Review

Chromatography, Vol.23 No.1 (2002)

Review

LC Packing Materials for Pharmaceutical and Biomedical Analysis
Jun Haginaka*
Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68, Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179, Japan
Received for review November 14, 2001.

Accepted November 28, 2001.

Abstract
The author has prepared novel liquid chromatography (LC) packing materials for pharmaceutical and biomedical analysis. Those include LC
packing materials for direct serum injection assays of drugs and their metabolites, LC packing materials for resolution of enantiomeric drugs,
and uniformly sized molecularly imprinted polymers for drugs and their metabolites. This review article deals with the preparation of thosematerials and the pharmaceutical and biomedical applications of them in recognition of The Society of Chromatographic Sciences Award.
Keywords: LC stationary phase; direct serum injection assay, restricted access media; chiral stationary phase; protein; molecularly imprinted
polymers

accumulation because of restricted access to some surfaces, while

1. Introduction
Many liquid chromatography (LC) packing materials haveallowing small molecules such as drugs and their metabolites tobeen prepared since the introduction of HPLC. One type is for gen-reach the hydrophobic, ion-exchange or affinity sites and to beeral purposes such as octadecyl-silyl silicas. The other type is forseparated. We prepared two RAM, internal-surface reversed-phase

specific purposes such as chiral stationary phases (CSPs) and re-(ISRP) and mixed functional phase (MFP) materials.

stricted access media (RAM). The author has prepared LC packing
2.1. Internal surface reversed-phasematerials for direct serum injection assays of drugs and their metabolites, LC packing materials for resolution of enantiomeric

The ISRP silica materials, produced from porous silica gelsdrugs, and uniformly sized molecularly imprinted polymers (MIPs)

with 8 nm average pore diameter, have hydrophilic exterior and hy-

for drugs and their metabolites. This review article deals with the

drophobic interior surfaces, as shown in Figure 1. Hagestam andpreparation of those materials and the pharmaceutical and biomedi-

Pinkerton [2] prepared an ISRP support from covalently modified

cal applications of them in recognition of The Society of Chroma-

glycerylpropyl (i.e. diol) phases by attachment of the tripeptide

tographic Sciences Award.

glycine-L-phenylalanine-L-phenylalanine (GFF), bound via theamino groups to the glycerylpropyl phases. The phenylalanine
moieties were then cleaved from the external surface of the silica

2. Restricted Access MediaFor the LC determination of drugs and their metabolites in se-with carboxypeptidase A [2] which is too large to enter the small

rum or plasma, tedious and time-consuming pretreatment proce-pores. After this enzymatic treatment, the glycerylpropyl-glycine

dures such as the removal of proteins by precipitation, liquid-liquid

phases remained on the external surfaces, while the internal sur-extraction or solid phase extraction (SPE) have been required in thefaces remained uncleaved. Thus, the external and internal surfaces

past. Recently, direct serum injection analysis of drugs in biologi-

consisted of hydrophilic and hydrophobic phases, respectively.

cal fluids by HPLC has been explored. One of the direct serum in-

Further, on the negatively charged GFF, analytes were retained byjection methods is the use of RAM. Many RAM have been pre-a unique combination of mechanisms; π-electron interactions andpared and used for enrichment and pretreatment of the analytes inweak cation-exchange properties [3]. This GFF ISRP material,

proteinaceous samples by HPLC [1]. With RAM large molecules

which is commercially available, had disadvantages in that it could

such as proteins are eluted in the void volume without destructive

not retain certain classes of hydrophilic drugs such as the ampho-*Tel, +81-798-45-9949; fax, +81-798-41-2792;
e-mail, haginaka@mwu.mukogawa-u.ac.jp―１―Chromatography, Vol.23 No.1 (2002)

Figure 1. Schematic representation of an internal-surface reversedphase (ISRP) material. Proteins do not adsorb on the hydrophilic exterior surfaces and do not penetrate into thehydrophobic interior surfaces, while analytes can reachthe interior surfaces and be separated.
Figure 2. Separation of cephalosporins in human serum by direct
injection onto the ISRP materials designed by (A) Hagestam and Pinkerton, and (B) us. Peak assignments: 1 =cefotaxime (10 µg mL-1); 2 = cefmenoxime (10 µg mL-1);
3 = cefamandol (20 µg mL-1). HPLC conditions: eluent,
100 mM phosphate buffer-acetonitrile (12:1, v/v) (final
pH 6.9); flow-rate, 0.8 mL min-1; detection, 254 nm; injection volume, 20 µL. Dotted lines indicate serum
blank.

teric drugs, cephalosporins and penicillins, the recommended eluent pH range is limited to 6.0-7.5, and it is difficult to obtain thedesired batch-to-batch reproducibility of its properties. We designed a new ISRP support having N -octanoylaminopropyl and N (2,3-dihydroxypropyl)aminopropyl groups on the internal and external surfaces by using a novel enzyme, polymyxin acylase [4].
This enzyme, a monomer (with molecular mass 62,000), has an affinity for long fatty acid groups and can cleave acyl groups of C2
to C16, phenyl and substituted phenyl residues [5]. The neutral
ISRP support prepared by us is more effective than that designed

