Solid-phase extraction on molecularly imprinted polymers

I.D. Wilson (Ed.), Bioanalytical Separations Handbook of Analytical Separations, Vol. 4 92003 Elsevier Science B.V.All rights reserved

45

CHAPTER 2

Solid-phase extraction on molecularly imprinted polymers Lars I. Andersson ~ and Leif Schweitz 2

2

Research DMPK, AstraZeneca R&D S6dertiilje, S-151 85 SOdertiilje, Sweden Analytical Development, AstraZeneca R&D MOlndal, S-431 83 MOlndal, Sweden

2.1 INTRODUCTION Despite the recent progress in mass spectrometry, providing both high separation power and impressive detection sensitivity, selective sample preparation and efficient chromatographic separation will remain indispensable to a robust analytical method. The extent of sample pre-treatment and separation required depends on the complexity of the analytical problem where, in particular, trace and ultra-trace analysis of complex biological and environmental matrices relies on efficient sample enrichment prior to a selective assay. Also, for high-concentration level analysis a more selective sample clean-up simplifies the total analytical protocol. Solid-phase extraction (SPE) is continuously growing in importance [1-5], and various formats of SPE are currently routine sample preparation techniques employed in numerous environmental and bioanalytical applications. Many current SPE sorbents retain, however, not only the target analytes but also other matrix components, and often a considerable amount of method development work is spent on optimising the extraction protocol. Whereas for most materials the separation is based on physicochemical retention on the functionalised surface, more selective SPE materials, such as immunosorbents [6-7] and molecularly imprinted polymers (MIPs) [8-11 ], rely on affinity interactions. Characteristic for both affinity sorbents are their high ligand selectivity and affinity, which potentially yield a higher degree of sample cleanup. Selectivity can be pre-determined for a particular analyte or structural class of compounds by, respectively the choice of antigen used for antibody generation and the choice of template used for polymer preparation. Recent years have seen an increasing research activity into molecular-imprint based solid-phase extraction (MISPE). In parallel with genetic research into methodology development an increasing number of investigations into the use of MIPs for analysis of drugs and pollutants in biological and environmental samples are being reported. This References pp. 69-71

46

Chapter 2

review discusses the potential scope and some pitfalls of using MIPs for solid-phase extraction as well as some fundamental aspects of the molecular imprinting technology for such applications.

2.2 IMPRINT PREPARATION

Imprinting of molecules occurs by the polymerisation of functional and cross-linking monomers in the presence of a template (Fig. 2.1) [8-11]. The monomer system is chosen such that pre-polymerisation complexes of one or several functional monomers per template molecule are formed. During the polymerisation reaction these complexes become spatially fixed in the highly cross-linked polymer network and subsequent removal of the template molecules leaves behind imprints, or "memory sites", in the solid polymer. These imprints possess a topological (size and shape) and chemical (spatial arrangement of complementary functionality) memory for the template, and enable the polymer to selectively rebind the imprint species from a mixture. Two principally different approaches to molecular imprinting may be distinguished: The noncovalent, or self-assembly, protocol [12] where complex formation is the result of non-covalent or metal ion coordination interactions, and the covalent, or pre-organised, protocol [13], which employs reversible covalent bonds, usually involving a prior chemical synthesis step to link the monomers to the template. While the non-covalent strategy is the one being by far the most widely employed for applications in the analytical field, both approaches have their strengths and weaknesses. Whereas it is generally perceived that covalent imprinting yields more defined and more homogenous binding sites, non-covalent imprinting is more flexible in terms of the range of chemical functionalities that can be targeted and thus the range of templates that can be used. The latter is also much easier practically, since complex formation occurs on mixing template and monomers in solution prior to polymerisation. Recent developments have seen the advent of the covalent sacrificial spacer imprinting with non-covalent recognition (semi-covalent) protocol [14,15] and the stoichiometric non-covalent imprinting protocol [16,17]. Both techniques combine the advantages of well-defined imprint formation characteristic of covalent imprinting with rapid association/ dissociation kinetics of non-covalent interaction-based recognition. Although, at present, they suffer from the same limitation as the covalent protocol of "individualised" monomer design with a consequent lack of general applicability, both protocols show great promise to improve on current imprinting efficiency. In analytical chemistry, MIPs are increasingly being employed in application areas as diverse as liquid chromatography [18-20], capillary electrochromatography and capillary electrophoresis [21-23], pseudo-immunoassay [24,25], chemical sensing [26-29] and solid-phase extraction [25,30-35], where each application area has its own specific demands on the properties and formats of the MIP preparations used. The following discussion will, however, concentrate on issues critical for MIP preparation for SPE applications, such as removal of template molecules, choice of template, and format of the polymer. A discussion on imprint preparation generally is beyond the

,,,,.i

%

Fig. 2.1. Schematic depiction of a molecular imprinting protocol. The imprint molecule is mixed with monomers, which have the ability to interact non-covalently with chemical functionality of the template, and cross-linking monomer in an apolar organic solvent. Typically one or several of functional monomers, such as methacrylic acid, trifluoromethylacrylic acid, 2- or 4-vinylpyridine, methacrylamide, or hydroxyethylmethacrylate, are used together with a cross-linking monomer, such as ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate or divinylbenzene. Following addition of an azobis-nitrile initiator, the polymerisation is conducted either by elevation of temperature or by irradiation of UV-light. Extraction removes the imprint substance and leaves behind imprints, which have a chemical and topological memory for the original template.

t%

~,~~

t,,~o

t%

~,,,i ~

48

Chapter 2

scope of this review, instead consultation of the excellent reviews by Sellergren [ 12] and Wulff [ 13] is recommended.

