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[ 32 ] S. Plunkett, F. Arnold, J. Chromatogr. A 708 (1995) 19.[ 33 ] A.G. Mayes, K. Mosbach, Anal. Chem. 68 (1996) 3769.[ 34 ] K. Hosoya, Y. Shirasu, K. Kimata, N. Tanaka, Anal.

Chem. 70 (1998 ) 943.[ 35 ] M. Walshe, E. Garcia, J. Howarth, M.R. Smyth, M.T.

Kelly, Anal. Commun. 34 (1997) 119.[ 36 ] J.M. Lin, T. Nakagama, K. Uchiyama, T. Hobo, J. Liq.

Chrom. Rel. Technol. 20 (1997) 1489.[ 37 ] S.-Y. Hjerteèn, M. Li, J.L. Lao, K. Nakazato, J. Moham-

mad, G. Pettersson, Nature 356 (1992) 810.[ 38 ] S. Nilsson, L. Schweitz, M. Petersson, Electrophoresis 18

(1997) 884.[ 39 ] L. Schweitz, L.I. Andersson, S. Nilsson, J. Chromatogr. A

792 (1997) 401.[ 40 ] J.M. Lin, T. Nakagama, K. Uchiyama, T. Hobo, J. Pharm.

Biomed. Anal. 15 (1997) 1351.[ 41] O. Bruëggemann, R. Freitag, M.J. Whitcombe, E.N. Vulf-

son, J. Chromatogr. A 781 (1997) 43.[ 42 ] D. Kriz, O. Ramstroëm, K. Mosbach, Anal. Chem. 69

(1997) 345A.[ 43 ] R. Levi, S. McNiven, S. Piletsky, S.-H. Cheong, K. Yano,

I. Karube, Anal. Chem. 69 (1997) 2017.[ 44 ] M. Senholdt, M. Siemann, K. Mosbach, L.I. Andersson,

Anal. Lett. 30 (1997) 1809.[ 45 ] G. Chen, Z. Guan, C.-T. Chen, L. Fu, V. Sundaresan, F.H.

Arnold, Nature /Biotechnol. 15 (1997) 354.[ 46 ] H. Bengtsson, U. Roos, L.I. Andersson, Anal. Commun.

34 (1997) 233.[ 47 ] G. Vlatakis, L.I. Andersson, R. Muëller, K. Mosbach,

Nature 361 (1993) 645.

[ 48 ] L.I. Andersson, R. Muëller, G. Vlatakis, K. Mosbach, Proc.Natl. Acad. Sci. USA 92 (1995) 4788.

[ 49 ] M.-P. Marcos, S. Gee, B.D. Hammock, Trends Anal.Chem. 14 (1995) 341.

[ 50 ] B. Hock, A. Dankwardt, K. Kramer, A. Marx, Anal.Chim. Acta 311 (1995) 393.

[ 51] M. Wlashe, J. Howarth, M.T. Kelly, R. O'Kennedy, M.R.Smyth, J. Pharm. Biomed. Anal. 16 (1997 ) 319^325.

Dr. P.K. Owens completed his Ph.D. studies on thedevelopment of chiral methods in spectroscopic andseparative studies at Pharmaceutical Chemistry, Schoolof Pharmacy, University of Bradford, UK. He currentlyholds a postdoctoral position at Astra Haëssle AB, Moë lndal,Sweden.

Dr. E.S.M. Lutz completed her Ph.D. studies on thedevelopment of af¢nity-based detection systems for liquidchromatography at the Leiden /Amsterdam Center forDrug Research, Division of Analytical Chemistry, LeidenUniversity, The Netherlands. She currently holds apostdoctoral position at Astra Haëssle AB, Moë lndal,Sweden.

Dr. L.I. Andersson obtained his Ph.D. from LundUniversity, Sweden. He holds a position as AssociateDirector at the department of Bioanalysis, Astra PainControl AB, Soëdertaë lje, Sweden. His main researchinterest concerns molecularly imprinted materials.

Dr. L. Karlsson obtained his Ph.D. from Lund University,Sweden. He holds a position as Associate Director atProduct Analysis I, Astra Haëssle AB, Moë lndal, Sweden.His main research interest concerns sample preparationand chromatography of pharmaceutical formulations.

Molecular imprinted polymers forsolid-phase extractionDerek StevensonSchool of Biological Science, University of Surrey, Guildford GU2 5XH, UK

Solid-phase extraction (SPE) has become themethod of choice in many laboratories for theanalysis of complex samples. Recently,highly selective extraction based on antibodycolumns or molecular imprinted polymers(MIPs) has been developed. To date biologicalantibodies have shown better speci¢city butMIPs are easier to produce. Several proce-dures using MIPs for SPE are reviewed alongwith the preparation and evaluation of a MIPfor SPE of atenolol. z1999 Elsevier ScienceB.V. All rights reserved.

