Chitosan beads as molecularly imprinted polymer matrix for selective separation of proteins

Two kinds of molecularly imprinted polymers were prepared using hemoglobin as the imprinting molecule, acrylamide as the functional monomer, chitosan beads and maleic anhydride-modified chitosan beads as matrixes, respectively. Static adsorbing experimental results showed that an equal class of adsorption was formed in the imprinted polymers and the adsorption equilibrium constant and the maximum adsorption capacity were evaluated. Chromatographic characteristics showed that the column bedded with the hemoglobin imprinted beads could separate hemoglobin and bovine serum albumin effectively from their mixture, which indicates that the imprinted beads have very higher selectivity for hemoglobin than the non-imprinted with the same chemical composition.     

Shell-core imprinted polyacrylamide crosslinked chitosan for albumin removal from plasma

Molecularly imprinted polymeric beads were prepared using albumin as the imprinted molecule, acrylamide as the functional monomer, and epichlorhydrin crosslinked chitosan beads as the supporting matrix. The recognition and binding of the imprinted beads was also tested with human plasma for the targeted removal of HSA. Plasma is a rich source of biochemical products that can act as biomarkers of disease or physiological status of a patient. The application of current proteomic technologies in the search for potential diagnostic/prognostic indicators in the plasma of patients is limited by highly abundant albumin that constitute >60% of the total plasma proteins. Removal of abundant proteins will help in the discovery and detection of less abundant proteins that may prove to be informative. The adsorption capacities of the imprinted polymeric beads for pure human serum albumin and plasma albumin were estimated as 92% and 80%, respectively. The easy preparation protocol of derivatised beads and good protein recognition properties make them attractive for biotechnologic approaches.     

Protein recognition onto silica particles using chitosan as intermedium substrate

A novel molecular imprinting method was used to prepare twice-coated silica particles with specific recognition sites for hemoglobin. Chitosan was used as an intermedium to be coated on silica particles via phase inversion process, and the abundance of exposed amine groups (NH2) were active sites for introducing aldehyde groups. After hemoglobin was covalently immobilized by forming imine bonds with the aldehyde groups, acrylamide was then polymerized onto chitosan-coated silica particles to form the recognition sites. The obtained hemoglobin imprinted [molecularly imprinted polymer (MIP)] beads were characterized by FTIR spectroscopy, thermogravimetric analysis (TG), and scanning electron microscopy (SEM). The MIP particles exhibited selectively adsorption for the imprinted protein compared to the nonselectively adsorption for most of proteins of the nonimprinted (NIP) beads.     

Synthesis of creatinine-imprinted poly(beta-cyclodextrin) for the specific binding of creatinine

An artificial receptor for creatinine was synthesized by the method of molecularly imprinted polymer (MIP). beta-Cyclodextrin was used as a monomer cross-linked with epichlorohydrin in the presence of creatinine, which was a template for the imprinting. Different molar ratios of monomer to template were used to synthesize the polymers so that better specific adsorption ability towards creatinine could be achieved. The results showed that to carry out the polymerization with a molar ratio of monomer to template of 3:2 and monomer to cross-linking agent of 1:10 was proper. N-hydroxysuccinimide and 2-pyrrolinidone were used as the analogues of creatinine in the adsorption experiments of multi-component solutions to reveal the specific recognition ability of the molecularly imprinted poly(beta-cyclodextrin) (poly(beta-CD)) for the template molecule, creatinine. One such detection interference of creatinine was creatine, which also co-exists in serum. Hence, adsorption experiments of creatinine/creatine binary mixture were also carried out to investigate and confirm the specific binding of the creatinine-imprinted poly(beta-cyclodextrin) towards creatinine. The hydroxyl groups of the imprinted poly(beta-CD) was further capped by chlorotrimethylsilane (CTMS) to investigate the interaction between creatinine and the imprinted poly(beta-CD). The adsorption resulting from the mixture solution by MIPs suggested that the creatinine-imprinted poly(beta-CD) demonstrated superior binding effect for the target molecule, creatinine, rather than creatine, N-hydroxysuccinimide and 2-pyrrolinidone.     

