1. ROLE OF SAMPLE PREPARATION IN ISO/IEC 17025
- Why sample preparation is critical: sensitivity, accuracy, reproducibility; reduces matrix interference/ion suppression; ensures sample integrity; prevents instrument contamination
- Relevant ISO/IEC 17025 clauses: sample handling, storage, traceability; method validation; nonconforming work; technical competence
2. SYSTEMATIC APPROACH TO LC–MS SAMPLE PREPARATION
Structured workflow
- Define matrix and analytes; identify interferences; select extraction/clean‑up (protein precipitation, SPE, LLE, QuEChERS, dilution); filter/centrifuge/concentrate; verify LC‑MS compatibility; document workflow
Trend‑based troubleshooting
- Monitor ion suppression/enhancement, peak shape deterioration, sample stability
Risk‑based decision making
- When to re‑prepare/re‑extract; when to reject a compromised sample; when matrix effects invalidate data
3. SAMPLE COLLECTION AND STORAGE
- Collection guidelines: contamination‑free containers, avoid reactive plastics
- Storage conditions: temperature, light protection, solvent compatibility
- Stability considerations: short‑term vs long‑term, freeze‑thaw cycles
- Labelling and traceability: unique IDs, chain‑of‑custody, audit‑ready documentation
4. COMMON SAMPLE PREPARATION TECHNIQUES
Protein Precipitation (PP)
- For plasma, serum, protein‑rich matrices; solvents: ACN, MeOH, acidified; removes proteins causing fouling/ion suppression
Solid‑Phase Extraction (SPE)
- Targeted clean‑up, concentration, fractionation; chemistries: C18, HLB, mixed‑mode, ion‑exchange; applications: environmental, food, pharmaceuticals
Liquid‑Liquid Extraction (LLE)
- Selective partitioning into immiscible solvents; applications: non‑polar drugs, lipids, pesticides
QuEChERS
- Multi‑residue analysis in complex matrices (food, feed); extraction, salt‑induced phase separation, dSPE clean‑up
Filtration and Centrifugation
- Removal of particulates; use low‑binding, MS‑compatible filters
5. MATRIX CONSIDERATIONS AND MINIMIZING ION SUPPRESSION
- Identify co‑eluting matrix components causing suppression/enhancement
- Dilution, SPE, or HILIC to mitigate matrix effects
- Evaluate using post‑column infusion or matrix‑matched calibration
6. SAMPLE DERIVATIZATION (IF APPLICABLE)
- Convert analytes to MS‑friendly, detectable forms; common for small polar molecules or reactive compounds
- Ensure complete reaction, minimal excess reagent; document conditions, reagents, safety
7. SAMPLE QUALITY CONTROL
- Use Certified Reference Materials (CRMs) where possible
- Spike/recovery studies to evaluate extraction efficiency
- Include blanks, duplicates, QC standards in batches
- Track sample handling, storage, preparation steps
8. ONLINE PRACTICAL COMPONENT
- Interactive exercises: selecting appropriate preparation technique for complex matrices
- Simulating protein precipitation, SPE, QuEChERS workflows
- Evaluating chromatograms for matrix effect indications
- Logging sample prep steps, deviations, corrective actions for ISO 17025 compliance
9. PREVENTIVE STRATEGIES
- Use standardized protocols and SOPs
- Pre‑check solvent and reagent quality
- Filter and centrifuge before injection
- Avoid repeated freeze‑thaw cycles
- Maintain detailed sample preparation logs
10. SYSTEM SUITABILITY AND POST‑PREPARATION VERIFICATION
- Evaluate recovery, reproducibility, MS response
- Compare with reference standards or CRMs
- Confirm absence of contaminants affecting MS sensitivity or column life
11. ISO/IEC 17025 DOCUMENTATION
- Document method, deviations, rationale for preparation steps
- Record QC outcomes and corrective actions
- Trace sample lineage from collection to analysis
- Maintain audit‑ready logs for sample integrity, storage, extraction
12. COMMON ISO/IEC 17025 NON‑CONFORMITIES (LC‑MS SAMPLE PREP)
- Improper storage leading to analyte degradation
- Incomplete documentation of sample preparation steps
- Failure to assess matrix effects
- Using incompatible solvents or filters with LC‑MS system