Keywords: Molecular imprinted polymers; Antibodies;Atenolol; Solid-phase extraction

1. Introduction

The analysis of trace organics, such as drugs,metabolites, endogenous compounds, food additives,pesticides and other analytes of environmental con-cern remains a very important analytical task. Clientsof analytical laboratories (whether internal or exter-nal ) desire rapid, cost-effective and accurate results.

0165-9936/99/$ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 0 9 4 - 6

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In many cases these criteria are in con£ict and there isno one simple approach for solving all or even themajority of analytical challenges. The most dif¢cultanalyses involve complex matrices such as thoseencountered in the analysis of biological and environ-mental samples. It is generally accepted [ 1] that themost important step in such analytical methods is sam-ple preparation.

Many analytical procedures are based on the use ofliquid-liquid extraction, solid-phase extraction (SPE)or combinations of these followed by an analyticalseparation method, typically high performance liquidchromatography (HPLC), gas chromatography (GC)or capillary electrophoresis (CE). Method selectionfor a particular problem is a matter of personal choice,based on experience and techniques available, as wellas analyte and matrix properties. Nonetheless the pop-ularity of SPE has increased in recent years as it iseasily automated and a wide range of phases is avail-able. It is also regarded as environmentally morefriendly as large volumes of solvents are not used asin liquid^liquid extraction.

Commercially available phases for SPE based onsilica and bonded silicas have been used for a widerange of analytes. Early problems with batch-to-batchvariation in analyte recovery inhibited their use butthese have been addressed by manufacturers. One ofthe biggest problems was the presence of residual sila-nols on the most popular reversed phase materials.These could give separations dependent on morethan one mechanism of separation and greater vulner-ability to variations between different batches. SPEcolumns based on polymers have been developed toovercome the uncertainty caused by such secondaryinteractions.

2. Speci¢c sorbents for SPE

Another recent trend has been the development ofsorbents designed to show high speci¢city to the ana-lyte of interest. This has been achieved using biolog-ical antibodies covalently bound to a suitable supportsuch as silica or controlled pore glass. These haveshown the possibility of selectively extracting drugs[ 2 ] and pesticides [ 3^5 ] from biological and environ-mental matrices. The major disadvantage of thisapproach is the supply of biological antibodies.Their production can be uncertain and costly. How-ever once available it has been shown that extractionprocedures based almost entirely on antibody^antigeninteractions can be obtained [ 3 ]. Furthermore these

methods involve the use of aqueous-based environ-mentally friendly solvents.

The dif¢culty and cost of obtaining biological anti-bodies has led to attempts to synthesise antibodymimics in the chemistry laboratory. One suchapproach has been the development and evaluationof molecularly imprinted polymers (MIPs ). Thisinvolves the synthesis of cross-linked polymersaround a template molecule ( the analyte ). Once thepolymer has been formed the template is removed bywashing, leaving an `imprint' of the analyte template.Ideally this gives a sorbent on which highly selective,reversible binding of the analyte can achieved.

There have been two main approaches to the syn-thesis of MIPs. Wulff [ 6 ] and co-workers producedMIPs by synthesising speci¢c sugar or amino acidderivatives which contained a polymerisable functionsuch as vinylphenylboronate. After polymerisationthey hydrolysed the sugar moiety and used the poly-mer for selective binding. This approach is usuallyreferred to as covalent molecular imprinting. Mosbach[ 7 ] and co-workers developed the so-called non-covalent approach. They used a monomer such asmethacrylic acid along with a cross-linker such as eth-ylene glycol dimethacrylate mixed with the template(analyte molecule ). After polymerisation the analyteis washed out of the polymer leaving a cavity whichcan selectively bind the template.

Such sorbents allow several possibilities for use inanalytical methods. These include the production ofHPLC columns [ 8,9 ], capillary electrochromatogra-phy media [ 10 ], selectively permeable membranes[ 11,12 ], radioligand binding assays [ 13,14 ], sensors[ 15^17 ] and SPE columns [ 18^23 ].

3. MIPs for SPE

The use of MIPs for solid phase extraction is thetopic of this article. The main perceived advantageof MIPs over biological antibodies for SPE is theease with which they can be obtained and the conse-quent lower cost and speed. One of the main disadvan-tages with the MIP approach to SPE is the dif¢culty inremoving all of the template analyte molecule. Evenafter extensive washing it has proven dif¢cult toachieve this. This leads to leaching of the analyte inactual samples being processed and subsequent inac-curate results. The problem is exacerbated by the factthat comparatively large amounts of template ( mglevels ) are used to prepare the polymer, but individualsamples may contain only a few pg^ng of analyte.

