Determination of Theophylline in Serum by Molecularly Imprinted

Jul 15, 1998 - Synthesis and Study of Guest-Rebinding of MIP Based on MAA Prepared using Theophylline Template. T Nurhayati , Yanti , I Royani , Widay...
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Anal. Chem. 1998, 70, 3636-3641

Determination of Theophylline in Serum by Molecularly Imprinted Solid-Phase Extraction with Pulsed Elution Wayne M. Mullett and Edward P. C. Lai*

OttawasCarleton Chemistry Institute, Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada

The technique of molecular imprinting is used to produce an extensively cross-linked poly(methacrylic acid-co-ethylene dimethacrylate) material that contains theophylline as a print molecule. After Soxhlet extraction of the theophylline, binding sites are formed in the polymer with complementary size, shape, and positioning of chemical functionalities. The molecularly imprinted polymer’s (MIP) high theophylline selectivity, chemical stability, and physically robust nature make it an ideal stationary-phase material in columns for HPLC separation of theophylline from other structurally related drug compounds. Mobilephase tests confirm that a retention mechanism typical of normal-phase chromatography governs the separation, and selectivity of the MIP column can be controlled by a combination of the mobile phase and the sample solvent. Under optimal conditions, the MIP column functions like a solid-phase sorbent for theophylline extraction. Rapid elution of the bound theophylline can be accomplished in a pulsed format through injection of 20 µL of a solvent that has the proper polarity and protic nature to disrupt the electrostatic interactions and hydrogen bonding between theophylline and binding sites. A concentration detection limit of 120 ng/mL is obtained using direct UV absorption detection at 270 nm, which corresponds to a mass detection limit of 2.4 ng. This new technique, molecularly imprinted solid-phase extraction with pulsed elution (MISPE-PE), permits on-line preconcentration of theophylline from a large volume of dilute sample solution. Using a sample volume of 300 µL, a 40 ng/mL standard solution produces a detectable peak signal. Application of MISPE-PE in serum analysis further demonstrates the high capability of the MIP column to selectively isolate theophylline from other matrix components for fast, accurate determination. Theophylline has long been used to treat asthmatic symptoms in children and adults as well as apnea in premature infants.1 The safe and effective use of theophylline requires careful dosage adjustment based on measurement of theophylline concentration in the blood serum. The use of standard chromatographic techniques, such as GC and HPLC, has been established for this analysis.2,3 However, these approaches can be seriously disad(1) Schreiber-Deturmeny, E.; Bruguerolle, B. J. Chromatogr., B 1996, 677, 305312.