Serum proteins were completely recovered from them at the firstby Hagestam and Pinkerton, when separating hydrophilic drugs

injection by changing the balance of hydrophobic and hydrophilic(Figure 2) [4] and when an eluent pH of 3 to 7 is employed [6].

phases. The obtained MFP materials could tolerate over 500 injec-

Further, the neutral ISRP support prepared by us was applied to di-tions of a 20-µL serum sample with no change of back pressure

rect serum injection assays of anticonvulsant drugs and methylxan-

and column efficiency. The MFP packing materials having β-CD-

thine derivatives combined with a column-switching technique [7].

diol groups had been prepared using an 8-nm pore size silica. Bovine serum albumin (BSA) was eluted from the material only
bonded with β-CD as a tailing peak with protein recovery of 90%.

2.2. Mixed functional phase (MFP)
We prepared MFP materials for direct serum injection assays

However, from the β-CD-diol material BSA was eluted with a pro-of achiral [8-12] and chiral [13, 14] drugs, having a β-cyclodextrin

tein recovery of almost 100% [13]. This is due to low surface cov-(CD)-diol phase and a hydrophobic (phenyl, butyl or octyl)-diolerage of the material bonded only with β-CD. The β-CD-diol mate-phase bonded to porous silica particles. The MFP materials wererial can be used for over 400 injections of a 20-µL serum sample.

synthesized from porous silicas in three steps: introduction of hy-

Figure 3 shows chromatograms of direct injection analysis ofdrophobic or β-CD groups, introduction of 3-glycidoxypropyl

chlorpheniramine in human serum on the β-CD-diol packings [13].

groups, and hydrolysis of the oxirane ring to diol groups. The MFP

It was found that protein-based CSPs worked as MFP materi-materials having hydrophobic-diol phases were prepared fromals because a protein, especially a glycoprotein, has hydrophilic ex-

starting silica materials with average pore diameters of 6-10 nm.

ternal surfaces [15]. Ovomucoid-based CSP could be used for di-―２―

Chromatography, Vol.23 No.1 (2002)

Jun Haginaka

Figure 4. Chromatograms of benzoin on the BSA (A) and BSAfragment (B) columns.
HPLC conditions: column, 2.1 mm i.d. × 100 mm; eluent, 50 mM phosphate buffer (pH 7.5)-1-propanol (96:4,v/v); column temperature, 25°
C; flow-rate, 0.2 mL min-1;
detection, 254 nm; injected amount, 0.1 µg each.

Figure 3. Chromatograms of (A) control serum and (B) serum
spiked with chlorpheniramine by direct injection on the
β-CD-diol silica. Peaks 1 and 1’ are (R,S) chlorpheniramine (25 µg mL-1). Chromatographic conditions are as follows: eluent, 99 mM NaH2PO4 + 1 mM
H3PO4-acetonitrile (6:1, v/v) (final pH 4.3); flow-rate,
0.6 mL min-1; detection, 264 nm; injection volume, 20µL.

disadvantages of protein-based CSPs as described below.
3.1. Chiral stationary phases based on serum albumin fragments
The BSA fragment columns obtained by other investigators
could have less capacity and enantioselectivity, and be less stable
than the intact BSA column. We isolated the BSA fragment of molecular mass 35,236 estimated by electrospray ionization mass

rect serum injection assays of chiral drugs [15].

spectra [19, 20]. The estimated molecular mass was in good agreement with the predicted molecular mass 35,234 of the half-cystinyl

3. Protein-based chiral stationary phases
A protein and glycoprotein, respectively, consist of amino ac-

BSA fragment, which is ascribable to amino acid sequences 1-307

ids, and amino acids and sugars, both of which are chiral. Thus, allhaving eight disulfide bonds and one half-cystinyl bond in the 34th.

proteins have the ability to discriminate a chiral molecule. How-

Next, the intact and fragmented BSA were bound to N,N’ -ever, only a limited number of proteins have been investigated as

disuccinimidyl carbonate (DSC)-activated aminopropyl-silica gels.

HPLC CSPs. Protein-based CSPs are of special interest because of

The bound amounts of the BSA fragment were 2.2-2.7 times moretheir unique, enantioselective properties and because they are suit-than that of the intact BSA. Chiral resolution of 2-arylpropionicable for separating a wide range of enantiomeric forms [16-18].

acid derivatives, benzodiazepines, warfarin and benzoin was at-

The advantages of protein-based CSPs generally include the use oftained with the BSA fragment columns. Figure 4 shows chroma-an aqueous mobile phase, as for reversed-phase HPLC, enantiose-

tograms of benzoin on the BSA and BSA-fragment columns. The

lectivity for a wide range of compounds and direct analysis without

BSA fragment columns gave higher enantioselectivity for loraze-

derivatization. The disadvantages have included low capacity, lackpam and benzoin because of the larger bound amounts, and lowerof column ruggedness and limited understanding of the chiral rec-enantioselectivity for other compounds tested, compared with theognition mechanism. A few trials were made to overcome these