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Hence retention in the polymer (and subsequent leach-ing) of even a fraction of a per cent of the template isvery signi¢cant [ 20 ]. This problem has been tackledby using a structural analogue to the analyte of interestas the template [ 22 ]. It relies on the fact that the MIPwill have some af¢nity to closely related compoundsjust as many biological antibodies often do. If the tem-plate continues to leach out this does not matter as longas it can be separated by the chromatographic end step.It must also separate from the internal standard and anymetabolites if these are also to be measured.

The selection of washing and elution steps is crucialfor optimisation of selectivity when developing a MIPbased extraction procedure [ 20,21 ]. With many pro-cedures it has been found that optimum selectivity isfound if the solvent in which the polymer was formedis used for retention and elution studies. This is exem-pli¢ed in the preliminary work reported below.

4. Preparation of a MIP for extraction ofatenolol

4.1. Preparation of MIP

Atenolol (0.53 g, 2 mmol) (Fig. 1 ), methacrylicacid (0.68 g, 16 mmol), ethylene glycol dimethacryl-ate (8.0 g, 81 mmol ) and 2,2P-azobis-(2-methylpro-pionitrile ) (0.08 g, 1 mmol), were dissolved in 7 ml ofacetonitrile containing 200 Wl of tri£uroacetic acid(TFA). The mixture was sparged with nitrogen for5 min. The simple apparatus used is shown in Fig. 2.The mixture was then polymerised under nitrogen for12 h at 60³C. The resulting polymer was ground intoparticles of s 100 Wm diameter using a mortar andpestle. The polymer was washed six times with meth-anol containing 0.1% TFA to remove monomer, ini-tiator and template. The polymer was then washedwith 0.1% TFA in methanol using a Soxhlet apparatusfor 16 h to try to remove all traces of atenolol template.The polymer was then left in a fume cupboard to airdry at room temperature. A control polymer withoutthe atenolol template was prepared in an identicalmanner for comparison. For subsequent experiments

0.5 g of each polymer was placed in empty SPEsyringe cartridges.

4.2. Optimisation of elution and washing solvent

Before each experiment the polymer was condi-tioned with 5 ml of washing and 5 ml of elution sol-vent. Atenolol solution in methanol or acetonitrile (1ml of 10 Wg / ml ) was loaded into the column. Thecolumn was washed ¢ve times with 5 ml of washingsolvent ( methanol or acetonitrile ) and then for theelution step ¢ve times with 5 ml of elution solvent,1% triethylamine (TEA) or 1% TFA in methanol oracetonitrile. All fractions, breakthrough, washing andelution were collected, evaporated to dryness undernitrogen, re-dissolved in mobile phase and analysedby HPLC.

4.3. HPLC conditions

The column was Sphereclone 5 Wm ODS, 250U5mm. The mobile phase was 70% acetonitrile, 30% pH5 0.01 M phosphate buffer. Detection was UV at 235nm.

4.4. Results

The results of this preliminary evaluation of thepolymer are shown in Table 1. With this type ofexperiment the ideal results would show no atenololin the wash and breakthrough fractions and quantita-tive recovery in the elution solvent for the work on theMIP. Selectivity would be obtained if the control poly-mer under the same conditions allowed most of theatenolol to elute in the breakthrough and wash frac-tions. This would allow the possibility of washing

Fig. 2. Apparatus used for polymer synthesis.

Fig. 1. Structure of atenolol.

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interfering compounds from the MIP while retainingthe analyte. It can be seen that of the two organicsolvents used better selectivity was found with aceto-nitrile, the solvent used to prepare the polymer, andthat results were more selective under basic condi-tions.

The polymer was easy to prepare which is in con-trast with the time required to obtain biological anti-bodies. In the case of the MIP recoveries in someexperiments were greater than 100% suggesting thatthere was some template leakage. Under the optimumconditions (using acetonitrile washing and acetoni-trile 1% TEA for elution) there was a clear differencebetween the behaviour of the MIP and the controlpolymer.

These results were in agreement with our earlierexperiment to produce a MIP for SPE of tamoxifen[ 20 ]. This work concluded that optimised selectivitywas obtained using acetonitrile as washing solvent andacetonitrile /acetic acid as elution solvent. Compari-son of MIP and control polymer does demonstrate thatan imprint has been made. Larger volumes of elutionsolvent (15 ml ) were needed for the MIP protocoldescribed above for atenolol and for the tamoxifenwork than for the immunoaf¢nity work (1 ml ).

