Today, researchers have multiple techniques, such as MSD immunoassays, for studying analytes in complex study matrices. LC-MS is one such powerful tool for quantifying small molecules in biological samples. However, this technique requires sample preparation during LC-MS method development before analysis. With numerous sample preparation options available, identifying an ideal cleanup method for individual applications can be challenging for novice users.
A better understanding of sample preparation principles coupled with a well-designed approach to developing and validating an assay protocol can save time and generate more robust and accurate LC-MS testing results. This current article explores factors LC-MS laboratories should consider while selecting and optimizing an LC-MS sample preparation strategy.
Approaches to optimize sample preparation during LC-MS method development
Small molecule analysis used in different sample types is standard in a clinical research setting. These sample types include serum, plasma, whole blood, urine, etc. The primary reason for employing sample preparation techniques in research and experiments is to remove protein components and other constituents that may precipitate in the LC mobile phase and clog the chromatography column.
As samples may get contaminated or degraded over time, the primary strategy of sample preparation is collecting and storing study samples properly. Depending on the source and nature of study samples, researchers may employ different approaches such as refrigeration, thawing, freezing, or adding stabilizer. Besides, they should document and label each sample clearly and minimize exposure to heat, light, or moisture and several freeze-thaw cycles.
After securing study samples, the next step includes extracting and cleaning them. First, separate the target analytes from the study matrix and remove any interfering components. Researchers have several methods to extract and clean up the sample, including solid phase extraction, protein precipitation, liquid-liquid extraction, and derivatization. The choice of extraction method depends on the LC-MS conditions, sample type, and target analyte properties. Besides, optimizing parameters such as extraction time, pH, volume, and solvent type is critical to achieving maximum selectivity and recovery.
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The final step for optimizing sample preparation is to dilute or concentrate the study samples. This step may influence the linearity and sensitivity of LC-MS drug testing. LC-MS labs may need to concentrate study samples if the target analytes are in low concentration or if the cleanup and extraction method generates large volumes. However, LC-MS laboratories can employ techniques that suggest lyophilization, evaporation, or solid phase extraction to minimize sample volume and increase the concentration of target analytes. On the other hand, researchers might need sample dilution for analytes present in high concentrations or in cases when solvent composition becomes incompatible with the LC-MS method. Researchers can use approaches such as solvent exchange, buffer addition, or serial dilution to alter solvent composition and sample concentration.
By considering these approaches, LC-MS laboratories can optimize LC-MS sample preparation and generate accurate, reliable, and high-quality data. However, they should consider that LC-MS sample preparation is unique for each approach and will require tailored solutions to meet specific goals and needs. Besides, validating sample preparation techniques and monitoring their performances remains crucial for the success of robust LC-MS method development and validation.
