Steroids were extracted from the
medium (1 ml) with 3 ml methylene-chloride (GC) or 3 ml
methylene-chloride}methanol (9 :1) extraction (TLC). For
HPLC analysis, samples were diluted ®ve times with methanol}
water (70:30) and ®ltered (0¡À45 lm). Steroids were
analysed by HPLC [column, reversed phase Lichrosorb
10RP18, SS 250¬3 mm (Varian Chrompack International)],
UV#&% detection [liquid phase, methanol}water (60: 40),
35 ¡ãC], GC [column, J&W DB-5MS 30 m¬0¡À25 mm
i.d.¬0¡À25 lm(Alltech) : pre-column, 2 m¬0¡À25 mmi.d. (Inter-
Sciences) : FID-40 detection at 300 ¡ãC] or TLC [Kieselgel 60
F#&% 10¬20 cm (Merck) in toluene}ethylacetate 1: 1]. Retention
times for HPLC (¡¥ow rate 0¡À8 ml min−" were
tR,*OHAD¡¥4¡À2 min, tR,ADD¡¥7¡À0 min and tR,AD¡¥10¡À5 min, and
for GC (carrier N#, 100 kPa) tR,AD¡¥10¡À8 min and tR,ADD¡¥11¡À1
min. Retention factors for TLC were RF,*OHAD¡¥0¡À23,
RF,ADD¡¥0¡À44 and RF,*AD¡¥0¡À56. Substrates used (AD,
9OHAD, ADD) were supplied by Diosynth.
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