2.2.1 Removal of template molecules MIPs are made in the presence of high concentrations of template and despite exhaustive washing, trace amounts of the imprint species may remain in the MIP and may later leak during use, so-called template bleeding. While being less critical for most chromatographic and other continuous-flow applications, near-quantitative removal of the imprint species is crucial for a sensitive SPE application, where even a small amount of bleeding may greatly affect the assay result. Template bleeding has repeatedly been observed [36-43]. Generally, a more thorough extraction yields a MIP where more of the high-avidity sites are free, leading to a material better equipped to bind analyte from highly diluted samples and less prone to bleed template molecules at use. Complete extraction requires extensive washing using solvents with strong ability to swell the MIP, such as chlorinated solvents, and solvents with strong elution power, such as aqueous methanol or ethanol, containing acid or base. Alternating acid and base washings may be beneficial. Heat-treatment of the polymer accompanied with excessive washes with strong eluents has been tried and this treatment was claimed to greatly reduce or even eliminate bleeding of template from the MIP phase [44]. Since in this study relatively high sample concentrations were extracted it remains, however, to be seen how useful this procedure will be for trace analysis. In a comprehensive investigation Ellwanger et al. studied the influence of various post-polymerisation treatments, such as thermal annealing, microwave assisted extraction, Soxhlet extraction and supercritical fluid desorption, on the level of bleeding [45]. While microwave assisted extraction using trifluoroacetic acid or formic acid was found to be the most efficient extraction technique, also polymer degradation and loss of selectivity were observed. Neither of the treatments eliminated template bleeding completely and it was concluded that, at present, the bleeding problem therefore appears to be best solved by the use of an analogue of the analyte as the template [45].

2.2.2 Choice of template The obvious choice is, of course, to use the target analyte as the template and this approach is employed in the vast majority of molecular imprinting studies. In at least two situations, however, a different approach may be required: (i) when the template is insoluble in the polymerisation solution and (ii) when template bleeding causes problems (see above). To circumvent these problems an alternative template, which is a structural analogue of the analyte(s) of interest, can be used for the MIP preparation. The analyte and the alternative template should possess common structural features such that the template used gives rise to imprints that have the ability to bind the target analyte(s) (Fig. 2.2). The wealth of data already available on template structure-imprint selectivity relationships will often aid the selection of a suitable structural analogue. A

"-3

%

Fig. 2.2. Imprinting of a close structural analogue generates imprints with a chemical complementarity for the target analyte.

@

4~

t~

~,,~o

~..,d.

~,,~~

50

Chapter 2

typical example of difficult templates is peptides, which due to their low solubility in the pre-polymerisation mixture normally are not amenable to non-covalent molecular imprinting. In order to circumvent the limited solubility Leu-enkephalin anilide has been substituted for the target ligand for the preparation of a Leu-enkephalin selective MIP [46]. Similarly, amino acids are insoluble in organic solvents and L-phenylalanine anilide has been used for the preparation of a MIP, which recognized the enantiomers of the parent amino acid phenylalanine [47]. In both instances, conversion of the carboxylic acid functionality into an anilide improved solubility while the sub-site formed for the amide functionality also bound a carboxylic acid. Hence, the imprints were able to rebind the parent structure with a free acid in this position. The resultant MIPs were used, respectively for fundamental investigations into molecular recognition of enkephalin peptides and for capillary electrochromatographic separation of the enantiomers of phenylalanine. For solid-phase extraction purposes, where even low levels of template bleeding would interfere with the quantification, the alternative-template species approach is often used for preparation of the MIE Should template bleeding occur, the subsequent analytical separation will resolve the template from the analyte(s) and the quantification will still be accurate. This was first demonstrated by the preparation of a MIP for extraction of sameridine using a close structural analogue [36]. Another example is the imprinting of trialkylmelamines for the preparation of MIPs capable of recognition of the related triazine structures [48]. In the LC mode these MIPs retained atrazine and related triazines in a group selective manner, whereas structural unrelated agrochemicals were not adsorbed to the imprinted polymer, and in a subsequent study the MIP was used for selective SPE of atrazine from a mixture of herbicides [49]. The alternative template protocol has been used in many more investigations (Table 2.1), however, the availability at reasonable cost of a suitable analogue has to be evaluated on a case-bycase basis.

2.2.3 Format of polymer Bulk polymerisation, followed by grinding and particle sizing is the most commonly used technique for preparation of non-covalent MIPs [12]. The grinding process produces, however, irregular particles as well as a considerable quantity of fine particles which have to be removed, for instance by sedimentation. Typically about 20-60% of the ground polymer is recovered as useable particles. While the procedure is simple and requires no specialist knowledge or speciality equipment, and works well for MIP synthesis in the research laboratory, it is probably not suitable for preparation uniform, high quality particles on a large scale. Furthermore, ground particles are irregular and non-uniform in size, which renders them technically difficult to pack into columns. Means to improve particle shape have included dispersion polymerisation, which starts with a homogenous solution of monomers and template, where, as the polymerisation proceeds, the growing polymer becomes insoluble and precipitates out as particles. This technique has been used for the preparation of MIP particles for pentamidine extraction [50]. Monodisperse imprinted beads can be made by a two-step swelling technique [51 ].

Solid-phase extraction on molecularly imprinted polymers

51

TABLE 2.1 SOME EXAMPLES OF MIPs PREPARED USING A STRUCTURAL ANALOGUE AS THE TEMPLATE Analyte

Template

MIP system j

H2N~ ~ / N

~N

4-Aminopyridine

NH~ 2-Aminopyridine

N

H Atrazine

LN ~

~ N ~ N ~ NH

H

N"

"N

I

.~.