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vantaged in the presence of a complex sample matrix containing many interferences, such as blood serum. In these instances, a lengthy sample extraction of ∼30 min is necessary before the analysis can be performed. Molecular imprinting is a technique based on creating cavities, in a highly cross-linked polymer matrix, that correspond to the size and shape of the print molecule. The method involves preorganization of the print molecule with functional monomers followed by a polymerization reaction fixing the monomers in the polymer network. Removal of the print molecule by Soxhlet extraction leaves behind functional monomer groups at defined positions in a spatial arrangement that is complementary to the structure of the original print molecule. Intermolecular interactions (such as hydrogen bonding, dipole-dipole, and ionic) between the print molecule in a sample solution and the functional groups of the molecularly imprinted polymer (MIP) can subsequently drive the specific molecular recognition process that causes the selective binding of the print molecule. The design of functional polymers that can selectively recognize target molecules has become an area of active research in recent years.4-8 Molecular imprinting has seen huge growth, and its extensive applications to numerous areas of scientific research (including chromatography, antibody and receptor binding mimics, artificial enzymes, and biosensors) have been recently reviewed.9-12 More specific to drug analysis, the ability of MIP columns to separate racemic mixtures13,14 represents a major potential application for the resolution of over 500 optically active drugs on the market. MIP columns are generally resistant to mechanical stress, heat, acids, bases, water, and organic solvents. Recently, MIPs have (2) Theinpont, L. M.; Van Nieuwenhove, B.; Stockl D.; De Leenheer, A. P. Clin. Chem. 1994, 40, 1503-1511. (3) Delbeke, F. T.; De Backer, P. J. Chromatogr., B 1996, 687, 247-252. (4) Dhal, P. K.; Arnold, F. H. Marcromolecules 1992, 25, 7051-7059. (5) Beach, J. V.; Shea, K. J. J. Am. Chem. Soc. 1994, 116, 379-380. (6) Matsui, J.; Doblhooff, O.; Takeuchi, T. Chem. Lett. 1995, 6, 489-489. (7) Kugimiya, A.; Takeushi, T.; Matsui, J.; Ikebukuro, K.; Yano, K.; Karube, I. Anal. Lett. 1996, 29, 1099-1107. (8) Matsui, J.; Kaneko, A.; Miyoshi, Y.; Yokoyama, K.; Tamiya, E.; Takeuchi, T. Anal. Lett. 1996, 29, 2071-2078. (9) Mosbach, K.; Ramstrom, O. Biotechnol. Rev. 1996, 14, 163-170. (10) Kriz, D.; Ramstrom, O.; Mosbach, K. Anal. Chem. 1997, 69, A345-A349. (11) Mayes, AG.; Mosbach, K. TrAc, Trends Anal. Chem. 1997, 16, 321-332. (12) Levi, R.; McNiven, S.; Piletsky, S. A.; Cheong, S. H.; Yano, K.; Karube, I. Anal. Chem. 1997, 69, 2017-2021. (13) Ficher, L.; Muller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360. (14) Kempe, M.; Mosbach, K. J. Chromatogr., A 1994, 664, 276-279. S0003-2700(98)00264-9 CCC: $15.00

© 1998 American Chemical Society Published on Web 07/15/1998

shown significant promise as a solid-phase extraction (SPE) sorbent.15-20 However, none of these methods were on-line techniques and therefore disadvantaged in terms of time and sample handling requirements.21 In addition the large elution solvent volume requirements (>0.5 mL) may compromise detection sensitivity. In this work, the objective is to develop a new method for the rapid analysis of theophylline in blood serum. This method uses a MIP column for on-line, SPE of the theophylline. An appropriately chosen mobile-phase composition can provide exceptionally strong binding of theophylline to the MIP column. Subsequent injection of a 20-µL aliquot of a protic polar solvent onto the column will produce a rapid, pulsed elution (PE) of the theophylline for direct UV detection. This PE band is very narrow, thus providing great analytical sensitivity. The present technique, molecularly imprinted solid-phase extraction with pulsed elution (MISPE-PE), also allows for analyte enrichment or preconcentration through injection of a relatively large volume of dilute sample solution, thus further improving the detection limit. In comparison with existing HPLC methods of theophylline analysis, the MISPEPE technique offers a very competitive detection limit and greatly reduced time requirements.22 Compared to enzyme-modified immunoassay techniques (EMIT), MISPE-PE requires a lower cost for each sample analysis.23 EXPERIMENTAL SECTION Chemicals. Methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA), 2,2′-azobis(2-isobutyronitrile) (AIBN), and theophylline were supplied by Pfaltz and Bauer (Waterbury, CT). Caffeine, xanthine, dyphylline, and (β-hydroxyethyl)theophylline were purchased from Sigma (St. Louis, MO). All solvents were HPLC grade from Caledon (Georgetown, ON). Molecularly Imprinted Polymer. The polymer preparation was previously reported by Mosbach and co-workers.24 The monomer MAA (0.90 g), the print molecule theophylline (0.47 g), and 25 mL of chloroform were placed in a 100-mL three-necked round-bottom flask. The mixture was allowed to preorganize until equilibrium overnight. The cross-linker EDMA (9.4 g) was then added, followed by the reaction initiator AIBN (0.12 g). The mixture was degassed under vacuum in a sonicating water bath while being purged with nitrogen for a period of 5 min. While a flow of nitrogen was maintained, the reaction flask was removed from the sonicating bath, fitted with a condenser, and placed inside a 60 °C water jacket to begin the reaction. The reaction was continued for 24 h under these conditions. The product polymer, after drying in air overnight, was white in color and possessed a rigid structure. It was ground into fine particles using a mortar and pestle. (15) Andersson, L. I.; Paprica, A. Chromatography 1997, 46, 57-62. (16) Martin, P.; Wilson, I. D.; Morgan, D. E.; Jones, G. R.; Jones, K. Anal. Commun. 1997, 34, 45-47. (17) Matsui, J.; Okada, M.; Tsuruoka, M.; Takeuchi, T. Anal. Commun. 1997, 34, 85-87. (18) Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803-808. (19) Rashid, B. A.; Briggs, R. J.; Hay, J. N.; Stevenson, D. Anal. Commun. 1997, 34, 303-305. (20) Sellergren, E. Anal. Chem. 1994, 66, 1578-1582. (21) McMahon, G. P.; Kelly, M. T. Anal. Chem. 1998, 70, 409-414. (22) Schreiber-Deturmeny, E.; Bruguerolle, B. J. Chromatogr., B 1996, 677, 305312. (23) Juarezolguin, H.; Floresperez, J. Arch. Med. Res. 1998, 29, 45-50. (24) Vlatakis, G.; Anderson, L. I.; Muller. R.; Mosbach, K. Nature 1993, 361, 645-647.

