Why Hyphenation of LC and MS?

• MS is not only an attractive detection method but a powerfull separation technique
• MS relies on a different physical property of the analyte to effect separation, i.e. on the mass-to-charge ratio (m/z) of ions derived from the target analyte(s)
• MS is orthogonal to LC
• Coupling of LC with MS results in a multidimensional separation / detection system
• LC-MS has a very high separation power (peak capacity)

The question is: Can we just dilute and shoot? So, is there any need for sample clean-up ? ! The answer is definitely: yes we need to clean up!

• Fractionate - Switch - Separate

SPE - LC is an attractive combination.

• Relatively low separation power of conventional analytical columns
  Interferences by LMW matrix components
• Mobile phases with acidic pH and relatively high amount of organic modifier
• Incompatible with biofluids
• Precipitation or unspecific adsorption of proteins
• Irreversible deterioration of separation column
• MS compatible buffers restrict pH range for separation to pH 3 – 6 (ESI)

MS-Detector

• Ionisation of target analyte(s) can be supressed or enhanced by low molecular weight (LMW) and/or high molecular weight (HMW) matrix components
  irreproducible signal(s)
• LMW and HMW matrix components not beeing removed or fully separated can cause: 
• high baseline noise 
• worse signal to noise ratio (reduced sensitivity)
• contamination of MS-source
• Sample or buffer salts not easily volatilised contaminate MS-source causing a loss in sensitivity

Understanding and eliminating ion suppression effects is essential for LC-MS analysis of complex biofluids.

In mass spectrometry

• Ion suppression is the result of sample components that diminish the ionization of or compete in the ionization process with the analyte(s) of interest 
• Also components of the sample that do not appear in the mass spectrum can cause ion suppression
• In biological fluids, natural variation in endogenous compound concentrations from one sample to another can cause varying levels of ion suppression
• This variation in turn contributes to unacceptable variability in the signal response for the target analyte

Ion suppression in bioanalytical MS

Ion suppression phenomena (“matrix effects”) are directly related to

• unselective sample clean-up and / or
• insufficient / inappropiate chromatographic separation

Monitoring Ion Suppression (Matrix Effects)  

Postcolumn Infusion Experiment  

1. Infuse a dilute solution of the analyte at a constant rate into the  effluent flowing from the LC system to the mass spectrometer.

2. After obtaining a steady baseline, inject a blank sample extract into the LC system Any eluted material that suppresses ionization in the MS will cause a drop in the baseline of the “Infusion Chromatogram”

Infusion Chromatograms with matrix effects: examples of different sample clean-up methodsFigure kindly provided by Dr. Richard King, Merck and Co., Inc., Drug Metabolism; West Point, PA, USA Matrix: Human plasma; Analyte: Phenacetin; Mode: ESI-MS/MS
Infusion Chromatograms: Plasma samples originating from different species Polson C. et al., J. Chromatogr. B 785 (2003) 263-275Precipitating agent:  Acetonitrile; Mobile phase :  MeOH / H2O (50/50; v/v); Sample:  Plasma/Precipitant (33/66; v/v)

Infusion Chromatograms: Effect of Protein Precipitating Agents on Ion SuppressionPolson C. et al., J. Chromatogr. B 785 (2003) 263-275Mobile phase: MeOH/H2O (50/50; v/v); Sample:  human plasma / precipitant (33/66; v/v)