Prevention of co-elution of steroid sulfates with serum proteins from pre-column in column-switching HPLC system.
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 ion-pair 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 microl) 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/microl 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.
Tagawa
N
N
Clinical Chemistry Laboratory, Kobe Pharmaceutical University, Japan.
Tsuruta
H
H
Fujinami
A
A
Kobayashi
Y
Y
D001798:Blood Proteins / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019314:Dehydroepiandrosterone Sulfate / Q000097:blood* / Q000737:chemistry; D004964:Estriol / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry; D006801:Humans; D011284:Pregnenolone / Q000097:blood* / Q000737:chemistry; D000644:Quaternary Ammonium Compounds
D001798:Blood Proteins; D000644:Quaternary Ammonium Compounds; C018370:pregnenolone sulfate; C027974:estriol 3-sulfate; D019314:Dehydroepiandrosterone Sulfate; D011284:Pregnenolone; C009405:tetrabutylammonium; D004964:Estriol