Dibutylmelamine

~H

Reference

MAA/EGDMA

[76]

MAA/EGDMA, suspension polymerisation

[49]

4: ~ Bupivacaine OH

H2

[41,57]

OH

H2N

8r

Brombuterol

CI

Clenbuterol

MAA/EGDMA on silica fiber

[79]

2-VPy/EGDMA

[91 ]

4-VPy/EGDMA, multistep swelling polymerisation and modification of surface with hydrophilic layer

[42]

MAA/EGDMA

[36]

O II O--P--OH

O II O-P--OH

Diphenylphosphate

~

MAA/EGDMA

Pentycaine

Ditolylphosphate

OH

~ o ~ ~ O

S-Ibuprofen

OH

S-Naproxen

L.. Sameridine

\ N,N-dimethyl-analogue of sameridine

MAA, methacrylic acid; 2-VPy, 2-vinylpyridine;4-VPy, 4-vinylpyridine;EGDMA, ethylene glycol dimethacrylate

References pp. 69-71

52

Chapter 2

Aqueous two-step swelling employs a suspension of latex seed particles, which are first swelled with solvent and then with monomers and template to the desired particle size prior to polymerisation. In a more recent study, the surface was made hydrophilic through a second polymerisation of a mixture of glycerol mono- and dimethacrylate onto the beads. Chromatographic evaluation showed that, in contrast to the 10% recovery for the unmodified surface, bovine serum albumin was not retained on the hydrophilic beads and was recovered quantitatively, and the internal chiral recognition sites for S-naproxen remained unchanged [52]. This material was used in a coupledcolumn system for direct injection of rat plasma [42]. Potential problems with aqueous and polar solvent suspension media are their partial distribution into the organic droplets where they interfere with monomer-template complex formation, as well as the risk of less lipophilic monomers and templates distribute into the suspension phase. Both processes were attenuated in a novel suspension polymerisation protocol by which small droplets of imprinting mixture were polymerised in a continuous phase of an inert liquid perfluorocarbon [53]. Like water the perfluorocarbon phase is immiscible with the monomers and solvent used, however, should some partition occur the perfluorocarbons do not participate in hydrogen bonding and therefore leave the monomer-template complexes intact. A promising recent approach is the grafting of thin films of imprinted polymer in the pores of preformed well-defined silica particles [54], which may be useful in particular for online extraction using coupled-column systems.

2.3 MISPE METHOD DEVELOPMENT STRATEGIES

Efficient MISPE method development requires an understanding of the selective and non-specific binding modes to the MIE Generally, a MIP is best characterised as being a mixed-mode separation material containing, in addition to the imprinted affinity sites, both polar and lipophilic surface functionality. Hence, under normal-phase conditions non-specific physicochemical retention is due mainly to polar interactions and under reversed-phase conditions to hydrophobic interaction. Also, the selectivity of the imprint-analyte interactions is tuned by the solvent properties of the surrounding medium. For each compound, analyte as well as all other components of the sample, the observed retention on a MIP column is the sum of selective and non-specific retention modes through interaction with imprints and polymer surface, respectively. If the nonspecific retention mechanisms dominate, the analyte will be retained mainly through adsorption to the polymer surface and any selectivity shown by the imprints may remain undetected. The optimisation of the sample loading conditions and of the wash and elution steps of the MISPE method should be based on an understanding how the strength and nature of imprint-analyte and polymer surface-analyte interactions, respectively, vary with the type of solvent or buffer employed. The following paragraphs discuss problems encountered with the use of MIPs for SPE and highlight some issues relevant to MISPE method development.

Solid-phase extraction on molecularly imprinted polymers

53

2.3.1 Non-specific adsorption Due to the hydrophobic nature of the polymer, extraction of aqueous samples often results in moderately and highly lipophilic compounds being non-specifically adsorbed to the MIE This hydrophobic driven adsorption can be reduced by the addition of an organic modifier [55,56], such as ethanol, methanol or acetonitrile, or a detergent to the sample. The upper limit of the organic modifier content is, however, in part dependent on the type of sample, as protein precipitation may occur for plasma samples. Detergents tested and found useful include Tween 20, Triton X-100 and Brij 35 [57]. Also, buffer pH may influence the extent of non-specific adsorption, with an increase with increased pH being seen for adsorption of basic compounds to MAA-type polymers [55,56] and a decrease with increased pH for adsorption of acidic compounds to VPy-type polymers [58]. Non-specific adsorption may also be reduced by the use of small amounts of MIP, thereby reducing the polymer surface area available for lipophilic adsorption. For off-line extractions using columns or cartridges polymer amounts ranging from 2 g down to 15 mg have been used. As most applications deal with trace analysis, binding capacity does not seem to be a problem.

2.3.2 Solvent switch Following extraction of aqueous samples analyte molecules are retained both imprintbound and adsorbed non-specifically through hydrophobic interactions, and a wash step is required to improve MIP selectivity and to remove all other adsorbed sample components. A solvent switch, e.g. to dichloromethane or acetonitrile, changes the retention conditions to the normal-phase mode, which leads to re-distribution of the analyte to the imprinted sites and washing off of non-related structures (Fig. 2.3). In apolar solvents the selective imprint-analyte binding, which is due mainly to hydrogen bonding and electrostatic interactions, is strong and non-specific hydrophobic adsorption is weak. For environmental analysis hydrophobic, non-specific adsorption can be employed for capturing the analyte from a large volume of water passed through the column. A subsequent solvent switch assures a selective MISPE method. This protocol was first introduced by Takeuchi and co-workers [59] and later used by others [60-63].

2.3.3 Elution Due to the often, strong imprint-analyte affinity, difficulties in effecting quantitative elution of the analyte have been encountered in some cases [36,41,64-65]. This is most pronounced for extraction of strong bases, such as compounds with amino functionalities, on MAA type MIPs where typically eluents, consisting of acetonitrile or methanol containing (sometimes high percentages of) acetic acid, TFA or TEA are used [37,40-41,44,64,66,67]. Sometimes very harsh elution conditions are required, an example of this is the batch-mode extraction of sameridine which used a mixture of 5M References pp. 69-71

9

Q

Fig. 2.3. Loading of an aqueous sample results in the analyte, as well as other compounds, are retained through hydrophobic adsorption to the polymer surface, reversed-phase mode retention. A solvent switch to an organic solvent changes the retention conditions to the normal-phase mode, which leads to re-distribution of the analyte into imprinted sites and removal of hydrophobically bound contaminants. A second switch to a more polar solvent elutes the analyte.

OO

t,o

4~

Solid-phase extraction on molecularly imprinted polymers

55

sodium hydroxide, ethanol and heptane [36]. For neutral compounds, weak acids and weak bases, however, complete elution may occur simply by treatment with polar solvents or mixtures of polar solvents and water [42,49,59,60,68-71 ].

