Development of a semiautomated procedure for the synthesis and evaluation of molecularly imprinted polymers applied to the search for functional monomers for phenytoin and nifedipine

Development of a semiautomated procedure for the synthesis and evaluation of molecularly imprinted polymers applied to the search for functional monomers for phenytoin and nifedipine
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  Analytica Chimica Acta 435 (2001) 91–106 Development of a semiautomated procedure for the synthesis andevaluation of molecularly imprinted polymers applied to the search forfunctional monomers for phenytoin and nifedipine F. Lanza a , ∗ , A.J. Hall a , B. Sellergren a , A. Bereczki b , G. Horvai b ,S. Bayoudh c , P.A.G. Cormack  c , D.C. Sherrington c a  Institut für Anorganische und Analytische Chemie, Johannes Gutenberg Universität, Duesbergweg 10-14, 55099 Mainz, Germany b  Division of Chemical Information Technology, Technical University of Budapest, Gellért tér 4, H-1111 Budapest, Hungary c  Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, UK  Received 24 July 2000; received in revised form 31 January 2001; accepted 31 January 2001 Abstract A previously described scaled-down version of the established monolith procedure, where molecularly imprinted polymers(MIPs) are prepared on the bottom surface of chromatographic vials [Anal. Chem. 71 (1999) 2092] has been here furtheroptimised with respect to its full automation. The protocol results in savings of time and reagents compared to the monolithprocedure, allowing ca. 60 polymers ( ∼ 50mg each) to be synthesised in parallel. Both blank and imprinted polymers are thenevaluated in situ by equilibrium batch rebinding tests. Each step in the synthesis and evaluation was considered with the aimof achieving an automated method with wide applicability with regards to template targets and monomer compositions. Asystembasedonthermalinitiationwasconsideredeasiertoimplementandapplicabletoalargernumberoftemplatesthanonebased on photo-initiation. For the purpose of choosing a suitable initiator, azo-initiators with different dissociation energieswere compared. 2,2  -Azobis(2,4-dimethylvaleronitrile) (V-65) was selected as the initiator of choice based on the observedrebinding selectivity and the low temperature of use (45 ◦ C). The time of degassing and polymerisation were also considered.With respect to the reproducibility of the automated procedure, confidence values of the mean rebinding percentage of 12 and8 were found, respectively, for the blank and imprinted polymers when five parallel batches of ametryn blanks and imprintswere submitted to rebinding tests. The small-scale protocol was then applied to the search for functional monomers for twofurther templates of interest: phenytoin and nifedipine. The results of the rebinding experiments on the small scale were foundto be in agreement with the equilibrium rebinding evaluation of the regular scale batches. However, the equilibrium rebindingresults cannot be used as a general predictor for the chromatographic selectivity of the MIPs. © 2001 Elsevier Science B.V.All rights reserved. Keywords:  Molecularly imprinted polymers; MiniMIPs; Phenytoin; Nifedipine; Search for functional monomers 1. Introduction The success of any imprinting protocol has beenreliant more on chemical intuition than on rational ∗ Corresponding author.  E-mail address: (F. Lanza). design. In view of the many synthetic parametersthat need to be optimised in order to obtain the de-sired recognition properties, practical techniques forthe rapid synthesis and screening of large groups of molecularly imprinted polymers (MIPs) are desirable.A small scale procedure for the rapid synthesis andscreening of large groups of MIPs was previously 0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003-2670(01)00905-9  92  F. Lanza et al./Analytica Chimica Acta 435 (2001) 91–106  described for the case of terbutylazine [1]. The proto-col consists of a scaled down version of the monolithprocedure [2,3] allowing rapid in situ template extrac-tion and batch rebinding evaluation. It is based on twoscreening steps: the first where the amount of templatereleased from the polymers in the porogenic solventis evaluated (release test) and the second based on theamount of