Molecularly imprinted polymer hydrogels displaying isomerically resolved glucose binding

Non-covalent molecular imprinting of poly(allylamine hydrochloride) (PAA HCl) with glucose phosphate mono-sodium salt produced molecularly imprinted polymer (MIP) hydrogels capable of quantitative, isomerically specific binding of glucose. By ionic association of a template molecule, glucose phosphate mono-sodium salt, to the polymer prior to covalent crosslinking, MIP hydrogels were created with an affinity for binding glucose. In this study we have synthesized MIPs using epichlorohydrin, ethylene glucol diglycidyl ether, and glycerol diglycidyl ether as crosslinkers in order to evaluate their effectiveness with respect to molecular imprinting for glucose. MIP hydrogels were also synthesized with the different crosslinkers and varying amounts of the template molecule in an attempt to elucidate the impact of imprint quantities on the effectiveness of the imprinting technique. Batch equilibration studies, using each of the MIPs and similar non-molecularly imprinted polymers were performed to determine their binding capacities with respect to glucose and fructose. The binding capacity data are discussed and employed in the evaluation of the specificity imparted by the imprinting procedure. MIP hydrogels with binding capacities in excess of 0.5 g of glucose per gram of dried gel were synthesized. Isomeric specificity in hydrogels imprinted for glucose was demonstrated by higher binding capacities of glucose than those of fructose in the same polymers.     

Binding specificity of alpha-bilirubin-imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate) toward alpha-bilirubin

alpha-Bilirubin is an important index to determine the liver's functions. Poly(methacrylic acid-co-ethylene glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was proposed and shown to be able to bind alpha-bilirubin specifically. The extraction condition was also discussed. Polymers prepared by imprinting bilirubin in poly(MAA-co-EGDMA) and in poly(beta-cyclodextrin-co-EGDMA) were compared. In this work, binding specificity of molecularly imprinted polymer (MIP) toward the target template, alpha-bilirubin, is discussed. Adsorption profile of alpha-bilirubin by bilirubin-imprinted poly(MAA-co-EGDMA) was measured as a function of time, from where the equilibrium could be determined. Two hours was determined to be the proper time for adsorption. Biliverdin as a rather similar analog compound of bilirubin was chosen for comparison of binding specificity in this study. Progesterone as well as testosterone was also chosen for study because they both co-exist in serum with bilirubin and might interfere with binding capacity of imprinted poly(MAA-co-EGDMA) toward alpha-bilirubin. Specificity of this polymer for bilirubin was thus confirmed by tasks carried out in mixture solutions comprised of compounds above. It is true that biliverdin contained in the binary mixture did affect the binding capacity of bilirubin. Nevertheless, polymer's binding specificity for bilirubin was essentially sufficient for recognition of alpha-bilirubin in the presence of other compounds. How MIP identified the target template molecule, alpha-bilirubin, is also elucidated.     

A flow-injection chemiluminescent biomimetic immunoassay method using a molecularly imprinted polymer as a biomimetic antibody for the determination of trichlorfon

In this study, a molecularly imprinted polymer (MIP) was synthesized using trichlorfon as a template molecule. The MIP exhibited a high adsorption capacity toward trichlorfon. Using bovine serum albumin as a carrier, trichlorfon hapten was indirectly labeled with luminol, which competed with free trichlorfon for the combination with a biomimetic antibody of MIP. Based on this direct competitive format, a high throughput flow-injection chemiluminescent biomimetic immunoassay method was developed for fast detection of trace trichlorfon. The influencing factors were investigated and optimized. Under the optimal condition, a lower limit of detection (IC₁₅) of 0.0024?mg/L was obtained using this method. This method was also used to detect the trichlorfon spiked in carrot and cabbage samples, and the results were also verified using the method of gas chromatography.     