2.3.4 Template bleeding Each method development must include a confirmation that template bleeding does not interfere with the assay and gives rise to poor accuracy and precision. Control over template bleeding is particularly important when dealing with trace analysis. The risk is most severe for off-line extractions protocol using fresh material for each extraction, whereas for on-line protocols the MIP column is constantly washed by the continuous flow.

2.4 SOLID-PHASE EXTRACTION APPLICATIONS MISPE has been demonstrated in a number of proof of concept studies as well as been applied to pre-concentration of drug compounds in plasma and urine samples (Table 2.2), pollutants in environmental water and soil samples (Table 2.3) and controlled substances in tissue and urine (Table 2.4). The following paragraphs summarise publications according to the experimental set-up used.

2.4.1 On-line extraction systems On-line systems reported include both systems where the sample is injected directly on the MIP column and those where the MIP column is placed after a trapping column, where the latter system uses a pre-column to capture the analyte from an aqueous sample and transfer it to a solvent in which MIP-analyte binding is selective. An ,example of the second type is a MIP-C18 coupled-column system described by Bjarnason et al. for detection of four triazine herbicides, simazine, atrazine, propazine and terbuthylazine [71 ]. Samples, consisting of urine, apple extract or water containing humic acid, were spiked with the triazines and injected into a C18 pre-column which t~rapped the analytes from the aqueous sample. Elution with acetonitrile transferred the analytes into the MIP column, which under these conditions selectively bound the triazines. Subsequent elution with water and C18-based analytical reversed phase liquid chromatography completed the separation. Whereas humic acid was efficiently removed, urine and apple extract had some tendency to be retained by the MIP column. Enrichment, with enrichment factors of up to 100, was observed in all cases. Koeber et al. used an analogous solvent-switch approach for determination of nine triazines in river water samples [72]. Following injection of the water sample on a restricted access column, elution with acetonitrile transferred the analytes into the MIP column. A restricted access material (RAM) combines size exclusion and adsorption chromatography [73]. The material has a hydrophilic outer surface, which permits high molecular References pp. 69-71

Type of sample

Tamoxifen

Sameridine

Hydroxycoumarin Nicotine and oxidation products Phenytoin Propranolol

Chlorphenamine Darifenacin

Caffeine

Brombuterol

Human plasma Human urine Acetonitrile standards

Plasma Dog plasma Rat bile Human urine Aqueous standards Toluene standards Human plasma

Urine Chewing gum

Cola beverage Water Plasma

Human urine Buffer standards Acetonitrile standards Human plasma

Off-line mode extraction Atenolol Aqueous standards Bupivacaine Human plasma

Analyte

[36] [37,88]

2.5 ~g/mL 2.5-20 ixg/mL 10 txg/mL 8-120 nmol/L 500-1000 ng/mL

GC-NPD LC-UV

Extraction in heptane and addition of ethanol None

2.5 ~g/mL

2.5-40 txg/mL 2.5 txg/mL

[87] [38,64,65, 67,80,81]

[86] [44]

[39,85]

[84]

LC-UV LC-UV

[82] 10 ixg/L

None None

[79]

100 ng/mL

CZE-UV diode array LC-UV diode array

[41,571

[83]

Reference

10 I~g/mL 160-1000 nmol/L

Concentration range

0.02-1 txmol/L - 50 ~zg/mL, 25-1600 pg/mL 10-50 ~g/mL 0.04 mg/mL

LC-UV LC-APCI-MS-MS

LC-UV

LC-electrochemical detection

GC-NPD

Analytical separation and detection

Protein precipitation with methanol + acetonitrile and dilution with water Dilution with water. None Protein precipitation with acetonitrile None Extraction in ethyl acetate

None Dilution with citrate buffer pH 5 None

Treatment prior to MISPE

TABLE 2.2 BIOANALYTICAL AND PHARMACEUTICAL APPLICATIONS OF MISPE

t,o

, ,Iq

t% r~

%

Human serum Tobacco

Urine

Human serum Chloroform standards

Pentamidine

Theophylline

Rat plasma Rat plasma Human serum Human plasma

Type of sample

4-Aminopyridine Nicotine

Extraction with direct detection

Ibuprofen Naproxen Propranolol Tramadol

On-line mode extraction

Analyte

TABLE 2.2 CONTINUED

Extraction in chloroform Extraction in methanol -0.1N NaOH (1:1) and dilution with methanol Dilution with phosphate buffer pH 5 and acetonitrile Extraction in chloroform

None None None Extraction on RAM-SPE and elution with acetonitrile

Treatment prior to MISPE

0.25-1000 ~g/mL

10-60 nM

UV UV

2.5-100 txg/mL 1.8-1000 Ixg/mL

0.2-50 ~g/mL n.d. 0.5-100 p~g/mL 10 ng/mL 10 ~g/mL

Concentration range

UV UV

LC-UV LC-UV LC-UV LC-fluorescence LC-UV

Analytical separation and detection

[68-70]

[50]

[76] [77]

[42] [42] [75] [74]

Reference

--.,I

%-,

r~

%

Aqueous standards

Soil

Water

Methanol standard Drinking water Agricultural soil Human serum

Corn

Soil

Ground water Sediment samples Tap and groundwater

Aqueous standards Aqueous standards pH 5 River water

Type of sample

On-line mode extraction Aquacheck samples Chlorotriazine River water pesticides

Simazine

Nerve agent degradation products Phenylurea herbicides

Diphenylphosphate Diquat

Off-line mode extraction Atrazine Bentazone Chlorophenoxyacetic acids Chlorotriazine pesticides Chlorotriazine pesticides

Analyte

TABLE 2.3 ENVIRONMENTAL APPLICATIONS OF MISPE

Extraction on C18 RAM-SPE and elution with acetonitrile

Extraction on PS-DVB SPE disks and re-dissolution in toluene Extraction in acetone and re-dissolution in toluene None

Soxhlet extraction with methanol Extraction on PS-DVB SPE disks and re-dissolution in toluene Extraction in acetone and re-dissolution in toluene Extraction in acetonitrile and re-dissolution in toluene None None Suspension in water Extraction in acetonitrile