BSA column. The lower enantioselectivity might be due to changes―３―Chromatography, Vol.23 No.1 (2002)

Figure 5. Chromatograms of pranoprofen (A) and warfarin (B) on HSA and HSA-FG 75
columns.
HPLC conditions: column size, 2.0 mm i.d. × 100 mm; eluent, 50 mM sodium
phosphate buffer (pH 7.5)-1-propanol (94:6, v/v); flow rate, 0.2 mL min-1; detection, 210 nm; temperature, 25°
C; loaded amount, 100 ng.

and flurbiprofen) participate in enantioselective binding interactionin the globular structure of the BSA fragment and/or changes in thewith the N -terminal half of HSA, despite the fact that the primary

local environment around the binding sites.
Similarly, a human serum albumin (HSA) fragment was iso-binding sites of these compounds are on the other half of HSA.

lated by size-exclusion chromatography following peptic digestion

Figure 5 shows the enantioseparations of pranoprofen and warfarinof HSA [21]. Matrix-assisted laser-desorption ionization time-of-on the HSA and HSA fragment columns. Though the HSA column+

flight (MALDI-TOF) mass spectra indicated three (M + H) ion

showed higher enantioselectivity than the HSA fragment column,

peaks, whose molecular masses were estimated to be 34,816,

the enantioseparations of pranoprofen and warfarin were attained

29,178 and 44,134. The molecular masses estimated were in goodwith a shorter analysis time on the HSA fragment column. Thisagreement with the predicted molecular weights of amino acid se-

could be due to removal of the non-specific binding sites of HSA,

quences 1-308, 49-308 and 1-387. The fragment protein, whosechanges in the globular structure of the HSA fragment and/or

molecular mass was about 35,000, was mainly observed. Next, thechanges in the local environment around the binding sites [21].

intact and fragmented HSA were bound to DSC-activatedFurther, the HSA fragment column was as stable as the intact HSA

aminopropyl-silica gels. Though the retentivity and enantioselec-

column for repetitive injection of samples. These results suggest

tivity of all solutes tested were lower on the HSA fragment columnthat the HSA fragment column could be useful for the enantiosepa-

thanration of solutes having strong affinities for HSA.

the

HSA

column,benzodiazepine(oxazepam),2-arylpropionic acid derivatives (ibuprofen, ketoprofen, pranoprofen
3.2. α1-acid glycoprotein

and flurbiprofen) and warfarin were still enantioseparated on the
HSA fragment column. However, no enantioseparation of benzoin

Since the molecular mass of α1-acid glycoprotein (AGP) fromwas attained. As reported by He and Carter [22], primary binding

human plasma ranges from 38,800 to 48,000, it is generally as-sites for benzodiazepines and 2-arylpropionic acid derivatives are

sumed to be 40,000 [23]. Recently, we reported that the average

located in the subdomain IIIA, while that of warfarin in the subdo-

molecular mass of AGP could be ca. 33,000 by MALDI-TOF mass

main IIA. It is interesting that benzodiazepine (oxazepam) and 2-

spectra, and that the sugar content of AGP could be estimated to bearylpropionic acid derivatives (ibuprofen, ketoprofen, pranoprofen

34% [24]. There is no precise information on the chiral recognition―４―

Chromatography, Vol.23 No.1 (2002)

Jun Haginaka

sites and mechanism of AGP because of the lack of information onage of a sugar chain(s), pd-AGP could become more hydrophobic

the tertiary structure. Further, the role of sugar moieties in enantio-

than AGP, and/or that a solute could be easily accessible to the spe-selective bindings by AGP has not been investigated precisely. We

cific and/or non-specific binding sites of pd-AGP [24]. Further

prepared partially deglycosylated (pd)-AGP by removal of a sugar

study is required to clarify the chiral recognition mechanism ofmoiety of AGP by treatment with N -glycosidase [24]. The average

AGP and the role of the sugar moiety on chiral recognition.

molecular mass of pd-AGP was estimated to be ca. 30,600 by
3.3. Ovomucoid and ovoglycoprotein

MALDI-TOF mass spectrometry. Figure 6 A and B shows chromatograms of ethotoin enantiomers on the AGP and pd-AGP col-CSPs based on ovomucoid from chicken egg whites (OMCHI)

umns, respectively. The retentivity and enantioselectivity of thewere developed by Miwa et al. [25]. The OMCHI column can re-neutral, acidic and basic solutes tested on the pd-AGP column weresolve a wide range of acidic, basic and neutral enantiomers.