Core–shell molecularly imprinted polymer nanoparticles with assistant recognition polymer chains for effective recognition and enrichment of natural low-abundance protein

Core–shell molecular imprinting of nanomaterials overcomes difficulties with template transfer and achieves higher binding capacities for macromolecular imprinting, which are more important to the imprinting of natural low-abundance proteins from cell extracts. In the present study, a novel strategy of preparing core–shell nanostructured molecularly imprinted polymers (MIPs) was developed that combined the core–shell approach with assistant recognition polymer chains (ARPCs). Vinyl-modified silica nanoparticles were used as support and ARPCs were used as additional functional monomers. Immunoglobulin heavy chain binding protein (BiP) from the endoplasmic reticulum (ER) was chosen as the model protein. The cloned template protein BiP was selectively assembled with ARPCs from their library, which contained numerous limited-length polymer chains with randomly distributed recognition and immobilization sites. The resulting complex was copolymerized onto the surface of vinyl-modified silica nanoparticles under low concentrations of the monomers. After template removal, core–shell-structured nanoparticles with a thin imprinted polymer layer were produced. The particles demonstrated considerably high adsorption capacity, fast adsorption kinetics and selective binding affinities toward the template BiP. Furthermore, the synthesized MIP nanoparticles successfully isolated cloned protein BiP from protein mixtures and highly enriched BiP from an ER extract containing thousands of kinds of proteins. The enrichment reached 115-fold and the binding capacity was 5.4 μg g⁻¹, which were higher than those achieved by using traditional MIP microspheres. The advantageous properties of MIP nanoparticles hold promise for further practical applications in biology, such as protein analysis and purification.     

Optimization of Molecularly Imprinted Polymers of Serotonin for Biomaterial Applications

We prepared molecularly imprinted polymers (MIPs) of serotonin (5-hydroxytryptamine or 5-HT), a neurotransmitter and mood modulator, using a combination of neutral (methacrylamide or acrylamide) and positively charged (methacrylic acid) functional monomers. Water, PBS, acidified methanol and sodium dodecyl sulfate were compared as rinsing solvents for the removal of serotonin from the MIPs. Methacrylamide MIPs rinsed in acidified methanol (92% serotonin removal) produced the highest imprinting factor (3.1) in equilibrium batch rebinding experiments using a combination of 2% water and 98% MeCN as the rebinding solvent. For the first time, these MIPs were assessed for cytocompatibility using mouse mesencephalon neural progenitor cells (NPC) and NIH 3T3 fibroblasts. Although MIP particles decreased NPC viability to <70%, MIP particles did not significantly reduce fibroblast viability when incorporated into a hyaluronic acid biomaterial. MIP microparticles were incorporated into a cross-linked hyaluronic acid biomaterial to present one way in which molecularly imprinted polymers may be used in vivo in future biomaterials or biosensors applications.     

Protein recognition via molecularly imprinted agarose gel membrane

Agarose gel membranes (AGMs), which could selectively recognize bovine serum albumin (BSA) and bovine hemoglobin (Hb), were prepared by molecular imprinting technique under moderate preparation conditions. Four imprinting processes, including gelation without any treatment, second gel-melting, and two glutaraldehyde crosslinking processes of fumigation or direct addition of the crosslinking agent, were developed to investigate the protein-recognition behavior of the AGMs. Results showed that the preparation processes affected the adsorption capacity and selectivity of the imprinted AGMs. Both BSA- and Hb-imprinted AGMs exhibited higher adsorption abilities for the targeted proteins (3.77-5.72 times for BSA, 1.31-2.18 times for Hb) than the nonimprinted ones. And the selectivity of BSA-imprinted AGMs for BSA molecules (the selective factor K = 3.29-4.90) was higher than that of Hb-imprinted AGMs for Hb (K = 0.32-1.17). The optimal adsorption capacity of BSA- and Hb-imprinted AGMs was 25.90 and 117.45 mg/g, respectively, when the membrane was crosslinked by glutaraldehyde with a fumigation process; the optimal selectivity of BSA- and Hb-imprinted AGMs was 4.91 when the membrane was prepared by second gel-melting process, and 0.76 when the membrane was prepared without any treatment. These findings demonstrate that the molecularly imprinted AGMs are hopeful to be used in specific protein analysis.     

