The LC-MS method is a great way to get faster, more reliable results. This method brings the separation powers of Liquid Chromatography (LC) together with the detection ability of Mass Spectrometry (MS). As is evident, creating a suitable assay that can work with LC and MS is challenging.
Setting up an LC MS method is an iterative process that can be very time-consuming. There is no one-size-fits-all approach. Factors ranging from the laboratory’s situation to properties of the assay, sample, and/or analyte should be considered.
Improvements in technology have brought changes to LC-MS analysis, though the name remains unchanged. This includes using High-performance liquid chromatography (HPLC) or Ultra HPLC (UHPLC) systems and tandem Mass Spectrometry.
Considering Analyte Physical and Chemical Properties
Factors that affect the analyte, such as polarity (Log P, Log D), charge (pKa), and others should be considered. For example, some analytes adsorb into the plastic, and therefore, plastic plates and containers are avoided for such analytes. Some solvent mixtures can dissolve molecular components from the container and make the sample more complex.
The thermal stability of analyte is another crucial aspect. Depending on this, a choice may be made for reduced temperature or amber glass vials.
The Concentration of the Analyte
LC/MS can detect the analyte even in low concentrations. However, the LC-MS assay cannot entirely ignore the strength of the analyte. It is necessary to achieve the desired lower limit of quantitation (LLoQ) in a sample.
For quantitation using LC/MS, the most common approach is to isolate the analyte, thus increasing the potential sensitivity selectively.
In a qualitative approach, there may be minimal information about the analyte. In such cases, the common approach is to reduce other components selectively. Removal of the components that can potentially interfere with the results helps in boosting the analyte concentration.
In a general sense, this also affects assay quality. Ionization techniques like Atmospheric pressure ionization (API) convert analytes in the liquid phase to gas for detection by the MS. However, the presence of substances like phospholipids in the sample creates matrix effects. Cleaning the sample for the assay can help reduce this interference.
Handling Known Challenges
Analytes, assays, and specific applications often have associated difficulties. In most cases, the challenges become known with the preparation of the assay. Some analytes can pose issues with sensitivity and/or selectivity. Others may affect robustness. Assay preparation should focus on specific known challenges and make allowances to handle them.
Setting up the assay and related operations are dependent on the laboratory resources and expected workload. Often, these are the deciding factors for assay set up, as compared to other aspects like analyte properties.
It’s important to consider throughput, constraints on the sample volume, and batch size. The presence of automation, e.g. liquid handling or multiplexing techniques, can greatly affect the preparation of the assay.
Analysis using panels is alluring since it reduces the number of extraction protocols and offers a more efficient laboratory workflow. However, it negatively affects instrument efficiency in the shape of lost analytical time.