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Speakers
Introduction to Analytical Method Validation

Scott Fletcher
Technical Business Development Manager Crawford Scientific
Dr Mike Swartz
Principal Scientist Ariad Pharmaceuticals
Moderator
Alasdair Matheson
Editor LCGC Europe
www.CHROMacademy.com
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Aims
The validation process
Guidelines on Analytical Method Validation (AMV) Analytical performance characteristics; Precision Accuracy Limit of Detection Limit of Quantification Specificity / Selectivity
Linearity and Range

Robustness
Objectives
Understand what encompasses analytical method validation and how this fits into the overall validation process Review the various government and other agencies who issue guidelines on method validation Define the analytical performance characteristics investigated during method validation
Describe which characteristics are required to be investigated by method type
Why Validate?
Method validation is completed to insure that an analytical methodology is accurate, reproducible and rugged over the specific range that an analyte will be analyzed. Method validation provides assurance of reliability. FDA Compliance
"The process of providing documented evidence that something does what it is intended to do."
General Analytical Method Validation
Initial Method Validation Guidance Issued in 1987 Guideline for submitting samples and analytical data for methods validation. Food and Drug Administration, February 1987. US Government Printing Office:1990-281794:20818. Updated in August 2000 (Draft Guidance!) Analytical Procedures and Method Validation. Fed. Reg. 65(169), 52,776-52,777, 30 August 2000 Incorporated ICH Guidelines Software Validation HPLC System Validation 1994 Position Paper All Guidances are on FDA Web site www.fda.gov/cder/guidance
The Process of Validation

Methods
Hardware
Validation
Validation
System
Software
Suitability
HPLC Method Validation Process

Step 1: Software Validation
Step 2: Chromatographic System Qualification

Step 3: Method Validation Using Validated Chromatographic System Step 4: Total System Validation Using System Suitability
FDA Draft Guidance/ICH Guidelines Analytical Performance Characteristics

Precision Accuracy Limit of Detection Limit of Quantitation
Method Validation
Specificity
Linearity Range Robustness
Accuracy: Definition
The closeness of test results obtained by the method to the true value. Established across the range
Accuracy: Determination
Drug Substance Analysis of reference material Compare results to a second, well-characterized method Determined concurrently with precision, linearity and specificity
Drug Product Analysis of synthetic mixtures spiked with known quantities of components Compare results to a second, well-characterized method Determined concurrently with precision, linearity and specificity
Accuracy: Determination (Cont.)

Impurities (Quantitation) Analysis of samples (Drug substances/Drug product) spiked with known amounts of impurities If impurities are not available, see specificity Recommended Data Minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g. 3 concentrations/3 replicates each) Reported as % recovery of known, added amount, or difference between the mean and true value, with confidence intervals Example Acceptance Criteria 97-103% of nominal value
Precision: Definition
Precision The measure of the degree of agreement among test results when the method is applied repeatedly to multiple samplings of a homogeneous sample Expressed as %RSD for a statistically significant number of samples
Precision: Definition
Precision Should Be Performed at Three Levels Repeatability Intermediate Precision Reproducibility
Precision: Definition/Determination
Repeatability (Generally the criterion of concern in USP analytical procedures) Same operating conditions, short time interval Inter-assay precision Minimum of 9 determinations covering specified range of procedure (3 levels, 3 reps each), or Minimum of 6 determinations at 100% test conc. Intermediate Precision (Experimental design recommended) Within-lab variations (Random events) Different days, analysts, equipment Reproducibility Precision between labs Collaborative studies
Precision - Acceptance Criteria

Less than 2% relative standard deviation is often recommended.
Less than 5% RSD can be acceptable for minor components.
Up to 10% RSD may be acceptable near the limit of quantitation.
Specificity: Definition
Specificity (Selectivity)
The ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the matrix The degree of interference Active Ingredients Excipients Impurities Degradation Products Placebo Ingredients
Specificity (Selectivity)
Separation Resolution Determination of separation between peaks Plate Count Determination of a systems efficiency Tailing Factor Calculation referencing peak shape
Specificity: Determination
Impurities Are Available Demonstrate that the assay is unaffected by the presence of spiked materials (impurities and/or excipients). Impurities Are Not Available Compare test result to a second well-characterized procedure For Assay, compare the two results For Impurity Tests, compare impurity profiles Peak Purity Test ("diode array, MS")
Detection Limit: Definition