Acidification to pH 4

None

Treatment prior to MISPE

LC-APCI-MS

LC-UV diode array

LC-UV diode array

LC-ESI-MS Differential pulse voltammetry CE-UV

MEKC-UV diode array

LC-UV diode array

LC-UV LC-UV CZE-UV

Analytical separation and detection

0.1-2 ng/mL

[72]

[591

[94]

1 ~g/mL

0.1 lxg/mL

[93]

[91] [92] 0.2-10 ~xg/g

2.1 ~g/mL 3-18 Ixmol/L

100 ~g/L

100 ~g/L

[90]

[60]

20 ~g/L 0.1-0.5 p~g/L

[49] [89] [62]

Reference

0.1 Ixg/mL 10 mg/L 2-10 tzg/L

Concentration range

I

%

River water Water containing humic acid Apple extract

4-Nitrophenol Triazine herbicides

Pirimicarb

Tap water, spring water, fiver water and sea water

Extraction with direct detection

Urine

Type of sample

Analyte

TABLE 2.3 CONTINUED

Differential pulse voltammetry

LC-UV LC-UV

Acidification to pH 2.5 Extraction C 18 SPE column and elution with acetonitrile Extraction in methanol, re-dissolution in buffer, extraction on C18 SPE column and elution with acetonitrile Extraction C18 SPE column and elution with acetonitrile None

Analytical separation and detection

Treatment prior to MISPE

71.5 ~g/L

20 ng/mL

20 ng/mL

10 txg/L 0.5 ng/mL

Concentration range

[78]

[61,63] [71]

Reference t...,.

~..~~

r~

Type of sample

None

Milk replacer, Bovine urine Bovine liver Liver samples

Chloroform standard

Merlot red wine

Clenbuterol

Indoleacetic acid Quercetin

Clenbuterol

Extraction on Extrelut 20 and residue resuspended in phosphate buffer (pH 3.4)-acetonitrile (3:7; v/v) Matrix solid-phase dispersion on C18-sorbent and elution with acetonitrile- 1% acetic acid None

Calf urine

Clenbuterol None

Beef liver homogenate

Extraction in chloroform

Treatment prior to MISPE

Atrazine

Off-line mode extraction

Analyte

TABLE 2.4 AGRICULTURAL AND FOOD CONTROL APPLICATIONS OF MISPE

[96] [97]

250 Ixg/L 8.8 mg/L

LC-UV LC-UV

LC-electrochemic al detection

[95]

[40]

[66]

Reference

[43]

0.005-0.5 ppm 0.005-0.5 ppm 100 ng/mL

Concentration range

5 ng/mL 5 ng/mL 20 ng/g 5 ng/g liver

LC-UV ELISA LC-electrochemical detection LC-UV diode array

Analytical separation and detection

t,~

r

Solid-phase extraction on molecularly imprinted polymers

61

weight humic substances and large proteins to flow though the column without retention, while smaller molecules, such as triazines, are retained on the hydrophobic inner surface. The RAM column reduces the concentration of matrix components and the MIP column selectively retains the triazines whereas the residual matrix molecules are not retained and separated completely. The cleaned and enriched extract is subsequently eluted with methanol-water-acetic acid to a C18 column and analysed by LC-MS. The accuracy of the RAM-MIP-LC-MS system was checked using a certified reference material (Aquacheck). The deviation from the certified value was below 9% and the relative standard deviation was better than 8%. The applicability of the method to the clean-up of real samples was demonstrated by injection of contaminated fiver water samples and the results gave good agreement with previous determinations. Boos and Fleischer used a very similar system for tramadol analysis [74]. Serum samples were injected on a RAM column, transferred with acetonitrile into the MIP column and eluted with buffer at low pH for the final reversed phase analytical separation. Detection with fluorescence or UV revealed that all biological matrix components were eliminated. The purpose of the water-to-solvent switch is to first quantitatively trap the analyte from the aqueous sample and then change the solvent to one in which the MIP binds the analyte in a highly selective manner, and in which non-specific MIP-analyte adsorption is weak or absent. The solvent switch can also follow the (non-specific) adsorption of the analyte on the MIP, thus permitting direct injection of the sample onto the MIP column. This was first demonstrated by Takeuchi and co-workers for the extraction of triazines in the off-line mode [59] (see below) and later employed by Masque et al. for the selective on-line MISPE of nitrophenol [61,63]. Spiked river water samples were acidified to pH 2.5 and applied to the MIP-column. An intermediate dichloromethane wash removed non-specifically bound compounds, including other phenolic structures, and strengthened the selective irnprint-nitrophenol binding. Finally, the analyte was eluted and transferred to the analytical column by acetonitrile containing 1% acetic acid. Three different MIPs were evaluated: two prepared by the non-covalent approach using either methacrylic acid or 4-vinylpyridine as monomer and one prepared by the semicovalent approach where the monomer was 4-nitrophenyl methacrylate. The 4-VPy-MIP gave best recovery and was the most selective one of the three. The MIP was compared with a commercially available highly cross-linked poly(styrenedivinylbenzene) resin (LiChrolut EN) for extraction of fiver water and the former yielded cleaner extracts (Fig. 2.4). Haginaka and co-worker developed a column-switching system, consisting of a RAM-MIP and a conventional C18-column, for direct injection of serum and determination of ibuprofen and naproxen [42]. The RAM-MIP was prepared by an initial multistep swelling and thermal polymerisation protocol using 4-vinylpyridine as the functional monomer, followed by coating of the polymer outer surface with an external hydrophilic layer by a second polymerisation of an equimolar mixture of glycerol monomethacrylate and glycerol dimethacrylate. Plasma samples could be injected directly on the RAM-MIP column, which previously had been equilibrated with a mixture of phosphoric acid pH 2.2 and 20% acetonitrile, and proteinaceous components washed away. Elution and transfer of the analyte to the analytical reversed References pp. 69-71

Chapter 2

62

18000

2

3-4

IIEK)O0

14000 8..9

6

12000

7' ^

10.

10000

8OOO

6OOO i

10

.

.

.

.

,

2O

-

(n~)

......

