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Enhancing Mass Spectrometry Sample Preparation with Automated Solid-Phase Extraction Wednesday, December 18, 2013

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Rohit Shroff

Tecan Schweiz AG

Application Specialist

Mass spectrometry (MS) instrumentation has advanced tremendously over the past

few years, placing considerable sample preparation demands on the laboratory.

Significantly, sample preparation methodologies have progressed at a much slower

rate, becoming the major bottleneck in high-throughput mass spectrometry protocols. This, along with the limitations

associated with manual processing, has hindered the uptake of MS innovation in the life science industry.

Solid-phase extraction explained

Laboratories working in the small-molecule field generally must be able to handle large numbers of samples from

several different biological matrices, including blood, urine, plasma and serum. They may use a range of different MS

sample-preparation techniques—solid-phase and liquid-liquid extraction, enzymatic hydrolysis, protein precipitation,

‘dilute and shoot’ and protein purification—with the final choice depending on the application, matrix, analyte(s) of

interest and sensitivity required.

Solid-phase extraction (SPE) is a popular means of purifying and concentrating samples prior to analysis by liquid

chromatography-mass spectrometry (LC-MS). SPE is used widely in the pharmaceutical industry, clinical labs and

academia for toxicology testing, food testing and environmental analysis. It can be performed in several different formats

(cartridge, disk and 96-well plate) on a variety of biological matrices, but in all cases the result is the same: extraction of

the compounds of interest by retention of the analyte(s) on the solid phase and removal of matrix interferences, or vice

versa.

Designing a successful experiment

A typical SPE experiment involves four steps: (1) conditioning the solid phase, (2) sample application, (3) washing the

solid phase to remove impurities and (4) elution of the analyte(s).

SPE method development begins with the selection of the most suitable solid phase for the analyte(s) of interest.

Options include reverse-phase, normal-phase, ion-exchange and mixed-mode SPE. With a range of materials available

in each of these categories, developing a successful extraction methodology is highly dependent on selecting the correct

phase, and the properties of the analyte(s) of interest must be carefully considered.

Reverse-phase SPE is ideal for isolation of nonpolar analytes from polar matrices, and normal-phase SPE generally is

used to extract polar analytes from non-polar matrices. For charged analytes, ion-exchange SPE is the method of

choice; this may be cation or anion exchange, depending on whether the analyte is positively or negatively charged.

Mixed-mode SPE typically is used when a combination of neutral, acidic and basic compounds needs to be isolated from

a single complex matrix for subsequent separation.

Having selected the most appropriate SPE phase for the analyte or analytes of interest, it is important to establish the

correct bed weight for optimal extraction. As a rule of thumb, the mass of the analytes to be extracted should not exceed

4% to 5% of the mass of packing in the SPE consumable to avoid overloading the phase. For example, a maximum of 5

mg of analyte can be loaded onto a 1 ml SPE cartridge containing 100 mg of solid phase.

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Sample pH is another crucial consideration when selecting the solid phase, playing a key role in efficient interaction

between the phase and the analyte. Silica-based SPE phases are generally only stable between pH 2.5 and 7.5, and

sample pH may need to be adjusted prior to loading onto these phases. Alternatively, if experiments must be conducted

at extreme pH values outside this range, the more stable polymer-based phases may be a better choice.

Depending on the matrix, some form of sample pretreatment may be necessary prior to SPE extraction. Urine samples,

for example, may require straightforward dilution and/or hydrolysis to ensure solvation of the analytes of interest. For

other biological matrices, such as plasma, serum and whole blood, a protein-precipitation step, either by simple pH

adjustment or addition of a precipitant, may be required to release protein-bound analytes. Water samples typically do

not need any pretreatment beyond simple filtration, if a lot of solid material is present. Pretreated samples are then

applied to the SPE device, where careful choice of wash solvents enables any interfering compounds to be removed

without loss of analyte from the solid phase. Finally, the analytes are eluted from the solid phase for LC-MS analysis by

changing the elution strength of the solvent or, in the case of ion-exchange SPE, the pH.

A carefully developed SPE protocol offers several benefits. It is ideal for analyte enrichment, as large sample volumes

can be applied to the solid phase and the retained analytes eluted in a small volume of solvent, increasing the analyte

concentration in the aliquot for analysis. SPE also allows matrix interferences that would affect chromatographic analysis

to be removed, eliminating many co-eluting compounds that would otherwise mask the compounds of interest and make

detection and quantitation difficult or even impossible. In some cases, undetectable interferences can cause ion

suppression during LC-MS analysis; an optimized mixed-mode SPE protocol provides a very clean extract, minimizing

this effect.

Drawbacks of manual SPE

Very often, SPE is performed by hand. But manual SPE has some major limitations. It is cumbersome, time consuming

and prone to errors, resulting in poor reproducibility and sample recoveries as well as reduced productivity.

As mass spectrometry and chromatography have accelerated in recent years, slow sample-preparation procedures have

become a major bottleneck for high-throughput laboratories. Automation of SPE protocols can help overcome this

hurdle, offering many advantages. A diverse range of analytes and laboratory protocols can be accommodated,

increasing versatility and analytical flexibility, and enhancing throughput considerably. Typically, one liquid-handling

system can prepare sufficient samples for up to 10 mass-spectrometry systems on a daily basis, allowing laboratories to

meet high-throughput demands without compromising data quality. Additionally, the adoption of built-in security features,

such as sample tracking, satisfies even the most stringent quality requirements. Consequently, a variety of automated

devices have emerged, from small workstations processing six samples in parallel through to large, high-throughput

liquid-handling platforms—such as Tecan’s Freedom EVO® workstations—that are capable of performing parallel

extractions in 96-well plate format, enabling SPE protocols to be semi- or fully automated.

Typically, laboratories are looking for maximum throughput with excellent precision, seeking to minimize the number of

false positive and negative results and repeat analyses. SPE offers a simple means of isolating and concentrating

analytes from biological matrices, generating a clean extract for LC-MS analysis, and automation is the key to enhanced

sample throughput. By harnessing the power of automation, laboratories can enjoy benefits including full sample

traceability, increased throughput, improved turnaround times and enhanced reproducibility, as well as the almost

complete elimination of manual errors. That means fewer repeat analyses and, with full walk-away processing assured,

analysts are free to focus on what they do best: data interpretation.

Rohit Shroff is an Application Specialist at Tecan Schweiz AG

Related Products from: Tecan

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