Fig. 1. Structures of cholesterol and stigmasterol used as an internal standard (I.S.).

Fig. 2. Cyclic voltammograms in acetonitrile-2-propanol (9:1, v/v) containing 50 mM LiClO₄ in the absence (A) or presence of 0.45 mM of cholesterol (B): scan rate, 0.1 V/s; working electrode, glassy carbon; counter electrode, platinum wire; reference electrode, Ag/AgCl.

Fig. 3. Hydrodynamic voltammograms of cholesterol and oxysterols. Cholesterol (●), (24S)-24-OH-Chol (○), 26-OH-Chol (■), or 4β-OH-Chol () dissolved in the mobile phase (20 μM each except 150 μM of 4β-OH-Chol) was injected into HPLC-ED. HPLC conditions used were: degasser, DG-980-50 vacuum degasser; pump, PU-880 pump; injector, 7125 Rheodyne injector fitted with a 5 μl injection loop; column, Develosil C30-UG-3 microbore column (150 mm × 4.6 mm i.d., 3 μm); column oven, CTO-10AS column oven; detector, LC-4C electrochemical detector; mobile phase, acetonitrile-2-propanol (9:1, v/v) containing 50 mM LiClO₄; flow rate, 1 ml/min; and column temperature, 20 °C. To measure the oxidation current of cholesterol and oxysterols, charging current (background) at each potential was automatically subtracted to zero after stabilization for 30–60 min. The values of charging current subtracted were: 1.635 μA at 1.5 V vs. Ag/AgCl; 2.318 μA at 1.6 V vs. Ag/AgCl; 3.536 μA at 1.7 V vs. Ag/AgCl; 5.030 μA at 1.8 V vs. Ag/AgCl; 7.005 μA at 1.9 V vs. Ag/AgCl; and 9.440 μA at 2.0 V vs. Ag/AgCl.

Fig. 4. Chromatogram of cholesterol, (24S)-24-OH-Chol, 26-OH-Chol, and 4β-OH-Chol. These sterols were dissolved in the mobile phase (40 μM each except 150 μM of 4β-OH-Chol) was injected into HPLC-ED. HPLC conditions used are the same as in Fig. 3.

Fig. 5. Chromatogram of TC obtained from the human serum (SRM909b, Level 1). The preparation of the serum was described in Section 2. HPLC conditions used are the same as in Fig. 3.

Linear range of calibration curve, detection limit, and relative standard deviation of cholesterol by the present method

To draw the calibration curve, various concentrations of cholesterol and 50 μM of stigmasterol (I.S.) were injected into HPLC-ED. The value of y was calculated on the basis of the ratio of the current peak heights of cholesterol and I.S. r indicates the correlation coefficient. RSD indicates the relative standard deviation. To obtain RSD, 100 μM of cholesterol was repeatedly injected into HPLC-ED (n = 8).

Effects of minor changes of applied potential and flow rate on current peak height ratio of cholesterol and I.S. and retention time of cholesterol

Cholesterol (100 μM) and 50 μM of stigmasterol (I.S.) in the mobile phase were injected into HPLC-ED (n = 3). HPLC conditions used are the same as in Fig. 3 except for the applied potentials and the flow rate.

Contents of TC and FC in Control Serum II Wako/B and recovery of TC and FC from the plasma spiked with standards

For the recovery test, each concentration of cholesteryl linoleate (for TC) or cholesterol (for FC) was spiked into the plasma and analyzed as described in Section 2.

Contents of TC and FC in SRM909b (Levels 1 and 2) and recovery of TC and FC from the plasma spiked with standards

For the recover test, each concentration of cholesteryl linoleate (for TC) or cholesterol (for FC) was spiked into the plasma and analyzed as described in Section 2.