By Agnieszka Sitarska
Choosing a method and developing a protocol for small molecule LC-MSMS sample preparation can be a complex process. An effective shortcut is to use an extraction plate built for automation. With fewer processing steps and an automation-ready format, the use of extraction plate technology can help make tedious LC-MS sample prep processes more routine, whether it’s the analysis of testosterone in serum or anti-depressants in whole blood.
Boost your LC-MS/MS productivity with AC Extraction Plate from Tecan
Why consider an extraction plate?
Sample preparation prior to LC-MS can be a complex process that can introduce error, as well as consuming valuable time and resources. The best way to save time, ease your workload and ensure success is to minimize the number of preparation steps. An extraction plate can provide a rapid means of purifying and concentrating analytes, in a multi-well format that is amenable to automation.
Using an automated extraction plate can simplify your LC-MS sample prep routine for apolar small molecules, such as vitamin D, certain therapeutic drugs and testosterone.
For example, the AC Extraction Plate from Tecan* has a special immobilized extraction coating (called TICE™) that enables a straightforward three-step “pipette and shake” process. The three simplified steps include extraction of analyte from one of a range of biological fluids, followed by washing to remove matrix materials, and finally elution.
This eliminates the need for sample transfers, vacuum systems, filtration, centrifugation, and solvent evaporation steps. The resulting eluate is so pure that it can be injected directly into an LC-MS system for analysis.
Raising your standards for LC-MS/MS sample prep
When using LC-MSMS for diagnostic analysis, the results need to be absolutely consistent – from day to day, operator to operator, and lab to lab. Standardization is the key to success.
Sample preparation is easily standardized and automated using an extraction plate to eliminate random errors and increase consistency of results. It is important to choose an extraction plate that offers an open and ANSI/SLAS standard format that can be applied to sample preparation for a broad range of small molecule analytes from biological fluids.
Using this type of extraction plate, you’ll have a convenient, automation-friendly solution that can handle almost any LC-MS sample preparation challenge. In addition you should be able to use it immediately, without the need for conditioning or other pretreatments.
Case study: which extraction method is best for testosterone analysis?
Routine diagnostic analysis of testosterone by LC-MS/MS requires rapid and reliable extraction to obtain a sample that is pure enough for direct injection. For successful application in a busy clinical lab, the method also needs to be sensitive, easy to use, cost-effective and readily automatable.
Testosterone is a thermally stable, non-polar, uncharged steroid (Log P = 3.32) with a molecular weight of 288.42. Extraction protocols that work particularly well with such non-polar small molecules (<1,000 molecular weight) include liquid-liquid extraction (LLE), supported-liquid extraction (SLE) and the AC Extraction Plate.
To choose between these options, as well as a few less suitable options such as protein precipitation and solid-phase extraction, three key parameters to consider are:
- Detection limits: Detection of low-level analytes in bodily fluids can present a significant challenge that often necessitates sample concentration prior to LC-MS/MS. When testosterone has to be measured in pediatric and female patient samples,much greater sensitivity is necessary than for samples from adult males. A lower limit of quantitation (LLOQ) of 1–5 ng/dL is required
- Workload: LLE has the lowest materials cost and works well for small batches, but demands an excellent manual pipetting technique, is too labor intensive for large workloads, and can be difficult to automate. While SLE and the AC Extraction Plate are good options when using manual pipetting, they can be readily used to increase throughput by combining with high-throughput automated liquid handling. When comparing for ease of automation, the AC Extraction Plate uses less equipment (only an orbital shaker) and doesn’t require a positive pressure or vacuum manifold, as does SLE.
- Resources: This is usually the decisive factor, where personal preference, prior experience, and availability of equipment for extraction and automated liquid handling will all come into play. This means that diagnostic laboratories use a large range of methods, as well as the most recent option, the AC Extraction Plate, for testosterone analysis. Integrated use of automated liquid handling, LC-MSMS and the AC Extraction Plate is desirable because this combination could be expected to deliver good precision, better selectivity than automated immunoassays, and ease of automation. The option to test pediatric, female and male samples with a single AC Plate assay may be more cost-efficient per test, even for laboratories testing only 100–300 testosterone samples per week (see References 1 and 2).
Taking all of these factors into account, the AC Extraction Plate is an excellent choice for testosterone sample preparation prior to LC-MS/MS diagnostic analysis. An optimized and extensively validated method (1) delivers excellent precision, accuracy and robustness, making it a successful alternative to the automated immunoassays commonly used.
As illustrated by this data driven selection process, AC Extraction Plate offers an easy and flexible sample extraction solution that can significantly boost your productivity. It is a perfect match to the Tecan Freedom EVO and Fluent liquid handling platforms.
Could AC Extraction Plate* be the sample preparation solution you’ve been searching for?
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* for research use only. Not for use in diagnostic procedures.
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.
1. Automated Sample Preparation Enables LC-MS/MS as a Routine Diagnostic Analysis for Serum Testosterone. Judith A. Stone, Dave R. van Staveren, Robert L. Fitzerald, Journal of Applied Laboratory Medicine: An AACC Publication. DOI: 10.1373/jalm.2016.022772. Published online May 2017.
2. Application Note: Tecan AC Extraction Plate. Automated sample preparation for the determination of testosterone in serum by LC-MSMS.