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4 Steps to Successful Compound Optimization on LC-MS/MS

4 Steps to Successful Compound Optimization on LC-MS/MS content piece image
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In a modern world filled with complexities, we are exposed to millions of novel contaminants. The unknown compounds are not only strangers to analytical laboratories, but also the instrument used for detection. Chemical instrumentation is not magical and requires guidance parameters to relate the target compound with instrumental data. Therefore, chemists need to determine and optimize parameters for instruments to perform the analysis.

Chromatography coupled with mass spectrometry is a common instrumentation choice for analytical detection. In particular, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is often used to identify and quantify trace amounts of chemicals with high sensitivity.1

As the name suggests, LC–MS/MS consists of liquid chromatography (LC) and mass spectrometry (MS/MS) components. The LC separates the targeted compound from the sample while the MS breaks it down into fragments for identification and quantitation.

While many chemists may long be familiar with LC-MS/MS for routine analysis, method development with compound optimization requires another set of considerations and operation. This guide provides an easy reference for compound optimization with LC-MS/MS.

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Step 1: Dilution of the chemical standard

If we want to teach the instrument of an unknown compound, we need a chemical standard that is free from other compounds. This is to ensure the compound optimization is free from interference caused by other chemicals present in the solution we used for optimization. Therefore, a pure chemical standard is often used for optimization. Depending on the instrumental sensitivity, the standard is diluted to a suitable concentration (50 ppb-2 ppm) with an appropriate solvent. This solvent should be able to dissolve the compound whilst not damaging the instrument. For a starter, the solution could be a mixture of the prospective mobile phases for analysis.

Step 2: MS/MS Optimization

Although a sample containing a mixture of compounds first enters the LC compartment, compound optimization starts with MS/MS. The signal we obtained from the spectrum is mostly a result of the MS/MS operation. In order to ensure the signal we finally obtained is coming from our target compound and not something else running out of the LC, it is necessary to first get the MS/MS parameters of our target compound.

The MS/MS compartment breaks down the compound into fragments by both ionization and collision. The identity of the compound is determined by the fragments detected. Therefore, compound optimization involves specifying the energy required to direct the compound to the detector and the energy for fragmentation.

Optimization of ionization energy of the parent ion

When the compound enters the first MS compartment, it turns into an ion (called the parent ion) by either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). To optimize the parameters for the parent ion, one needs to know its mass. As a rule of thumb, the mass is usually its molecular weight plus or minus a proton from the original molecule ([M+H]or [M–H] ). Some useful resources to find the mass of the parent ion are the NIST Chemistry WebBook2 and academic journal articles. Once the mass of the parent ion is known, one can optimize the orifice voltage. This is achieved by scanning through a range of voltages to select the optimum value that gives the maximum response of the parent ion.

In some cases the responses are low for both [M+H]+ or [M–H]. One of the possible causes is that the ion has formed an adduct with additives in the mobile phase or solvent. For example, ammonium formate is a commonly used additive that may result in adduct formation. In such cases, one could try optimization with the mass [M+NH4]+.

After entering the first MS compartment, the parent ions arrive at the collision chamber. Through collision with gas molecules, the parent ions are broken down into fragments. But what are the fragments

Optimization of collision energy of the fragmented ion

Different fragments are formed from different collision energies applied and in turn different spectra are formed. By scanning a range of collision energies and overlaying the spectra obtained, one can identify the most abundant fragments (or daughter ions). Each compound has its own characteristic distribution of daughter ions. With LC-MS/MS, one can select multiple reaction monitoring (MRM) to monitor several daughter ions from one parent ion at the same time.

One of the key parameters in MRM optimization is the collision energy. It is the energy required to break down the parent ion into specific daughter ions. By scanning the collision energy that gives rise to the maximum response of each daughter ion, one can optimize the collision energy for each MRM pair.

It is a common practice and criteria to have at least two MRM pairs for each compound. The first pair is usually for quantification whilst the second pair is for confirmation. Since the optimization makes use of a standard, the first and second ion pairs come in a particular ratio. This ratio is also used in confirmation of the compound.

A compound is said to be found only if:

  1. It contains both MRM pairs as with the standard
  2. The MRM pairs have the same ratio as of the standard

It is therefore suggested to optimize at least two MRM pairs for each compound. In some cases, the mass of the daughter ion may be the same as a commonly used solvent, or susceptible to interference in sample preparation. It is always useful to optimize three or four MRM pairs (if possible) to ensure accuracy.

Step 3: Chromatography optimization

After the optimization in the MS/MS compartment, the next step is to optimize the LC conditions.

LC separation relies on the retention of the compound due to the competition between the column (stationary phase) and the mobile phase (the dynamic phase). Column selection depends on the physical and chemical properties of the compound of interest. For example, a C18 column is usually used for non-polar compounds. It is always useful to familiarize yourself with columns specifications3 or obtain a column selection guide from the column manufacturer. As for the mobile phase, methanol, acetonitrile and water or a mixture of the solvents are commonly used. An addition of acids or ammonium salts may be used to enhance peak resolution and shape.

After selecting a suitable column and mobile phase, one needs to optimize LC conditions including the flow rate, mobile phase gradient, column temperature. This is to ensure the compound can be identified and quantified as a nicely resolved peak. A distorted peak or tailing could occur if the LC conditions are not suitable for the compound. For example, while a faster flow rate may increase the efficiency of a run, a flow rate too high may cause several peaks to merge together. Therefore, if a broadened peak is observed, it would be useful to slow down the flow rate and check whether the peak is due to the overlapping of two peaks. Another tip is to modify the mobile phase gradient. Some interfering compounds may elute with one of the mobile phases easily due to its solubility. Increasing the percentage of a mobile phase used may help to get rid of those interferences and yield a nice resolved peak for the target compound. A third tip is to use a uniform column temperature. While most LC runs can be used under ambient conditions, fixing the column temperature can prevent peak broadening due to the uneven distribution of the temperature inside the column.4

Step 4: Verification with a calibration curve

Before getting to sample preparation, it is important to confirm the optimized conditions are true for the targeted compound. One method for verification is to use solutions at different concentrations, or test with a calibration curve. If the response obtained is in proportion with the compound concentration with a nice resolved peak, the instrumental method should be ready to go.


  1. Tuzimski, T.; Sherma, J. (2015) High Performance Liquid Chromatography in Pesticide Residue Analysis. CRC Press
  2. NIST Mass Spectrometry Data Center. Retrieved from https://chemdata.nist.gov/ on 10-10-2018.
  3. Dolan, J. W.; Maule, A.; Bingley, D.; Wrisley, L.; Chan, C. C.; Angod M.; Lunte, C.; Krisko, R.; Winston, J. M.; Homeier, B. A.; McCalley, D. V.; Snyder, L. R. (2004) Choosing an equivalent replacement column for a reversed-phase liquid chromatographic assay procedure. J. Chromatogr. A 1057, 59–74.
  4. Dolan, J. W. (2014) The Importance of Temperature. Retrieved from http://files.pharmtech.com/alfresco_images/ pharma/2014/08/22/c91e07c2-9442-47b5-b2d1-c9d4f7e635db/article-23766.pdf on 29-10-2018.