template rebound to the polymers after anexhaustive extraction. In the rebinding step, the sepa-ration factor of the MiniMIPs is calculated based on areference, which can be an internal standard [4] or therebinding to a blank, non-imprinted polymer. Thus farthe polymers showing high separation factors in therebinding test have been shown the same behaviourin the chromatographic mode, as demonstrated in thecase of the terbutylazine model system [1], but thiscannot be generally assumed as is shown in the cur-rent study. Owing to the low consumption of reagentsand the absence of any manipulation of the polymers,the small scale protocol is well suited for automa-tion [4]. This allows combinatorial synthesis of a largenumber of polymers and efficient screening of thevarious factors influencing the recognition propertiesof MIPs.In the present study, the optimisation of the smallscale synthesis is addressed the choice of the initiation(photo- or thermal-chemical) and of the azo-initiatoras well as the degassing and polymerisation times arediscussed in terms of rebinding data for the terbuty-lazine model system. The reproducibility of the opti-mised protocol is given in terms of confidence valuesof the mean percentage of rebinding for the ametrynmodel system. The advantages and limitations of theprotocol are then highlighted by a study aimed at find-ing suitable functional monomers for the imprintingof two difficult templates: phenytoin and nifedipine.In particular, the issue of whether the batch rebind-ing assay can be used as general predictor for the be-haviour of the MIPs in the chromatographic mode isaddressed. 2. Experimental 2.1. Materials Triazine samples of technical grade were do-nated by Novartis (Basel, Switzerland). Phenytoin(5,5-diphenylhydantoin)andnifedipine(dimethyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate) were purchased from Sigma-Aldrich.The azo-initiators dimethyl-2,2  -azobisisobutyrate(V-601), 2,2  -azobis(4-methoxy-2,4-dimethyl)valero-nitrile V-70, 2,2  -azobis(2,4-dimethyl)valeronitrile(V-65) were provided by Wako Pure Chemical Indus-tries (Osaka, Japan), AIBN (2,2  -azobisobutyronitrile)was purchased from Janssen. Methacrylic acid(MAA), methylmethacrylate (MMA), acrylamide(AAM), methacrylamide (MAAM) 2-trifluorometha-crylic acid (TFM), 4-vinylpyridine (4VP), 2-vinyl-pyridine (2VP),  p -methoxystyrene (PMS), 3,4-dimethoxystyrene (MS), acrylonitrile (AcrN),  N  -vinylcarbazole (NVC), hydroxyethyl methacrylate(HEMA),diethylaminoethylmethacrylate(DEAEMA)andethyleneglycoldimethacrylate(EDMA)werepur-chased from Sigma-Aldrich. 2-Naphtylmethacrylateand 9-anthracenylmethyl methacrylate were fromPolysciences (Eppelheim, Germany) and 4-amino-styrene from Lancaster Synthesis (Mülheim am Main,Germany). The monomers 9-(3/4-vinylbenzyl)adenine(VBA), and 2,6-bis(acrylamido)pyridine (DAP) weresynthesised as described in a forthcoming report [5].Dichloromethane (DCM), acetonitrile, tetrahydrofu-ran (THF), dimethylformamide (DMF) and toluene,all of HPLC grade, were purchased from Acros Or-ganics NV/SA (Geel, Belgium). No further purifi-cation of commercial monomers and solvents wasperformed prior to the MiniMIP synthesis but allsolvents used in this case were of HPLC grade.In the case of the synthesis of MIPs, solvents andmonomers were purified prior to use. Glass vials(1.5ml) with silicone/teflon septa (Supelco) wereused as polymerisation reactors. 2.2. Instrumentation The UV lamp used in the photo-polymerisationswas a high-pressure mercury vapour lamp (Philips,HPK 125W). All the solutions were prepared,dispensed and degassed automatically by the pro-grammed liquid handler 233XL (Gilson) equippedwith the 735 software (Fig. 1). The HPLC evaluationof the samples was also performed by the programmedliquid handler 232XL coupled to an HPLC HP1100system equipped with a binary pump and a variablewavelength UV detector and an HP workstation.  F. Lanza et al./Analytica Chimica Acta 435 (2001) 91–106   93Fig. 1. Semiautomated system for the synthesis and evaluation of MiniMIPs. 