A control polymer was also prepared when polymerization was carried out in the absence of theophylline. This control polymer was used to determine the presence of any nonspecific binding of the target molecule. Extraction of Theophylline from the MIP. Removal of the theophylline print molecule from the MIP particles was accomplished through a Soxhlet extraction. A sample of the theophylline-imprinted polymer (5.500 g) was placed inside the cellulose extraction thimble. The extraction solvent (150 mL) was a mixture of methanol and acetic acid (9:1). Heat was applied to the flask containing the solvent, at a rate that caused a filling and eventual emptying of the extraction chamber every 45 min. The extraction was continued for 24 h, to produce the anti-theophylline polymer particles. Quantification of the extracted theophylline was accomplished by HPLC using UV detection at 270 nm. Soxhlet extraction was not performed on the control polymer particles. Batch Binding Assay. A 0.100-g sample each of the antitheophylline and control polymer particles was placed in separate glass vials containing 5.00 mL of a 20 µg/mL theophylline standard solution prepared in either methanol or chloroform. Each suspension was magnetically stirred for 24 h and then passed through a 0.45-µm filter. The concentration of theophylline in the filtrate was analyzed by HPLC using UV detection at 270 nm and compared to the original standard concentration. Preparation of Columns. The anti-theophylline polymer particles were sieved, and the smallest size fraction (e63 µm) was used. A small volume of methanol was added to the polymer particles to produce a slurry. This was dispensed into a stainless steel column measuring 8 cm in length and 0.4-cm i.d. until the column bed was fully packed. A 10-µm frit was used in the end column fitting to ensure no loss of the anti-polymer. Methanol (10 mL) was run through the column to ensure uniform particle packing. The column was capped and set up in a HPLC system. The preparation of a control polymer column was achieved in a similar manner. High-Performance Liquid Chromatography. HPLC analyses for theophylline using the anti-theophylline polymer column were performed isocratically at room temperature with 1.0% methanol in chloroform as the mobile phase. An Eldex model 9600 solvent delivery system (San Carlos, CA) was operated at a flow rate of 1.0 mL/min. A Valco C6W switching valve (Houston, TX) containing a 20-µL sample loop was used for sample injection. Detection was accomplished with a Gilson 115 UV detector (Middleton, WI) set at 270 nm, and the absorbance signal was recorded by a Dionex 4270 integrator (Sunnyvale, CA) for retention time and peak area measurements. Various mixtures of theophylline, caffeine, xanthine, dyphylline, and (β-hydroxyethyl)theophylline (all prepared in acetonitrile) were analyzed on the column. The void time, t0, was determined by injection of acetonitrile. Molecularly Imprinted Solid-Phase Extraction. Pulsed Elution. The anti-theophylline column and HPLC system were used to perform new MISPE-PE studies. The mobile phase was changed to 100% chloroform. A theophylline standard solution in acetonitrile was injected onto the column using the Valco C6W switching valve with a 20-µL sample loop. For sample preconcentration, multiple injections of a dilute sample were made to Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

