Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography–triple quadrupole linear ion trap mass spectrometry

Abstract

Background The determination of steroids is important for the diagnosis and monitoring of endocrine diseases and infertility workup. We developed a rapid and reliable mass spectrometric method for the simultaneous quantification of steroid patterns in human serum. Methods An on-line solid phase extraction (SPE)-liquid chromatography–triple quadrupole linear ion trap (LC-QTrap) method utilizing atmospheric pressure chemical ionization was developed. Following protein precipitation of 100 µL serum, on-line SPE and chromatographic separation was performed for 13 steroids in 1.8 min. Analytes were confirmed by the characteristic fragment patterns. Results The total run time of the method was 4 min. Detection limits ranged between 0.02 µg/L (testosterone) and 9 µg/L (dehydroepiandrosterone sulfate). The method was linear up to 7000 µg/L for dehydroepiandrosterone sulfate, 500 µg/L for cortisol, 125 µg/L for 11-deoxycortisol, and 25 µg/L for aldosterone, 17-hydroxyprogesterone, progesterone, testosterone, androstenedione and β-estradiol, respectively. Accuracy ranged between 80 and 114%. Between-day variance at three different concentration levels was < 15%. Excellent correlations with immunoassays were observed for testosterone, cortisol and β-estradiol with Pearson's correlation coefficient r = 0.967, 0.963, and 0.998, respectively. Conclusion The novel on-line SPE-LC-MS/QTrap platform offers a very fast, reliable, and sensitive quantification of steroid patterns and fulfils the quality criteria for routine laboratory application.