Developing New Methods for LC-MS Analysis

LC-MS methods employed today must cover a much wider range of sample types and complexities than ever before. Compared to isocratic elution, gradient elution chromatography allows much faster elution of strongly retained analytes without loss of resolution of the more polar analytes.

Gradient elution chromatography forms the corner stone for the majority of LC-MS methods developed to date. The choice of buffer, its concentration and type of organic modifier used are important considerations when using MS as the detection system, as does the choice of HPLC column.

The LC-MS Interface

The basic principle of MS is the production of ions which are subsequently separated according to their mass-to-charge ratio (m/z) and detected. The technique provides information that is both wide ranging and accurate.

Atmospheric Pressure Ionization (API) has greatly expanded the range of compounds that can be analysed by LC/MS. The analyte is brought to the interface via a solvent typically at a rate of 1.0ml/min, depending on column diameter and method requirements. The API interface must separate the analytes from the solvent, ionize the analyte molecules and maintain a vacuum in the mass detector. The method by which this is done differentiates the API processes. See Table 1 for information on what flow rates should be used for each LC-MS interface. Electrospray (ES) gives the widest range of application both in terms of molecular mass and analyte polarity. Atmospheric Pressure Chemical Ionization (APCI) may be the method of choice where slightly more hydrophobic analytes need to be analyzed (Figure 1).

Figure 1 – The Suitability of Different Techniques for Compounds of Different Molecular weight


Figure 2 shows basic compounds analyzed in positive ion mode. The data allows for highly specific identification of analytes and is more sensitive than traditional UV methods of detection. The total ion current (TIC) gives relative abundance of all ions detected. Those that co-elute can still be isolated and quantified by plotting their relative abundance as a function of time (mass chromatograms).

Figure 2 – A Comparison of Different Mass Spectral Information


Proportion of Organic Solvent in the Mobile Phase

The ratio of aqueous-to-organic solvent is particularly important in electrospray ionization. The efficiency of the electrospray process depends on the conductivity and surface tension of the liquid being nebulized. When the conductivity and/or the surface tension are too high (i.e. highly aqueous), it is difficult to produce a stable spray and it is difficult to vaporize the droplets formed by the action of the high voltage and nebulizing gas. The percentage of water used should not be too high since surface tension of water is much higher than the surface tension of methanol or acetonitrile. Additive concentration will have considerable effect on conductivity of the nebulized liquid and consequently should be kept low. These factors are most important when working at high flow rates since there is more solvent to be nebulized and vaporized. One alternative is to use a sheath liquid which is highly organic (e.g. IPA) to help the spray and vaporization. However, this involves a more complex setup, and may not be suitable for high throughput applications. In general, it helps the LC-MS sensitivity to have at least 20-30% organic. 100% or very high organic content may also lower the sensitivity, especially where no additive is used. This is primarily because the conductivity of organic solvent is too low. A small percentage of water in the mobile phase helps the droplet formation.

Buffers / Additives

Buffers serve two purposes in LC-MS:

to act as a buffer for the chromatographic process in the traditional way, i.e. control and maintain the pH of the mobile phase in order to keep the ionization state of an analyte constant

to adjust the pH of the carrier solvent (mobile phase) in such a way as to present the analytes to the MS already in ionic form.

The most compatible buffers are ammonium formate, ammonium acetate, and ammonium hydroxyde at concentrations of 10 to 50mM. The preferred additives are formic and acetic acids (0.01 to 1%v/v) because they improve protonation of basic samples in positive ionization. Other additives occasionally used include trifluoroacetic acid and trialkylamine type bases, but these need to be used at low concentrations (<0.1% v/v) since they may cause ionisation suppression. Non-volatile salts, such as phosphates and borates, ion pairing agents and inorganic acids should be used with precaution.

Modern orthogonal (off-axis) sources are more robust, and have been designed to operate with unvolatile buffers / additives and minimal sample clean-up. In order to achieve the best response in LC/API-MS, mobile phase composition must be such that the solution and gas phase chemistries are optimized to maximize ionization and eliminate any components which will cause ion supression.

Additives for Optimum MS sensitivity

Some additives provide more sensitivity than others when used with MS. The additive used can greatly effect the sensitivity of the MS ionization source and hence the sensitivity of the method. Careful consideration is therefore required on which additive will give the greatest MS sensitivity. A recent study of volatile additives showed formic acid to give the greatest sensitivity

Figure 3 – A Comparison of the Sensitivity of an LC-MS Separation With Different Mobile Phase Additives



Reducing the concentration of each additive gave significant increase in sensitivity. For example, improved sensitivity was observed when the TFA concentration was reduced from 0.1% to 0.01% TFA. Chromatographic performance conditions are shown in Figure 4. In this example, the additive employed was 0.01% TFA. High levels of additive concentration lead to ion suppression that in turn will lead to loss in sensitivity for the method.

Balancing UV detection with MS sensitivity requirements

In the same study diode array detection was used in series with the MS system. The aim was to use the chromatographic data from the diode array to help identify analytes that either gave rise to poor MS detection or which were present only as minor peaks barely visible in the noise of the total ion chromatogram. Diode array is ideal for this purpose because it can operate across a wide wavelength range. Sensitivity of each additive system towards diode array detection was therefore investigated in the 190nm to 280nm wavelength region. The chromatograms in Figure 5 show clearly the difficulties that can arise at low wavelength where an unstable baseline can often lead to loss in sensitivity. In this case, TFA was found to be a more attractive additive than formic acid as it gives better UV transparency, especially at the lower wavelengths. A compromise was made whereby the final method used TFA as the additive in place of formic acid in order to maximize UV sensitivity, but used at low concentrations (i.e. 0.01%), to maximize MS sensitivity with TFA.

Figure 4 – Comparison of Two Additives with UV Detection


Figure 5 shows how the method was used to analyze some in-vitro incubation samples. Note how the UV diode array chromatogram can be used to help identify regions of the total ion chromatogram where sensitivity is poor. Identification of metabolites can often be a time consuming process when the peaks in the total ion chromatogram are lost amidst the noise of the baseline. On these occasions identification can often be aided by the diode array chromatogram where a sensitive LC method may allow for increased sample detection at low UV wavelength as shown for pethidine.



Figure 5 – A UV (A) and MS (B) Comparison Pethadine and Its Metabolites


In this example, the diode array provides a clear indication for the presence of a metabolite. The lambda max (not shown) can provide further information in support of MS data that can aid in structural elucidation and peak location on the MS total ion chromatogram.

HPLC columns that can provide optimum performance for LC-MS

As mentioned earlier, low additive concentrations offer the reward of increased sensitivity for LC-MS. This is an important consideration when trying to identify trace quantities of a drug compound or impurity that may normally disappear into the noise of the base line.

The choice of HPLC column used is of key importance as the quality of the bonded phase and underlying silica can strongly influence the concentration of additive required. In order to maintain good peak shape of basic drug