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Table 1. Batch Binding Assay of 20 µg/mL Theophylline in Methanol and Chloroform with Anti-Theophylline and Control Polymers sample solvent

theophylline concn % theophylline % cavities binding in filtrate (µg/mL) bound theophylline

methanol chloroform

Anti-Theophylline Polymer 16.5 17.5 0.42 97.9

methanol chloroform

19.78 19.54

Control Polymer 1.1 2.3

0.18 2.27 0.02 0.05

introduce a total volume of 300 µL. Approximately 1 min after the sample solvent peak was detected, 20 µL of a selected polar and protic solvent was injected through the same C6W valve to cause sample elution. To optimize the elution process, various pure solvents and a mixture of 1% water in methanol were tested with the MISPE-PE protocol. Serum Analysis. A 500-µL human serum sample was spiked with theophylline (in water) to provide a working concentration of 4.0 µg/mL. The serum sample was placed into a glass vial with 500 µL of chloroform, vortexed for 10 s, and centrifuged at 4000 rpm for 5 min. A 20-µL aliquot of the chloroform layer was injected onto the anti-theophylline column. After the elution of sample solvent and matrix constituents, the theophylline bound to the column was eluted in a pulsed format with a 20-µL injection of methanol for direct determination of theophylline. Safety considerations: Human serum samples are a potential biohazard. Unused serum samples should be treated with Javex before disposal as hazardous waste. RESULTS AND DISCUSSION Soxhlet Extraction of Theophylline from the MIP. A total of 210 mg of theophylline was extracted from a mass of 5.500 g of the MIP. Therefore, the percent by weight extracted was calculated to be 3.8%. By comparison, the maximum possible extraction would be 4.3%. The extraction efficiency was therefore 89%, which was sufficiently high to ensure the presence of selective cavities in the anti-theophylline polymer. Batch Binding Assay. The functionality of the anti-theophylline polymer was confirmed in a batch binding assay. Table 1 summarizes the theophylline concentration in the filtrate and the percentage of theophylline bound to the polymers in methanol and chloroform. Since the binding occurred over a 24-h period, the percentage of theophylline bound would represent the maximum binding capacity. This binding capacity was highly dependent on the sample solvent used in the assay, with an almost quantitative uptake of theophylline occurring in chloroform. This observation corresponds well with previous results for a methacrylic acid MIP in its ability to bind atrizine. Under the batch binding conditions, the theophylline uptake was smaller in methanol, due to the solvent’s polar and protic nature. The target molecule binding property of a MIP is known to be influenced by the solvent used in the polymer synthesis and the solvent used in the binding application of the MIP.25 Since chloroform provides maximal interactions between the theophylline print molecule and (25) O’Shannessey, D. J.; Ekberg, B.; Andersson, L. I.; Mosbach, K. J. Chromatogr. 1989, 470, 391-399.

