Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Prevention of Co-elution of Steroid Sulfates with Serum Proteins from Pre-column in Column-switching HPLC System
Noriko TAGAWAHisao TSURUTAAya FUJINAMIYoshiharu KOBAYASHI
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1998 Volume 21 Issue 11 Pages 1211-1214

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Abstract
A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ionpair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 μl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25 μl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.
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© The Pharmaceutical Society of Japan
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