22
September 2022

A step-by-step guide to developing a robust assay in bioanalysis using LC-MS/MS

A step-by-step guide to developing a robust assay in bioanalysis using LC-MS/MS

LC-MS/MS assay development within Bioanalysis is driven by developing accurate, precise and robust methods within the shortest time frame possible.
Within our department we have employed a generic protocol to enable a consistent and efficient process to reach these goals.

LC-MS/MS Conditions

Initial Screening

Determine: Log D and pKa to assist in initial decision making

Infusion of analyte into MS

  • Pos/Neg, ESI/APCI
  • M+H or M-H or Adducts
  • Minimise adducts or maximise

Scouting HPLC Gradients

  • Acidic & Basic & buffered mobile phases for retention and signal:noise (S:N)
  • Gradient for sharper peaks and sensitivity
  • Isocratic for resolution

 

Limit of Quantitation

  • Dilution series made in solution
  • Provides an idea of what sort of sample extraction technique is needed
  • S:N of 20:1 to enable an ↑chance it will be suitable in matrix

Carryover

If observed will need to locate the source: 

 

Sample Extraction

Binding

Non-specific Binding
Binding test performed as follows in 3 types of materials:

  • Glass
  • Polypropylene
  • Polyethylene

Analyte 1

Analyte 2

Binding in Urine

Conclusion

Utilising this generic guide as a starting point for method development of each new compound streamlined our processes and highlights issues at an early stage and creates a solid basis for future troubleshooting.