Selective polymers speed sample preparation

Molecularly imprinted polymers offer a new extraction method for sample preparation. Christine Widstrand, Johan Billing, Brian Boyd and Ecevit Yilmaz of MIP Technologies explain the potential applications

Molecularly imprinted polymers offer a new extraction method for sample preparation. Christine Widstrand, Johan Billing, Brian Boyd and Ecevit Yilmaz of MIP Technologies explain the potential applications

Sample preparation continues to be the bottleneck for analytical chemists and is the most labour-intensive and time-consuming step as well as the biggest source of errors in an analytical method. There is, therefore, a constant need for new sample preparation methods that are faster, easier and give more reliable and reproducible results.

Solid-phase extraction (SPE) was developed to simplify sample preparation compared with traditional liquid-liquid extraction (LLE). Lately the popularity of polymer-based SPE has grown, but these conventional SPE techniques lack the high selectivity that can be obtained using molecularly imprinted polymers (MIPs).

The use of MIPs in SPE results in cleaner extracts with minimised chemical noise and more reliable analytical results. This is particularly important for demanding clean-up of bioanalytical samples in the pharma industry or for detection of veterinary drug residues in food samples.

Figure 1 shows sample preparation techniques with a range of selectivities from non-selective, such as protein precipitation, to highly selective such as SPE using MIPs.

MIPs are a class of highly cross-linked polymer-based molecular recognition elements engineered to bind a target compound or a class of structurally related target compounds with high selectivity. Selectivity is introduced during MIP synthesis in which a template molecule, designed to mimic the analyte, guides the formation of specific cavities or imprints that are sterically and chemically complementary to the target analytes.

As illustrated in Figure 2, MIPs are prepared by first mixing the template molecule with one or more functional monomers that spontaneously form complexes around the template. Upon complex formation, cross-linking monomers and a suitable porogen (a solvent that aids in the role of pore formation) are added and the polymerisation is initiated. An extensive wash procedure is used to remove the template from the polymer leaving behind imprints or binding sites in the polymer that are sterically and chemically complementary to the template.

By careful choice of the template, either by molecular modelling, experimental design, or screening methods, the binding cavities can offer multiple interactions with the analytes of interest. Multiple non-covalent interaction points (ion-exchange, reversed-phase with polymer backbone, and hydrogen bonding) between the MIP phase and analyte functional groups lead to stronger and more specific analyte retention, which allows for improved selectivity to be introduced through the use of more rigorous wash conditions during the sample preparation. Because extraction selectivity is significantly improved, lower background is observed allowing analysts to achieve lower detection limits.

It is known that MIPs can show selectivity for a whole class of molecules. One such example is the commercially available MIP SupelMIP SPE-Beta-agonist (Sigma-Aldrich) that has been shown by Kootstra to selectively extract around 25 different beta-agonists.1 Kootstra has also compared MIP extraction with mixed-mode conventional SPE and found that the amount of extraction solvent used in MIP extraction is only 30% compared with mixed-mode extraction, and also that limits of quantitation are lower using MIPs and the extracts are cleaner.

The imprinted sites may also recognise molecules that differ more significantly from the template molecule used for imprinting. Two such examples from the literature are the study by Haupt et al who imprinted 2,4-dichloro phenoxyacetic acid and were able to use the non-related molecule 7-carboxy methoxy-4-methyl coumarine as a fluorescent probe2 (Fig. 3) and the study by Martin et al who imprinted the beta antagonist propranolol and found that the MIP also bound tamoxifen, a compound used in the treatment of breast cancer3 (Fig. 4).

Even though the recognised molecule is significantly different from the template in these two cases, they have common features (marked in yellow in figs 3 & 4). 2,4-dichloro phenoxyacetic acid and 7-carboxy methoxy-4-methyl coumarine both have large portions in common, and even though propranolol and tamoxifen differ more, both contain the O-CH2-CH2-NH moiety.

Borad selectivity

These findings suggest that MIPs can have a broader selectivity than just for the template molecule and closely related analogues, and that it is the placement of a few key recognition elements that matters. This is analogous to the concept of 'pharmacophores' in medicinal chemistry, where different molecules can have the same function as long as they contain the pharmacophore that is recognised by the natural receptor.

