PHA6122 LEC: Instrumental Methods of Analysis and Validation of an Analytical Procedure

Classical/Wet-Chemical Method

measurement depends on the chemical properties of the sample

Classical/Wet Method

reagent is made to react completely with the analyte

Classical/Wet-Chemical Method

relationship between the measured signal and analyte concentration is determined by chemical stoichiometry

Techniques in Classical/ Wet-Chemical Method

(1) Separation

(2) Qualitative analyses

(3) Quantitative analyses

(1) Separation

(2) Qualitative analyses

(3) Quantitative analyses

Techniques in Classical/ Wet-Chemical Method

separation in classical method

precipitation, extraction, or distillation

qualitative analyses in classical method

colors, boiling or melting points, solubility, odors, OA, or RI

quantitative analyses in classical method

gravimetric or volumetric

(1)More suitable for analysis of major constituents

(2)Used to certify analytical standards

(3)Generally cheaper

(4)More accurate and precise

(5)More robust and less susceptible to environmental fluctuations

Advantages of Classical/Wet-Chemical Method

Instrumental method

measurement of physical or chemical properties of the analyte

separation in instrumental method

chromatography and electrophoretic techniques

quantitative analyses in instrumental method

conductivity, electrode potential, light absorption or emission, mass- to-charge ratio and fluorescence

(1)Ability to perform trace analysis

(2)Most are multi-channel techniques

(3)Shorter analysis time

(4)Amenable to automation

(5)More samples may be analyzed quickly

(6)Less skill and training required

Advantages of Instrumental method

(1)Spectrometric Methods

(2)Chromatographic Methods

(3)Electrochemical Methods

(4)Miscellaneous Methods

Types of Instrumental Methods (4)

(1) Emission

(2) Absorption

(3) Scattering

(4) Refraction

(5) Diffraction

(6) Rotation

Spectrometric Methods (6)

Emission

[Spectrometric Methods]

Emission spectroscopy (X-ray, UV, visible, electron, Auger)

Fluorescence

Phosphorescence and Luminescence (X-

ray, UV and visible)

Absorption

[Spectrometric Methods]

Spectrophotometry and photometry

(X-ray, UV, IR)

Photoacoustic spectroscopy

Nuclear magnetic resonance and electron spin resonance spectroscopy

Scattering

[Spectrometric Methods]

(1)Turbidimetry

(2)Nephelometry

(3)Raman spectroscopy

Refraction

[Spectrometric Methods]

(1)Refractometry

(2)Interferometry

Diffraction

[Spectrometric Methods]

(1)X-ray

(2)Electron diffraction methods

Rotation

[Spectrometric Methods]

(1)Polarimetry

(2)Optical rotary dispersion

(3)Circular dichroism

Chromatographic Methods

(1) GC - Gas Chromatography

(2) HPLC - High-Performance Loquid Chromatography

Electrochemical Methods

(1) Conductometry

(2) Coulometry

(3) Polarography

(4) Potentiometry

Conductometry

[Electrochemical Methods]

resistance

Coulometry

[Electrochemical Methods]

charge

Polarography

[Electrochemical Methods]

current

Potentiometry

[Electrochemical Methods]

potential

Miscellaneous Methods

[Types of Instrumental Methods]

(1)Mass-to-charge ratio

(2)Thermal characteristics

(3)Radioactivity

Mass-to-charge ratio

[Miscellaneous Methods]

Mass spectrometry

Thermal characteristics

[Miscellaneous Methods]

Thermal gravimetry and titrimetry

Differential scanning calorimetry

Differential thermal analyses Thermal conductometric methods

Radioactivity

[Miscellaneous Methods]

Activation and isotope dilution methods

Analytical Instruments

converts information about the physical or chemical characteristics of the analyte to information that can be manipulated and interpreted by man

(1) Signal Generator

(2) Transducer or Detector

(3) Signal Processor

(4) Read-out Device

General components of Instrumental Measurement

Signal generator

[Analytical Instruments]

consists of the chemical system interacting with the stimulus from the energy source

