Sensitivity of MS #

Sensitivity definition: signal-to-noise

From HPLC system hardware perspective, what could affect the sensitivity? #

contamination

HPLC system settings (dead volume)

From the perspective of sample introducting to mass spectrometer #

1. less carry-over
2. less sample volume [50ng is plenty for MS]
3. solution: pH, low boiling point, low dielectric constant, low surface tension.

From mass spectrometer hardware perspective, what could affect the sensitivity? #

Asymmetric spray

matrix effect

We won’t get ion currents if they are under the Scan Line. We need to do tuning to raise the Scan Line in order to improve resolution and separation. Do not let the ions hit the quadrupole.

compartment temperature does not help resolution, but repeatability. temperature vs. solvent viscosity.

baseline resolution.

Ion source options #

1. select an ionization method between ESI and APCI (according to the residue of compounds).
2. select positive ion mode or negative ion mode.

There are chemical noise there, use APCI instead of ESI; or negative ionization mode.

Electrospray #

 Electrospray makes droplets, ions are in the droplets. We put high voltage on it. It undergoes the ion evaporation model.

 The liquids coming through at 200 microliters per minute. If the electrosprayer was blocked, you would have unstable ion current noise that ion current will be noisy. We need to have asymmetrical spray.

 The spray first takes place, the ion evaporation takes place to produce gas-phase ions.

 A combination of heat, nebulizing gas, and time can get the large droplet to sub micro droplet.

 Only one in 10,000 ions get inside the mass spectrometer.

 Check the the voltage potential between the sprayer and the interface plate (orifice??), if the voltage is decreased. The voltage potential makes ions go where we want them to go. The ions could go other places with the reduced voltage potential so that the sensitivity suffers.

 What does it take to increase evaporation? optimum temperature and flow rate.

 If the spray is structured as a pneumatic systems,

 It is a concentration sensitive detector, , matrix effects. optimize flow rate, lower the voltage, pH in mobile phase to improve ionization. clean the source.

APCI #

Electron (not electronic) multiplier #

The very weak ion current exits the quadrupole, ions hit the electron multiplier surface, and produce electrons. They are amplified by 10 to the six. Don’t jack up the voltage on the multiplier, but to clean the source, or re-tune it. Monitor the EMV.

Scan modes #

SIM or MRM can improve sensitivity comparing to full scan. SIM is preferred over full-scan.

Misc #

) capillary. temperature, voltage

From LC hardware perspective #

Samples are diluted in proportion to the cross-sectional area of the column and therefore, smaller ID columns yield less dilution.
Reduction of particle size causes an increase in the sensitivity because of more narrow and higher peaks.
In order to have a high-efficiency separation, it is important to minimize the instrument’s dead volume. This can be achieved by optimizing the entire HPLC system using smaller I.D. and/or shorter connection capillaries, smaller injection unit, and smaller detector cell.

From mobile phase perspective #

formic acid concentration decreased, will lower the sensitivity, because formic acid promotes

systematic approach to expectation:

1. Proactive approach: monitor the mass (exact mass for HRMS) daily, and plot it.
2. monitor back pressure., and plot it.
3. build troubleshooting database (knowledge transfer).

From sample preparation perspective #

A loss in sensitivity can indicate problems with either the sample preparation or the analytical system. First, confirm that the sample preparation procedure was followed correctly, including storage temperature and time, and verify that system parameters are set correctly and functioning properly. Always check for obvious problems (e.g., calculation/dilution error, autosampler needle not reaching sample, incorrect injection volume, wrong detector settings, no mobile phase flow, detector or lamp turned off, integrity of the reference standard, etc.) before looking for more complex issues. Having a coworker double check your work can be invaluable because it is often easier for an independent person to spot problems than it is for someone who is already familiar with the work.

Summary of sensitivity reduction #

1. Simple potential causes:
   1.1) sample preparation,
         abnormal storage temperature,
     storage time,
     abnormal extraction,
     abnormal clean-up,
     Was matrix be cleaned-up in sample preparation?
   1.2) LC system,
     Dilution error,
     autosampler needle not reaching sample,
     incorrect injection volume,
     abnormal LC back pressure,
     Leak,
     mobile phase unstable (modifier, prepared date, )
   1.3) Mass spectrometer system,
     non-asymmetric spray (due to clog)
     Electron multiplier voltage error,
     the declustering potential error,
     the collision energy error
     the collision exit cell potential error
     GS1 pressure - nebulizer gas error
     GS2 pressure - turbo gas error
     Curtain gas pressure error
     Ion spray voltage error
     test
   1.4) System monitoring,
      monitoring charts of internal standards, blank, calibrators,
   1.5) Data processing,
     Calculation error (dilution factor),
     Peak integration error (baseline issue, noise),
     Retention time shift,
           

2. Complex potential causes:
     Analyze new known standard
     Analyze blank
     Infuse standard in 50-50 mobile phase solution to the mass spectrometer
     Dry run
           

During the routine analysis #

What if the sensitivity dropped?

The troubleshooting strategy. What cause the dropped sensitivity?

What tools could we use?

During the method development #

How to increase resolution of MS? #

Quadrupole (Q1). , needs tuning (autotune or manual tune) to keep the instrument in the best condition.