Molecularly imprinted polymer doped with Hectorite for selective recognition of sinomenine hydrochloride

In this work, a new and facile method was introduced to prepare molecularly imprinted polymers (MIPs) based on nano clay hectorite (Hec) for sinomenine hydrochloride (SM) analysis. Hec was firstly dissolved in distilled water in order to swell adequately, followed by a common precipitation polymerization with SM as the template, methacrylic acid as monomer, ethylene glycol dimethacrylate as a crosslinker and 2,2-azobisisobutyronitrile as an initiator. Hec@SM-MIPs were characterized by Fourier transform infrared spectrometer, transmission electron microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and X-ray diffraction. The maximum binding capacity of Hec@SM-MIPs, SM-MIPs and non-imprinted polymers (NIPs) (Hec@NIPs) was 57.4, 16.8 and 11.6 mg/g, respectively. The reason for this result may be that Hec@SM-MIPs have more binding sites and imprinted cavities for template molecule. Equilibrium data were described by the Langmuir and Freundlich isotherm models. The results showed that the Hec@SM-MIPs adsorption data correlated better with the Langmuir equation than the Freundlich equation under the studied concentration range. In vitro drug release experiment, Hec@SM-MIPs have a better ability to control SM release than SM-MIPs. Therefore, Hec@SM-MIPs were successfully applied to extraction of SM and used as the materials for drug delivery system.     

Biomimetic glucose recognition using molecularly imprinted polymer hydrogels

Non-covalent molecular imprinting of poly(allylamine hydrochloride) (PAA.HCl) with D-glucose 6-phosphate monobarium salt (GPS-Ba) produced molecularly imprinted polymer hydrogels (MIP) having an affinity to glucose over fructose. The hydrogels were formed by ionic association of the template molecule, GPS-Ba, to the polymer, prior to covalent crosslinking using epichlorohydrin (EPI). The template was removed by an aqueous base wash. Batch equilibration studies using different MIP hydrogels and non-molecularly imprinted polymers (NIPs) were performed in aqueous and buffered media to determine the binding capacities and isomeric selectivities with respect to the sugars, glucose and fructose. MIP glucose hydrogels exhibited binding capacities in excess of 0.6g of glucose per g of dry gel in a 100% DI H(2)O glucose solution, and in a 50-50% glucose-fructose solution mixture. Equilibrium binding capacities of fructose were lower than those observed with respect to glucose, indicating an isomeric preference for the binding of glucose over fructose. These hydrogels demonstrated a remarkable degree of biomimetic sugar recognition to specifically and selectively bind glucose in their swollen state in environments mimicking physiological conditions.     

Synthesis and characterization of albumin imprinted polymeric hydrogel membranes for proteomic studies

Abstract

In this presented study, a novel molecularly imprinted polymeric hydrogel membranes (PHMs) were developed to use for the albumin depletion studies. For this, albumin imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl-(L)-phenylalanine methyl ester) polymeric hydrogel membranes [p(HEMA-MAP) PHMs] were synthesized by the photopolymerization technique, and then characterized by SEM, EDX, FT-IR and swelling studies. Synthesized PHMs had spherical structure and the MAP monomer incorporation onto the PHMs was determined by EDX analysis by using nitrogen stoichiometry. Also, the swelling ratio of the albumin imprinted p(HEMA-MAP) PHMs was determined as 215%. The optimum albumin adsorption condition (adsorption capacity, medium pH, adsorption rate, temperature, ionic strength) were studied and the maximum albumin adsorption capacity was found to be as 34.28?mg/g PHMs. Selectivity experiments were also carried out with the presence of the competitive proteins such as lysozyme and amylase, and the results demonstrated that the albumin imprinted p(HEMA-MAP) PHMs showed high affinity towards the BSA molecules than the competitive proteins of lysozyme and amylase. Adsorbed albumin was desorbed from the PHMs by 1.0?M of NaCl, and the reusability of the imprinted PHMs was also demonstrated for five successive adsorption-desorption cycles without any significant loss in the albumin adsorption capacity. As an application, sodium-dodecyl sulfate polyacrylamide gel electrophoresis was used to indicate the albumin depletion efficiency of albumin imprinted p(HEMA-MAP) PHMs. This presented study showed that, these imprinted membranes are promising for proteomic studies and applications, and can be used for the investigations for human diagnostics.     

