The analytical lab has experienced an increase in demand for precise measurement of microgram and sub-microgram amounts of targets, sometimes in complicated matrices, in anything from medicines and food to body fluids and dirt. While this is not a simple operation in and of itself, the probable necessity to swiftly evaluate hundreds of samples while maintaining data quality adds extra difficulties.
Liquid chromatography (LC) and mass spectrometry (MS) integration has given analytical scientists a potent instrument to satisfy these demanding requirements. Instruments for liquid chromatography-mass spectrometry (LC-MS) have gained popularity in many contemporary analytical laboratories due to their adaptability and effectiveness.
What is LC-MS?
In the LC-MS analytical procedure, target chemicals (or analytes) are physically separated before being detected by mass spectrometry. Despite being a relatively new technology, its sensitivity, selectivity, and accuracy have made it a preferred method for detecting microgram or even nanogram levels of a wide range of analytes, including drug metabolites, pesticides, food adulterants, and natural product extracts.
How does LC-MS work?
LC separation
In a liquid sample or a solution of a solid sample, LC causes a physical separation of the analytes. A little amount of sample solution is injected into the mobile phase, which is a stream of a solvent. While the appropriate injection volume depends on the specifics of the experiment, an autosampler may correctly inject any amount of the sample, from as little as 0.1 L to as much as 100 L. 1 The stationary phase, which is often packed with silica particles covered with another liquid, is pushed continuously through the column, which is a stainless-steel tube.
Depending on their chemical makeup or physical characteristics, the components of the sample solution-mobile phase mix will interact differently with the stationary phase (which stays in the column) when they reach the column. LC separations have been divided into many modalities based on the mechanism of interaction between the analyte and stationary phase, including:
Partition chromatography: based on the differing solubility and hydrophobicity of the analytes in the stationary phase as compared to the mobile phase.
Ion-exchange chromatography: separates the analytes on the basis of their ionic charges.
Size-exclusion chromatography: exploits the differences in the sizes of the analyte molecules to separate them.
Affinity chromatography: separates the analytes based on their ability to bond with the stationary phase.
As the analytes go along the column, they will separate because some of them will interact with the stationary phase more strongly than others. First to leave the column are the analytes that interact with the stationary phase the least. The remaining analytes are flushed out progressively as the mobile phase continues to flow through the column, with the analytes with the strongest interactions emerging last. Retention time refers to the amount of time an analyte stays in the column after being added to the mixture (RT).
LC detection
- Eluent, the mobile phase that flows out of the column, passes through a detector that "responds" to a certain physical or chemical characteristic of the analytes contained within, such as refractive index or light absorption. This reaction is recorded as a signal or "peak" whose strength (peak area or peak height) reflects the concentration of the component in the sample.
- The detector's RT is the moment when it "sees" the analyte. By comparing a chemical's RT to the RT of a known compound, it is possible to identify a compound in a sample. Even though this approach of chemical identification is not precise, it works better when some a priori knowledge about the sample is available.
Using MS for LC detection
- The mass spectrometer has evolved as a selective, sensitive, and all-purpose detector, despite the fact that many other detectors of various technologies and sensitivities have been linked with LC for the analysis of various sample types.
- The LC eluent containing the separated analytes is not permitted to flow into the mass spectrometer, in contrast to other detectors. The mass spectrometer operates in a vacuum while the LC system operates at atmospheric pressures, and the two are connected through an interface. Heat is used to evaporate the solvent as the column eluent runs into the interface, which causes the analyte molecules to vaporize and become ionized. This step is essential since the mass spectrometer can only detect and measure ions in the gas phase.
- As the analyte ions are generated at atmospheric pressure in the interface, the process is called atmospheric pressure ionization (API) and the interface is known as the API source. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most commonly used sources in LC-MS analysis.
- The analyte ions are drawn into the mass spectrometer where they are subjected to electric fields and/or magnetic fields. The flight paths of the ions are altered by varying the applied fields which ensure their separation from one another on the basis of their mass-to-charge (m/z) values. Post-separation, the ions can be collected and detected by a variety of mass detectors, of which the most common one is the electron multiplier.
- When the separated ions strike the surface of the electron multiplier (a dynode), secondary electrons are released. These secondary electrons are multiplied by cascading them through a series of dynodes. The amplified current generated by the flow of the secondary electrons is measured and correlated to the ion concentrations in the mass spectrometer at any given instant in time (above Figure).
Why Use LC-MS for Quantitation?
