About HFL. Markets & services. Markets & services. Qualitative MS: Pharmaceutical Applications. Regulatory Framework

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1 Certainty of Identification in Pharmaceutical and Forensic Mass Spectrometry Phil 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 Markets & services Government Sports Healthcare Pharma Sports Drug surveillance Animal sports Human sports Sports research WADA HBLB NGRC UK Sport Veterinary Sports Pharma Government Healthcare Pharma In vitro discovery Bioanalysis Biomarkers Central clinical laboratory Pharmaceutical analysis 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 1

2 Forensic Applications Illicit drug identification (including source) Sample characterisation and comparison Toxicology (poisoning and illicit use) Sports / residue testing Regulatory Framework ISO 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 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. 2

3 R.A. of matched ion in reference spectrum Full-scan single-stage MS: Acceptable R.A. in test spectrum (10% absolute or 30% relative) Full-scan MS/MS & related techniques: Acceptable R.A. in test spectrum (20% absolute or 40% relative) 100% (90-100) (85-100) 2 90% (80-100) (75-100) 80% (70-90) (65-95) 70% (60-80) (55-85) 60% (50-70) (45-75) 50% (40-60) (62-38) 40% (32-48) (30-50) 30% (24-36) (23-37) 20% (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-MS 2 HFL all other techniques Example 1. Ethamsylate Ethamsylate 4-HBSA Issues Small highly polar analyte Extraction Chromatographic retention Ionisation efficiency Ethamsylate standard LC-MS/MS using ESI and MRM 3

4 3. Extract 3. Extract 4. Bio-blank rpt Relative abundance 3. Extract 4. Bio-blank rpt 5. Standard Std 5.6% Ext 5.5% Std 47% Ext 48% Std 50% Ext 45% 189>80 base-peak Relative abundance 189>189 base-peak Std 1.0% Ext1.1% Example 2. Std 8.6% Ext 9.5% Std 9.1% Ext 9.1% Std 18% Ext 20% Betamethasone 4

5 Example 2. Confirmatory Extract m/z 345 RA AORC HFL Dex std 92% Beta std 49% Conf extract 70% LC-MS 3 Betamethasone LC-MS 3 on Hypercarb LC-MS 3 on Hypercarb Betamethasone Confirmatory Extract.it s the way that you use them EC criteria for residue testing. 2 MS 2 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. 5

6 Thanks Simon Hudson Simon Biddle HFL Drug Surveillance Group Staff 6