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Quick LC-MS Method Optimization

The most significant choice in many LC–MS methods is the ionization mode. The generally accepted rule is that electrospray ionization (ESI) works best for higher-molecular-weight compounds that are more polar or ionizable, and atmospheric pressure chemical ionization (APCI) is best for lower-molecular-weight, less-polar compounds.

Atmospheric pressure photoionization (APPI) was originally designed to work with less-polar analytes; however the careful choice of dopant or the use of direct ionization sources can significantly extend the capability of APPI techniques (Figure 1). 

LC-MS ionization mode based on analyte polarity and molecular weight

Figure 1: LC-MS ionization mode based on analyte polarity and molecular weight.

Although these are good rules to help guide you towards the correct ionization technique you should always treat each analyte independently and (where practical) perform an infusion of your analyte(s) to select the correct ionization technique and polarity (positive or negative ion mode), and then optimize the critical parameters.

To do this you need a 10 mM ammonium formate buffer adjusted to both pH 2.8 and 8.2.  Carry out an infusion of your standard or sample through a tee piece, at the analytical flow rate using a 50/50 mix of organic/buffer at pH 8.2 and 2.8 using both negative and positive ionization modes.  Use the instrument autotune routine first and then carry out a manual tune on the key parameters i.e. voltages, temperatures, and gas flows to give you the optimum signals under each set of conditions (Table 1).  From these spectra you will be able to select the optimum ionization mode and eluent composition.

Parameter Non-Assisted ESI Assisted ESI APCI
Eluent flow rate 1-5 μL/min 0.1-1 mL/min 0.5-2.5 mL/min
Nebulizing gas (N2, L/min) 0 1-10 1-10
Desolvation gas (N2, L/min) 1-1.5 3-9 2-4
Ion source temperature (°C) 100-150 100-400 150-200
Capillary (ionization) voltage (kV) 4-5 4-5 2.5-3
Cone voltages (kV) 10-60 10-60 10-60

Table 1: LC-MS source parameter settings.

When tuning key parameters which can generate a response curve (e.g. ESI source voltages, flow rates, and temperature) setting the value to a maximum may not give you the most robust method, instead set values on a maximum plateau where small changes in that particular variable will not produce a large change in instrument response.

If you are carrying out a selected reaction monitoring (SRM) experiment you can now optimize the SRM conditions.  Using the optimum ionization mode and eluent composition adjust the collision energy (CE) voltage to give the product ions, you should be left with 10-15% of the parent ion to get the product ions.  You will want to use the product ions that give you the highest response.

Some further points of note when optimizing MRM experiments are:

  • Identify unique and /or abundant precursor ion and optimize instrument parameters to gain maximum sensitivity
  • Identify product ion(s) (fragments) giving highest signal abundance - this may be achieved using a scan function on Q3
  • Pay attention to uniqueness of the fragment ions (larger mass difference between parent and precursor tend to be more specific)
  • Dwell time – affects the abundance of each ion / transition so if multiple transitions monitored issues can arise where dwell times differ
  • Dwell time alters the overall MRM transition time
  • Collision energy (ΔV across Q2)
  • Collision gas pressure
LC-MS ionization mode based on analyte polarity and molecular weight

Figure 2: Collision gas optimization.

Now to optimize the HPLC-MS method.  Start with a high concentration of your standard (for example 1 μg/mL).  Run a gradient from 5-100%B using the optimized mobile phase (i.e. MeCN/ammonium formate buffer at the optimum pH, either 2.8 or 9), ionization mode, and SRM transitions.

At this point you should hopefully get a good total ion chromatogram (TIC) and optimum MS spectra for your analytes.  The method can be further optimized to reduce analysis time by calculating values for initial %B, final %B. gradient time (tg), and re-equilibration time (Equations 1-4).

Where, ti and tf are the elution time of the initial and final peaks (min.),
Δ%B/min. is the rate of change of the mobile phase during the gradient,
VD = dwell volume (mL),
F = flow rate (mL/min.),
tg is the gradient time (min.),
k* is the gradient retention factor (use 5 as a starting point),
S is the shape selectivity factor (for small molecules use 5 or calculate using the molecular weight S=0.25MW0.25),
ΔΦ is the change in %B expressed as a decimal,
and VM is the column interstitial volume (mL).

LC-MS still relies on a good chromatographic separation before MS detection.  When using single ion monitoring (SIM) or SRM many quantitative problems are caused by co-eluting substances entering the ion source with the analyte of interest, causing ionization efficiency issues.  Therefore, it is a good idea to run a full scan acquisition on a representative sample to visualize any potential co-elution problems. Quantitation issues do not solely lie with the mass spectrometer but are often due to ineffective sample preparation or chromatographic separation.


There may be other causes of these problems, so for further troubleshooting tips visit the CHROMacademy GC Troubleshooter »

Watch the associated webcast here and check out some of our other great CHROMacademy content…

Webcasts & Tutorials

What LC-MS Operators Need to Know »

Optimizing LC-MS and LC-MS/MS »

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New to LC-MS? 10 Practical Tips That Will Make a Real Difference »

My LC-MS isn’t behaving! Where do I start? »

10 Ways to Break Your LC-MS »

Optimizing Gradient HPLC Parameters »

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Dr. Dawn Watson

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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