BENEFITS |
Selectivity | Combining the two separation mechanisms of LC and MS allows the analysis of complex mixtures. The resulting selectivity allows a particular analyte or analytes to be isolated from the mixture and gives confidence that the correct component is being measured. Since analytes are separated by their mass-to-charge ratio (m/z) the technique allows for the use of isotopically labeled internal standards, which may not separate by LC but can be separated by their mass difference. The use of stable isotopically labeled (SIL) internal standards can help control variability in a quantitative assay. |
Speed | Since the MS will distinguish compounds based on mass, the chromatographic method does not have to separate every single component in the sample, so co-elution of non-isobaric analytes is possible. This allows fast LC analysis times and reduced sample preparation, which helps with method development and high throughput sample analysis. |
Sensitivity | Mass spectrometry is an inherently sensitive technique. Good selectivity also leads to reduced noise, allowing very low levels (fg mL-1) to be detected. |
DISADVANTAGES |
Expense | Mass spectrometers that can couple to LC systems are expensive to buy and run. Regular servicing is also required, adding to the cost. The environmental conditions in the laboratory need to be well controlled to ensure system stability. |
Complexity | In their own right, both LC and MS can be difficult to optimize. Combining the two leads to a complex co-dependent synergy. The ionization mechanism can be especially complicated – often several species are formed in the ionization source and multiple charging can occur. Care must be taken to choose conditions for optimum sensitivity and reproducibility. Sufficient training is also needed to allow analysts to run the systems effectively. |
Limited dynamic range | Compared to other quantitative techniques LC-MS can have a limited range where the response is linear with respect to concentration. Typically, ranges should not exceed 500-fold concentrations. |
Excessive selectivity | In quantitative analysis, it is usual that the MS is set to only detect specific analytes. This results in a very ‘clean’ looking chromatogram and it is easy to forget that there can be a lot of components still present, but not seen. These components can cause problems with reproducible quantitation and can be difficult to trace if they are not being looked for. |
The key stages of quantitative analysis
Sample collection
For any quantitative analysis, it is crucial to ensure that a representative and a sufficiently homogeneous sample is taken for analysis. The storage conditions between sampling and analysis must also be controlled to ensure that the samples do not degrade. Stability is usually tested prior to sampling.
Calibration and quality control samples
In order to quantify unknown concentrations of analyte, samples containing known concentrations must be prepared first. For quantitation this normally involves adding a range of concentrations to blank samples, and providing a set of calibration solutions that can be used to generate a calibration plot or line. The blank samples must be as close as possible in
composition to the samples being analyzed (matrix matching). QC samples are typically prepared in bulk and analyzed at regular intervals to monitor assay precision and bias.
Acceptance of data usually depends on QC samples being successfully quantified within predefined limits. It is becoming routine to reanalyze a proportion of test samples to demonstrate that the precision of the assay is under control (incurred sample reanalysis – ISR). This is because test samples are often subtly different to control samples. For example, biological samples may contain metabolites, whereas QC samples will not. Predefined criteria must be in place to allow the assessment of replicated data sets.
Sample preparation and extraction
Direct analysis of samples using LC-MS is possible but it is usually the case that samples will need to be cleaned up to remove the worst interferences and also to concentrate the sample if the analyte is only present at very low concentrations. Typically an internal standard is added to control for variations in recovery, matrix effect, and ionization. The endpoint of extraction will need to be compatible with the chosen LC method.
Analysis
Calibration standards, QCs, and samples are injected into the LC-MS system. It is expected that the assay will have been thoroughly assessed and validated to establish that its performance is fit-for-purpose. The assay should be sensitive enough to detect the lowest sample concentration and selective enough to ensure that interfering components do not compromise quantitation.
Before analyzing samples it is expected that a system suitability test (SST) is carried out. This typically involves injecting a known solution and comparing its response to previous data. Analyzing a blank sample and a sample just containing an internal standard is used to demonstrate selectivity.
Data processing
Correct data processing is a fundamental step in generating good-quality quantitative data. Most modern software packages contain automated algorithms for integrating peaks and these are preferred over manual integration. Each chromatogram must be inspected to ensure that the baseline is correctly drawn and that the analyte is resolved from any close eluting peaks.
The key point here is that integration should be consistent. Typically the analyte: internal standard response ratio is used to create the calibration line and quantify the QC samples and unknowns. Predefined acceptance criteria should be applied for key aspects such as repeatability or calibration linearity.
Reporting
Most modern quantitation software allows the direct export of results into word processing packages or spreadsheets. LIMS systems are also designed to integrate with most LC-MS instruments.
