1. Multi-Analyte LC-MS/MS Methods – Best Practice.
Martin Danaher

2. Contents LC-MS/MS Overview LC-MS/MS Optimisation Method Validation
Specificity and selectivity
Stability studies
WLr and WLR
Data quality checks
Conclusions

3. LC-MS/MS Overview Sample manager Column oven Injector Pumps MS/MS
(QqQ) 3 3

4. LC-MS/MS instrumentation (HT)
Column manager: 4 columns
Sample organizer:
10 trays
Injector : 48 positions

5. Electrospray Ionisation

6. MS Optimisation MS Optimisation Rafoxanide: EF = C19H11Cl2I2NO3
MW: g/mol (average value)
Monoisotopic
Which polarity?
Optimum cone voltages
ESI voltage
Temperatures

7. Information sources

8. Isotopic Distribution of Rafoxanide

9. 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.

10. 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

11. Start method validation

12. 2002/657/EC Criteria

13. 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

14. Isobaric interference

15. Cross-talk phenomenon

16. Isotopic Distribution of Rafoxanide

17. Selectivity – Matrix components
HPLC-FLD separation of analyte from the matrix peak.

18. 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

19. Matrix Effects Study – Approach I
Post-column infusion of standards with blank matrix samples

20. Matrix Effects Study Example

21. 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

22. Importance of Chromatography

23. Importance of Chromatography

24. 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

25. 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.

26. Data quality Checks (qualitative)
Identification
RT (5%)/RRT (2.5%)
S/N >3
Identification points (3 or 4)
Ion ratio

27. 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.

28. 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.

29. 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.