2.3. Semiautomated synthesis of MiniMIPs Two mother solutions (with and without template)were prepared for the scaled down version of thepolymerisation. Both were obtained by mixing all thecomponents of the pre-polymerisation mixture exceptfor the one variable component (functional monomeror azo-initiator). A 1:4:20 ratio (template:functionalmonomer:crosslinker) was used in all cases. The samevolume of solvent was used throughout (5.6ml foreach 20mmol of crosslinking monomer).In the case of the screening of functional monomersfor terbutylazine, the mother solution was prepared bymixing 4.75ml of EDMA (25mmol), 50mg of AIBN(0.30mmol), 286mg of terbutylazine (1.25mmol) in7ml of CH 2 Cl 2 . In this case, the functional monomerwas the factor to be screened. A total of 95  l of each mother solution was automatically dispensed into1.5ml glass vials, which had been previously spargedwith argon. After addition of 25  l of a 2M solutionof the screened monomer in the porogen, all the vialswere again sparged with argon for 5min at 15 ◦ C andfinally sealed with rubber septa. The polymerisationwas thermally initiated at 45 ◦ C and the samples wereallowed to polymerise for 24h. 2.4. Semiautomated extraction and rebindingexperiments A 1ml volume of the porogen was pipetted intoeach of the vials containing the blank and imprintedpolymers. The vials were then sonicated for 1h with-out heating and the concentration of the releasedtemplate was quantified in each extract by HPLC–UVusing an external standard. The HPLC evaluation wasperformed using a C18 reversed-phase column (for theevaluationconditionsseethescreeningprotocols).Thesupernatant was directly sampled by the programmedliquid handler and injected onto the column. The vialswere then left overnight at room temperature, soni-cated again for 15min and then the amount of releasedtemplate was analysed by HPLC–UV. The supernatantwas thereafter removed from each vial and the poly-mers were submitted to several washing steps (see the  94  F. Lanza et al./Analytica Chimica Acta 435 (2001) 91–106  screening protocols for details). The concentration of the released template was quantified in most of thewashing fractions by HPLC–UV. A rebinding exper-iment was then performed by addition of 1ml of asolution of the template in the porogen (1/10 of theconcentration of the template in the polymerisationmixture) followed by sonication of the vials for 1h.The polymers were allowed to stand for 24h andthe concentration of free (unbound) template wasdetermined by reversed-phase HPLC. The rebindingpercentage was calculated for blank and imprintedpolymers by subtracting the concentration of the tem-plate in the supernatant from the initial concentration. 2.5. Synthesis of MIPs A previously described procedure was followed forthe preparation of MIPs as monoliths for evaluationin LC [3]. EDMA (20mmol, 3.8ml), the functionalmonomer (4mmol), the template (1mmol in the caseof nifedipine, 0.5mmol in the case of the less solublephenytoin) and the azo-initiator (0.24mmol, 1% w/w)were mixed in 5.6ml of the porogen and the solutionwas transferred to a glass tube (14mm i.d.). The poly-merisation mixture was then degassed with nitrogenfor 5min. The tubes were flame sealed, while cooledon ice and transferred to a thermostated water bath(15 ◦ C for the photo-polymerisations, 45 ◦ C for thethermal ones). In the case of photopolymerisation, allthe tubes were placed at ∼ 10cm distance from the UVsource and then turned at regular intervals during thefirst 30min to obtain a more even exposure. In bothcases, after 24h the tubes were crushed, the polymerswere ground and sieved under water and the particlesize fraction 25–36  m was collected. This fractionwas then repeatedly washed with 3 × 50ml aliquots of MeOH/H 2 O 1/1, MeOH, MeOH/HOAc 80/20 (v/v),and MeOH and then submitted to chromatographicevaluation. 2.6. Chromatographic evaluation of MIPs Stainless steel HPLC columns (125mm  × 4mm)were slurry packed using MeOH/H 2 O 80/20 (v/v) aspushing solvent at 300bar pressure. A preliminaryevaluation of the polymer selectivity was performedusing MeCN/H 2 O/HOAc (92.5/2.5/5 v/v/v) as the mo-bile phase. Thereafter, the polymers were evaluatedusing MeCN and MeCN/KH 2 PO 4  mixtures at variouspH.After the HPLC evaluation, some polymer batcheswere also submitted to a batch rebinding test: 1g of the 25–36  m size particles were suspended in 10mlof a 1mM solution of the template in the porogen. Thesuspension was sonicated for 1h and left overnightunder stirring. The supernatant was then separatedfrom the polymer particles and analysed by HPLC.The amount of template rebound to the polymer wasthen calculated from the concentration of the freetemplate in the solution. 