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Shiseido's Semi-Microcolumn LC
Since the 1990s, the development of new drugs has shown a tendency toward drugs designed to act in a lower concentration. Also, there are increasing environmental concerns and requests to quantify lower-concentration substances. In liquid chromatography, a response to such social needs is required. However, the superior sensitivity of small-bore columns is for absolute amounts of substances; therefore, the idea of "reducing the column bore" is incompatible with that of "dealing with diluted samples (to increase the injection amount for increased sensitivity)." From this viewpoint, Shiseido proposes the "column switching method" capable of utilizing the concentrating effect of the semi-microcolumn LC, by improving the columns and constitution of the devices according to the target compound. Fig. 5 shows an example of the column switching method wherein a pre-treatment "CAPCELL PAK MF"8),9) column with a larger inner diameter having a protein removal function, the "CAPCELL PAK C18" of a semi-microcolumn of a reversed-phase system, and an intermediate column between the two columns are combined10). Through this method, biological samples can be directly injected into the pretreatment column, and unwanted high molecular-weight components, such as proteins, can be removed by the size-exclusion principle. Then, target compounds are introduced into the semi-microcolumn. Mobile phases are arranged so that the targert comounds are forcussed on the top of the semi-microcolmn. The general idea of this method is that the "dirty components" are taken care of in the inexpensive pretreatment column with a limited separation efficiency, while only the "refined" fraction is fed to the semi-microcolumn for the final separation.
With the rising abuse of controlled drugs among young people, the authorities are trying to develop quick and highly sensitive methods for analyzing drugs in urine in order to cope with the current situation. The Narcotics Control Department of the Ministry of Health, Labor & Welfare and Shiseido developed direct analysis of methamphetamine (ME) in urine using semi-microcolumn LC and the column switching method, and obtained results enabling quantification with high sensitivity. As a further problem, two stereoisomers (d-type for abuse purposes and l-type for medical treatment) of ME had to be clearly distinguished from each other. Fig. 6 shows a part of the successful results (using a photodiode array detector12) and using a mass spectrometer13)) for discriminating the stereoisomers by using a CHIRAL DRUG, the column especially for chiral separation of ME.

Through launching of the "NANOSPACE", both the instrumental and column technologies have been improved, and semi-microcolumn LC is now being a matured method. The high sensitivity earned from the concentrating effect of semi-micro LC is an attraction not found in the conventional LC. Shiseido, as stated above, has succeeded in minimizing the dead volume the instrument system, and has made it possible to keep a satisfactory peak profile even for those with small retention volumes. These features can lead to high-quality separations in "high-speed analysis" (small-retention-volume analysis), too.
【Fig. 5】  Column switching system for direct analysis of biological samples
【Fig. 5】 Column switching system for direct analysis of biological samples 10)
【Fig. 6】  Analysis of methamphetamine’s optical isomers
【Fig. 6】 Analysis of methamphetamine's optical isomers
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5) O. Shirota and H. Otsu: Shiseido Research Center, unpublished data
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7) O. Shirota and H. Otsu: Abstracts of liquid chromatography research group, p. 10 (1995)
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12) A. Suzuki, O. Shirota, Y. Ohtsu, Y. Makino, S. Ohta, T. Nagano : Chromatography, 17, 21 (1997).
13) Y. Togashi and O. Shirota: Shiseido Research Center, unpublished data.
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