Designing a molecularly imprinted polymer as an artificial receptor for the specific recognition of creatinine in serums

In this study, molecularly imprinted polymers (MIP) synthesized from two different functional monomers, beta-cyclodextrin (beta-CD) and 4-vinylpyridine (4-Vpy), were prepared. The crosslinkers used for these two monomers were epichlorohydrin (EPI) and divinylbenzene (DVB), respectively. It was attempted to adsorb the target molecule, creatinine, from its mixture solutions. A proper molar ratio of monomer/crosslinker for the preparation of the imprinted poly(beta-CD) was 1:10. Between both polymers mentioned above, the affinity of the imprinted poly(4-Vpy-co-DVB) towards creatinine was comparably superior. The imprinted poly(4-Vpy-co-DVB) for creatinine could reach a specific binding ratio of 3.11. The imprinted poly(4-Vpy-co-DVB) was further utilized to bind creatinine from human serum samples. The binding capacity of the imprinted poly(4-Vpy-co-DVB) for creatinine from the serum samples was plotted against the creatinine concentration. From the correlation, the feasibility of the imprinted poly(4-Vpy-co-DVB) thus prepared for the target analyte, creatinine, was experimentally confirmed.     

Detection of ciprofloxacin through surface plasmon resonance nanosensor with specific recognition sites

The novel nanosensor based on Surface Plasmon Resonance (SPR) was developed for sensitive and selective detection of ciprofloxacin via molecularly imprinted nanoparticles (MIP/NPs). NPs were synthesized through miniemulsion polymerization technique with methacrylic acid as a functional monomer. FTIR, SEM, zetasizer and contact angle measurements were used for the characterization of MIP/NPs. After modification the SPR chip surface, the nanosensor was used for detection of ciprofloxacin in aqueous solution. According to selected concentration range, the correlation coefficient and limit of detections were obtained as 0.993 (R²) and 3.21 and 7.1 ppb in ultrapure water and SWW, respectively. Association kinetic analysis, Scatchard, Freundlich, Langmuir and Freundlich-Langmuir isotherms were also performed on the data to investigate adsorption behaviour of ciprofloxacin on the surface of nanosensor. Tetracycline and enrofloxacin were used as competitor agents to examine the selectivity of nanosensor. Performance of the SPR nanosensor was also investigated by using synthetic wastewater (SWW) for detection of CPX. The reusability of nanosensor was investigated and good repeatability was obtained with 5.81% RSD. As a result, selective, simple and low-cost method to detect ciprofloxacin in aqueous solution was developed by combining MIP/NPs and SPR.     

Superporous hydrogels containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks

Superporous hydrogels containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks (SPH-IPNs) were prepared by cross-linking O-carboxymethyl chitosan (O-CMC) with glutaraldehyde (GA) after superporous hydrogel (SPH) was synthesized. The structures of the SPH-IPNs were characterized with FT-IR, 13C-NMR and DSC. SEM, CLSM and light images revealed that the SPH-IPNs possessed both the IPN network and large numbers of pores and the cross-linked O-CMC molecules were located on the peripheries of these pores. The swelling behavior of SPH-IPNs was dependent on the O-CMC content, GA amount and cross-linking time. Due to the cross-linked O-CMC network, in vitro muco-adhesive force and mechanical properties, including compression and tensile modulus, of the SPH-IPN were greatly improved when compared with the CSPH. An enhanced loading capacity for insulin could be obtained by the SPH-IPNs as compared to non-porous hydrogel and CSPH, and more than 90% of the insulin was released within 1 h. With the improved mechanical properties, in vitro muco-adhesive force and loading capacities, the SPH-IPN may be used as a potential muco-adhesive system for peroral delivery of peptide and protein drugs.     

Via zinc(II) protoporphyrin to the synthesis of poly(ZnPP-MAA-EGDMA) for the imprinting and selective binding of bilirubin