significantly or not significantly larger in most solutes than those

Various ovomucoids such as ovomucoid from turkey egg

on the native AGP column. A possible explanation is that by cleav-

whites (OMTKY) and OMCHI exist as three tandem, independent
domains. In order to gain information regarding the enantioselective recognition mechanism of ovomucoid proteins, it is of interest
to know whether chiral recognition is expressed by all three domains, by only one domain, or by a combination of domains. Eachdomain and combination domains, first and second, second andthird domains, were isolated, purified and characterized [26]. Further, columns were made with purified OMTKY and OMCHI domains to test chiral recognition properties [25, 26]. The third domain of OMTKY and OMCHI consisted of glycosylated (OMTKY
3S and OMCHI3S) and unglycosylated domains (OMTKY3 andOMCHI3). The third domains of the OMTKY and OMCHI domains were found to be enantioselective for at least two classes ofcompounds, benzodiazepines and 2-arylpropionic acid derivatives
as shown in Tables 1 and 2, respectively. Glycosylation of the third
domain did not affect chiral recognition. Further, the chiral recognition mechanism of the OMTKY3 was elucidated by using NMR
measurements, molecular modeling and computational chemistry[26]. Figure 7 illustrates the binding orientations of the enantiomers of U-80413, which is one of 2-arylpropionic acid derivatives, in each of the two surface regions identified on the OMTKY3

Figure 6. Chromatograms of ethotoin enantiomers on AGP (A)and pd-AGP (B) columns.
HPLC conditions: column size, 2.0 mm i.d. × 100 mm;
eluent 10 mM phosphate buffer (pH 5.0); flow-rate, 0.2
mL min-1; detection, 210 nm; temperature, 25°
C; loadedamount, 100 ng.

[26]. The first group of amino acids, Val 6, Arg 21, Pro 22, Leu 23,
Ly 34 and Phe 53, is on the left, while the second group, Val 41,
Val 42, Leu, 48 and Lue 50, is on the right. The tubular structure
represents the protein backbone, and side chains of selected aminoacid residues (Arg 21, Lys 34 and Phe 53) are shown. The peptide

Table 1. Retention factors and enantioseparation factors of benzodiazepines and 2-arylpropionic acidderivatives on OMTKY domain colunmsa.

―５―Chromatography, Vol.23 No.1 (2002)

Table 2. Retention factors and enantioseparation factors of benzodiazepines and 2-arylpropionic acidderivatives on OGCHI domain colunms.

Figure 8. Enantiomers of U-80413 in enantioselective binding siteof OMTKY3.

Figure 7. Molecular modeling simulation of U-80413 enantiomers
bound to OMTKY3.
Ligands are labeled according to their R or S chirality
and numbered according to their position among the 100
lowest energy minimized binding orientations.

the proximity of the phenyl group of (R) -U-80413 and Phe 53.
However, neither the first, second nor a combination domain of
OMTKY gave appreciable chiral recognition ability [26].

strand is wrapped around an α-helix and held in place by three di-

Recently, we isolated and characterized a new protein from

sulfide bonds. The N -terminal is at the top right and the C-terminal

chicken egg whites [27, 28]. It was termed ovoglycoprotein fromis in the back. The dockings in the second site to the right were ofchicken egg whites (OGCHI). In addition, it was found that 10% of

higher energy and without apparent points of interaction that could

OGCHI was included in crude OMCHI preparations [28]. More-produce chiral recognition. This site could account for the nonspe-

over, OMCHI and OGCHI columns were made from isolated, purecific binding site. The selected, specific binding model for each ofproteins and were compared with regard to chiral recognition abili-the (R) - and (S) -U-80413 with OMTKY3 is shown in Figure 8

ties. It was found [28] that the chiral recognition ability of OMCHI[26]. One can see similarities and differences in orientation and in-reported previously [25] came from OGCHI, and that OMCHI had

termolecular interactions between the (R) - and (S) -U-80413. The

no chiral recognition ability. Further, it was found that OGCHI iscarboxyl groups of each enantiomer engage in electrostatic interac-

preferentially bound to DSC-activated aminopropyl-silica gelstions with the positive charge on Arg 21. The carbonyl group on U-

compared with OMCHI, even though the average molecular mass

80413’s central ring share a hydrogen bond with the NH3+ group ofof OGCHI and OMCHI are 30,000 and 27,000, respectively [29,

Lys 34. The distinguishing difference between the enantiomers is30]. This is why CSPs based on crude OMCHI, which are now―６―Chromatography, Vol.23 No.1 (2002)

Jun Haginaka

OGCHI existed on the protein domain of OGCHI.

commercially available, show moderate chiral recognition ability.
The effects of the sugar moieties of OGCHI on the enantio-

OGCHI was bound to aminopropyl-silica gels via an amino or

separations of various solutes were investigated. The retentive andcarboxyl group(s) of OGCHI [34]. The OGCHI materials prepared

enantioselective properties of the OGCHI, asialo OGCHI and

via a carboxyl group(s) of OGCHI are suitable for chiral resolution

asialo-agalacto OGCHI columns were compared. Generally, re-of acidic solutes, and those via an amino group(s) of OGCHI are

moval of a sialic acid or sialic acid-galactose group of OGCHI re-suitable for chiral resolution of basic solutes. It was suggested that

sulted in no change or an increase of the retention factors and enan-electrostatic interaction between an amino or carboxyl group oftioseparation factors of solutes tested. It was concluded that sialic

