certainty of identification in pharmaceutical and forensic mass …aamg/mscertofident.pdf ·...

1

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

2

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.



3

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



4

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



5

Example 2. Dexamethasone


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


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.



6

Thanks

• Simon Hudson • Simon Biddle• HFL Drug Surveillance Group Staff



certainty of identification in pharmaceutical and forensic mass …aamg/mscertofident.pdf ·...

Documents