A sensitive approach for simultaneous quantification of carbonyl and hydroxyl steroids using 96-well SPE plates based on stable isotope coded-derivatization-UPLC-MRM: method development and application

Abstract

Steroid hormones are crucial substances that mediate a wide range of vital physiological functions. Because of the important biological significance of steroids, this paper presents a new targeted metabolic method based on adding stable isotope tags to hydroxyl containing and carbonyl containing steroid hormones with two pairs of synthesized derivatization reagents: deuterium 4-(dimethylamino)-benzoic acid (D 4 -DMBA), and D 5 -Girard P (D 5 -GP) using of ultra performance liquid chromatography-multiple reaction monitoring (UPLC-MRM). Firstly, an Oasis PRiME hydrophilic-lipophilic balance (HLB) 96-well solid phase extraction plate was used to pretreat a number of biological samples simultaneously. Secondly, hydroxyl and carbonyl steroids were labeled using two pairs of synthetic reagents, namely DMBA and D 4 -DMBA, and GP and D 5 -GP, respectively. Thirdly, the mixed products were detected using UPLC-MRM and the mass spectroscopy conditions were optimized. Methodology development showed that the sensitivity was enhanced 1 to >500-fold. Finally, the new method was applied to analysis of urine samples of healthy males, females and rats. The results revealed that the method can be sensitive and reliable for simultaneous quantification of steroid hormones containing hydroxyl and carbonyl groups in 12 min in a single run. This method provided a powerful tool for studying the metabolic mechanism of steroids and contributed to the development of targeted metabolomics.

Challenges and recent advances in quantitative mass spectrometry‐based metabolomics

“…Many studies have used CIL to quantify metabolites in complex biological matrices. Liu et al 88 used this approach to quantify steroids in rat urine with LC-MRM. Two different reagents, 4-dimethylaminobenzoic acid and Girard's reagent P were used for derivatization, with their deuterated analogues for quantitation.…”

“…Many studies have used CIL to quantify metabolites in complex biological matrices. Liu et al 88 . used this approach to quantify steroids in rat urine with LC‐MRM.…”

“… 83 Many techniques have been developed to address this problem, including the use of chemical derivatization to improve the detectability of some more problematic substances and enable their analysis using more generic workflows. Some compounds lend themselves particularly well to derivatization, and this technique is now common for the analysis of amino acids, 84 , 85 , 86 steroids 87 , 88 and short‐chain fatty acids (SCFA), 89 , 90 , 91 among others. In addition to these advantages, incorporating isotope labels through the derivatization process provides access to a quantitative dimension.…”