ANALYTICAL METHOD DEVELOPMENT

BY

BHUPENDRASINH F. CHAUHAN Research Scholar

PERD Centre, Ahmedabad

Use of Analytical Method in Pharma Industries

Drug synthesis,Separation Med. Chem. Dept.

Raw Drug quality control & Impurities profiling Preformulation of Drug & Dosage Form Development

Q C Dept.

F & D Dept.

Preclinical and Clinical Studies, BE/BA Studies

Pharmacology Dept.
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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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Method Development

The process of finding a set of conditions that adequately separates and enables the quantification of the analyte with acceptable accuracy, precision, sensitivity, specificity, cost and speed.

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GC

HPLC

GC-MS

LC-MS

UV-Visible Spectrophotometer

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

Chromatogram containing three peaks Qualitative analysis (identification) and Quantitative analysis (determination) Can be performed using the information contained in the chromatogram
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Identification
What is component

Component A elutes at specific Rt

Component A is identified

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Determination
What 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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Separation techniques
HPL Chromatographic separation is based on interaction 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

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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 phase remains the same throughout the separation.

Gradient: It involves changing the relative amounts of usually two, but occasionally three or four mobile phase during a chromatographic separation.

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Reference Standard
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. Low 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.

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Method Development Approach

 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

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Steps to Develop Method

Step 1: Define method objectives. Step 2: Understand the chemistry of the analytes/ drug product. Step 3: Develop initial conditions to achieve minimally acceptable 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.
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Step 1: Define the Objective

Analytical vs. Preparative

Analytical Requirements Linearity Precision Accuracy Sensitivity Assay reproducibility

Robustness

Preparative Requirements Recovery Product purity Capacity Costs

Scale up Process throughput Speed

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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 point and 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
The number of theoretical plates N is given by:

N = 16 (tR/ W4W )2

N = 25 (tR/ W5W )2

N = 5.545 (tR/ W0.5)2

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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 interval between peaks N : Column efficiency produces narrow peaks
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Parameters and selectivity

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Common Mistake..
Most analysts focus too much on the chromatographic conditions and 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 Cleaner Sample = Better Results Integration: Repeatability, Reliability, Accuracy.
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Extraction Techniques
Many extractions practices are based on classical methodologies of liquid-liquid or liquid-solid extraction using different practices. CLASSICAL Protein precipitation Liquid-liquid extraction Membrane extraction Soxhlet NEW TECHNOLOGIES Solid phase extraction Solid phase micro extraction Supercritical fluid extraction Microwave-assisted extraction

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Protein Precipitation
Probably the most popular and usually the first choice for sample preparation used in the pharmaceutical industry. Pipette 200 Ql of plasma Pipette 400-600 Ql of MeCN/MeOH Vortex Centrifuge Collect supernant Inject It is faster method but the samples are crude and dirty. Not useful for very low concentration.

1. 2. 3. 4. 5. 6.

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Liquid-Liquid Extraction
Solvent extraction is defined as the process of separating one constituent from 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. 2. 3. 4. 5. 6. Pipette 1 ml of plasma Pipette 2-5 ml of organic solvent (e.g. CH2Cl2) Shake for 5 min. Remove organic layer Evaporate organic layer to dryness Reconstitute in mobile phase Overall, 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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Solid Phase Extraction

In SPE, the analyte of interest gets completely absorbed onto the solid phase to be subsequently desorbed by an appropriate solvent.

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Solid Phase Extraction

Prepare Sample Solution Condition/Equilibrate Load
Pre-treat sample for optimized flow rate (to improve contact time with sorbent and to prevent plugging of wells) Food/ Tissue homogenate - Blend with buffers - Centrifuge and collect supernant for analysis Plasma/serum samples - Eliminate protein binding Check stability of analyte at pH used in method
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Wash Elute Dilute / Evaporate & Reconstitute

Solvent Selectivity E

pH

Columns

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MeOH Solvent MeCN Selectivity E

High pH Low

Columns Non-Polar
C18 C8

Mid-Polar
Phenyl Cyano

Polar
Silica Alumina
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Effect of pH

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Effect of pH
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 ionizable function 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.
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Solvent Selection
Common solvents: Methanol Aacetonitrile Less common: Isopropanol Ethanol THF (Tetrahydrofuran) The use of different solvents provides changes in selectivity as well as elution strength.

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Effect of Column Chemistry

Length of packing materials carbon chains and retention time.

Min.

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Tailing Problem
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.

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By increasing the coverage of the endcapping through more efficient bonding technology, less silanols are available to interact 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

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.

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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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Calibration /Standard Curve

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.

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Sensitivity
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 with acceptable precision and accuracy under the stated experimental conditions.

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Selectivity
Selectivity is the ability of an analytical method to differentiate 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.

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Accuracy

Accuracy is the measure of how close the experimental value is to the true value. Accuracy should be measured using a minimum of five determinations 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.

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Precision
Precision is the measure of how close the data values are 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 the LLOQ where it should not exceed 20% of the CV. A) Intra-day precision B) Inter-day precision
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Extraction Efficiency and Recovery

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.

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Stability of Drug
Stability procedures should evaluate the stability of the analytes during 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)
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Validated Method

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Solve nt use d in a na lytica l me thod deve lopme nts Solve nt Pola rity 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 W ater 10.2 0.95 0.89

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Further Readings
Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. Practical HPLC Method Development, 2nd ed. John Wiley & Son: New York, 1997. Venn, Richard F.. (2000) Principle and Practice of Bioanalysis, London: Taylor & Francis.

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

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THANK YOU

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