OGCHI and a charged solute should play an important role inacid and sialic acid-galactose groups of OGCHI did not participate

chiral recognition of the solute [34]. Further, chiral recognitionin chiral discrimination of the solutes tested [31]. Moreover, pd-

properties of OGCHI and ovoglycoprotein from Japanese quail egg

OGCHI and completely deglycosylated OGCHI (cd-OGCHI) werewhites (OGJPQ) were compared [35]. The average molecularobtained by treatments of OGCHI with N -glycosidase, and a mix-

masses of OGCHI and OGJPQ were estimated to be about 30,000

ture of endoglycosidase and N -glycosidase, respectively [32, 33].

and 27,400. The OGCHI column is suitable for chiral resolution of

The average molecular masses of pd-OGCHI and cd-OGCHI werebasic compounds, while the OGJPQ column is suitable for that ofestimated to be about 28,400 and 21,400, respectively. It was foundacidic compounds. With regard to chiral resolution of neutral com-that the pd-OGCHI column showed excellent chiral recognition

pounds, it is dependent on the compound to be resolved which col-abilities comparable to the OGCHI column, and that the retentivity

umn could be suitable. The results obtained revealed that chiraland enantioselectivity of basic solutes tested on the pd-OGCHI col-recognition of various solutes should be efficiently attained by us-

umn were higher than those on the OGCHI column, while those ofing both columns complementarily.

acidic solutes on the pd-OGCHI column were lower. Further, cd3.4. Chiral stationary phases based on other proteins

OGCHI still showed chiral recognition abilities for various solutes

CSPs based on lysozyme [36] and pepsin [37] were developed

tested. These results revealed that the chiral recognition site(s) for

Figure 9. Schematic diagram of preparation of non-covalent MIPs.

―７―

Chromatography, Vol.23 No.1 (2002)by us. By using a mixture of phosphate buffer and organic modifier

glycol dimethacrylate (EDMA) as the functional monomer andas an eluent, basic and uncharged enantiomers were resolved oncrosslinker, respectively. The retentive and enantioselective proper-

both CSPs, while no resolution of acidic enantiomers was ob-

ties of these two drugs and their structurally related compounds onserved. Further, the pepsin and OMCHI were mixed-immobilized

the MIPs were evaluated using an aqueous eluent. Hydrophobic

onto the same porous aminopropyl-silica gels [38]. The retentive

and ionic interactions could mainly be responsible for the retention

and enantioselective properties of the mixed-protein-based CSP

and enantioseparation of propranolol and chlorpheniramine in an

were compared with those of pepsin- and OMCHI-based CSPs.

aqueous eluent. The MIP showed the highest recognition for the

The pepsin-OMCHI-based CSP showed a similar enantioselectivity

template molecules and slight recognition for their structurally re-with the pepsin-based CSP because OMCHI has no enantioselec-

lated compounds. Further, the MIPs for 2-arylpropionic acid de-tivity. In addition, the pepsin-OMCHI-based CSP was more stablerivatives, (S) -naproxen [46-48] and (S) -ibuprofen [49] were pre-than the pepsin-based CSP for repetitive injections of samples andpared using 4-vinylpyridine (4-VPy) and EDMA as the functional

continuous flow of an eluent. The pepsin-OMCHI-based CSP wasmonomer and crosslinker, respectively, by a two-step swelling andfurther stabilized by crosslinking with glutaraldehyde [38].

polymerization method. The obtained MIP for (S) -naproxen,
evaluated in aqueous eluent, gave similar enantioselectivity fornaproxen, compared with that prepared by the bulk polymerization

4. Uniformly sized molecularly imprinted polymers
Since molecular imprinting techniques can afford complemen-

method and evaluated in an organic eluent [47]. The baseline reso-

tary binding site(s) for a template molecule (Figure 9), the MIPs

lution of naproxen enantiomers was attained by optimization of theare used for chromatographic separations, SPEs, membranes,

preparation method of the uniformly sized MIP and separation pa-

antibody-mimics and sensors for the purpose of specific recognition of the target molecule [39, 40]. Usually, non-aqueous bulk polymerization methods have been utilized to obtain MIPs. The disadvantage of the method is that the obtained block polymers had tobe crushed, ground and sieved to produce packing materials. The
MIPs obtained are unsuitable for HPLC packing materials owing torandom shape and size distribution. Uniformly sized MIPs havebeen prepared through a combination of a typical multi-step swelling and polymerization method and molecular imprinting technique [41, 42]. The advantages of the method are as follows: it iseasy to prepare uniformly sized and monodispersed particles, easy
to perform in situ modification, and suitable for preparing HPLC
packing materials. We have prepared uniformly sized MIPs for basic and acidic drug enantiomers, and applied the obtained MIPs forresolution of drug enantiomers by HPLC.
Recently, selective enrichment and pretreatment of analytes incomplex matrixes have been attained with SPE based on MIPs. The
SPE based on MIPs has been generally carried out by an off-line
mode. We have prepared a RAM-MIP, a uniformly sized MIP selectively modified with a hydrophilic external layer, through a
combination of molecular imprinting and hydrophilic surface
modification techniques. Further, the obtained RAM-MIP for 2arylpropionic acid derivatives was applied for the direct serum injection assays of the drug by a column-switching system, consist-