Detection Limit (LOD) Lowest Concentration of Analyte in a Sample That Can Be Detected (not necessarily quantitated) Limit Test: Above or Below a Certain Level Expressed as Concentration (%, ppb) Almost never necessary to determine actual detection limit Rather, limit is shown to be sufficiently low (e.g. 0.1%)
Detection Limit (DL/LOD): Determination

Visual (Non-Instrumental Methods) Signal To Noise Ratio (3 or 2:1 Generally Accepted)
Detection limit may be based on the standard deviation of the response and slope:
DL = (3.3)STD/S
STD = standard deviation of the response S = slope of the calibration curve
Quantitation Limit (QL/LOQ): Definition

Limit of Quantitation (LOQ) Lowest Concentration of Analyte in a Sample That Can Be Determined With Acceptable Precision and Accuracy Under Stated Operational Conditions Expressed as the Concentration of Analyte Accuracy Precision
Quantitation Limit (QL/LOQ): Determination

Visual (Non-Instrumental Methods) Signal To Noise Ratio (10:1 is Typical) Quantitation limit may be based on the standard deviation of the response and slope:
QL = (10)STD/S
STD = standard deviation of the response S = slope of the calibration curve Both DL and QL are validated by analyzing a suitable number of samples. Method should be documented.
HPLC Columns and Effect on LOQ

0.0005
A) Waters Symmetry C18 Signal to Noise = 11

AUFS
5.00
Minutes
10.00
0.0005
B) Brand X C18 Signal to Noise = 6.5

AUFS
Sample: 0.25 g/mL Tamoxifen Injection volume and linear flow velocity compensated for on both columns
5.00
Minutes
10.00
System Noise and Effect on LOQ
Linearity and Range: Definition

Linearity The Ability of the Method to Elicit Test Results That Are Directly Proportional to Concentration Within a Given Range Expressed as the Variance of the Slope of the Regression Line
Range Interval between upper and lower levels of analyte demonstrated by the method Precision and Accuracy Expressed in the same units as the test results
Linearity: Determination
Established across the Range of the method Dilutions Separate Weighings Evaluate by Appropriate Statistical Methods (e.g. Regression) Include Correlation Coefficient, y-Intercept, Slope, Residual Sum of Squares, Plot Itself
Minimum 5 Concentrations
Determination of Appropriate Range

Minimum Specified Ranges Assay 80-120% Impurity Test From QL to 120% of spec. Toxic or more potent impurities: commensurate with the controlled level Content Uniformity 70-130% of test concentration Dissolution Testing +/- 20% over specified range
Robustness: Definition
Robustness Measure of The Capacity to Remain Unaffected by Small (Deliberate) Variations in Method Parameters Indication of Reliability During Normal Use
Robustness: Determination
Consider during development of method Shows reliability of method with respect to deliberate changes
If measurements are susceptible to variations in analytical procedures, these conditions should be controlled and a precautionary statement included. Establish System Suitability parameters to ensure the validity of the method
Robustness Ruggedness
Ruggedness refers to parameters external to the method Operator Day of week Robustness refers to parameters internal to the method % Organic in mobile phase Temperature
Why is Robustness Important?

Adjustments to methods are allowed
Changes to methods invite a whole new level of scrutiny! E.g. Revalidation..
Determining Robustness Factors

Chosen symmetrically around a nominal value, or the value specified in the method Form an interval that slightly exceeds the variations that can be expected when the method is implemented or transferred
E.G. for 60% methanol, the high (1) and low (-1) factors might be 58% and 62% methanol Range expected to bracket the variability a properly trained analyst can be expected to measure using proper laboratory apparatus For instrument settings, manufacturers specifications can be used to determine variability.
The range evaluated shouldnt be selected to be so wide that the robustness test will purposely fail, but to represent the type of variability routine encountered in the laboratory.
Challenging the method to the point of failure is not necessary!
Determining Robustness Factors: Isocratic Method

Factor Organic Solvent Concentration Limit Range (+/-) 2-3%
Buffer Concentration
Buffer pH (if adjusted) Temperature Flow rate Wavelength
1-2%
0.1-0.2 pH units 3C 0.1-0.2 mL/min. 2-3nm for 5nm bandwidth
Injection Volume
Column Lots
Injection type and size dependant

