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Shiseido's Carbohydrate Analysis
Osamu Shirota, Ph.D.
Vice Chief Researcher
Laboratory of Chromatographic Technologies
Shiseido Research Center
【Fig. 1】  Carbohydrates in strong base
【Fig. 1】 Carbohydrates in strong base
Fig. 2  Detection limit of glucose
【Fig. 2】 Detection limit of glucose

Since carbohydrates have no chromophore to be utilized in detection, they could only be detected with a refractive index (RI) detector. The RI detector, however, has several disadvantages, such as low selectivity, low sensitivity, and inapplicability to gradient elution. Therefore, derivatization chemistry have been extensively explored for optical detections of carbohydrates. In particular, a method using fluorescent derivatization and a laser-induced fluorescence detection allowed attomole-level detection1. However, there are still a lot of demands for direct analysis of carbohydrates in nanomole to picomole regions without complex pretreatment. In response to these demands, Shiseido developed a system3 dedicated for carbohydrate analysis, using "SUCREBEAD I, a column for carbohydrate analysis" and a "pulsed amperometric detector (PAD)"2 for analyzing underivatized carbohydrates with high sensitivity.

Hydroxyl groups of carbohydrates are known to dissociate in a strongly basic environment, and then have anionic properties (Fig. 1). The resultant negatively charged carbohydrates can be separated in anion exchange mode. SUCREBEAD I allows the use of strong base and is packed with strong anion exchanging polymer with uniform particle size. The column has a inner diameter of 2 mm (semi-microcolumn), and allows high-sensitivity detection. PAD can detect the electrochemically active hydroxyl groups on a gold electrode. (PAD and SUCREBEAD can be incorporated into any HPLC system to build the carbohydrate system.)

Figure 2 shows an brief idea of detection limit of unmodified glucose. At a concentration of 50 ppb, a 20-microlitter injection resulted in a recognizable peak (1 ng, equivalent to 5.6 pmol). In comparison with a method with an RI detector and a conventional normal phase NH2 column, this amount corresponds to a sensitivity increase of 10,000 fold.

Figure 3(c) shows another example4 of PAD applications, analyzing ibuprofen metabolites in urine. Here, PAD was connected in series with a UV detector ( Fig. 3(a)). The system distinguishes metabolites with conjugated glucuronic acid from other substances.

 

(a) System drawing of UV and PAD in series
   【Fig. 3】 Analysis of ibuprofen metabolites
     (a) System drawing of UV and PAD in series
(c) Overlayed chromatogram
   【Fig. 3】 Analysis of ibuprofen metabolites
   (c) Overlayed chromatogram
(b) Metabolic pathway of ibuprofen
【Fig. 3】
Analysis of ibuprofen metabolites
(b) Metabolic pathway of ibuprofen

(d) Analytical conditions
【Fig. 3】
Analysis of ibuprofen metabolite
(d) Analytical conditions
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