VALIDATION PLAN INFORMATION
Organization Name
Division Performing Validation
Method Validation for
Standard Operating
Procedure (SOP) Title
Equipment
Validation Number MVAL-01-2019-001
Number of Revision None
Authors
Reviewed by: Signature Date Signed
Approved by: Signature Date Signed
(TITLE) Method Validation Plan for
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1. Scope and Purpose of Validation
To establish a qualitative Liquid Chromatograph-Quadrupole Time of Flight (LC-
QTOF) Mass Spectrometer method for positive identification of Cocaine in seized
samples in the Philippines.
2. Types of materials for which the method will be applied
Standard Cocaine (Solid)
Unknown powders and other solid materials which are suspected to contain
Cocaine.
3. Equipment, Chemicals, and Supplies
3.1 Equipment
Agilent 6530 QTOF LC/MS equipped with Agilent 1260 Infinity II high speed
pump, multi-sampler, and multi-column thermostat with Mass Hunter Software.
3.2 Chemicals and Sample Materials
3.2.1 Reagents
Distilled water, LC-MS grade (Honeywell)
Acetonitrile, LC-MS grade (Sigma-Aldrich)
Methanol, LC-MS grade (Sigma-Aldrich)
Isopropanol, LC-MS grade (Sigma-Aldrich)
Formic Acid, Reagent grade (Agilent)
ES-L low concentration tuning mix (Agilent)
ES-TOF biopolymer analysis kit (Agilent)
API-TOF reference mass solution kit (Agilent)
HPLC flushing solvent
3.2.2 Reference Standard
Cocaine, solid, 50mg (Australian Government National Measurement Institute)
3.2.3 Actual Sample
Cocaine powder samples without on-going case.
4. Supplies
2
LC vials (amber) with septum and cap (1.5ml)
Volumetric flasks-glass (2ml, 5ml, 10ml, 100ml, 500ml, 1000ml)
Volumetric flasks-plastic (2ml, 5ml, 10ml, 100ml, 500ml, 1000ml)
Autopipettors (0.1-2µl, 1-10µl, 10-100µl, 100-1000µl)
Pipette tips (0.1-2µl, 1-10µl, 10-100µl, 100-1000µl)
Disposable plastic syringe (10 ml)
Filter membrane units (0.22 µm)
Nalgene bottles (100ml, 500ml, 1000ml)
Amber bottle with plastic hose and plastic cap for LC
Ultra high purity Nitrogen gas
Vortex mixer
Sonicator
5. Procedure
5.1 Preparation of standard solutions
Certified reference material of Cocaine dissolved in acetonitrile will be used for
standard stock solution preparation. Standard stock solutions will must contain a
concentration in parts per billion (ppb) and will be placed in a closed flask in a
refrigerator if not needed. Pipette accurately appropriate amounts of the stock standard
solution into appropriate volumetric flasks. Dilute solutions in appropriate amount of
acetonitrile up to mark and calculate the concentration of each subsequent standard
solution in ppb. Then transfer prepared standard solutions into a LC vial and inject 1-2
μL into the LC-QTOF instrument. On the other hand, all Cocaine standard and sample
solutions will be analyzed with a minimum of 3 trials in every validation parameter
conducted, and all values will be reported up 2 to 4 decimal places.
5.2 Instrument Parameters
5.2.1 LC Conditions
Column Agilent ZORBAX Eclipse Plus C18
2.1mm x 50mm, 1.8µm
Column Temperature 60oC
Mobile Phase A: 0.1% Formic acid B: 0.1% Formic acid in
in water acetonitrile
Flow Rate 0.5ml/min
Isocratic Program 85% A and 15% B
Analysis Time 4 minutes
Post Time 2 minutes
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Overall Cycle Time 8 minutes
Gas Ultra High Purity Nitrogen
Injection Volume 1µl (with 5 seconds needle wash in flushport)
5.2.2 QTOF Conditions
Electrospray AP-ESI
Source (Using Agilent Jet Stream Technology)
Ionization Polarity Positive
Sheath Gas Temperature and 380oC at 12L/min
Flow
Nozzle Voltage 500 V
Drying Gas Temperature and 320oC at 8L/min
Flow
Nebulizer Gas Pressure 27psi
Capillary Voltage 3.750 V
Fragmentor Voltage 150 V
Collision Energy 10 V
Personal Compound
Database and Library Forensics (ForTox PCDL)
(PCDL)
5.2.2 MS Acquisition Method Parameters
Reference Ion Masses 121.050873 and 922.009798
Enabled
Acquisition Mode MS1
Minimum Mass Value 50 m/z
Maximum Mass Value 1000 m/z
Scan Rate 3 Hz
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5.2.3 Mass Calibration, Tuning and Corresponding Accepted Values
Corrected Residual Must be less than 0.5ppm
Mass Error
Resolution 2GHz: 118 ion > 4,500
1521 ion > 13,000
Abundance 200,000 to 500,000
Note: Check tune and mass calibration must be performed before the start of each
analysis for the day. Check tune results must be accepted in order to proceed with the
validation analysis.
6. Validation Parameters
Peak Symmetry
Precision (Repeatability/Reproducibility)
Robustness
Stability
Carryover
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7. Validation Procedure and Acceptance Criteria
Acceptance Proposed
Parameter Validation Procedure Criteria Duration
1. Peak Symmetry
-a measure of
chromatographic
peak’s symmetry.
2. Precision
- Measure of the
closeness of the
analytical results
obtained from a
series of replicate
measurements of the
same measure under
the conditions of the
method.
3. Robustness
- Performance of an
analytical method in
the presence of
minor variation of
some experimental
conditions of the
method within the
laboratory.
4. Stability
- a method that
demonstrates the
extent to which the
analytes are stable
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during the whole
analytical
procedure, including
storage before and
after analysis.
5. Carryover
- a type of test to
check possible
significant peaks in
the blank run after
testing a sample of
typically higher
concentration.
8. References
8.1 United Nations Office on Drugs and Crime, Validation of Analytical Methodology
and Calibration of Equipment used for Testing Illicit Drugs in Seized Materials and
Biological Specimens, ST/NAR/41 2009, pp. 5-50.
8.2 United Nations Office on Drugs and Crime, Recommended Methods for the
Identification and Analysis of Cocaine in Seized Materials, ST/NAR/7/REV.1 2012,
pp. 17-34.
8.3 Health Sciences Authority, Singapore, Training Course on Qualitative Method
Validation, April 05, 2017, Training Manual, pp. 1-15.
8.4 University of the Philippines- Natural Sciences Research Institute, Training Course
on Quality Assurance in Chemical Analysis for the Philippine Drug Enforcement
Agency (PDEA) Laboratory Service, February 17-19, 2016, Training Manual, pp.
155-178.
8.5 Agilent Technologies, The Secret of Good Peak Shape in HPLC, Available:
https://www.agilent.com/cs/library/eseminars/Public/secrets%20of%20good
%20peak%20shape%20in%20hplc.pdf.
8.6 SWGDRUG Supplemental Document SD-2, Quality Assurance/Validation of
Analytical Methods, SWGDRUG 2006-02-09, pp. 6-8, 18-20, 47-51.
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9. Appendices
9.1 Appendix A:
9.1.1 Peak symmetry
9.1.2 Precursor ion and two (2) candidate ions and their corresponding % abundances
9.1.3 Mass spectrum and library search score
9.1.4 Mean, standard deviation and relative standard deviation
9.2 Appendix B:
9.2.1 Proposed Validation Report Format
Note: Proposed submission period of validation report which includes calculation,
interpretation and reporting will be on ___________.