2-3 Different
These limits are examples only and should be chosen according to expected laboratory and instrument variations.
Determining Robustness Factors: Gradient Method

Factor Initial hold time* Slope and length Limit Range (+/-) 10-20 % of segment time The slope is set by the initial %B and the final %B, as well as the gradient length. It is recommended to adjust the lengths by 10-20% and allow the slope to vary. Adjusted according to the last eluting compound and varied accordingly
Final hold time
Factors and limits listed here are in addition to many of the factors considered in an isocratic method. * It is increasingly common for gradient methods to have an initial hold time to accommodate transfer to instruments with different dwell volumes.
System Suitability
System Suitability The checking of a system, before or during analysis of unknowns, to insure system performance.
No sample analysis is acceptable unless the requirements for system suitability have been met. (USP Chapter 621)
Plate Count, Tailing, Resolution Determination of reproducibility (%RSD)

For %RSD < 2.0%, Five replicates For %RSD > 2.0%, Six replicates
System Suitability "Sample" A mixture of main components and expected by-products utilized to determine system suitability
Whenever There is a Significant change in Equipment or Reagents System Suitability Testing Should be Performed (USP Chapter 621)
Recommendations From FDA 1994 Guideline: System Suitability

Capacity factor k' > 2
Precision/Injection repeatability RSD </= 1%, n >/= 5 Resolution Rs >/= 2 (Major peak and closest eluting)
Tailing factor T </= 2

Theoretical Plates In general N > 2000
How Does Validation Change According to the Type of Method?

Different Methods Have Different Requirements Amount or Extent of Validation Will Change Depending On: Methods intended use Stage of development
Data Elements Required For Assay Validation: USP 24

Category 1 Analytical methods for the quantitation of major components or active ingredients Category 2 Analytical methods for the determination of impurities or degradation compounds Category 3 Analytical methods for the determination of performance characteristics
Category 4 Identification tests

Validation Characteristics Vs. Type of Analytical Method

Analytical Performance Parameter Accuracy Precision Specificity LOD LOQ Linearity Range Robustness
Category 1: Assays Category 2: Impurities
Quant. Limit Tests
Category 3: Category 4: Specific Tests I.D.
Yes Yes Yes No No Yes Yes Yes
Yes Yes Yes No Yes Yes Yes Yes
* No Yes Yes No No No No
* Yes * * * * * Yes
No No Yes No No No No No
* May be required, depending on the nature of the specific test.
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Webcast Notes

Introduction to Analytical Method Validation

Interactive HPLC Troubleshooter

Linearity and Range

Describe which characteristics are required to be investigated by method type

General Analytical Method Validation

The Process of Validation

HPLC Method Validation Process

Step 2: Chromatographic System Qualification

FDA Draft Guidance/ICH Guidelines Analytical Performance Characteristics

Accuracy: Determination (Cont.)

Precision - Acceptance Criteria

Less than 5% RSD can be acceptable for minor components.

Up to 10% RSD may be acceptable near the limit of quantitation.

Detection Limit: Definition

Detection Limit (DL/LOD): Determination

Quantitation Limit (QL/LOQ): Definition

Quantitation Limit (QL/LOQ): Determination

HPLC Columns and Effect on LOQ

A) Waters Symmetry C18 Signal to Noise = 11

B) Brand X C18 Signal to Noise = 6.5

System Noise and Effect on LOQ

Linearity and Range: Definition

Determination of Appropriate Range

Why is Robustness Important?

Determining Robustness Factors

Determining Robustness Factors: Isocratic Method

Injection type and size dependant

Determining Robustness Factors: Gradient Method

Final hold time

Plate Count, Tailing, Resolution Determination of reproducibility (%RSD)

Recommendations From FDA 1994 Guideline: System Suitability

Tailing factor T </= 2

How Does Validation Change According to the Type of Method?

Data Elements Required For Assay Validation: USP 24

Category 4 Identification tests

Validation Characteristics Vs. Type of Analytical Method

Category 3: Category 4: Specific Tests I.D.

Yes Yes Yes No No Yes Yes Yes

Yes Yes Yes No Yes Yes Yes Yes

* May be required, depending on the nature of the specific test.

CHROMacademy & CHROMmunity

All questions will be answered within CHROMmunity: http://chrommunity.chromacademy.com

Question and Answer

We Need Your Feedback!

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