There are 3 three method previously applied to Determine pregnenolone and 17 -hydroxypregnenolone: the use of gas chromatography–mass spectrometry (GC–MS), immunoassay. However, the GC–MS method requires a derivatization procedure, while immunoassay is difficult to optimize for simultaneous determination of both analytes.HPLC system is used to separate Pregnenolone and 17 alpha -hydroxypregnenolone(do not absorb UV radiation) that are converted to progesterone and 17 -hydroxyprogesterone (absorb UV radiation), respectively at the pre-oxidase column.
UV-detector: 240 nm, Linear relationship in the range of 0.4–10 ug/ml for pregnenolone and 0.3–10 g/ml for 17 alpha -hydroxypregnenolone.
a correlation coefficient of 0.9993 and 0.9998
Pre-column reaction
The cholesterol oxidase used for this synthesis(enzymatic conversion) is 3-β-HSD
The advantages of this methods
- eliminate the derivatization procedure as done in GC-MS
- short detection time about 15min
- large linear range(0.4-10 ug/ml and 0.3-10 ug/ml) so it is possible to make a calibration curve
The drawbacks
The system for separation is quite complicated. It requires two pumping systems.
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