Another example from our laboratory is the finding that theophylline binds to a MIP that was imprinted against nicotinamide. If theophylline and nicotinamide are placed next to each other (Fig. 5), one can see that the best hydrogen-bond acceptors in theophylline match the best hydrogen-bond acceptors in nicotinamide, which may explain that using nicotinamide as the template leads to binding sites where also theophylline can bind.

This cross-reactivity can be ex-ploited. MIPs with suitable selectivity can be found through the screening of MIP libraries against the target molecule and the 'hit' MIPs can then be used to develop an analytical method based on that MIP or as a starting point for further MIP development. This can be a very effective way to find quickly a selective SPE phase that will suit a particular need in pharma or in other industries.

A range of SupelMIP phases are commercially available to detect banned veterinary drug residues such as clenbuterol, beta-agonists, chlor-amphenicol and fluoroquinolones.

The benefits of these SupelMIP phases are:

  • Low detection limits
  • Minimised ion suppression in
  • LC-MS
  • Time saving and reduced cost
  • Minimal method development.

The following examples will illustrate some of these general benefits.

superior selectivity

SupelMIP SPE - chloramphenicol was used to extract chloramphenicol (CAP) from shrimp according to the recommended SupelMIP application for shrimp extraction.4 The MIP method was compared against a published method using a hydrophilic polymer SPE phase.5 Analysis was conducted via LC-MS/MS.

Fig. 6 shows that the background is considerably lower when using the described SupelMIP SPE procedure versus the conventional hydrophilic polymer SPE approach. The decrease in background observed in the SupelMIP SPE approach was significant enough to reveal trace level chloramphenicol contamination in a shrimp sample purchased from the local supermarket intended to be used as a blank in a study.

The SupelMIP chromatogram shows a cleanly resolved chloramphenicol peak with a very low level of interferences. By virtue of higher recovery and improved selectivity (lower background), SupelMIP SPE can achieve lower limits of quantitation and detection relative to conventional SPE techniques.

Fig. 7 depicts multiple reaction monitoring (MRM) chromatograms of extracted shrimp samples spiked with CAP at the level of 50ng/kg. From this figure, it can be seen that CAP peak response and the signal-to-noise ratio of samples extracted using the SupelMIP SPE procedure was considerably higher than with conventional hydrophilic polymer SPE.

ion suppression

Matrix effects, such as ion suppression, are often a problem in trace analysis of compounds from complex matrices. This is due to lack of sufficiently high selectivity of conventional SPE and LLE methods: matrix components are co-extracted that cause reduction of the signal intensity in the MS source. Ion-suppression often leads to poor assay reproducibility, accuracy and sensitivity and such deleterious effects are often most notable near the lower limit of quantitation. Fig. 8 shows an example of ion suppression.

Sample preparation is often the rate-limiting step within the analytical process, and can often take up to 10 times as long as the analysis itself. It is therefore critical for analysts to develop simple, robust and rapid extraction techniques that are selective enough to achieve the sensitivity, precision and accuracy limits required.

fast clean-up

According to the authors, the use of molecularly imprinted polymers for sample clean-up meant that 18 samples could be processed within three hours, whereas it took eight hours with the classical SPE method.

Sample preparation methods are often developed using a variety of schemes such as referring to published methods of similar/identical applications; implementation ofgeneric methodology; requesting support from a chromatography vendor; and screening of techniques, phase chemistries and method conditions.

These approaches are often effective; however, more often than not, the development of a sample preparation method can be frustrating and time consuming.

minimal development

Unlike many traditional sample preparation techniques, SupelMIP is developed and tailored for very specific applications. Therefore, each SupelMIP SPE phase comes with a detailed protocol and analytical technique for its respective application.

In summary, the concept molecular imprinted polymer SPE technology has been presented along with the benefits of the degree of cross-reactivity that MIPs exhibit. Because selectivity is engineered during the development of the MIP phase itself, the MIP allows for a binding site that is sterically and chemically complementary to the target analytes. The multiple interactions that take place between the imprint binding site and analytes of interest offer strong interactions enabling the use of harsher wash conditions during the SPE process.

Benefits that are achieved by using MIPs are lower limits of detection and quantitation, less matrix effects such as ion suppression when MS is used as detection method. It also leads to faster and simpler methods with reduced analytical costs and with a higher sample throughput.