraction is defined as the process of separating one constituentfrom a mixture by dissolving it into a solvent in which it is soluble but in

which the other constituents of the mixture are not.

1. Pipette 1 ml of plasma

2. Pipette 2-5 ml of organic solvent (e.g. CH2Cl2)

3. Shake for 5 min.

4. Remove organic layer

5. Evaporate organic layer to dryness

6. Reconstitute in mobile phaseOverall, liquid-liquid extraction offers a better clean up than protein

precipitation, by using added variants such as back washing, back

extraction, evaporation and drying. The technique, on the other hand, is

extremely time consuming.


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In SPE, the analyte of interest gets completelyabsorbed onto the solid phase to be subsequently

desorbed by an appropriate solvent.

Solid Phase Extraction


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Solid Phase Extraction

Pre-treat sample foroptimized flow rate (toimprove contact time with

sorbent and to preventplugging of wells)

Food/ Tissue homogenate

- Blend with buffers

- Centrifuge and collectsupernant

for analysis

Plasma/serum samples

- Eliminate protein binding

Check stability of analyte atpH used in method

Prepare Sample Solution

Condition/Equilibrate

Load

Wash

Elute

Dilute / Evaporate & Reconstitute


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pHSolvent

Columns

Selectivity

E


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pHSolvent

Columns

Selectivity

E

High

Low

MeOH

MeCN

Non-Polar

C18

C8

Mid-Polar

Phenyl

Cyano

Polar

Silica

Alumina


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Effect of pH


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Affects only analytes with ionizable functional groups:

amines, carboxylic acid, sulphonyl group, phenol.

(pH does not impact compounds which do not ionize)

Most pharmaceuticals contain one or more ionizablefunction

Strongest selectivity effects caused by pH changes.

Below pH 2.5 Hydrolysis of the bonded phase.

Above pH 7.0 Silica support starts to dissolve.

Effect of pH


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Common solvents:

Methanol

Aacetonitrile

Less common:

Isopropanol

Ethanol

THF (Tetrahydrofuran)

The use of different solvents provides changes inselectivity as well as elution strength.

Solvent Selection


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Effect of Column Chemistry

Min.

Length of packing materials carbon chains and retention time.


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Tailing is caused by

1. Free silanol acidic groups react with protonated

bases.

2. Hydrogen bonding between protonated species in

mobile phase and residual alkali metals in the silica.

3. Completion of column life.

Tailing Problem


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By increasing the coverage of the endcapping through more

efficient bonding technology, less silanols are available tointeract with solute molecules. With fewer silanols to interfere

with the chromatography, peak tailing is reduced and

reproducibility improves.


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Analytical Method Validation


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Analytical method validation includes all of the

procedures, checks and balances required to prove

the reliability of a particular method for the

quantitative determination of the concentration of an

analyte (or a series of analytes) in a particular

biological matrix for the intended application.

Analytical Method Validation


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Validation Parameters

Calibration and Linearity

Sensitivity and the Limit of Detection

Selectivity

Accuracy and Precision

Extraction efficiency and Recovery

Stability of Drug


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A calibration (standard) curve is the relationship between

instrument response and known concentrations of the analyte.

Concentrations of standards should be chosen on the basis of the

concentration range expected in a particular study.

A calibration curve should consist of a blank sample (matrix

sample processed without internal standard), a zero sample

(matrix sample processed with internal standard), and six to

eight non-zero sample covering the expected range, including

LLOQ.

Calibration /Standard Curve


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SENSITIVITY AND LIMIT OF DETECTION

Detection limit is the lowest concentration of analyte in a

sample that can be detected, but not necessary

quantitated, under the stated experimental conditions.

LIMIT OF QUANTIFICATION

Quantitation limit is the lowest concentration of the

analyte in a sample that can be determined withacceptable precision and accuracy under the stated

experimental conditions.

Sensitivity


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Selectivity is the ability of an analytical method todifferentiate and quantify the analyte in the presence of

other components in the sample.

For selectivity, analytes of blank samples of the

appropriate biological matrix (plasma, urine or other

matrix) should be obtained form at least six sources.

Potential interfering substances in a biological matrix

include endogenous matrix compounds, metabolites,decomposition products, concomitant medications and

etc.

Selectivity


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Accuracy is the measure of how close the experimental

value is to the true value.

Accuracy should be measured using a minimum of fivedeterminations per concentration.

The mean value should be within 15% of the coefficient

of variation (CV) the actual value except at LLOQ,

where it should not deviate by more than 20% of CV.

Accuracy


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Precision is the measure of how close the data valuesare to each other for a number of measurements under

the same analytical conditions.

Precision should be measured using a minimum of five

determinations per concentration.

Precision determined at each concentration level should

not exceed 15% of the coefficient of variation (CV)

except for theLLOQ

where it should not exceed 20% ofthe CV.

A) Intra-day precision

B) Inter-day precision

Precision


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Recovery experiments should be performed by

comparing the analytical results for extracted samples

at three concentrations (low, medium and high) with

unextracted standards that represent 100 % recovery.

Extraction Efficiency and Recovery


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Stability procedures should evaluate the stability of the analytesduring sample collection and handling, after long-term (frozen at

the intended storage temperature) and short-term (bench-top,

room temperature) storage, and after going through freeze and

thaw cycles and the analytical process.

1. Freeze and Thaw Stability

2. Short-Term Stability

3. Long-Term Stability

4. Stock Solution Stability

5. Post-Preparative Stability (Autosampler stability)

Stability of Drug


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ValidatedMethod


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Solvent Polarity B.P. Viscosity UV cut off Isooctanol 0.1 99 0.47 197

L. chain Hexane 0.1 69 0.3 190

Cyclohexane 0.2 81 0.9 200

Triethylamine 1.9 89 0.36

Isopropylether 2.4 68 0.38 220

Toluene 2.4 110 0.55 285

Ethyl ether 2.8 35 0.24 218

Benezen 2.7 80 0.6 280

Methylene chloride 3.1 40 0.41 233

n-butanol 3.9 118 2.6 210

n-propanol 4 97 1.9 240

Tetrahydrofuran 4 66 0.46 212

Ethyl acetate 4.4 77 0.43 256

Isopropanol 3.9 82 1.9 205

Chloroform 4.1 61 0.53 245

Methylethyl ketone 4.7 80 0.38 329

Dioxane 4.8 101 1.2 215 Acetone 5.1 56 0.3 330

Ethanol 4.3 78 1.08 210

Acetic acid 6 118 1.1

Acetonitrile 5.8 82 0.34 190

Dimethylformamide 6.4 153 0.8 268

Dimethylsulfoxide 7 189 2

Water 10.2 0.95 0.89

Solvent used in analytical method developments


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Further Readings

Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. Practical HPLCMethod Development, 2nd ed. John Wiley & Son: New

York, 1997.

Venn, Richard F.. (2000)Principle and Practice ofBioanalysis, London: Taylor & Francis.

html://www.fda.gov/cder/guidance/index.htm


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THANK YOU

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Analytical Method Develop