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GLP Training
Extraction and Analysis
Wayne Jiang, PhD
IR-4, Michigan State University, jiangwa@msu.edu
Michael Braverman, PhD
IR-4 headquarters, Rutgers University
Contents
Sample Receipt
Sample Handling and Processing
Sample Storage
R f S d d d S l i Reference Standards and Solutions
Reference Method and Working Method
Method Validation and Sample Analysis
Storage Stability
Raw Data
Analytical Summary Report
Definitions
Lowest level of Method Validation (LLMV) is the
lowest fortification concentration level at which
the method is validated for a particular matrix.
Limit of Detection (LOD) is the smallest amount Limit of Detection (LOD) is the smallest amount
of the analyte that can be reliably detected from
the background for a particular matrix.
Limit of Quantitation (LOQ) is the smallest
amount of the analyte that can be quantified with
a certain degree of reliability.
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Reference Methods
Section 29.1 in an IR-4 protocol includes a reference
method (registrant reference method) that mainly
should be followed.
Working Method
Due to the nature of sample matrices and
availability of analytical instrumentation, a
working method can be developed with
minor modifications.
The working method is needed to verify the
validity of the method for different matrices.
The SD approves the minor modifications.
Working Method
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Working Method Example
Minor vs Major Modifications
Minor Modifications are generally:
Changes in sample size, which should not lead to sample integrity
and homogeneity concerns
Changes in extract volumes; if it is a change in sample size, the
change of the rest procedure should be changed accordingly and is
typically proportional to those the original method yp y p p g
Modification of cleanup steps
Removal of cleanup steps
Optimization of instrument analysis parameters
Major Modifications are:
Changes in extraction method or extraction solvent
Changes in chemistry at major steps (extraction,
derivatization, hydrolysis, detection nature, etc.) .
Questions: Minor or Major ?
Change in extraction method?
Homogenizing QuEChERS
Change in extraction solvent?
Acetonitrile Acetone
?
Change in analytical Instrument?
GC/MS* HPLC/MS*
LC/UV LC/MS/MS
*Reason: GC equipped with ECD, NPD, or MS
detection is for detection of volatile, semivolatile,
and thermally stable compounds; while HPLC with
UV, FL, or MS detection for nonvolatile and/or
thermally labile compounds.
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Approval by SD
Any major change, such as a change from GC to
LC method or visa versa, or a change in
extraction method from homogenizer to shaker,
or to QuEChERS, will require independent MV , q p
and is therefore NOT recommended.
All changes/modifications should be discussed
with and approved by the Study Director (SD)
prior to sample analysis.
QuEChERS : Quick Easy Cheap Effective Rugged Safe
Weighing
Balance annual calibration (certificate)
Check balance using standard weights
Make sure all weights are bracketed by standard weights
Weighing
Sample jars are well labeled
Double sample ID before weighing
Weigh frozen samples (do not let it thaw)
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Balance Log
Minimum 1 control
Minimum 1 QC
Field samples
Calibrate pipet
(if pipet is used)
Check balance
before weighing
weigh vessle to
bracket weighings
Fortification
Pipet/syringe calibration
Fortify samples
Method Validation (MV)
Three (3) recovery spike concentrations in
triplicates are required as per protocol.
Lowest level of method validation (LLMV) and
two higher concentrations Bracket anticipated two higher concentrations. Bracket anticipated
range of residues.
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Inform SD with MV Data
The SD is informed of the
recovery results and the
working method.
f % Recoveries of 70 - 120%
are accepted.
If outside this range, SD
approval or additional MV
recovery spikes are
required.
Question: GLP Starts in Lab
When GLP starts in an analytical lab?
SD signs the protocol
Analytical reference standard is received and the first
application is performed in the field
First field sample is received by lab
MV is completed and SD approves to start sample
analysis
Sample Analysis
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Analytical Set
An analytical set
contains:
- Minimum 1 control
- Minimum 1 QC
(concurrent recovery spike)
- Field treated samples.
Double Injection
Double injection (also called dual injection) is
required for analyzing weathered samples,
including control, concurrent spike and field treated
samples in the same analytical set For example samples in the same analytical set. For example,
each sample vial is injected for analysis by two
separate injections (as one after the other) to
ensure the quality of analytical data.
Double injection is not required for analytical
standards, solvent blank, samples of MV set, or
samples of SS sets.
Worklist (Sequence)
Calibration standards
1
Solvent blank
2
Control sample
3
C t ik l ( )
4
Concurrent spike sample(s)
4
Field treated samples
5
Solvent blank
2
Calibration standards
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1
Generally 5-6 calibration standards (minimum 3), starting and ending the worklist
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Minimum one blank after standards; may add more to ensure minimized carryover
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Double injection is require for control analyzed with treated samples Order may change
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Double injection is required for QC sample (minimum 1 QC at LLMV)
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Double injection is required for treated samples
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Worklist (Sequence)-Example
Calculation Sheet (Example1)
Calculation Sheet (Example2)
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Sample Analysis
During the course of residue sample
analysis, adequate concurrent recovery
spikes bracketing the actual residues have
to be analyzed to be analyzed.
If recoveries outside of 70 - 120%, set will
first be re-injected and if still not
satisfactory another set will be extracted
and reanalyzed in agreement with the SD.
Sample Analysis
Controls with interference or
contamination as well as field
samples with unusual
residues must be reported to
the SD promptly.
During MV as well as during
field sample analysis
instrument parameters and
conditions should stay
constant and are recorded in
an instrument log book.
Data Acceptance Criteria
Spike recovery is between 70 - 120%
1
Double injection on weathered samples
3
Meet linearity requirements (R
2
0.98 or 0.99)
2
Five (5) calibration levels or more
2
Five (5) calibration levels or more
2
Difference between double injection 15%
2,3
Days between extraction and analysis 14 days
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Residues in treated bracketed by spike levels
1,3
Residue in control LOD
3
1
Required by protocol
2
Required by Lab SOPs
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IR-4 Laboratory Guidelines
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Storage Stability
Storage Stability
One time analyses are
carried out to show that
samples are stable under
the conditions stored.
St t bilit l i Storage stability analysis
is performed after the
longest interval between
harvest and extraction of
a field sample.
Storage Stability
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Calculation of LOD/LOQ
Before completion of
the analytical phase
of the study a
minimum of six minimum of six
recovery spikes at
the LLMV are
required in order to
calculate the LOD
and LOQ.
LOQ Calculations
Analytes of Interest?
Residue Expression will determine which
compounds (Parent, metabolites)will need to
be analyzed for risk assessment and
f enforcement
Determined by Regulatory authorities after
extensive review of metabolism and toxicology
data
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Questions ?
Any questions?
Thank
Y ! You!
jiangwa@msu.edu