3O

Fig. 2.4. LC-UV chromatogramsobtained by on-line SPE with a 4-nitrophenol-MIP(a, c) and LiChrolut EN (b, d) of 10 mL of Ebro fiver water spiked at 10 Ixg/L with 11 phenolic compounds. (a, b) with a washing step using dichloromethane and (c, d) without a washing step. Peak 2 is 4-nitrophenol. Reproduced with permission from [61].

phase column was effected with a mixture of phosphate buffer pH 7.3 and 25% acetonitrile. Leakage of imprint molecules prevented accurate determination of the drug, a problem that could be overcome by the use of a RAM-MIP prepared against naproxen for determination of ibuprofen. By this approach ibuprofen was determined in rat plasma with good precision and accuracy in the concentration range of 0.2-50 I~g/mL, which was sufficient for pharmacokinetic measurements. The column could be used for up to 500 injections. An automated and on-line MIP solid-phase micro extraction (SPME) method has been developed by Mullett et al., and its versatility was demonstrated by the determination of propranolol in biological fluids [75]. The system consisted of a propranolol MIP packed in a fused-silica capillary column, preconditioned with

Solid-phase extraction on molecularly imprinted polymers

63

acetonitrile, in which the serum sample was repeatedly drawn and ejected 10 to 20 times through the capillary for extraction. The extracted analytes were directly desorbed from the capillary by water-methanol containing 0.2% trifluoroacetic acid for transport to the LC column. Excellent method reproducibility and column reusability ( > 500 injections) were observed over a fairly wide dynamic range of 0.5-100 ~g/mL in serum samples. The method showed improved selectivity in comparison to alternative in-tube stationary-phase materials, and the MIP extraction was claimed to overcome present selectivity limitations of existing SPME coating materials.

2.4.2 Extraction systems with direct detection Provided the extraction is sufficiently selective, the downstream analytical separation can be omitted and the eluent directed directly to the detector for analyte detection. Sample pre-concentration with direct in-line UV-detection of the analyte following elution from the MIP-column was first shown by Sellergren in a model study on the determination of pentamidine in urine [50]. A mixture of spiked urine, phosphate buffer pH 5 and 70% of acetonitrile was loaded onto a pentamidine MIP-column and elution was effected by lowering the pH to 3, using the same buffer-acetonitrile composition. An enrichment factor of up to 54 was achieved. In a series of papers, Mullett and Lai explored the use of a MIP micro-column and direct in-line UV-detection for determination of theophylline in serum [68-70]. Human serum was extracted with an equal volume of chloroform and an aliquot of the organic layer was injected into the MIP micro-column heated at 60~ Non-specific adsorption of interfering drugs was eliminated by an intermediate wash with a pulse of acetonitrile, followed by quantitative desorption of the bound theophylline by a pulse of methanol and in-line UV determination. The method showed good accuracy and precision over the linear dynamic range of 2-20 ~g/mL, which was concluded adequate for therapeutic monitoring of the drug. This protocol, which was termed MISPE with differential pulsed elution, has also been applied to the determination of 4-aminopyridine in human serum [76] and nicotine in tobacco [77]. Due to its more polar nature and therefore stronger binding to the imprints, acetonitrile could be used as the mobile phase for extraction of nicotine. In this instance, the sample was injected dissolved in methanol, the column was washed with a pulse of methanol and the analyte eluted with 1% TFA in water. Recently, a similar approach was employed for MISPE of the analyte primicarb from water samples, in this instance quantification was done by differential pulse voltammetry [78]. 25 mL of spiked water samples including tap, spring, fiver, and sea water was loaded onto the MIP-microcolumn. Elution with an enrichment factor of 50 was effected with methanol containing 20% water and 10% acetic acid.

2.4.3 Off-line extraction systems Despite great success with on-line coupled-column systems, various off-line SPE techniques are still the more popular approaches with respect to the number of samples processed in routine sample pre-concentration. Likewise, the majority of studies into References pp. 69-71

64

Chapter 2

MISPE have used cartridges or columns off-line from the downstream analytical separation. A study by Andersson addressed mainly problems encountered with direct extraction of bupivacaine from human plasma [41 ]. The template was a structural analogue. The conditions for efficient and quantitative binding of the analyte from the plasma sample, intermediate wash steps and elution were optimised through a series of radioligand binding experiments. The final MISPE protocol consisted of adjusting pH of the human plasma samples by addition of citrate buffer pH 5, containing ethanol and Tween 20, prior to sample loading, washing with 20% methanol in water followed by acetonitrile, and elution of the analyte using 2% TEA in acetonitrile. The low recovery of 65-75% was due mainly to incomplete elution. TFA-acetonitrile based elution did not improve recovery and the eluates were found less pure. A direct comparison with conventional sample pre-treatment methods showed the MISPE method resulted in cleaner chromatographic traces than were obtained both after liquid-liquid extraction and C18based SPE (Fig. 2.5). To identify suitable solvents for loading, washing and elution, Ensing and co-workers undertook a feasibility study into the use of a clenbuterol-MIP for pre-concentration of this compound from calf urine [40]. Neat urine was loaded onto the MIP column, which was washed with 1% acetic acid in acetonitrile prior to elution of the analyte using 10% acetic acid. Freeze-drying of the urine sample and re-dissolution in acetonitrile prior to loading increased recovery up to 100%. Template bleeding was found to prevent accurate determination of trace levels of clenbuterol and it was concluded that future studies should use a MIP made against a structural analogue. Hence, the next study employed a bromoclenbuterol-MIP for determination of residue clenbuterol in liver tissue [43]. The combination of MISPE with matrix solid-phase dispersion (MSPD) was investigated for its potential to simplify post-MSPD sample clean-up treatment. Liver samples were ground in a mortar together with C 18 sorbent, the homogenised mixture packed into an SPE cartridge and placed on top of a MISPE cartridge. Elution with acetonitrile containing 1% acetic acid, which previously was found a good solvent for selective imprint-clenbuterol binding, transferred the analyte to the MIP column. Finally, clenbuterol was eluted with acidic methanol and determined by LC electrochemical detection (LC-ECD) with a recovery for the complete extraction exceeding 90%. Using LC ion trap mass spectroscopy (LC-IT-MS) analysis the method detection limit was <0.1 Ixg/kg, which satisfies regulatory requirements for food control of this substance. Furthermore, MISPE has been employed in the solid-phase microextraction mode with silica fibres coated with a 75-1xm layer of clenbuterol-MIP [79]. Extraction efficiency was evaluated for five structural analogues of clenbuterol, which could all be extracted selectively from acetonitrile and through non-specific adsorption from phosphate buffer pH 7. Brombuterol was extracted by non-specific adsorption from human urine by immersion for 45 minutes, then a solvent switch through immersion for 5 minutes in acetonitrile re-distributed the analyte to the selective imprint sites and removed most matrix components. Finally, the analyte was desorbed by methanol 10% acetic acid and analysed by LC-ECD. Several investigators have evaluated the potential to use MISPE prior to multi-residue analysis of triazine type herbicides in environmental water samples. The group of