Signal generator

[Analytical Instruments]

producing an analytical signal reflecting the presence and usually the concentration of the analyte

Transducer or Detector

[Analytical Instruments]

transforms the analytical signal produced by the signal generator into an electrical signal

Signal Processor

[Analytical Instruments]

modifies and "cleans up" the electrical signal to make it more convenient to interpret

Read-out Device

[Analytical Instruments]

converts the electrical signal to a form usable to the analyst

Calibration

determines the relationship between the analytical response and the analyte concentration

Calibration of Instrumental Methods

1.Comparison with standards

2.External standard calibration

3.Standard addition method

4.Internal standard method

direct comparison

[Comparison with standards]

concentration of the analyte was then equal to the concentration of the standard after dilution

direct comparison

[Comparison with standards]

property of the analyte is compared with standards such that the property being tested matches the standard

Titration

[Comparison with standards]

amount of the standardized reagent needed to achieve chemical equivalence can then be related to the amount of analyte present

External standard

[External standard calibration]

-used to calibrate instruments and procedures when there are no interference effects from matrix components in the analyte solution

-prepared separately from the sample

-series of such external standards containing the analyte in known concentrations is prepared

-response signal is obtained (absorbance, peak height, peak area) as a function of known analyte concentration

calibration curve

[External standard calibration]

prepared by plotting the data or by fitting them to a suitable mathematical equation, such as the slope-intercept form used in the method of linear least squares

response signal

[External standard calibration]

_ is then obtained for the sample and used to predict the unknown analyte concentration from the calibration curve or best- fit equation

Standard addition method

useful for analyzing complex samples in which the

likelihood of matrix effects is substantia

spiking

[Standard addition method]

adding one or more increments of a standard solution to sample aliquots containing identical volumes

Internal standard

substance that is added in a constant amount to all samples, blanks, and calibration standards in an analysis

Internal standard method

- calibration involves plotting the ratio of the analyte signal to the internal-standard signal as a function of the analyte concentration of the standards

Signal

analytical measurement that carries information about the analyte that is of interest to the scientist

Types of Signal

(1) absorbance

(2)peak area

(3)peak location

(4)peak height

(5)retention time

Noise

-analytical measurement made up of extraneous information that is unwanted

-it degrades the accuracy and precision of an analysis and also places a lower limit on the amount of analyte that can be detected

Chemical noise

arise from a host of uncontrollable variables that affect the chemistry of the system being analyzed

Chemical noises

1.undetected variations in temperature or pressure 2.fluctuations in relative humidity

3.vibrations that lead to stratification of powdered solids 4.changes in light intensity

5.laboratory fumes

Instrumental noise

associated with each component of an instrument

Signal-to-noise (S/N) ratio

equation that indicates the magnitude of an experimental effect above the effect of experimental error due to chance fluctuations

International Council for Harmonisation (ICH)

an international non-profit organization that aims to develop guidelines via a process of scientific consensus with regulatory and industry experts working together

Out-of-control process

a process in which variations among the observed sampling results cannot be attributed to a constant system of chance causes

Out of specifications (OOS) result

a result that falls outside established acceptance criteria which have been established in official compendia and/or by company documentation

Out of trend (OOT) result

a time-dependent result which falls outside a prediction interval or fails a statistical process control criterion

Standard

the metric, specification, gauge, statement, category or physical product sample against which the outputs of a process are compared and declared acceptable or unacceptable

Specification

- a list of tests, references to analytical procedures, and appropriate acceptance criteria that are numerical limits, ranges, or other criteria for the test described

- establishes the set of criteria to which a material should conform to be considered acceptable for its intended use

System suitability test

- used to verify that the test system will perform in accordance with the criteria set forth in the procedure

- the tests are based on the concept that the equipment, electronics, analytical operations, and samples analyzed constitute an integral system that can be evaluated as such

VALIDATION OF AN ANALYTICAL PROCEDURE

A process by which it is established, by laboratory studies, that the performance characteristics of the procedure (test method) meet the requirements for the intended analytical application.