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the MAA functional monomers during the preorganization, strong binding of theophylline can be expected when chloroform is used as the binding medium. If a polar solvent such as methanol serves as the binding medium, the specific binding of theophylline is greatly weakened. A small amount of nonspecific binding (1.12.3%) was elucidated by the control polymer. With a theoretical number of 1.31 × 1020 cavities/g of polymer, the percentage of cavities binding the print molecule was determined to be 2.27% in chloroform and 0.18% in methanol. These percentages should be evaluated in consideration of the theoretical number of cavities calculated, which makes the assumption that every print molecule added to the polymerization reaction mixture produces a useful binding site. In practice, extensive cross-linking around the print molecule must occur in order to produce cavities of the correct size and shape. Since there is a potential for the production of sites containing varying degrees of cross-linking, varying degrees of recognition ability may result. HPLC. Various solvents that stretch across a rather broad range of polarities were evaluated for use as the mobile phase in the HPLC separation of theophylline from different drug molecules on the anti-theophylline polymer column. Generally, all the experimental results confirmed that a retention mechanism typical of normal-phase chromatography rules the separation. Chloroform was found to enhance the specific molecular recognition to such an extent that total retention of theophylline occurred on the column. Conversely, other mobile phases that are more polar and protic than chloroform could not generate adequate retention to even separate theophylline from the solvent peak. The best mobile-phase composition was a mixture of 1% methanol and chloroform. It was also observed that theophylline solutions could not be prepared in any protic solvent to result in a good separation. This observation suggests that protic solvent molecules effectively compete for the hydrogen-bonding and ionic interactions with imprinted sites on the stationary phase.26 A recent paper by Babbiani et al evaluated the chromatographic performance of a MIP, selective for theophylline, in aqueous buffers and reached a similar conclusion.27 Theophylline (1,3-dimethylxanthine) was successfully separated from caffeine (1,3,7-trimethylxanthine) on the anti-theophylline column under mobile-phase conditions of 1.0% methanol in chloroform. Similar results were noted for the separation of theophylline and xanthine. A typical chromatogram illustrating the separation of theophylline from a mixture with caffeine is shown in Figure 1. The peak profile for theophylline elution is characterized by a prolonged retention time of 12.4 ( 0.2 min accompanied by peak broadening. When the control polymer was packed in a column for the HPLC, it was unable to retain theophylline, caffeine, or xanthine. The overall HPLC performance characteristics of the anti-theophylline column are summarized in Table 2 for comparison with those for the control column. There was a very high capacity factor (k′) for theophylline and a good separation factor (R) between theophylline and the weakly retained caffeine. As a result, quantification of the individual drug molecules was readily attained with good linearity (R2 ) 0.9999 for peak areas and 0.9955 for peak heights) up to (26) Mathew, J.; Buchardt, O. Bioconjugate Chem. 1995, 6, 524-528. (27) Baggiani, C.; Trotta, F.; Giraudi, G.; Moraglio, G.; Vanni, A. J. Chromatogr. A 1997, 786, 23-29.

Table 4. Retention of Structurally Similar Drugs on Anti-Theophylline Column

Figure 1. HPLC of a 1.00 mg/mL theophylline and 0.15 mg/mL caffeine mixture on an anti-theophylline polymer column: mobile phase, 1% methanol in chloroform; flow rate, 0.5 mL/min; sample injected, 20 µL; detection, λ ) 270 nm. Table 2. Chromatographic Performance of Anti-Theophylline Polymer and Control Polymer Columns capacity factor (k′) analyte theophylline caffeine

selectivity factor (R)

anti-theophylline control anti-theophylline control column column column column 5.3 0.2

0.3 0.4

26.5

0.8

Table 3. Peak Heights, Areas, and Retention Times for the HPLC of Theophylline and Caffeine Standards Using Anti-Theophylline Polymer Column concn (mg/mL) theophylline 2 1 0.5 0 caffeine 0.1 0

peak area (106 arbitrary units) 745 366 184 0.0 60 0.0

peak ht (cm) 12.5 5.6 2.5 0.0 8.8 ( 0.2 0.0

retention time (min) 9.68 12.3 14.5 0.0 2.3 ( 0.1 0.0

2.0 mg/mL, as shown in Table 3. HPLC results were also obtained for caffeine, xanthine, (β-hydroxyethyl)theophylline, and dyphylline, all of which were rapidly eluted from the anti-theophylline column close to the solvent peak (at 0.58 min) as presented in Table 4. These findings signify the ability of the anti-theophylline column to selectively bind and retain only theophylline, thereby separating the target molecule from a mixture of closely related compounds with similar chemical structures. Unfortunately, the HPLC separation efficiency in the isocratic mode was unacceptably low. The elution of theophylline was slow and diffuse, leading to a lengthy analysis time and poor detection limits. Column efficiency depends on a number of variables such as particle size, morphology, packing homogeneity, and pore size of the stationary phase. A recent report by Hosoya28 has outlined

sample

concn (µg/mL)

retention time (min)

