Method Validation Protocol

Study No.______

METHOD VALIDATION PROTOCOL

Method Validation Protocol for a

Chromatography based method(e.g., HPLC, GC, CE)

Determining the Concentration of Analyte in Matrix

Study No.Study#

Date Issued:Issue Date

SIGNATURE PAGE

This protocol has been performed and reviewed by the undersigned and to the best of their knowledge is correct and complete.

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Dept. Manager’s NameDate

Title

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Quality Person NameDate

Title

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Laboratory Manager NameDate

Title

TABLE OF CONTENTS

Page #

  1. Introduction………………………………………………………
  1. Experimental…………………………………………………….
  1. Validation Procedure…………………………………………
  1. Documentation…………………………………………………..
  1. Introduction

This Protocol covers the validation of Method Internal SOP#for assayingmatrix/matricesfor their content of analyte. (Basic description of the analyte,the method being validated and the validation concentration range). This protocol outlines the studies being undertaken to demonstrate the method’s applicability for the matrices in question and to provide method performance parameters for the method as performed by Laboratory’s staff.

  1. Experimental
  1. Test Materials (list standards, controls, testing samples)
  1. Methods (Internal method reference and published method that it is based upon).
  1. Validation Procedure
  1. Organization

Source of samples.

Source of Reagents and Standards.

  1. Testing Requirements

Table 1. Overview of Testing Requirements for Method Validation

Parameter / Description / Criteria
System Suitability / 5 consecutive replicate injections of a working standard / USP tailing factor of the standard peak ≤ ____
Resolution for standard from ?
must be ___
Peak with?
Std. peak area %RSD ≤ _____
Std. retention time %RSD ≤ _____
Theoretical Plates=
Linearity / Range of Std Concentrations; / Correlation Coefficient “R”______
ValidationRange / Concentration interval for which the method is being validated
Selectivity / Blank Control Samples -
Suggest one“Blank Control” sample for each matrix covered. / No peaks present in the region of interest which have a response of __% of the lowest standard.
Accuracy/Recovery (Spiked Samples) / At least one of the “blank control” samples is spiked with the equivalent of (number and levels of spikes) where possible (minimum 9 data points). / Spike recovery > __%
%RSD < __%
Repeatability (Control Samples) / At least 3samples containing the analyte of interest in the form normally received from clients (or spiked samples)are assayed using 2 separate sample preps within 1 run. This is repeated on 4 different days. / Establish median control value
Establish within run CV
Establish CV for all observations
LOD / LOQ / Spike “blank control” sample resulting in a final analyte concentrationclose to the estimated LOQ / Total 7 data points
LOD 3 x StdDev.
LOQ 10 X Std Dev.
Stability / Sampleextracts and a standard solutions containing approx (X, Y concentrations) are tested after (A,B,C,D,E) time of storage at ? ºC .
Matrix samples / Analysis of samples with known/estimated amount of analyte / Results must be compatible with known/estimated values.
Intermediate Precision / On a different Separation System including different reagents and column (to the extent possible), a 2nd analyst performs
  • System suitability
  • Linearity
  • Accuracy (Standard addition spikes, RMs)
  • Repeatability (Analysis of quality control sample(s) in pentaplicate)
/ System suitability criteria must be met
Spike recoveries must be between __% and __%
Reference Material results must be within __x CV of the mean value determined by Analyst 1
The average of the 5 values for the quality control sample must be within _ STDEV from the established control value
The within run CV must be within __ x the within CV established by Analyst 1.
Ruggedness / Perform ruggedness study challenging these seven factors:
  • A
  • B
  • C
  • D
  • E
  • F
  • G
/ There are no criteria for ruggedness studies. It will aid in determining Critical Control Points for the assay.
Uncertainty / 2 * %CV
  1. System Suitability

To show injection reproducibility, column and overall chromatography system suitability, perform five replicate injections of a standard with a concentration similar to those found in routine samples or near regulatory action levels when appropriate. Resulting peak retention time, peak area and peak shape are analyzed and Tailing factor, Resolution from ?, % RSD of peak area, %RSD on retention time and column theoretical plates are calculated.