Poly(zinc protoporphyrin-methacrylic acid-ethyl glycol dimethylacrylate) (poly(ZnPP-MAA-EGDMA)) imprinted with α-bilirubin can cause spectroscopic change in wavelength and absorption intensity due to the metal–ion coordination between ZnPP and bilirubin. The fluorescent imprinted polymer was able to selectively bind α-bilirubin. The corresponding imprinted polymer monolith was synthesized by using the functional monomer, methacrylic acid and the fluorescent monomer, zinc(II) protoporphyrin. Although the imprinted polymers (MIPs) using methacrylic acid, protoporphyrin, or zinc(II) protoporphyrin alone as the only functional monomer could bind bilirubin, the imprinting effects were all comparably inferior to the imprinted poly(ZnPP-MAA-EGDMA). Therefore, it revealed that via the combined utilization of ZnPP and MAA for the fluorescent and functional effect, the MIPs thus prepared were then able to create the highly selective cavities. The optimal condition for the heated polymerization of the imprinted poly(ZnPP-MAA-EGDMA) was found to be 60 °C for 6 h. The imprinting factor of 3.069 could be achieved from the fluorescent imprinted polymer by comparing the binding results obtained from the MIP and the NIP (non-imprinted polymer). The imprinting factor obtained from bilirubin/biliverdin mixture solution was reduced to 2.111 because of the presence of biliverdin. The selectivity toward bilirubin of 2.269 from the bilirubin/biliverdin mixture was obtained. Therefore, to utilize ZnPP for the preparation of the imprinted materials confirmed the selective binding and detection of bilirubin via the fluorescent approach.     

Adsorption of bilirubin by amine-containing crosslinked chitosan resins

Crosslinked chitosan resin, which has good blood compatibility, was prepared by crosslinking chitosan solution with glutaraldehyde. Polymeric adsorbents for bilirubin have been synthesized by chemical modification of crosslinked chitosan resins with di-, tri-, tetra-, and pentammines. The adsorption of bilirubin on the adsorbents was investigated in detail. The results indicated that electrostatic interaction and hydrophobic interactions are the main driving forces for the adsorption. Compared with crosslinked chitosan resin, the functionalized chitosan beads have higher adsorption capacity for unconjugated and conjugated bilirubin.     

Immobilization of lipase using hydrophilic polymers in the form of hydrogel beads

The purpose of this study was to immobilize lipase (triacylglycerol ester hydrolase, E.C. 3.1.1.3) from Candida rugosa using various polymers in the form of beads, to evaluate enzyme loading, leaching, and activity; and to characterize the beads. Agarose, alginate, and chitosan were the polymers selected to immobilize lipase by entrapment. Agarose beads exhibited undesirable swelling in the leaching and activity medium and the polymer was not used further. Alginate or chitosan beads were prepared by ionic gelation using calcium chloride or sodium tripolyphosphate, respectively, as the cross-linking agent in the gelling solution. Some hatches of beads of each polymer were freeze dried. The results show that alginate beads leached substantially more enzyme than did chitosan beads. Entrapment efficiency, however, was the same for different chitosan levels as well as different alginate levels (43-50%). Activity in alginate was low at 240 +/- 33 and 220 +/- 26, compared to 1,110 +/- 51 and 1,150 +/- 11 units/ml in chitosan, for fresh and freeze-dried beads, respectively. The higher lipase activity in chitosan beads compared to that in alginate beads could be attributed to an alginate-enzyme interaction. It can be concluded that chitosan is a polymer worthy of pursuit to immobilize lipase.     

Surface hydrophilic modification with a sugar moiety for a uniform-sized polymer molecularly imprinted for phenobarbital in serum

A uniform-sized polymer molecularly imprinted for phenobarbital, which is surface modified by a sugar moiety, has been prepared through a two-step swelling polymerization method using polystyrene beads as seeds, phenobarbital as the template, 4-vinylpyridine as a functional monomer, ethylene glycol dimethacrylate as a cross-linker and 2-O-meth-acryloyloxyethoxyl-(2,3,4,6- tetra-O-acetyl-β-d-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-β-d-glucopyranoside as a surface-modifying glycomonomer, respectively. After deprotecting the glycopolymer, a surface sugar moiety-modified, hydrophilic, molecularly imprinted polymer for phenobarbital (glyco-MIP) was obtained. The resulting polymer beads were packed into a stainless steel column to evaluate their chromatographic characteristics by high-performance liquid chromatography (HPLC). Good selectivity for phenobarbital was obtained with the glyco-MIP compared to the unmodified molecularly imprinted polymer, which revealed that the recognition sites of phenobarbital were unchanged with sugar moiety surface modification. Furthermore, bovine serum albumin was almost completely recovered from the glyco-MIP column, which indicates that the glyco-MIP materials can be used to separate and analyze drugs in complex samples, such as biological samples. The results of pretreatment with and analysis of phenobarbital in serum suggest that this material can be used to analyze phenobarbital in serum through a pretreatment and reverse-phase HPLC analysis process.