caffeine xanthine (β-hydroxyethyl)theophylline dyphylline theophylline

50 50 100 200 50

0.61 0.62 0.60 0.58 3.70

a method for the molecular imprinting polymerization of methacrylic acid that produces a uniform particle distribution. This new method of polymerization should be advantageous for HPLC applications. Another significant cause of peak broadening is the possibility of different types of binding sites found within the polymer. The nature of the preorganization process may give rise to the formation of different modes of interaction between the theophylline molecule and the functional groups on the monomer molecules. This will result in the creation of different recognition sites in the polymer. The variable binding energies or sorption kinetics of these sites would produce retention of theophylline to different extents and hence an overall appearance of peak broadening. Comparable peak broadening was also displayed in a previous work which prepared a rod-type affinity medium for liquid chromatography.29 Similar broad peaks were also observed by Sellergren et al in the elution profile of D,L-PhNHPh enantiomers on a L-PhNHPh MIP column.30 They addressed this issue in terms of dissociation constants; it was determined that the antipolymer contained binding sites that exhibited a variety of affinities for the print molecule. Furthermore, the use of a different crosslinker, trimethylolpropane trimethacrylate (TRIM), can improve column performance. TRIM has been shown to be superior in terms of load capacity, selectivity, and resolving capability of the resulting MIP stationary phases for liquid chromatography.31,32 Also, the temperature at which the polymerization is carried out affects the molecular imprinting process. The stability of the monomer and print molecule at low temperatures, due to a more favorable entropy term, can result in a MIP with superior recognition properties.33 Molecularly Imprinted Solid-Phase Extraction. Pulsed Elution. The anti-theophylline MIP was applied to the specific SPE of theophylline, to provide maximum separation of theophylline from different drug molecules. The selective retention of theophylline on the anti-theophylline column was controlled by electrostatic interactions and hydrogen bonding. Complete retention of theophylline could be achieved by using an aprotic solvent such as chloroform. Conventionally, elution of the analyte from a SPE cartridge requires several aliquots of 0.5 mL or larger volume of solvent. For on-line UV detection of the eluting analyte, even a 0.5-mL sample is too large a volume to be handled (28) Hosoya, K. J. Chromatogr. A 1996, 728, 139-147. (29) Matsui, J.; Miyoshi, Y.; Matsui, R.; Takeuchi, T. Anal. Sci. 1995, 11, 10171018. (30) Sellergren, B.; Lepisto, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 58535860. (31) Kempe, M.; Mosbach, K. Tetrahedron Lett. 1995, 36, 3563-3566. (32) Kempe, M. Anal. Chem. 1996, 68, 1948-1953. (33) Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 11791183.

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Figure 3. Correlation between normalized solvent polarity and normalized elution efficacy of 400 ng/mL theophylline from an antitheophylline polymer column: mobile phase, 100% chloroform; flow rate, 1.0 mL/min; sample injected, 20 µL; pulsed elution solvent, 20 µL; detection, λ ) 270 nm. Figure 2. MISPE-PE of a 500 ng/mL theophylline and 50.0 ng/mL caffeine mixture on an anti-theophylline polymer column: mobile phase, 100% chloroform; flow rate, 1.0 mL/min; sample injected, 20 µL; pulsed elution methanol, 20 µL; detection, λ ) 270 nm.