Criteria:

  • USP tailing factor of the standard peak ≤ ____
  • Resolution for standard from ?must be > ___
  • Peak with?
  • Std. peak area %RSD ≤ _____
  • Std. retention time %RSD ≤ _____
  • Theoretical Plates > ______
  1. Linearity

The standard curve for the method covers the range ___ - __ equivalent to a sample concentration of __ - __ (samples with higher concentrations (up to __) can be analyzed following appropriate dilution steps).Dilutions of the analyte are made in the appropriate solvent (e.g., mobile phase, blank matrix or extraction solution)and injected.From the response a correlation coefficient and y-intercept is calculated (the blank is not considered part of the standard curve). The standard curve should be made up by data points representing a minimum of 5 different concentrations covering the validation range of the assay with the lowest standard being close to the LOQfor linear curves and at least 7 different concentrations for non-linear curves.

Description of standard curve

and minimum correlation coefficient required for assay

Criteria:

  • Correlation coefficient R > 0.XXX
  1. ValidationRange

The validated range is the interval of analyte concentration within which the concentration can be regarded as validated. The range should be supported by assessment of precision at or near the extremes of the operating range.

Criteria:

  • None
  1. Selectivity

To assess the selectivity (or specificity) of the methoda minimum of one “blank control” sampleexpected to be free of analyte (if available) from each of the major matrices covered by the study will be analyzed.It is recommended to use at least five representative matrices.Detailed description.

There are situations where suitable “blank control” samples are not available e.g., Ginkgo tissue with no ginkgo. In such situations that authenticity and purity of the peaks observed can be verified by tools such as Diode Array Spectrophotometers (DAD) and MS detectors (checking if peak purity parameters change as the sample is spiked with the analyte of interest. It is also possible to check if the “background peak” moves similar to the analyte when changing chromatographic conditions (temperature, mobile phase, column, etc.) Mass spectral match to include 3-4 MS fragments, 10% match in SIM, 20% match in full scan or MSxMS.

Criteria:

  • No peak in the area of interest with a response > __% of the lowest standard.
  1. Accuracy/Recovery (Spiked Controls):

Spikes provide information about method recovery and precision. Oneor more of the blankcontrol samples (if available)will be spiked with analyte to a final concentration in the control sample of0,X,Y,Z units. The spiked controls will be prepared and assayed in triplicate. The study will be performed a total of three (3) times (Minimum of 9 data points per sample). Calculate %RSD at each level.

There are situations where suitable “blank control” samples are not available e.g., Ginkgo tissue with no ginkgo. In such situations samples containing the analyte of interest are used as the basis for spiking studies (Surrogate recovery – it is not ideal but better than nothing).

If a Certified Reference material (e.g., NIST material) is available such a material will be analyzed in duplicate.

Criteria:

  • Recoveries must be between __ % and __%
  • The mean value of duplicate analyses of a Certified Reference Material must be within the range given by the reference material provider.
  1. Repeatability (Control Samples)

Control Samples are created from samples where the matrix and analyte concentration is representative of the major part of samples received for this analysis. Control samples shall be representative of the range covered (e.g., low-, mid-, and upper range). A minimum of 3 such samples will be extracted and analyzed in duplicate as part of onerun. This analysis will be repeated on at least 3 different days (total of a minimum of 18 data points). In the absence of naturally fortified samples this analysis can be performed using matrices spiked at different levels (see also data points determined under “5.”)

Criteria:

  • Median value as well as average within-run CV (an expression of repeatability) and average CV of all results for the Quality Control sample(s)will be calculated for each
  1. Limit of Detection (LOD) & Limit of Quantitation (LOQ)

The determination of LOD and LOQ is not required if the method range is well above the LOQ/LOD. When challenging an assay at or below the range of the standard curvespike matrixBlank samples with analyteresulting in final concentrations close to the estimated LOQ (a minimum of 7 data points). The CV at the LOQ level should preferably be less than 20%.

