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Shiseido’s Pretreatment Strategy
Taketoshi Kanda, Ph.D.
Senior Researcher
Chromatography Team
Frontier  Science Laboratory
Shiseido Research Center
HPLC is widely used to measure drug concentrations in pharmaceuticals or in physiological liquids, such as blood, urine or saliva. In order to quantify a target substance in a complex matrix, pretreatments such as deproteinization, solvent extraction or concentration are necessary. The pretreatment process is, however, troublesome and time consuming, and can result in errors; therefore, a simple error-free method is required. A direct injection method and an automated sample pretreatment method for biological samples are described here.
【Fig. 1】
【Fig. 1】
Packing material designed for direct injection of biological samples
Proteins contained in in serum or plasma can cause serious problems in quantifying drugs. In general, direct injection of biological samples containing a large amount of proteins results in increased column pressure or deteriorated column performance, necessitating pretreatment such as deproteinization. A protein-coated ODS column1) is available to resolve these problems.

The protein-coated ODS column is prepared by coating the micro-porous ODS silica surface (6 nm,I.D.) with denatured plasma proteins. The column first lets proteins in an injected sample pass through by size exclusion mode using the proteins' large molecular size (proteins and other large molecules, do not enter micropores), and retains relatively small drug molecules or their metabolites by hydrophobic interaction (small molecules can diffuse unto the micropores.) However, since the packing was prepared by physical interactions with the proteins, the proteins are gradually eluted from the column. In addition, the pores of the packing were partially lossed upon the protein coationg, which makes it difficult to obtein a high separation efficiency.

【Fig. 2】 Inner surface reversed-phase packing material
【Fig. 2】 Inner surface reversed-phase packing material

Pinkerton et al.2) developed so-called "inner surface reversed-phase" type column that overcame the defect in the protein-coated ODS column (see Fig. 2). The packing material is prepared by modifying glycyl-L-phenylalanyl-L-phenylalanine (tripeptide), and then hydrolyzing with carboxypeptidase A, which is a macromolecule that cannot penetrate into the fine pores. Both types of packing materials have hydrophilic outer surfaces (causing no adsorption of proteins) and hydrophobic inner surfaces. Several other attempts have been reported , such as the dual zone type developed by Williams et al.3) and the semi-permeable surface type developed by Gluntz et al.4). The sealed hydrophobic phase type, another unique idea, was developed by Gisch et al.5), This is a monophasic packing material having a network structure of polyoxyethylene whose inner and outer surfaces of micropores contain hydrophobic groups. The biphasic packing material controls the penetration of proteins into the monopores using the characteristic large molecular nature of proteins, while in the monophasic packing material, even though proteins penetrate into the moicro pores, they elute without interacting with the hydrophobic group due to the network structure of the hydrophilic polyoxyethylene.

While several different attempts had been reported for treating protein-containing samples, Shiseido developed "CAPCELL PAK MF" based on its polymer coating technology that eliminates undesirable secondary effects of silica and enhances durability. The packing material is prepared by coating silica with the silicone polymer, then introducing hydrophilic and hydrophobic groups in a specific ratio. This is called mixed-function (MF) phase. CAPCELL PAK MF has the same surface in its inner and outer surfaces, as shown in Fig. 3 6,7). To provide an appropriate hydrophobicity accommodating various analyses, three types of MF phases, C1, Phenyl and C8 are available. Figure 4 shows chromatograms indicating elution behaviors obtained with these three types.
【Fig. 3】Mechanism of CAPCELL PAK MF packing material
【Fig. 4】
【Fig. 4】 Elution behavior obtained with three types of CAPCELL PAK MF columns
Chromatograms of human serum spiked with (1) phenobarbital (20 μg/mL), (2) carbamazepine (5 μg/mL) and (3) phenytoin (40 μg/mL) on (a) MF C1, (b) MF-Ph1 and (c) MF C8 packing materials. HPLC conditions: column size, 150 mm × 4.6mm I.D.; mobile phase, 100 mM phosphate buffer (pH 6.9): acetonitrile (80: 20, v/v); detection, 254 nm; injection volume, 20 μL.
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