VALIDATION OF AN ANALYTICAL PROCEDURE

Objective: to demonstrate that it is suitable for its intended purpose

ANALYTICAL PROCEDURE

Refers to the way of performing the analysis

ANALYTICAL PROCEDURE

• Should describe in detail the steps necessary to perform each analytical test.

ANALYTICAL PROCEDURE

Should define what the test results for the procedure are

IDENTIFICATION TESTS

• Spectroscopic identification Tests

• Infrared Spectroscopy 

QUANTITATIVE TEST FOR IMPURITIES’ CONTENT

Quantifies how much impurity is present.

QUANTITATIVE TEST FOR IMPURITIES’ CONTENT

Residue on Ignition

LIMIT TESTS FOR THE CONTROL OF IMPURITIES

Controls the amount of impurity in a particular sample.

LIMIT TESTS FOR THE CONTROL OF IMPURITIES

Limit of Free 4-aminophenol

QUANTITATIVE TESTS OF THE ACTIVE MOIETY

In samples of drug substance or drug product or other selected component(s) in the drug product.

TEST RESULT

The value of a characteristic obtained by carrying out a specified test method.

TEST RESULT

• Can be a result calculated from several observed values.

The observed value.

TEST RESULT

Can be, but need not be, the final, reportable value that would be compared to the acceptance criteria of a specification.

ACCURACY

• The closeness of test results obtained by that procedure to the true value. 

• Trueness

DRUG SUBSTANCE

Use analyte of known purity (e.g., RS or reference standard) or compare results of well-characterized procedure.

DRUG IN A FORMULATED PRODUCT (DP

Use synthetic mixtures of the drug product with known amount of the analyte, or to spike with the analyte.

IMPURITIES

Samples (of drug substance or drug product) spiked with known amounts of impurities.

PERCENTAGE OF RECOVERY

By the assay of the known added amount of analyte in the sample.

ACCURACY

CALCULATED AS:

DIFFERENCE BETWEEN THE MEAN AND THE ACCEPTED TRUE VALUE, TOGETHER WITH CONFIDENCE INTERVALS

 PERCENTAGE OF RECOVERY

ACCURACY

Minimum of nine determinations over a minimum of three concentration levels, covering the specified range (i.e., three concentrations and three replicates of each concentration).

PRECISION

The degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogeneous sample.

PRECISION

Expressed as the standard deviation or relative standard deviation (RSD) (coefficient of variation) of a series of measurements.

PRECISION

May be a measure of either the degree of reproducibility or of repeatability of the analytical procedure under normal operating conditions.

REPEATABILITY (INTRA-ASSAY PROCEDURE)

The use of the analytical procedure within a laboratory over a short period of time using the same analyst with the same equipment.

INTERMEDIATE PRECISION (RUGGEDNESS)

Expresses within-laboratory variation, as on different days, or with different analysts or equipment within the same laboratory.

REPRODUCIBILITY

The use of the analytical procedure in different laboratories, as in a collaborative study.

PRECISION

Aliquots of independent samples then calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation)

PRECISION

Minimum of nine determinations covering the specified range for the procedure (i.e., three concentrations and three replicates of each concentration) or using a minimum of six determinations at 100% of the test concentration

SPECIFICITY

The ability to assess unequivocally (no doubt) the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components.

IDENTIFICATION TESTS

Ensure the identity of the analyte.

IDENTIFICATION TESTS

The ability to select between compounds of closely related structure that are likely to be present should be demonstrated.

PURITY TESTS

Ensure that all of the analytical procedures performed allow an accurate statement of the content of impurities of an analyte (e.g., related substances test, heavy metals limit, or organic volatile impurities).

PURITY TESTS

Spiking the drug substance or product with appropriate levels of impurities and demonstrating that these impurities are determined with appropriate accuracy and precision.

ASSAYS

Provide an exact result, which allows an accurate statement on the content or potency of the analyte in a sample.

ASSAYS

Can be shown that the procedure is unaffected by the presence of impurities or excipients.