Criteria:

  • Determine STDEV.
  • Determine LOD as > 3 x STDEV
  • Determine LOQ as > 10x STDEV
  1. Solution stability

Store aliquots of stock and working standard solutions as well as fortified sample extracts (2x-5x the LOQ) at room temperature and refrigerated (around 4 C). At time intervals these solutions will be reassayed against freshly made up standards.

Criteria:

  • Will determine the stability of the solutions.
  1. Matrix samples

To prove the method is applicable to the matrices covered, samples with known/estimated amount covering one of more of the matrices will be analyzed in triplicate.

Criteria:

  • Results must be compatible with known or estimated values.
  1. Intermediate Precision

A 2nd analyst on a different separation system and with a different column and independently prepared solutions and standards, will repeat system suitability, linearity (1 set of standards), analysis of Quality Control Sample(s) (5 separate extractions analyzed together as 1 run) and Reference or control samples( if available, duplicate analyses). A spiking study involving the spiking of one of the blank control samples resulting in final concentrations in the extract equivalent to the lowest, middle and highest standard in the standard curve. Each spike will be performed in triplicate

Criteria:

  • System suitability criteria must be met.
  • The average of the 5 values from the quality control sample must be within _1_ STDEV from the established control value. The within run CV must be within _2_ x the within CV established by Analyst 1.
  • The spiking study must show recoveries between __% and ___%.
  1. Ruggedness

Perform ruggedness study challenging these seven factors. Factors to consider could include among many other factors: extraction solvent composition, extraction time, extraction temperature, extraction method; pH, SPE cartridge manufacturer, HPLC column manufacturer,blow down time and temperature, other concentration approaches, mobile phase, and chromatography conditions.

  • A
  • B
  • C
  • D
  • E
  • F
  • G

The factors will be evaluated using the Youden technique. The evaluation will be used to determine critical control points for the assay.Choose seven factors that may affect the outcome and assign values.

Factor / Value from Method / Challenge Testing
1 / A / a
2 / B / b
3 / C / c
4 / D / d
5 / E / e
6 / F / f
7 / G / g

Using the table above, conduct 8 assays, utilizing the specific combinations of factors as follows:

Table 6. Ruggedness Test Results
Trial Number / Factor Combinations / Result
TN 1 / A B C D E F G / T1
TN 2 / A B c D e f g / T2
TN 3 / A b C d E f g / T3
TN 4 / A b c d e F G / T4
TN 5 / a B C d e F g / T5
TN 6 / a B c d E f G / T6
TN 7 / a b C D e f G / T7
TN 8 / a b c D E F g / T8

Perform calculations using the Youden method. The numbers associated with each pair of factor (e.g. A vs a) indicates the importance of that specific aspect. The greater the absolute value, the more important the factor. A positive value means that compared to the challenge, the condition specified by the method gives higher result. A negative value means exactly the opposite.

Factor A = (T1+T2+T3+T4-T5-T6-T7-T8)/4 =

Factor B = (T1+T2-T3-T4+T5+T6-T7-T8)/4 =

Factor C = (T1-T2+T3-T4+T5-T6+T7-T8)/4 =

Factor D = (T1+T2-T3-T4-T5-T6+T7+T8)/4 =

Factor E = (T1-T2+T3-T4-T5+T6-T7+T8)/4 =

Factor F = (T1-T2-T3+T4+T5-T6-T7+T8)/4 =

Factor G = (T1-T2-T3+T4-T5+T6+T7-T8)/4 =

  1. Uncertainty

Uncertainty can be determined as 2* %CV of a control sample of reasonably similar matrix and analyte concentration.

  1. Protocol Amendments / Deviations:

All changes and revisions to this protocol and the reason for the change shall be documented, approved by the appropriate Business Unit quality personnel and Laboratory management and maintained with the protocol. If the protocol is sponsored by a client, the client must also approve all changes.

  1. Documentation
  1. Final Report

Attached to the report will be a final copy of method Internal SOP#, copy of Analyte Reference Standard CoA, and a listing of any protocol and method deviations occurring during the method qualification study.

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