Multi-Analyte LC-MS/MS Methods – Best Practice. Martin Danaher
Contents LC-MS/MS Overview LC-MS/MS Optimisation Method Validation Specificity and selectivity Stability studies WLr and WLR Data quality checks Conclusions
LC-MS/MS Overview Sample manager Column oven Injector Pumps MS/MS (QqQ) 3 3
LC-MS/MS instrumentation (HT) Column manager: 4 columns Sample organizer: 10 trays Injector : 48 positions
Electrospray Ionisation
MS Optimisation MS Optimisation Rafoxanide: EF = C19H11Cl2I2NO3 MW: 626.01 g/mol (average value) Monoisotopic 624.820496 Which polarity? Optimum cone voltages ESI voltage Temperatures
Information sources http://www.sisweb.com/mstools/isotope.htm
Isotopic Distribution of Rafoxanide
MS/MS Optimisation Collision induced dissociation with inert gas e.g. N2 or Argon. Identify most abundant?? daughter or product ions Product ions must be selective Avoid neutral losses -18 (H2O) and -17 (OH) Avoid non-specific fragments: 91 m/z, 105 m/z and 121 m/z. Consult literature, see what others are using.
Chromatographic development Generic scouting gradient M. Phase A 100% Aqueous M. Phase B Acetonitrile or Methanol Column: 100 × 2.1 mm Inject each mix at high concentration and optimise separation Evaluate the impact of different additives acids and salts Optimise additive concentrations
Start method validation
2002/657/EC Criteria
Specificity – Similar compounds Inject analyte standard and internal standard separately (highest concentration). Check for interference in each analyte or IS trace Isobaric interference Cross-talk Carry-over
Isobaric interference
Cross-talk phenomenon
Isotopic Distribution of Rafoxanide
Selectivity – Matrix components HPLC-FLD separation of analyte from the matrix peak.
Selectivity – Matrix components LC-MS/MS – Matrix peaks not visible Co-eluting peaks, late eluting peaks, etc. Ion suppression or enhancement Potential Solutions: Clean-up Chromatographic separation Matrix matched standards SILs
Matrix Effects Study – Approach I Post-column infusion of standards with blank matrix samples
Matrix Effects Study Example
Matrix Effects Study – Approach II Spike a range of representative samples post extraction and compare with solvent standards. Calculate enhancement or suppression effects Calculate the precision Evaluate the impact of the use of internal standards
Importance of Chromatography
Importance of Chromatography
Method Validation Stability studies Limit of detection Standard stability (3, 6, 12, 24, 36 months). Different storage conditions. Sample extracts – intermediate or in final injection solvent. Over 7 days or continuous injection Stability in matrix – spike samples and store for different periods of time (1, 2, 3, 4, 6, 8, 12, 26, 52 weeks) Limit of detection Limit of quantitation/Limit of Reporting
Method Validation Within laboratory repeatability 18 samples spiked at three different levels Repeat by the same analyst Within laboratory reproducibility Minimum of 18 “different” samples spike at three different levels Repeat on different days by the different analysts. Use different equipment if possible. CCα Calculate using WLR data.
Data quality Checks (qualitative) Identification RT (5%)/RRT (2.5%) S/N >3 Identification points (3 or 4) Ion ratio
Data quality Checks (quantitative) CCα Compare CCα with MRL. Big gap more precise method needed. Calibrations Use weighted linear regression not through (0,0) Inject at start and end of batch. Drift <30%. Inject LOQ as a response check throughout the run. Drift <30%. Residuals ±20%. Minimum of five points on curve.
Data quality Checks (quantitative) Trueness Precision For analyses carried out under repeatability conditions, the intra-laboratory CV would typically be between ½ and 2/3 of the above values.
Conclusions SANCO validation document presents complementary validation guidelines. Provides more practical information on routine analysis Interpretation of data quality. Elements in 2002/657/EC validation that should be retained. Good ideas e.g. Ccα Ambiguity around the validation approach Considered as being inflexible.