By Agnieszka Sitarska
In the previous article in this series it became clear that high productivity in small molecule LC-MSMS relies on effective sample prep that supports reproducible results and minimizes downtime for sensitive LC and MS equipment. The ideal sample prep protocol should be simple, cost-effective, and enable matrix depletion with the option to concentrate analytes independent of matrix components. The questions are, which method should you choose and how should you go about optimizing it? Let’s start by looking at the range of methods currently available.
Choosing the right method for small molecule LC-MSMS sample prep can be critical for productivity.
You may be tempted to keep it simple with dilution or protein precipitation. Certainly these methods are simple and low-cost, but they fail to deplete the matrix and cannot concentrate the analyte independent of the matrix. Dilution is only appropriate for low protein matrices such as urine, not serum or plasma. Phospholipid removal does enable matrix depletion but only selectively, and this method is also rather expensive. Liquid-liquid extraction is an alternative, but is complex.
More sophisticated small molecule extraction methods will help you to concentrate the analyte and deplete the matrix. These include supported-liquid extraction and solid-phase extraction, which can even be run online, but these methods are complex. There is, however, one cost-effective and relatively simple option that ticks all the boxes, enabling matrix depletion and analyte concentration without matrix concentration – AC Extraction Plate* from Tecan. More about that later.
When selecting a sample preparation technique for small molecule extraction, there is no one size fits all. These are the main factors to consider:
1. The chemistry of your analyte will steer method choice. Important parameters can include polarity (partition-coefficient, log P, and distribution coefficient, log D), charge (pKa), thermal stability, and molecular weight.
2. Do you need to concentrate or dilute the analyte(s) to reach your lower limit of quantitation (LLoQ)?
3. There may be specific challenges in your application to consider. For example, achieving an acceptable LLoQ for serum steroid hormones, sufficient selectivity for urine opiates/metabolites, or rapid throughput and robustness for large workloads such as serum 25-OH Vitamin D.
4. How is your workload? Do you have sample volume constraints, batch size expectations, or specific requirements for throughput and turn-around time?
5. What are the resources in your lab? Do you have automated or manual liquid handling, or experience with LC multiplexing and on-line solid-phase extraction automation? Do you have suitable ancillary extraction equipment, such as solvent evaporators, positive pressure or vacuum extraction modules, heating blocks, or multi-vortexers? Is there expertise available for sample preparation during development, validation and production?
Once you have settled on a small molecule sample prep method that suits your needs, you need to refine the protocol. Each type of sample preparation protocol has unique parameters to optimize, but there are some general aspects to consider for any small molecule extraction protocol development:
How to boost productivity
And what about the product I mentioned earlier that ticks all the boxes? AC Extraction Plate* from Tecan offers an easy and flexible solution that delivers highly reproducible results for a wide range of applications, supporting high throughput to maximize productivity. In the final article in this series, we will look at AC Extraction Plate in more detail, and find out why one researcher chose it for sample preparation for small molecule LC-MSMS.
Tecan would like to thank Judith Stone, Ph.D., MT(ASCP), DABCC for technical consultation about sample preparation for LC-MSMS. Judith Stone is the senior clinical laboratory scientist specialist at the University of San Diego toxicology laboratory in the Center for Advanced Laboratory Medicine.
*For research use only, not for use in diagnostic procedures.