Certainty of Identification in Pharmaceutical and Forensic Mass …aamg/MSCertofIdent.pdf · 2006-08-03 · 1 Certainty of Identification in Pharmaceutical and Forensic Mass Spectrometry
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Certainty of Identification in Certainty of Identification in Pharmaceutical and Forensic Pharmaceutical and Forensic
Mass SpectrometryMass SpectrometryPhil Teale, HFL Ltd.
About HFL• The only laboratory in the world engaged in sports doping
control & contract research• Long heritage in high integrity analysis & centralised laboratory
services (est. 1963)• 130 people• State-of-the-art facilities (1997) near Cambridge• Leader in development & validation of difficult analytical
methods in complex matrices• Quality focus
Markets & services
Sports• Drug surveillance
–– Animal sports Animal sports –– Human sportsHuman sports
• Sports research–– WADAWADA–– HBLBHBLB–– NGRCNGRC–– UK SportUK Sport
• Veterinary
Government
HealthcarePharma
Sports
Markets & services
Pharma• In vitro discovery• Bioanalysis• Biomarkers• Central clinical
laboratory• Pharmaceutical
analysis
Sports
GovernmentHealthcare
Pharma
Qualitative MS: Pharmaceutical Applications
• NCE synthesis and development• Metabolite identification /
characterisation• Impurity identification /
characterisation• Patent protection
Regulatory Framework
• FDA / GLP• Multiple analytical platforms
(accurate mass, NMR, etc.)• Verification against standards,
including isomers• Typically no industry generic MS
criteria • Expert user / Peer review
Certainty of Identification in Pharmaceuticaland Forensic Mass Spectrometry Applications
Phil Teale - HFL, Fordham
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Forensic Applications
• Illicit drug identification (including source)
• Sample characterisation and comparison
• Toxicology (poisoning and illicit use)• Sports / residue testing
Regulatory Framework
• ISO 17025
• Process driven (detailed SOP’s)• Consistent reporting• Criteria for identification are a
requirement
Process (Confirmatory)
• Instrumental criteria, maintenance, calibration, response, etc.
• Sample prep SOP• Sample type and running order• MS acceptance criteria
Industry Criteria
• FDA / EU residue testing (mandatory)
• WADA / AORC sports testing (advisory)
• Drive for ISO 17025 accreditation• Lab to lab consistency• Protection from legal challenge
AORC Criteria
• Retention time
• GC - 1% (RRT or RT) or 6 sec (RT)
• LC - 2% (RRT or RT) or 12 sec (RT)
MS Criteria
• Requires 3 diagnostic ions (criteria advise but are not specific).
• Single-stage MS: 10% absolute or 30% relative, whichever is the greater
• MS/MS & related techniques: 20% absolute or 40% relative, whichever is the greater.
Certainty of Identification in Pharmaceuticaland Forensic Mass Spectrometry Applications
Phil Teale - HFL, Fordham
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Full-scan
single-stage MS: Full-scan MS/MS
& related techniques:
R.A. of matched ion in reference
spectrum Acceptable R.A. in test spectrum (10% absolute
or 30% relative)
Acceptable R.A. in test spectrum (20% absolute
or 40% relative) 100% 70-100 (90-100)1 60-100 (85-100)2
90% 63-100 (80-100) 54-100 (75-100) 80% 56-100 (70-90) 48-100 (65-95) 70% 49-91 (60-80) 42-98 (55-85) 60% 42-78 (50-70) 36-84 (45-75) 50% 35-65 (40-60) 30-70 (62-38) 40% 28-52 (32-48) 20-60 (30-50) 30% 20-40 (24-36) 10-50 (23-37) 20% 10-30 (15-25) 0-40 (10-30) 10% 0-20 (5-15) 0-30 (1-20) 5% 0-15 (1-10) 0-25 (1-15) 1% 0-11 (1-6) 0-21 (1-11)
1 HFL EI GC-MS2 HFL all other techniques
Example 1. Ethamsylate
Ethamsylate4-HBSA
Issues
• Small highly polar analyte• Extraction• Chromatographic retention• Ionisation efficiency
• LC-MS/MS using ESI and MRM
Ethamsylatestandard
Running order
1. Bio-blank
Running order
1. Bio-blank2. System-blank
Certainty of Identification in Pharmaceuticaland Forensic Mass Spectrometry Applications
Phil Teale - HFL, Fordham
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Running order
1. Bio-blank2. System-blank3. Extract
Running order
1. Bio-blank2. System-blank3. Extract4. Bio-blank rpt
Running order
1. Bio-blank2. System-blank3. Extract4. Bio-blank rpt5. Standard
Relative abundance
Std 5.6% Ext 5.5%
Std 47% Ext 48%
Std 50% Ext 45%
189>80 base-peak
Relative abundance189>189 base-peak
Std 1.0% Ext1.1%
Std 8.6% Ext 9.5%
Std 9.1% Ext 9.1%
Std 18% Ext 20%
Example 2. Dexamethasone
Dexamethasone Betamethasone
Certainty of Identification in Pharmaceuticaland Forensic Mass Spectrometry Applications
Phil Teale - HFL, Fordham
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Example 2. Dexamethasone
Dexamethasone Betamethasone
LC-MS3
Confirmatory Extractm/z 345
RA AORC HFL
Dex std
92% 55-100 75-100
Beta std
49% 30-70 62-38
Conf extract 70%
LC-MS3 on Hypercarb
Dexamethasone Betamethasone
LC-MS3 on Hypercarb
Dexamethasone ConfirmatoryExtract
….it’s the way that you use them
• EC criteria for residue testing.• 2 MS2 ions. • >50% - 20% relative• 20-50% - 25% relative• <10% - 50% relative• Extensive validation required for the
matrix analyte combination.
Summary• Mass spec criteria are a valuable tool
for the analyst• They must be applied with due
consideration of the analyte, matrix and level of validation.
• They require appropriate managerial and procedural structures backed up by expertise.
Certainty of Identification in Pharmaceuticaland Forensic Mass Spectrometry Applications
Phil Teale - HFL, Fordham
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Thanks
• Simon Hudson • Simon Biddle• HFL Drug Surveillance Group Staff
Certainty of Identification in Pharmaceuticaland Forensic Mass Spectrometry Applications
Phil Teale - HFL, Fordham
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