Figure 10. Separation of naproxen enantiomers on the MIP for (S)-naproxen.
HPLC conditions: column size, 4.6 mm i.d. × 100 mm;
column temperature, 70°
C; eluent, 20 mM phosphatebuffer (pH 3.2)/acetonitrile = 50:50 (v/v); detection,
220 nm; flow rate, 0.2 mL min-1. Loaded amount, 250ng.

ing of a RAM-MIP and a conventional C18-silica column.
4.1. Chiral separation using molecularly imprinted polymers
We prepared uniformly sized MIPs for (S) -propranolol [43,44] and (S) -chlorpheniramine [45] by a two-step swelling and polymerization method using methacrylic acid (MAA) and ethylene―８―Chromatography, Vol.23 No.1 (2002)

Jun Haginaka

Figure 11. Effect of eluent pH on the retention properties of (S) -ibuprofen, benzoic acid, benzenesulfonic acid and benzene on non-imprinted
EDMA (A), styrene-EDMA (B) and 4-VPy-EDMA (C), and (S) -ibuprofen imprinted 4-VPy-EDMA (D) materials.
Keys: ○-○，(S) -ibuprofen; △-△, benzoic acid; ■-■, benzenesulfonic acid; ×-×, benzene. HPLC conditions: column size,
4.6 mm i.d. × 100 mm; column temperature, 25°
C; eluent, 20 mM phosphoric acid and/or sodium phosphate/acetonitrile = 60:40(v/v); detection, 220 nm; flow rate, 1.0 mL min-1. Loaded amount, 250 ng.

rameters such as flow rate and column temperature (Figure 10)

the EDMA and styrene-EDMA materials among the eluent pHs[48].

tested. It is thought that the apparent pKa value of benzenesulfonic
The retentive and enantioselective mechanism of the MIP foracid is < 2.3. On the other hand, benzenesulfonic acid was retained(S) -ibuprofen was precisely examined in aqueous media. Figureon the non-imprinted and (S) -ibuprofen imprinted 4-VPy-EDMA

11, parts A, B, C and D, shows the effects of eluent pH on the re-materials by decreasing the eluent pH. The retention of benzenesul-

tention properties of (S) -ibuprofen, benzoic acid, benzenesulfonic

fonic acid was due to ionic interactions of sulfonyl groups of ben-acid and benzene on non-imprinted EDMA, styrene-EDMA and 4-

zenesulfonic acid with the positively charged 4-VPy-EDMA mate-

VPy-EDMA, and (S) -ibuprofen imprinted 4-VPy-EDMA materi-rials. It was reported that the apparent pKa value of the pyridinylals, respectively [49]. Since the first two polymers had no ionizablegroup of the matrix was ~ 4.7 [50]. However, the retention data ofgroups in the polymer backbone, solutes were mainly retained withbezenesulfonic acid suggested the shift of the average apparent

hydrophobic interactions. Benzenesulfonic acid was not retained on

pKa value of 4-VPy-EDMA materials to < 3. Benzoic acid was―９―Chromatography, Vol.23 No.1 (2002)

Figure 12. Chromatograms of standard ibuprofen sample (A), control plasma sample (B), and control plasma sample
spiked with ibuprofen (C) using column-switching techniques.
HPLC conditions; Precolumn, RAM-MIP for (S) -ibuprofen (10 mm × 4.0 mm i.d); analytical column,
Cosmosil 5C18-MS (150 mm × 4.6 mm i.d.); eluent for pretreatment, 20 mM phosphoric acid-acetonitrile(78:22 (v/v), pH 2.24) at 1.0 mL min-1 for 5 min; eluent for analysis, 20 mM sodium phosphate bufferacetonitrile (75:25 (v/v), pH 7.34) at 1.0 mL min-1; detection, UV absorbance at 223 nm. Ibuprofen concentration is 5.0 µg mL-1 in (A) and (C).

more retained by hydrogen bonding interaction with a pyridinyl

dard ibuprofen sample (5.0 µg mL-1), control plasma sample andgroup on the non-imprinted and (S) -ibuprofen imprinted 4-VPy-

control plasma sample spiked with 5.0 µg mL-1 of ibuprofen, re-

EDMA materials than the EDMA and styrene-EDMA materials.

spectively, using column-switching techniques. Figure 12 illus-

The retention factor of (S) -ibuprofen increased with the order

trates the fact that ibuprofen is separated from the ordinary compo-

EDMA, 4-VPy-EDMA, styrene-EDMA and (S) -ibuprofen im-

nents of plasma samples, and that ibuprofen is almost completely

printed 4-VPy-EDMA materials. (S) -Ibuprofen could be retained

recovered from the serum samples. Further, (S) -naproxen, imprint

mainly with hydrophobic interactions with EDMA and styrene-

species, appeared on a chromatogram. However, it was completely

EDMA materials, and hydrophobic and hydrogen bonding interac-

separated from ibuprofen on a C18 column.

tions with 4-VPy-EDMA materials. Further, (S) -ibuprofen was

The developed method should have wide applicability for the

more retained by the molecular imprinting effect on the (S) -determination of a drug in biological fluids.

ibuprofen imprinted 4-VPy-EDMA materials than the nonimprinted ones.