5. An overview of related work

Other workers have shown the feasibility of usingMIPs for SPE. Andersson et al. [ 22 ] used a MIP forSPE of the drug sameridine. In order to overcome thepotential problems caused by template leaking theMIP was formed to a structural analogue of sameri-dine. They used the MIP added as a suspension after aninitial liquid^liquid extraction rather than as a column.The MIP suspension was added to heptane extractsand allowed to mix for 1 h. The MIP was washed

with heptane^ethanol 1:1 and then the drug elutedwith heptane^ethanol^sodium hydroxide. Templateleakage was observed but as it was subsequently sep-arated by GC from drug and internal standard this didnot interfere with the assay. They also demonstratedthat fewer interferences were co-extracted fromplasma than using liquid^liquid extraction alone.

Martin et al. [ 21 ] prepared a MIP for SPE of pro-pranolol in column format. They used radiolabelledcompounds for their investigation so problems withtemplate leakage were not encountered. Their resultsshowed that the MIP co-extracted other compoundsboth similar and dissimilar to propranolol unless care-ful choice of elution solvent is made. They obtainedoptimum speci¢city using methanol^water^1% TEAfor elution.

Muldoon and Stanker [ 19 ] prepared a MIP for SPEof the herbicide atrazine. They used this in columnformat for clean up of beef extracts before HPLC orELISA analysis. Their results showed that washingwith 3% acetonitrile in chloroform followed by elutionwith 50% acetonitrile in chloroform gave the optimumresults in terms of cleaning up samples, analyte recov-ery and allowed a lower limit of detection. The MIPwas used after chloroform extraction of the beef.

Sellergren [ 18 ] used a MIP for extraction of pen-tamidine from urine. This used acetonitrile /phosphatebuffer pH 5 for loading and washing, and lowered thebuffer pH to 3 for the elution step.

MIPs have not yet been demonstrated as exhibitingspeci¢city comparable to biological antibodies and inthe few experiments tried several structurally non-related compounds have also been extracted[ 21,24 ]. Presumably a lot of non-speci¢c bindingsites are present. Whitcombe et al. [ 25 ] have carriedout a series of calculations to predict the selectivity ofMIPs under various conditions and have referred to theexistence of speci¢c and non-speci¢c binding sites.

Table 1Distribution of atenolol (10 Wg) as a percentage between breakthrough, wash and elution solvent fractions

Elution solvent Polymer Breakthrough Wash Elution

Methanol^1% TFA Control 44 52 4MIP 48 40 12

Methanol^1% TEA Control 21 38 41MIP 2 33 65

Acetonitrile^1% TFA Control 56 36 8MIP 31 49 20

Acetonitrile^1% TEA Control 46 0 54MIP 1 4 95

In each experiment the same solvent ( methanol or acetonitrile ) was used for loading, washing and elution.

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6. Conclusions

SPE is a developing area for application of MIPtechnology. For the MIP approach to be of use insmall-scale analytical sample preparation there mustbe a demonstrable bene¢t over using commerciallyavailable SPE columns. Where the desired analyticalperformance ( in terms of limit of detection in partic-ular, but also reproducibility ) can be achieved with`off the shelf 'columns then the selectivity from MIPor antibody columns is not needed. Once an SPE phaseis available (MIP, biological antibody or `off theshelf'), the time to achieve optimum extraction con-ditions is similar in all three cases, at least in thisauthor's experience.

MIPs do have the advantage over biological anti-bodies that they are more stable to harsher conditionsof pH, organic solvents, pressure and temperature. Inthis respect they are comparable to `off the shelf'phases. The problem of template leakage with MIPsis partly circumvented by using a structural analogue,but only succeeds because of a lack of the very spec-i¢city that we are aiming to achieve. Such leakagedoes not arise when using phases based on biologicalantibodies.

Improved methods of synthesis [ 26 ] will allow pro-duction of MIPs with better selectivity and improvedmechanical properties. However, the technique muststill remain simple or it will not be an improvement oncurrent SPE methods other than in a few very speci¢capplications where conventional methods are not suc-cessful. In these cases biological antibodies, if avail-able, offer a feasible alternative. MIPs to a class ofcompounds such as a group of pesticides could alsobe an area of interest. The application of MIPs in otherareas is also worthy of further study.

References

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Dr Derek Stevenson is Head of the Analytical Centre in theSchool of Biological Sciences University of Surrey,Guildford, UK. He is immediate past President of theChromatographic Society and a member of the RoyalSociety of Chemistry Analytical Division Council.

Commercial reprintsWe can supply companies with reprints of any article in TrAC that directly or indirectly support their products atvery reasonable rates ( minimum order 100).Please apply for details to: TrAC Editorial Of¢ce, P.O. Box 330, 1000 AH Amsterdam, The Netherlands

158 trends in analytical chemistry, vol. 18, no. 3, 1999