efficiently by the 8-µL flow cell. The detection sensitivity will be greatly enhanced if the sample elution volume can be decreased. In the case of MISPE, this is feasible since the electrostatic and hydrogen-bonding interactions can be effectively disrupted with a very small volume of protic and polar solvent. A novel elution technique, coined pulsed elution by us, was designed whereby a 20-µL aliquot of the polar protic solvent was injected directly through the switching valve onto the anti-theophylline column. Upon testing different solvents for pulsed elution, methanol was found to produce quantitative elution of the theophylline in one single injection. Apparently, when this methanol solvent band was carried quickly down the column by isocratic chloroform, it ran into the theophylline solute zone where displacement of the analyte molecules from the binding sites in the stationary phase occurred. As the methanol solvent had sufficient polar and protic strength, it could keep the free theophylline molecules in the band for fast elution off the column. Since the elution band volume was small, the peak height and hence signal-to-noise ratio were large, thereby resulting in a low detection limit. Pulsed elution of theophylline could begin after the sample solvent and other component peaks were eluted. This could be as short as 4 min, and therefore, the overall analysis time would be 0.5 mL). In addition, there are several chemical limitations including pH sensitivity of the silicabased sorbent and requirement of a wetting solvent which must not dry out. A rapid MISPE method with direct UV detection was developed for the analysis of theophylline in blood serum in our laboratory, utilizing the anti-theophylline MIP column for on-line SPE of theophylline followed by pulsed elution. Chloroform extraction of the theophylline in blood serum samples was performed, which served to remove interferences such as proteins while simultaneously providing a solvent suitable for direct injection onto the MIP column. After any potential interferences passed through the column in ∼2 min, pulsed elution of the bound theophylline could be performed. Figure 4 shows a typical MISPE-PE analysis result for a 4.0 µg/mL spiked serum sample, where selective determination of theophylline in the serum extract was accomplished in less than 6 min. The peak area corresponds to a concentration of 0.78 µg/mL theophylline in the chloroform extract, which is in reasonable agreement with the estimated value based on the partition coefficient (0.29) for theophylline between water and chloroform. The anti-theophylline column only retained the target molecule while all the matrix constituents (e.g., lipids) and potential interferences (e.g., other drugs) that were not (37) Bouvier, E. S. P.; Martin, D. M.; Iraneta, P. C.; Capparaella, M.; Cheng, Y.; Phillips, D. J. LC-GC 1997, 15, 152-156.

recognized by the binding sites would rapidly elute through the column as one initial peak. Hence, the MISPE-PE technique with UV detection effectively accomplished all of the sample cleanup and analyte determination in one single continuous step. Column Stability. Many different samples and various solvents were injected in the anti-theophylline SPE column over the course of this study. Only very small changes in peak area responsivity were observed, with no significant changes in the column back pressure. Since MIP columns are chemically robust to be reusable for numerous runs, MISPE-PE incurs a lower cost for each sample analysis as compared to enzyme-modified immunoassay techniques (EMIT).22 In addition, the column’s molecular recognition capability is preserved over extensive time periods (>1 year) of dry storage. Rapid reconditioning of the column with chloroform and other solvents can easily be accomplished. All these properties clearly demonstrate the exceptional suitability of this anti-theophylline polymer column for use in the new MISPE-PE technique for the cost-efficient determination of theophylline in serum. CONCLUSION The excellent molecular recognition power of the anti-theophylline MIP material endows the analytical chemist with a functional column to perform SPE for the simple, selective preconcentration of theophylline. This is the first report of a MIP column utilized for on-line SPE in the pulsed elution mode. A concentration detection limit of 120 ng/mL theophylline has been achieved using direct UV absorption detection, which corresponds to a mass detection limit of 2.4 ng. The new MISPE-PE technique permits on-line preconcentration of theophylline from an increased volume of dilute sample solution. Using a larger sample loop to inject a volume of 300 µL, a 40 ng/mL standard solution has produced a detectable peak signal. In practice there is no limit to the sample loop size for preconcentration, provided that the load capacity of the MIP column is not exceeded. Application of MISPE-PE in serum analysis further demonstrates the high capability of the MIP column to selectively isolate theophylline from other matrix components for fast, accurate determination. Molecular imprinting technology represents a widely applicable strategy for producing MIP columns selective for target molecules over a range of clinically significant drugs. The MISPE-PE technique developed in this work can potentially be extended with versatility to a broad scope of selective, rapid drug screening efforts in clinical laboratories. ACKNOWLEDGMENT This work was funded in part by the Natural Sciences and Engineering Research Council of Canada and by the Faculty of Graduate Studies and Research, Carleton University. The authors thank Bob Burk and Fred Cassalman for assisting in the HPLC setup, Sandy Owega for his helpful discussions, and Dr. Hindmarch at the General Hospital, Ottawa, Ontario, for providing the serum samples.

Received for review March 9, 1998. Accepted May 29, 1998. AC980264S Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

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