Fig. 1 Representative calibration line and curve for HPLC and for microbiological assay methods. Left; Hayakawa K, Fujita T. Analysis of chondroitin sulfate in pharmaceutical preparations by high-performance liquid chromatography. Bunseki Kagaku (in Japanese) 35: 207-210, 1986. Right; Waller JR. Increased sensitivity of the microbiological assay for biotin by Lactobacillus plantarum. Appl. Microbiol. 20: 485-491, 1970.

Fig. 2 Inspirational graphic representation to show that the 1000-fold differences occur in the determination values between HPLC method and microbiological photometrical method for biotin. As shown in Fig. 4,, photometric assay gives about 3-fold lower values as compared to HPLC method; i.e., HPLC method measures chromatographically purified molecule, and photometric method measures non purified solution. Furthermore, method of biotin assay by using rat and egg-white by Umetaro Suzuki, which uses no photometer, gave the similar value to HPLC method as described in Hayakawa K, Katsumata N, Hirano M, Yoshikawa K, Ogata T, Tanaka T. Nagamine T. Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column. J Chromatogr B 869; 93-100, 2008.

Attenuation 2 Attenuation 2 Attenuation 3 Fig. 3 Typical separation chromatograms of fucoidan analysis by the Develosil 300 Diol-5 column (60 x 8.0 mm I.D.). Flow-rate was 0.8 mL/min. Detection was UV at 210 nm. Chart-speed was 5 mm/min. Left chromatogram; Analysis of 0 control. There was no peak at position Vo. Center chromatogram; A 0.005 mL of 0.01 mg/mL of Natural-fucoidan was injected; i.e., 50ng of Natural-fucoidan was injected. Fucoidan was appeared at the position of Vo (indicated by the arrow). Right chromatogram; A 0.005 mL of 0.01 mg/mL of fluorescent-fucoidan was injected; i.e., 50ng of fluorescent-fucoidan was injected.　　Fluorescent-fucoidan showed a 5.2-fold higher sensitivity than underivatized fucoidan at 210 nm. It is noteworthy that Brij-58 of Sigma Co. gives ghost peak (due to contamination of the unknown UV 210 absorbing high-molecular weight molecule at Vo), and calibration curve can not insert to zero. In this case, two-points calibration line should be used. 0 ng (control) 50 ng fucoidan 50 ng fluorescent-fucoidan

Photometric method (at visible 754 nm) At 1.0 mg/mL HPLC method (at UV 210 nm) Fig. 4 Comparison of the transported values as assessed by HPLC and photometric methods. HPLC method was done with the Shimadzu HPLC system. Photometric method was done with the Shimadzu UV-VIS Spectrophotometer (Model Uvmini-1240). Mean values (n = 2) were indicated. Photometric method (at visible 754 nm)