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Shiseido's HTS and High-Sensitivity LC-MS Analysis
Osamu Shirota, Ph.D.
Vice Chief Researcher
Laboratory of Chromatographic Technologies
Shiseido Research Center
Shiseido's HTS autosampler was designed to correspond to such a need. A maximum six plates of 96-well microplates are contained in a cooled region, and samples are run with a rapidly moving robot arm.
In the mid 1990s, "combinatorial chemistry"1) , a method to prepare a lot of candidate substances and test one after another rapidly, became the major process of drug development. High-throughput screening (HTS), computational chemistry, network formation by experimental facilities, etc., were developed in response and promoted the rise of combinatorial chemistry, leaving behind the traditional means of studying compounds one by one in the initial stage of drug development. High-throughput screening (HTS) mainly relying on biochemical means is to be also realized by chemical instrumental analysis to cover the entire drug development process.
Sample container closed Sample container opened
In rapid HPLC analysis, the NANOSPACE2) designed for semi-microcolumn (small diameter) demonstrates its ability to reflect the separation efficiency of the column to the final chromatogram, without any large instrumental band-broadening. Measurements even in a relatively short time with a short column provide large numbers of theoretical plates. For example, separation performance of CAPCELL C18 MG 3 micron, the phase for rapid separation, can be fully observed with the NANOSPACE (Figs. 1 and 2). This characteristic works very advantageously in a mass spectrometer, one of the flow- sensitive detectors (where the amount of chemical species supplied in a unit of time is proportional to the sensitivity).
【Fig. 1】  Flow rate and number of theoretical plates of CAPCELL PAK C18 MG S3
【Fig. 1】  Flow rate and number of theoretical plates of CAPCELL PAK C18 MG S3
【Fig. 1】 Flow rate and number of theoretical plates of CAPCELL PAK C18 MG S3
【Fig. 2】  Analysis time cut by 75%
【Fig. 2】 Analysis time cut by 75%
Using the LC-MS-MS with electrospray ionization (ESI), peak intensities were compared between another company's semi-microcolumn LC and Shiseido's NANOSPACE under the same conditions (Fig. 3). The comparison shows the advantage of NANOSPACE. This means that extra-column diffusion is minimized in NANOSPASE.
  Column: CAPCELL PAK C18 MG S5 2 mm i.d 20 mm
  Sample: Reserpine
【Fig. 3 】 Comparison in LC-MS-MS
   【Fig. 3】 Comparison in LC-MS-MS
【Fig. 4】  Analysis of methamphethamine optical isomers
【Fig. 4】  Analysis of methamphethamine optical isomers
【Fig. 4】  Analysis of methamphethamine optical isomers
    【Fig. 4】 Analysis of methamphethamine optical isomers
Semi-microcolumn LC, either by single column or column switching method, can provide the concentrating effect. In LC-MS, the concentrating effect reduces the relative background generated from the mobile phase, too. Several challenging applications have already been reported to date, such as analyses of environmental hormones in river water3), blockers (drug) in serum4), and abused stimulants in urine5).。
Another characteristic of the NANOSPACE is its performance against carryover (influence of former samples). Because of the spread of high- sensitivity LC-MS technology, carryover of a level not observed with conventional detection methods has become a serious problem. The HTS autosampler is equipped with four types of washing mechanisms as a solution to this problem. Use of these washing mechanisms reduces the influence of one injection on the next to a minimum6) (Fig. 5). This performance plays a very important role in conducting high-sensitivity analysis with LC-MS. Shiseido has studied a number of anti-carryover measures, and examined other companies' LC systems using various substances. Fig. 6 shows representative examples. Even though the degree of superiority of HTS autosampler differs depending on the substance, Shiseido's superiority has been confirmed in every comparison. We are still studying ways to improve the performance to cope with future development of the detector.
【Fig. 5】  Minimization of carryover by washing function 【Fig. 5】  Minimization of carryover by washing function
【Fig. 6】  Reduction of carryover in LC-MS analysis 【Fig. 6】  Reduction of carryover in LC-MS analysis
【Fig. 6】 Reduction of carryover in LC-MS analysis
1) Dolle, R.E., Nelson, K.H., J. Comb. Chem. 1999, 1, 235-282
2) Osamu Shirota, Review of Progress, Analysis 1997, 1.56
3) Akira Motoyama, Ayako Suzuki, Osamu Shirota, Ryujiro Namba,  Rapid Commun. Mass. Spec. 13, 2204-2208 (1999)
4) Akira Motoyama, Ayako Suzuki, Osamu Shirota, Ryujiro Namba J.Pharmaceutical Biomedical Analysis 28(2002)97-106
5) Muneo Katagi, Mayumi Nishikawa, Michiaki Tatsuno, Tadashi Miyazawa, Hitoshi Tsuchikashi, Ayako Suzuki, Osamu Shirota, Jpn. J. Toxicol. Environ. Health, 44(2) 107-115 (1998)
6) Yuki Togashi, Kaneko, Aya Ohkubo, Yutaka Ohtsu, Osamu Shirota, 2530-10, abstract of Pittcon 2003
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