内源性甾体激素GC/MS分析中酮基衍生规律的研究

Study on the Keto Groups Derivatization of Endogenous Steroid Hormones with GC/MS

本实验以甲氧胺(MOX)和N-甲基-N-(三甲基硅烷)三氟乙酰胺(MSTFA)为衍生化试剂,采用肟化-硅烷化方法对12种内源性甾体激素进行衍生,通过GC/MS解析鉴定衍生产物结构,研究甾体激素的衍生规律。结果表明,肟化-硅烷化反应可以提高甾体激素的信号,但孕酮(P)、雄烯二酮(ASD)、雌酮(E1)、睾酮(T)、皮质甾酮(B)、可的松(E)受共轭结构影响,产生多种同分异构体。[M-31]^+和[M-15]^+,[M-90]^+和[M-72]^+可分别作为肟化和硅烷化反应的鉴定依据。在80℃,加热40 min,酮基与MOX的摩尔比为1∶20是肟化反应的最佳条件。采用单反应离子检测(SRM)模式,衍生后甾体激素的检测灵敏度可达1μg/L,线性范围为1~200μg/L。该方法可为相关激素类成分的分析提供检测技术支持。

Steroid hormones are a kind of aliphatic hydrocarbon compounds.They are synthesized in central or peripheral nervous system which are considered as potential biomarkers.High sensitivity method was required for steroid hormones detection for their poor responses and low physiological concentrations.Gas chromatography-mass spectrometry(GC/MS)combined with pre-column derivatization is commonly used in detection of compounds such as steroid hormones.In this work,derivation of oximation conditions were studied based on 12 steroid hormones.Oximation-silylation method was applied for the derivatization of dehydroeppiandrosterone(DHEA),dihydrotestosterone(DHT),androsterone(ADL),epiandrosterone(Epian),pregnenolone(Preg),testosterone(T),estrone(E1),androstenedione(ASD),corticosterone(B),cortisone(E),progesterone(P)and allopregnanolone(AP).Twelve endogenous steroid hormones were processed with methoxyamine(MOX)and N-methyl-N-(trimethylsilane)trifluoroacetamide(MSTFA).Oximation-silanization reactions were carried out on ketone group and hydroxyl group separately which could avoid enolate reaction of ketone group.When processed with MOX,characteristic peaks of[M-31]^+and[M-15]^+were observed in the electron bombardment(electron-impact,EI)mass spectra,mainly due to the loss of[—OCH3]and[—CH3]groups.Therefore peaks of[M-31]^+and[M-15]^+could be considered as the identification for ketoximation.At the same time,characteristic peaks of[M-73]^+,[M-90]^+due to the loss of[—Si(CH3)3]or[—O—Si(CH3)3]groups from[M]^+were observed when steroid hormones were further silylated with MSTFA.Peaks of[M-73]^+and[M-90]^+could be considered as the basis for hydroxysilylation.Preg,DHEA,AP,ADL,Epian,DHT generated single derivatization with good peak shape.For the existence of conjugated groups in P,ASD and T structures,isomer products generated after derivatization.The ion peak shape was poor and noise was high.For B,and E six derivatives were observed in each derivatives after reaction.Then oximation conditions for endogenous steroid hormones were optimized.GC/MS detection method was obtained after optimization at 80℃ for 40 min with the molar ratio of ketone group to MOX 1∶20.With selective reaction monitoring mode(SRM),the detection sensitivity level could achieve to 1μg/L,and the linear range was 1-200μg/L.When processed with oximation and silylation,the response values of steroid hormones were greatly improved.GC/MS detection with oximation-silanization is suitable for the detection of steroid hormones without conjugated groups.This study can provide a detection method for the analysis of related hormonal components.