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Fig. 2.5. GC-NPD chromatograms of human plasma spiked at 735 nmol/L with bupivacaine and subjected to either MISPE on a pentycaine-MIE SPE on a nonimprinted reference polymer, SPE on a C18 column or liquid-liquid extraction. Reproduced with permission from [57].

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Takeuchi studied the use of an atrazine-MIE prepared by suspension polymerisation, for selective extraction of simazine from water [59]. Of an aqueous mixture of simazine and some structurally unrelated agrochemicals all compounds were found retained on the MIP column. A solvent switch to dichloromethane removed the contaminants whereas non-specifically bound simazine was re-distributed to the selective imprints, and finally methanol effected elution. The same group used dibutylmelamine as a structural analogue to prepare a MIP, which was assumed useful for selective extraction of triazines [49]. MISPE of the above aqueous mixture of herbicide standards, using the same solvent-switch protocol, confirmed that the MIP indeed selectively extracted atrazine with high recovery. Employing essentially the same solvent-switch protocol Barcelo and co-workers used a terbuthylazine MIP for enrichment of six chlorotriazines from natural water and sediment samples [60]. The analytes were captured by passing large volumes of water samples through the cartridge, again a solvent switch to dichloromethane removed non-specifically bound contaminants while the selective imprint-analyte binding was strengthened, and methanol eluted the analytes for subsequent LC-diode array analysis. Recoveries were better than 80% for most chlorotriazines and the limits of detection varied from 0.05 to 0.2 p~g/1. Furthermore, natural sediments samples containing atrazine and diethylatrazine were Soxhlet extracted and analysed by the novel method. The chromatograms recorded for sediment extracts following MISPE were cleaner, with better baselines than were obtained using conventional extraction on C18 cartridges. No significant sample matrix interferences were noticed and it was concluded that an additional clean-up step, which in general is necessary for sediment sample preparation, could be avoided. Turiel et al. applied MISPE to the clean-up of drinking water, ground water and soil prior to determination of six chlorotriazines by micellar eletrokinetic chromatography [90]. An atrazine MIP was evaluated by Muldoon and Stanker for its ability to clean up organic solvent extracts of beef liver [66]. The purification protocol consisted of extraction of beef liver with chloroform, loading of the organic layer, column wash using chloroform and elution using 10% acetic acid in acetonitrile. Purified and unpurified beef liver extracts were analysed by both LC and ELISA, and the MISPE was found to improve accuracy of both methods, and improve precision and lower limit of detection of the LC method. MISPE for the clean-up of chlorinated phenoxy acids from fiver water samples prior to CE analysis was investigated by Baggiani et al. [62]. Large volumes of acidified river water samples, spiked with chlorinated phenoxy acids, were loaded on a 2,4,5-trichlorophenoxyacetic acid-MIE the column was washed with methanol and the analytes eluted with methanol-20% acetic acid. Template bleeding was not detected and the MISPE system was concluded comparable to a conventional C18-SPE in terms of recovery and superior in terms of sample clean-up (Fig. 2.6). Martin et al. investigated the use of a propranolol-MIP for SPE of propranolol and analogues from a variety of aqueous matrices, as well as from toluene [64-65,67,80-81]. Extraction of propranolol from water, bile and urine was found complete, however, some losses of approximately 10% were observed for plasma samples [64]. Adsorption of a range of similar and dissimilar structures in addition to binding of propranolol was seen, and the conditions chosen for elution of the analyte was concluded important to ensure selectivity. Eluents based on methanol-water 1%

67

Solid-phase extraction on molecularly imprinted polymers

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TEA were found more selective than those based on methanol-water 1% TFA. Template leakage prevented accurate determination of propranolol and the MIP was explored for its ability to extract structural analogues [67]. Adsorption from water and TEA-based elution resulted in class selectivity for a series of structurally very similar compounds, although a slight correlation between extent of retention and structural similarity to propranolol was seen. A similar problem was encountered by the group of Stevenson for extraction of tamoxifen from human plasma and urine using a MIP prepared against the analyte [37]. Clean L C - U V traces were obtained, however, again template leakage prevented reliable quantification of low levels of the drug. Likewise, Venn and Goody pointed out the difficulty of quantitative removal of the template and the consequent effect of bleeding on assay precision and accuracy as a major obstacle to reliable trace

References pp. 69-71

68

Chapter 2

level determination [39]. A darifenacin MIP was found able to extract darifenacin directly from human plasma, protein-precipitated with an equivolume acetonitrile. During washing with acetonitrile, the drug was strongly retained on the MIP whereas early breakthrough was seen for a non-imprinted polymer. Furthermore, the MIP was found selective for sub-structure elements of darifenacin. A phenytoin-MIP was evaluated as a selective sorbent for determination of this substance in plasma [87]. Several washing solvents were studied for their ability to disrupt the non-specific interactions occurring on extraction from plasma and dichloromethane was found most optimal. Phenytoin could be determined in plasma with good precision and accuracy, and at the concentration range studied (2.5--40 Ixg/mL) template bleeding was not noticed. In a recent study, protein precipitated human plasma and diluted cola beverage samples were extracted using a caffeine-MIP followed by analysis of caffeine by HPLC [82]. An investigation into the MISPE conditions found that optimal sample clean-up was obtained through aqueous loading at high pH, followed by a first wash with buffer at high pH, a second wash with acetonitrile 1% TEA and elution with acetonitrile 1% acetic acid. Application of MISPE to selective off-line extraction and pre-concentration of red wine samples was studied by Mizaikoff and co-workers [97]. Imprinting of the flavoinid quercetin yielded a MIR which selectively retained this substance while C18based SPE co-extracted other phenolic compounds.