2.7. MiniMIPs protocols2.7.1. Experiment 1: comparison of azo-initiators •  Parameter: type and concentration of the azo-initiators (see Fig. 2 in Section 3). •  Composition of the mother solution: terbutylazine229mg (1mmol), MAA 344mg (4mmol), EDMA3.96g (20mmol), CH 2 Cl 2  5.6ml. •  Amount of azo-initiator dispensed into each vial:AIBN(0.5mg;1mg),V-601(0.7mg;1.4mg),V-65(0.75mg; 1.5mg), V-70 (0.94mg; 1.9mg). •  Degassing time: 5min. •  Polymerisation time: 12h.The polymerisation was thermally initiatedat different temperatures according to the Fig. 2. Comparison of azo-initiators: components of the polymeri-sation mixtures used (see experiment 1 in Section 2.7.1). TheWako tradenames and 10h half-life decomposition temperaturesare given for the azo-initiators.  F. Lanza et al./Analytica Chimica Acta 435 (2001) 91–106   95Table 1Comparison of initiators: properties of the azo-initiators used for the experiment and data relevant to an ocular inspection of the progressof polymerisationInitiator Half-life of 10h decom- Temperature of use ( ◦ C) Approximate gel point (min) Solidification (h)position temperature ( ◦ C)AIBN/thermal 65 (Toluene) 60 15 6V-601/thermal 66 (Toluene) 60 15 6V-65/thermal 51 (Toluene) 45 15 6V-70/thermal 30 (Toluene) 30 10 4AIBN/photo 15 10 2V-601/photo 15 10 2 dissociation energies of the initiators. Four polymerbatches (AIBN 1 and 2%, V-601 1 and 2%) werephoto-polymerised at 15 ◦ C. The time of gelationwas recorded for each polymer mixture as well asthe approximate time needed for complete solid-ification. In all cases, the batches containing thedouble concentration of initiator (2%) polymerisedmore quickly. In Table 1 the data relevant to the2% batches are shown. All the vials were allowedto polymerise for 24h. •  Release tests: addition of 1ml of DCM, 1h ultra-sonication, then quantification of the free terbuty-lazine in solution by HPLC–UV. The samples werethen allowed to stand overnight at room tempera-ture and were re-analysed by HPLC–UV. •  HPLC evaluation: column: Phenomenex Luna C18;mobile phase: CH 3 CN/CH 3 COOH/H 2 O 92.5/5/2.5,injection volume 10  l. •  Wash tests: 2  ×  (1ml DCM + 50  l acetic acid,30min ultrasonication, 2h standing), 2  ×  (1mlDCM, 30min ultrasonication, 2h standing), 1mlDCM, 30min sonication, overnight standing. •  Rebinding tests: addition of 1ml of a 1mM solu-tion of terbutylazine in DCM, overnight standingand then quantification of the free terbutylazine byHPLC–UV. 2.7.2. Experiment 2: optimisation of the degassingtime •  Screened parameter: degassing time. •  Composition of the mother solution: terbutylazine229mg (1mmol), MAA 344mg (4mmol), EDMA3.96g (20mmol), CH 2 Cl 2  5.6ml, AIBN (80mg). •  Degassing time (min): 0, 1, 2, 5, 10min. •  Polymerisation time: 24h.The polymerisation was photochemically initi-ated at 15 ◦ C. After 10min, all the polymers de-gassed for at least 2min showed gelation. After30min, gelation was also recorded for the mixturessubmitted to 1min degassing. The mixtures not sub-mitted to the degassing step never attained solid-ification. Release, wash and rebinding tests: as inexperiment 1. 2.7.3. Experiment 3: reproducibility of theautomated procedure •  Composition of the mother solution: ametryn227mg (1mmol), MAA 344mg (4mmol), EDMA3.96g (20mmol), CH 2 Cl 2  5.6ml, V-65 (120mg). •  Degassing time (min): 5min. •  Polymerisation time: 24h at 45 ◦ C, 1h at 60 ◦ C. •  Release tests: as in experiment 1. •  Wash tests: 2  ×  (1ml DCM + 50  l acetic acid,30min ultrasonication, 2h standing), 2  ×  (1mlCH 3 OH/CH 3 COOH/H 2 O 60/30/10, 30min ultra-sonication, 2h standing), 1ml DCM, 30min son-ication, overnight standing. The concentration of ametryn in each wash fraction was evaluated byHPLC–UV. •  Rebinding test: as in screening test 1. 2.7.4. Experiment 4: search for functional monomers for phenytoin •  Screened parameter: functional monomer (seeFig. 5 in Section 3 for the structures). •  Thecomponentsofthepolymerisationmixturewereinthiscasedirectlypipettedintoeachvial,sincedif-ferent monomer/template ratios and different poro-gens were used (see Table 2).
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