5. Conclusions

4.2. Restricted access media-molecularly imprinted polymers

rum injection assays of drugs and their metabolites, LC packing

The author has prepared LC packing materials for direct seWe have prepared a RAM-MIP material, a uniform-sized MIP

materials for resolution of enantiomeric drugs, and uniformly sizedfor (S) -naproxen selectively modified with a hydrophilic external

MIPs for drugs and their metabolites. These materials could be use-layer, through a combination of molecular imprinting and hydro-

ful for specific purposes; for the assays of drugs and their metabo-philic surface modification techniques [51, 52]. Further, we pre-lites and resolution of enantiomeric drugs. However, we requirepared RAM-MIP materials for (S) -naproxen and -ibuprofen, andfurther studies to clarify the chiral recognition site(s) and mecha-

tried to apply the respective RAM-MIP for direct serum injection

nism on OGCHI. In addition, MIPs for hydrophilic compounds,

assays of the drug by column-switching system, consisting of theproteins and peptides are the targets to be pursued. In the future, we

RAM-MIP material and a conventional C18-silica column. How-

will need further LC packing materials for high-performance sepa-

ever, leakage of the imprint molecule prevented accurate and pre-rations as well as for selective separations.

cise assays of the drug. Thus, the RAM-MIP material for (S) -Finally, the author would like to thank many people for theirnaproxen was applied for the assays of ibuprofen in rat plasma

help, discussions and encouragement. First of all, he thanks the[52]. Figure 12, parts A, B and C, shows chromatograms of stan-people who studied and are studying in his laboratory. Dr. H. Mat-

―１０―

Chromatography, Vol.23 No.1 (2002)

Jun Haginaka

215-223.

sunaga, Ms. H. Sanbe and Ms. C. Kagawa as colleagues in the[21] Matsunaga, H.; Fu, Q.; Haginaka, J. Anal. Sci. 2002, 18, 27-

laboratory are greatly appreciated for their talented assistance.

30.

Also, thanks are due to many coworkers for their help and discussions; Dr. T. Pinkerton (Pharmacia, Kalamazoo, MI, USA), Prof. J.

[22] He, X. M.; Carter, D. C. Nature 1992, 358, 209-215.

Markley (University of Wisconsin-Madison, Madison, WI, USA),[23] Kremer, J. M. H.; Wilting, J.; Janssen, L. H. M. Pharmaol.
Rev. 1988, 40, 1-40.

Prof. N. Tanaka (Kyoto Insitute of Technology, Kyoto, Japan),Prof. K. Hosoya (Kyoto Insitute of Technology), Dr. K. Kimata[24] Haginaka, J; Matsunaga, H. Enantiomer 2000, 5, 37-45.

(Nacalai Tesque, Kyoto) and Dr. H. Wada (Shinwa Chemical In-[25] Miwa, T.; Ichikawa, M.; Tsuno, M.; Hattori, T.; Miyakawa,

dustries, Kyoto). Last but not least the author thanks Professor T.

T.; Kayano, M.; Miyake, Y. Chem. Pharm. Bull. 1987, 35,

Uno (Professor Emeritus, Kyoto University, Kyoto, Japan) and

682-686.
[26] Pinkerton, T. C.; Howe, W. J.; Urlich, E. L.; Comiskey, J.

Professor T. Nakagawa (Kyoto University) for a lot of support at

P.; Haginaka, J.; Murashima, T.; Walkenhorst, W. F.; Wes-

the start of my scientific career.

tler, W. M.; Markley, J. L. Anal. Chem. 1995, 67, 23542367.

References[1]

Haginaka, J. Trends in Anal. Chem. 1991, 10, 17-22.

[2]

Hagestam, I. H.; Pinkerton, T. C. Anal. Chem. 1985, 57,[27] Haginaka, J.; Seyama, C.; Murashima, T. J. Chromatogr.
1995, 704, 279-287.
[28] Haginaka, J.; Seyama, C.; Kanasugi, N. Anal. Chem. 1995,

1757-1763.
[3]

67, 2539-2547.

Nakagawa, T.; Shibukawa, A; Shimono, N.; Kawashima, T.;[29] Haginaka, J.; Takehira, H. J. Chromatogr. A 1997, 773, 85-

Tanaka, H.; Haginaka, J. J. Chromatogr. 1987, 420, 297-

91.

311.