2.5 CONCLUSIONS The rapid increase in the number of articles published each year testifies to the growing interest in molecular imprinting technology generally, with molecular-imprint based solid-phase extraction, MISPE, arguably being the most advanced sub-area with respect to future use in routine work. As is seen in this review, already several examples of highly selective pre-concentration of biological and environmental samples have been reported. Essential for a successful development of selective MISPE methods is a generic understanding of imprint-analyte binding mechanisms as well as the various physicochemical binding modes to these extraction materials. Compared with immunoaffinity materials MIPs have the clear advantage of being highly stable under a large range of buffer pH, solvent, temperature and pressure conditions, allowing large opportunities for selection of the best experimental conditions for the most efficient use of MIPs. Also, the issue of template bleeding need to be addressed, however, it must be kept in mind that semi-irreversibly trapped template molecules are inherent to the imprinting process. While imprinting of an analyte analogue circumvents the problem rather than solves it, this approach may in many instances be vital to successful use of MISPE in trace analysis. Other urgent areas, and presently focus for intense research, are extension of the types of chemistry available for imprint formation and development of strategies for selection of the best recipe of template, monomers, cross-linkers and polymerisation conditions given a combination of analyte, sample matrix and analytical context. An interesting example is post-polymerisation hydrophilisation of the external surface of monodisperse MIP beads to render them more compatible with proteins in biosamples, thus allowing direct injection of serum samples into a highly selective MIP

Solid-phase extraction on molecularly imprinted polymers

69

p r e - c o l u m n in a c o u p l e d - c o l u m n system. M I S P E with direct d e t e c t i o n of the analyte f o l l o w i n g elution is a p p e a l i n g as it e l i m i n a t e s the n e e d for an analytical c o l u m n , w h i c h w o u l d simplify the overall separation s y s t e m and increase speed of analysis. T h e direct detection a p p r o a c h involves, however, the d e m a n d i n g r e q u i r e m e n t for c o m p l e t e r e m o v a l of all s a m p l e c o n t a m i n a n t s , and, secondly, a p r e s e n t limitation m a y be the large elution v o l u m e s often required. N o t w i t h s t a n d i n g , these p r o b l e m s are addressable, and given the high selectivity of i m p r i n t e d materials M I S P E with direct d e t e c t i o n will likely develop into a viable alternative for e n v i r o n m e n t a l and b i o - s a m p l e analysis.

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13

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39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

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86 87 88

89 90 91 92 93 94 95 96 97

71

W.M. Mullett, M.E Dirie, E.EC. Lai, H. Guo and X. He, Anal. Chim. Acta, 414 (2000) 123. W.M. Mullett, E.EC. Lai and B. Sellergren, Anal. Commun., 36 (1999) 217. M.L. Mena, E Martinez-Ruiz, A.J. Reviejo and J.M. Pingarron, Anal. Chim. Acta, 451 (2002) 297. E.H.M. Koster, C. Crescenzi, W. Den Hoedt, K. Ensing and G.J. de Jong, Anal. Chem., 73 (2001) 3140. P. Martin, I.D. Wilson and G.R. Jones, Chromatographia Suppl., 52 (2000) S-19. ED.Martin, T.D. Wilson, I.D. Wilson and G.R. Jones, Analyst, 126 (2001) 757. G. Theodoridis and E Manesiotis, J. Chromatogr. A, 948 (2002) 163. D. Stevenson, Trends Anal. Chem., 18 (1999) 154. W. Chen, E Liu, X. Zhang, K.A. Li and S. Tong, Talanta, 55 (2001) 29. R.E Venn and R.J. Goody, in: E. Reid, H.M. Hill & I.D. Wilson (Eds.), Drug development assay approaches including molecular imprinting and biomarkers (pp. 13-20). The Royal Society of Chemistry, Cambridge, U.K. (1998). M. Walshe, J. Howarth, M.T. Kelly, R. OI~ennedy and M.R. Smyth, J. Pharm. Biomed. Anal., 16 (1997) 319. A. Bereczki, A. Tolokan, G. Horvai, V. Horvath, E Lanza, A.J. Hall and B. Sellergren, J. Chromatogr. A, 930 (2001) 31. D. Stevenson, R. J. Briggs, J. Hay and B. Rashid, in: E. Reid, H. M. Hill & I. D. Wilson (Eds.), Drug development assay approaches including molecular imprinting and biomarkers (pp. 49-51). The Royal Society of Chemistry, Cambridge, U.K. (1998). C. Baggiani, E Trotta, G. Giraudi, C. Giovannoli and A. Vanni, Anal. Commun., 36 (1999) 263. E. Turiel, A. Martin-Esteban, P. Fernandez, C. Perez-Conde and C. Camara, Anal. Chem., 73 (2001) 5133. K. M611er, U. Nilsson and C. Crescenzi, J. Chromatogr. A, 938 (2001) 121. B.B. Prasad and S. Banerjee, Chromatographia, 55 (2002) 171. Z.-H. Meng and Q. Liu, Anal. Chim. Acta, 435 (2001) 121. A. Martin-Esteban, E. Turiel and D. Stevenson, Chromatographia, 53 (2001) $434. G. Brambilla, M. Fiori, B. Rizzo, V. Crescenzi and G. Masci, J. Chromatogr. B, 759 (2001) 27. A. Kugimiya and T Takeuchi, Anal. Chim. Acta, 395 (1999) 251. A. Molinelli, R. Weiss and B. Mizaikoff, J. Agric. Food Chem., 50 (2002) 1804.