Analysis of steroids in yeast-mediated cell culture by on-line solid-phase extraction coupled high-performance liquid chromatography electrospray-ionization/mass spectrometry and novel continuous postcolumn infusion of internal standard technique.
The reduction of 17-ketosteroid estrone or androstenedione to corresponding 17alpha- and 17beta-estradiol or testosterone and epitestosterone has been performed with Saccharomyces cerevisiae. In the analysis of the cell culture, the solid-phase extraction (SPE) method was on-line coupled to high-performance liquid chromatography electrospray-ionization/mass spectrometry (HPLC-ESI/MS) for sample pretreatment to eliminate the complicated matrix interference and preconcentrate of the analytes before chromatographic separation. A novel quantification method with the continuous postcolumn infusion of internal standard was developed for the determination of substrate and products. This novel quantitative method can stabilize and enhance the ionization of all analytes during analysis. The HPLC-ESI/MS analysis of estrone, 17alpha-, and 17beta-estradiol was operated with a negative ion mode and the analysis of androstenedione, testosterone, and epitestosterone was operated with a positive ion mode. The optimal concentration of the internal standard progesterone with the continuous postcolumn infusion technique was 3 microg mL(-1) for estrogen analysis and 1 ng mL(-1) for androgen analysis and both were at a constant infusion rate of 0.5 microL min(-1). All of the linear correlation coefficients of the standard calibration curves were over 0.99 and had a linear range from 0 to 50 ng mL(-1). The limit of detections (LODs) and the limit of quantitations (LOQs) for steroids analyzed were from 0.12 to 0.36 ng mL(-1) and from 0.4 to 1.2 ng mL(-1), respectively. The analysis accuracies and precisions were better than 94% and lower than 8.8% R.S.D., respectively. The developed method for the analysis of steroids in the cell culture was successful.
Cheng
Cheanyeh
C
Department of Chemistry, Chung Yuan Christian University, 200 Chung-Pei Road, Chungli 32023, Taiwan, ROC. chengce@cycu.edu.tw
Tsai
Hsiang-Rong
HR
D000735:Androstenedione / Q000032:analysis* / Q000378:metabolism; D016477:Artifacts; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004970:Estrone / Q000032:analysis* / Q000378:metabolism; D010084:Oxidation-Reduction; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004970:Estrone; D000735:Androstenedione