Today biomedical and pharmaceutical laboratories increasingly require robust techniques to measure minute quantities of targets in complex biological matrices. This task gets further complicated when analyzing hundreds of target analytes while maintaining accuracy and data quality. Combining liquid chromatography with mass spectrometry (LC-MS) has been a boon in meeting all these stringent demands.
Because of its efficiency and versatility in handling complex biological matrices, the LC-MS method has become the primary technique in modern bioanalytical testing. The liquid chromatography component separates the target compounds while the mass spectrometry unit detects them based on their mass-charge ratio. Researchers today heavily rely on LC-MS assays to detect analytes from various sources such as pesticides, drug metabolites, and food contaminants. The current article focuses on the different stages of LC-MS analysis. Let us first understand LC separation and detection and then move on to LC-MS detection.
Liquid chromatography separation
Liquid chromatography physically separates analytes from study samples. Researchers inject a few microliters of the study sample into the mobile phase, which is continually pumped through a column coated with the stationary phase. The stationary phase is usually filled with silica particles. Once the mobile phase and sample solution reach the column, the analytes will differentially interact with the stationary phase. Depending on the physical and chemical properties, some analytes will interact more strongly, and some will have less interaction. The analytes with the least interaction will elute from the column first. Accordingly, with continuous flow, the remaining analytes will elute sequentially.
Liquid chromatography detection
After the analytes elute through the column, it enters a detector. This detector responds to a specific property of the analyte, such as light absorption. The detector unit captures this property and corresponds the amount to the concentration of the analyte present in the sample. The moment the detector captures the analytes is called the retention time. Researchers compare the retention time of the analyte to the retention time of a known compound and identify the analyte of interest.
Detection using LC-mass spectrometry
Due to being a more selective and sensitive detector, the mass spectrometer has emerged as a robust detector with liquid chromatography techniques. While LC instruments operate at ambient pressures, mass spectrometry needs a vacuum. Hence, both these instruments are coupled through an interface. After eluting from the LC component, the analytes enter the interface, where they are vaporized and ionized. Only after ionizing the analytes, the MS unit can detect them from the study sample.
Once the analytes enter the MS unit, they are subjected to magnetic and/or electric fields. Varying these fields ensures that the ions are altered and separated from one another. Post-separation, the ions are collected and detected by MS detectors. Ideally, secondary electrons are released after the ions strike the MS surface. These electrons are multiplied, and the amplified current is measured and correlated to determine the ion concentration.
In conclusion, LC-MS testing has been employed increasingly in several biomedical and pharmaceutical settings. However, adequate focus on LC-MS method development and validation will be crucial for its evolution in LC-MS labs.