[30] Haginaka, J.; Takehira, H. J. Chromatogr. A 1997, 777, 281[4]Haginaka, J.; Yasuda, N.; Wakai, J.; Matsunaga, H.; Yasuda,[5]

Kimura, Y.; Yasuda, N. Agric. Biol. Chem. 1989, 53, 497-

[31] Haginaka, J.; Matsunaga, H. Chirality 1999, 11, 426-431.

504.

[32] Haginaka, J.; Matsunaga, H. Anal. Commun. 1999, 36, 39-[6]

Haginaka, J.; Wakai, J.; Yasuda, N.; Yasuda, H.; Kimura, Y.

-287.

H.; Kimura, Y. Anal. Chem. 1989, 61, 2445-2448.

41.
[33] Haginaka, J.; Matsunaga, H.; Kakehi, K. J. Chromatogr. B

J. Chromatogr. 1990, 515, 59-66.
[7].

2000, 731, 223-229.

Haginaka, J.; Wakai, J.; Yasuda, H.; Kimura, Y. J. Chroma-[34] Haginaka, J.; Okazaki, Y.; Matsunaga, H. J. Chromatogr. A

togr. 1990, 529, 455-461.
[8]

1999, 840, 171-181.

Haginaka, J.; Wakai, J. Chromatographia 1990, 29, 223-

[35] Haginaka, J.; Kagawa, C.; Matsunaga, H. J. Chromatogr. A

227.
[9]

1999, 858, 155-165.

Haginaka, J.; Wakai, J.; Yasuda, Y. J. Chromatogr. 1990,[36] Haginaka, J.; Murashima, T.; Seyama, C. J. Chromatogr. A

535, 163-172.

1994, 666, 203-210.

[10] Haginaka, J.; Wakai, J.; Yasuda, Y. J. High Resolut. Chro-[37] Haginaka, J.; Miyano, Y.; Saizen, Y.; Seyama, C.; Mu-

matogr. 1991, 14, 291-293.

rashima, T. J. Chromatogr. A 1995, 708, 161-168.

[11] Haginaka, J.; Wakai, J. J. Chromatogr. 1992, 596, 151-156.
[12] Haginaka, J.; Wakai, J. Anal. Sci. 1992, 8, 141-144.

[38] Haginaka, J.; Miyano, Y. Anal. Sci. 1996, 12, 727-732.

[13] Haginaka, J.; Wakai, J. Anal. Chem. 1990, 62, 997-1000.

[39] Takeuchi, T.; Haginaka, J. J. Chromatogr. B 1999, 728, 120.

[14] Haginaka, J.; Wakai, J. Anal. Sci. 1992, 8, 137-140.

[40] Haginaka, J. BioSeparation, in press.

[15] Haginaka, J.; Murashima, T.; Hiroya, F.; Wada, H. J. Chro-[41] Hosoya, K.; Yoshizako, K.; Tanaka, N.; Kimata, K.; Araki,

matogr. 1993, 620, 199-204.

T.; Haginaka, J. Chem. Lett. 1994, 1437-1438.

[16] Haginaka, J. Trend Glycosci. Glycotech. 1997, 9, 399-407.

[42] Hosoya, K.; Yoshizako, K.; Kimata, K.; Tanaka, N.; Hagi-[17] Haginaka, J. J. Chromatogr. A 2001, 906, 253-273.

naka, J. J. Chromatogr. A 1996, 728, 139-147.

[18] Haginaka, J. In Encyclopedia of Separation Science; Wil-[43] Haginaka, J.; Sakai, Y.; Narimatsu, S. Anal. Sci. 1998, 14,son, I. D.; Adlard, E. R.; Cooke, M; Poole, C. F., Eds.; Aca-

823-826.

demic Press: London, 2000; 2397-2406.

[44] Haginaka, J.; Sakai, Y. J. Pharm. Biomed. Anal. 2000, 22,[19] Haginaka, J.; Kanasugi, N. J. Chromatogr. A 1995, 694, 71-

899-907.

80.

[45] Haginaka, J.; Kagawa, C. J. Chromatogr. A, in press.

[20] Haginaka, J.; Kanasugi, N. J. Chromatogr. A 1997, 769,

―１
１―

Chromatography, Vol.23 No.1 (2002)

1999, 857, 117-125.

[46] Haginaka, J.; Takehira, H.; Hosoya, K.; Tanaka, N. Chem.

[50] Ramstrom, O; Ye, L; Gustavsson, P. E. Chromatographia

Lett. 1997, 555-556.

1998, 48, 197-202.

[47] Haginaka, J.; Takehira, H.; Hosoya, K.; Tanaka, N. J. Chro-[51] Haginaka, J.; Takehira, H.; Hosoya, K.; Tanaka, N. J. Chro-matogr. A 1998, 816, 113-121.

matogr. A 1999, 849, 331-339.

[48] Haginaka, J.; Sanbe, H. J. Chromatogr. A 2001, 913, 141-[52] Haginaka, J.; Sanbe, H. Anal. Chem. 2000, 72, 5206-5210.

146.
[49] Haginaka, J.; Sanbe, H.; Takehira, H. J. Chromatogr. A―１２―